LINGO-1 Structure
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 60/765,443, filed
February 3, 2006, which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
This disclosure relates to LINGO-1 polypeptides, LINGO-1 polypeptide/ligand
complexes, crystals of LINGO-1 polypeptides, crystals of LINGO-1 polypeptide/ligand
complexes, and related methods and software systems.
BACKGROUND
• LINGO-1 (LRR and Ig domain-containing, Nogo Receptor-interacting protein) is a
transmembrane coreceptor with p75. The coreceptors, with the Iigand binding Nogo-66 receptor
(NogoR), make up the Nogo receptor complex, which is commonly located on neurons.
Disabling the Nogo receptor complex in which LINGO-1 is located can induce myelin and nerve
fiber growth. LINGO-1 has been shown to be active in myelin-making cells known as
oligodendrocytes, and inhibition of LINGO-1 has also been shown to induce myelin formation.
Thus LINGO-1 signaling is predicted to be a negative regulator of myelin production.
SUMMARY
In one aspect, the invention features a crystallized LINGO-1 polypeptide.
In another aspect, the invention features a crystallized polypeptide-ligand complex that
includes a LINGO-1 polypeptide and a ligand that is an agonist or an antagonist of the LINGO-1
polypeptide.
In yet another aspect, the invention features a method that includes using a three-
dimensional model of a LINGO-1 polypeptide to design an agent that binds the LINGO-1
polypeptide.
In a further aspect, the invention features a method that includes using a three-
dimensional model of a LINGO-1 polypeptide-ligand complex to design an agent that binds the
LINGO-1 polypeptide.

In another aspect, the invention features a method that includes using a three-dimensional
model of a complex including a LINGO-1 polypeptide to design an agent that binds the LINGO-
1 polypeptide.
In another aspect, the invention features a method that includes selecting an agent by,
e.g., performing rational drug design with a three-dimensional structure of a LINGO-1
polypeptide, or a LINGO-1 polypeptide-ligand complex; contacting the agent with the LINGO-1
polypeptide; and detecting an ability of the agent to bind the LINGO-1 polypeptide. In
embodiments, the method further includes one or more of: obtaining a supplemental crystalline
complex that includes the LINGO-1 polypeptide and the agent; determining the three-
dimensional structure of the supplemental crystalline complex; selecting a second agent by, e.g.,
performing rational drug design with the three-dimensional structure of the supplemental
crystalline complex; contacting the second agent with the LINGO-1 polypeptide; and/or
detecting the ability of the second agent to bind the LINGO-1 polypeptide. The method can
further include one or more of: synthesizing the second agent; detecting an ability of the second
agent to inhibit LINGO-1 activity; and/or detecting an ability of the second agent to increase
myelin levels in vitro or in vivo.
la embodiments, the LINGO-1 polypeptide in the compositions and methods disclosed
herein includes (or consists essentially of) a leucine-rich repeat (LRR) domain, an
immunoglobulin-like (Ig-like) domain, and/or a stalk domain, or a combination thereof. In other
embodiments, the LINGO-1 polypeptide includes (or consists essentially of) the amino acid
sequence from a mammalian {e.g., human) or nonmammalian origin; for example, the LINGO-1
polypeptide can include (or consist of) the amino acid sequence of SEQ ID NO:1, or a variant
thereof (e.g., a conservative susbstitution thereof). In other embodiments, the LINGO-1
polypeptide is crystallized, e.g., is capable of diffracting X-rays, to a resolution of at least about
3.7 A. In embodiments, the crystallized LINGO-1 polypeptide has a space group a P21212 or
1222. In other embodiments, the crystallized LINGO-1 polypeptide has unit cell dimensions
chosen from one or more of: a=201.5 A, b=149.7 A, c=157.5 A, and α=β=γ= 90°, a=148.7 A,
b=158.6 A, c=200.0 A, and α=β=γ= 90°, and α=149.6 A, b=157.3 A, c=200.3 A, and/or α=β=γ=
90°. In yet another embodiment, the crystallized LINGO-1 polypeptide has a three-dimensional
structure that includes the structural coordinates according to Table 2, +/- a root mean square

deviation for alpha carbon atoms of not more than 1.5 A. In another embodiment, the
crystallized LINGO-1 polypeptide has a three-dimensional structure that includes the structural
coordinates of an atom chosen from one or more atoms of amino acids Aspl3, Arg20, Arg22,
Arg43, Glu60, Glu62, Leu94, Leul20, Metl23, Hisl76, Tyrl42, Hisl45, Hisl85, His209,
His233, Ala238, Trp346, Arg347, Asn349, Asn351, Arg352, Gln353, Phe396, Arg408, Leu420,
Leu426, Phe438, Asp440, Arg446, Tyr447 and/or Ile459 of the LINGO-1 polypeptide as set forth
in SEQ ID NO:l.
In other embodiments, the agent designed or screened using the methods disclosed herein
inhibits LINGO-1 activity. For example, the agent can bind LENGO-1 at a ligand binding site
and/or interfere with an interaction between LINGO-1 and a ligand. In embodiments, the ligand
binding site is located on a concave surface of an LRR domain, e.g.y a surface of the LRR
domain that includes one or more of Trp346 and Arg352; Hisl85, His209 and His233; Aspl3;
Glu60 and Glu62; or Arg20, Arg22 and Arg43, according to SEQ ID NO: 1. In other
embodiments, the ligand binding site is located on a convex surface of an LRR domain, e.g., a
surface of the LRR domain that includes one or more of Tyrl42 and Hisl45; Leu94, Leul20, and
Met l23;His l76; or Ala238, according to SEQ ID NO:1. In other embodiments, the ligand
binding site is located on an Ig domain, e.g., a domain that includes one or more of Arg446 and
Tyr447; Axg408, Phe438 and Asp440; Phe396; or Leu420, Leu426 and Ile459, according to SEQ
ID NO:1.
In embodiments, the methods disclosed herein include providing a composition including
a LINGO-1 polypeptide, ora LINGO-1 polypeptide-ligand complex, and/or crystallizing the
composition to form a crystalline complex that includes the LINGO-1 polypeptide, or the
LINGO-1 polypeptide-ligand complex. The crystalline complex can diffract X-rays to a
resolution of at least about 3.7 A. The methods disclosed herein can additionally include one or
more of the following steps: calculating a distance between an atom of the LINGO-1
polypeptide and an atom of the agent; determining the interaction of the agent with the LINGO-1
polypeptide; comparing the interaction of the agent with the LINGO-1 polypeptide to an
interaction of a second agent with the LINGO-1 polypeptide; selecting the agent via computer
modeling; synthesizing the agent; detecting the ability of the agent to inhibit LINGO-1 activity;
and/or detecting the ability of the agent to increase myelin formation in vitro or in vivo.

In another aspect, the invention features an agent designed or selected using the methods
disclosed herein.
In another aspect, the invention features a software system that includes instructions for
causing a computer system to accept information relating to the structure of a LINGO-1
polypeptide, accept information relating to a candidate agent, and determine binding
characteristics of the candidate agent to the LINGO-1 polypeptide. The determination of
binding characteristics can be based on the information relating to the structure of the LINGO-1
polypeptide, and the information relating to the candidate agent.
In yet another aspect, the invention features a computer program residing on a computer
readable medium having a plurality of instructions stored thereon, which, when executed by one
or more processors, cause the one or more processors to accept information relating to the
structure of a complex comprising a LINGO-1 polypeptide, accept information relating to a
candidate agent, and determine binding characteristics of the candidate agent to the LINGO-1
polypeptide. The determination of binding characteristics can be based on the information
relating to the structure of the LINGO-1 polypeptide and the information relating to the
candidate agent.
In another aspect, the invention features a method that includes accepting information
relating to the structure of a LINGO-1 polypeptide and modeling the binding characteristics of
the LINGO-1 polypeptide with a candidate agent. The method can be implemented by a
software system.
In yet another aspect, the invention features a computer program residing on a computer
readable medium having a plurality of instructions stored thereon, which, when executed by one
or more processors, cause the one or more processors to accept information relating to a structure
of a LINGO-1 polypeptide and model the binding characteristics of the LINGO-1 polypeptide
with a candidate agent.
In another aspect, the invention features a software system that includes instructions for
causing a computer system to accept information relating to a structure of a LINGO-1
polypeptide and model the binding characteristics of the LINGO-1 polypeptide with a candidate
agent.
In a further aspect, the invention features a method of modulating LINGO-1 activity in a
subject that includes: selecting, e.g., using the methods described herein (e.g., by rational drug

design), an agent that is capable of modulating; LINGO-1 activity and administering an effective
amount of the agent to the subject, such that the LINGO-1 activity is modulated.
In another aspect, the invention features a method of treating a subject having a condition
associated with LINGO-1 activity that includes: selecting, e.g., using the methods described
herein (e.g., by rational drug design), an agent that is capable of affecting LINGO-1 activity and
administering a therapeutically effective amount of the agent to a subject in need thereof.
In yet another aspect, the invention features a method of prophylactically treating a
subject susceptible to a condition associated with LINGO-1 activity that includes determining
that the subject is susceptible to the condition associated with LINGO-1 activity, selecting, e.g.,
using the methods described herein {e.g., by rational drug design),an agent that is capable of
effecting LINGO-1 activity, and administering a therapeutically effective amount of the agent to
the subject.
In embodiments, the agent used in the methods disclosed herein, e.g., the therapeutic and
prophylactic methods disclosed herein, inhibits LINGO-1 activity and/or increases myelin levels
in vivo. In embodiments, the condition is a demyelinating disease, e.g., multiple sclerosis.
Other features and advantages of the invention will be apparent from the description,
drawings and claims.
DESCRIPTION OF DRAWINGS
FIG 1 is the amino acid sequence of a histidine-tagged LINGO-1 polypeptide (amino
acids 1-516 plus 6-His-tag) (SEQ ID NO:1). The fragment used for crystallization is underlined.
FIG 2 is a 2fo-fc electron density map for the refined 2.7 A resolution LINGO-1
structure (see also Table I).
FIG 3 is a ribbon diagram of a LINGO-1 monomer.
FIG 4 is a space-filled model of a LINGO-1 monomer. The numbered residues denote
glycosylated asparagine residues.
FIGs. 5A-5C are structure diagrams illustrating the tetrameric organization of the
LINGO-1 crystal structure. FIG 5A is a top view of the tetrameric structure; FIG 5B is a view
of the interacting surfaces between the two monomers; and FIG 5C is a side view of the
tetrameric structure.
FIG 6 is a map of a pSMEG plasmid.

FIGs. 7A-1 to 7A-3 show the sequence of a pSMEG plasmid (SEQ ID NO:2).
FEGs. 8A-8B depict the full length amino acid and nucleotide sequence of human
LINGO-1 (SEQ ID NO:3 and 4, respectively). The coding sequence is encoded by nucleotides
53 to 1915 of SEQ ID NO:4.
DETAILED DESCRIPTION
In general, this disclosure relates to LINGO-1 polypeptides, LINGO-1 polypeptide/ligand
complexes, crystals of LINGO-1 polypeptides, crystals of LINGO-1 polypeptide/ligand
complexes, and related methods and software systems. Without wishing to be bound by theory,
it is believed that crystal structures of LINGO-1 polypeptides and crystals of LINGO-1
polypeptide/ligand complexes can be useful for designing or identifying ligands that can interact
with LINGO-1 polypeptides.
Ligands that interact with LINGO-1 polypeptides can inhibit or increase LINGO-1
activity. As an example, LINGO-1 inhibition has been shown to induce myelin formation.
Therefore, it is believed that identification of LINGO-1 inhibitors may be useful for treatment of
disorders characterized by myelin deficiencies, such as multiple sclerosis, Pelizaeus-
Merzbacher's disease, central nervous system trauma, leukodystrophies such as Krabbe disease
or Canavan Disease, or vitamin B12 deficiencies.
FIG 1 is the amino acid sequence (SEQ ID NO:1) of a fragment of human LINGO-1
polypeptide (LINGO-lK549his6) that includes a leucine-rich repeat (LRR) domain (amino
acids 1-382), an immunoglobulin-like (Ig-like) domain (residues 383-477), a stalk region (amino
acids 478-516), and a hexahistidine tag fused to the C-terminus of the stalk region. The
hexahistidine tag facilitated purification. Not present in the LINGO-lK549his6 polypeptide is a
33 amino acid signal sequence that is found at the N-terminus of the full-length human LINGO-
1. The wildtype sequence also includes a transmembrane domain and a short cytoplasmic tail at
the C-terminus of the protein (Mi et al, Nat. Neurosci. 7:221-228, 2004). FIG 2 is an electron
density map for a LINGO-lK549hiS6 structure solved to 2.7 A resolution (see Table 1). FIG 3 is
a ribbon diagram illustrating the structure of the LINGO-lK549his6 polypeptide. The coordinates
of a crystal structure of the LINGO-lK549his6 polypeptide are provided in Table 2.
To determine the structure of LINGO-1, a human LINGO-1-polypeptide can be prepared
and crystallized as described below. In general, the human LINGO-1 polypeptide can be

prepared as desired. For example, in some embodiments, the human LINGO-1 polypeptide is
expressed from a DNA plasmid. The expression can be driven by a promoter, such as an
inducible promoter or a constitutive promoter, such as a cytomegalovirus promoter. The human
LINGO-1 polypeptide can be expressed as a fusion protein with a suitable tag, such as a
polyhistidine (e.g., hexahistidine), glutathione-S-transferase (GST), myc, HA, Strep, or FLAG
tag. The tag can facilitate isolation of the human LINGO-1 polypeptide from cells, such as from
bacterial cells or from a mammalian cell line. For example, the human LINGO-1 polypeptide
can be expressed in and isolated from Chinese Hamster Ovary (CHO) cells. For example, the
human LINGO-1 polypeptide can be fused to a hexahistidine tag and isolated by contacting a
cell extract with a Nickel resin (e.g., a Nickel-nitrilotriacetic acid (Ni-NTA) resin) to bind the
hexahistidine tag, and then releasing the polypeptide by washing the resin with a buffer
containing imidazole. Further, a fusion LINGO-1 polypeptide can be cleaved at a protease site
engineered into the fusion protein, such as at or near the site of fusion between the polypeptide
and the tag.
The human LINGO-1 polypeptide can be placed in solution for collecting spectral data,
NMR data, or for growing a crystal. For example, the human LINGO-1 polypeptide can be
crystallized in the presence of a salt (e.g., a sodium salt and/or ammonium salt). Crystals can be
grown by various methods, such as, for example, sitting or hanging drop vapor diffusion. In
general, crystallization can be performed at a temperature of from about 4°C to about 60°C (e.g.,
from about 10°C to about 45°C, such as at about 12°C, about 15°C, about 18°C, about 20°C,
about 25°C, about 30°C, about 32°C, about 35°C, about 37°C).
In general, a crystal of the human LINGO-1 polypeptide can diffract X-rays to a
resolution of about 3.7 A or less (e.g., about 3.6 A or less, about 3.5 A or less, about 3.2 A or
less, about 3.0 A or less, about 2.7 A or less, about 2.4 A or less, about 2.3 A or less, about 2.2 A
or less, about 2.1 A or less, about 2.0 A or less, about 1.9 A or less, about 1.8 A or less, about 1.7
A or less, about 1.6 A or less, about 1.5 A or less, or about 1.4 A or less). In some embodiments,
a crystal of the human LINGO-1 polypeptide can diffract X-rays to a resolution of from about
1.7 A to about 3.7 A (e.g., the crystal of the human LINGO-1 polypeptide can diffract X-rays to
about 2.7 A, about 3.5 A or about 3.6 A).
In certain embodiments, a crystal of the human LINGO-1 polypeptide belongs to space
group 1222 with unit cell parameters a=148.7 A, b=158.6 A, c=200.0 A, and α=β=γ=90; In

other embodiments, a crystal of the human LINGO-1 polypeptide belongs to space group 1222
with unit cell parameters a=149.6 A, b=157.3 A, c=200.3 A, and α=β=γ=90°. In other
embodiments, a crystal of the human LINGO-1 polypeptide belongs to space group P21212 with
unit cell parameters a=201.5 A, b=149.7 A, c=157.5 A, and α=β=γ=90°. The space group refers
to the overall symmetry of the crystal, and includes point symmetry and space symmetry. In
certain embodiments, a crystal of the human LINGO-1 polypeptide belongs to space group 1222
and contains two molecules of the human LINGO-1 polypeptide in the asymmetric unit. In other
embodiments, a crystal of the human LINGO-1 polypeptide belongs to space group P21212 and
contains four molecules of the human LINGO-1 polypeptide in the asymmetric unit. The
asymmetric unit is the smallest unit from which the crystal structure can be generated by making
use of the symmetry operations of the space group. A crystal is generally made up of the motif
defined by the space-group symmetry operations on the asymmetric units, and a translation of
that motif through the crystal lattice.
Structural data describing a crystal can be obtained, for example, by X-ray diffraction.
X-ray diffraction data can be collected by a variety of sources, X-ray wavelengths and detectors.
In some embodiments, rotating anodes and synchrotron sources (e.g., Advanced Light Source
(ALS), Berkeley, California; or Advanced Photon Source (APS), Argonne, Illinois) can be used
as the source(s) of X-rays. In certain embodiments, X-rays for generating diffraction data can
have a wavelength of from about 0.5 A to about 1.6 A {e.g., about 0.7 A, about 0.9 A, about 1.0
A, about 1.1 A, about 1.3 A, about 1.4 A, about 1.5 A, or about 1.6 A). In some embodiments,
area detectors and/or charge-couple devices (CCDs) can be used as the detector(s).
X-ray diffraction data of a crystal of the human LINGO-1 polypeptide can be used to
obtain the structural coordinates of the atoms in the complex. The structural coordinates are
Cartesian coordinates that describe the location of atoms in three-dimensional space in relation to
other atoms in the complex. For example, the structural coordinates listed in Table 2 are the
structural coordinates of a crystalline human LINGO-1 polypeptide. The structural coordinates
of Table 2 describe the location of atoms of the human LINGO-1 polypeptide (SEQ ED NO:5),
(SEQ IDMO:6), (SEQ ID NO:7) and (SEQ ID NO:8) in relation to each other. The structural
coordinates can be modified by mathematical manipulation, such as by inversion or integer
additions or subtractions. As such, structural coordinates are relative coordinates. For example,

structural coordinates describing the location of atoms in the human LINGO-1 polypeptide are
not specifically limited by the actual x, y, and z: coordinates of Table 2.
The structural coordinates of the human LINGO-1 polypeptide can be used to derive a
representation (e.g., a two dimensional representation or three dimensional representation) of the
polypeptide or a fragment of the polypeptide. Such representations can be useful for a number of
applications, including, for example, the visualization, identification and characterization of an
active site of the polypeptide. In certain embodiments, a three-dimensional representation can
include the structural coordinates of the human LINGO-1 polypeptide according to Table 2, ± a
root mean square (rms) deviation from the alpha carbon atoms of amino acids of not more than
about 1.5 A [e.g., not more than about 1.0 A, not more than about 0.5 A).
RMS deviation is the square root of the arithmetic mean of the squares of the deviations
from the mean, and is a way of expressing deviation or variation from structural coordinates.
Conservative substitutions (see discussion below) of amino acids can result in a molecular
representation having structural coordinates within the stated rms deviation. For example, two
molecular models of polypeptides that differ from one another by conservative amino acid
substitutions can have coordinates of backbone atoms within a stated rms deviation, such as less
than about 1.5 A (e.g., less than about 1.0 A, less than about 0.5 A). Backbone atoms of a
polypeptide include the alpha carbon (Ca or CA) atoms, carbonyl carbon (C) atoms, and amide
nitrogen (N) atoms.
Various software programs allow for the graphical representation of a set of structural
coordinates to obtain a representation of the human LINGO-1 polypeptide or a fragment of the
polypeptide. In general, such a representation should accurately reflect (relatively and/or
absolutely) structural coordinates, or information derived from structural coordinates, such as
distances or angles between features. In some embodiments, the representation is a two-
dimensional figure, such as a stereoscopic two-dimensional figure. In certain embodiments, the
representation is an interactive two-dimensional display, such as an interactive stereoscopic two-
dimensional display. An interactive two-dimensional display can be, for example, a computer
display that can be rotated to show different faces of a polypeptide or a fragment of a
polypeptide. In some embodiments, the representation is a three-dimensional representation. As
an example, a three-dimensional model can be a physical model of a molecular structure (e.g., a
ball-and-stick model). As another example, a three dimensional representation can be a

graphical representation of a molecular structure (e.g., a drawing or a figure presented on a
computer display). A two-dimensional graphical representation (e.g., a drawing) can correspond
to a three-dimensional representation when the two-dimensional representation reflects three-
dimensional information, for example, through the use of perspective, shading, or the obstruction
of features more distant from the viewer by features closer to the viewer. In some embodiments,
a representation can be modeled at more than one level. As an example, when the three-
dimensional representation includes a polypeptide, such as a human LINGO-1 polypeptide, the
polypeptide can be represented at one or more different levels of structure, such as primary
(amino acid sequence), secondary (e.g., a-helices and (3-sheets), tertiary (overall fold), and
quaternary (oligomerization state) structure. A representation can include different levels of
detail. For example, the representation can include the relative locations of secondary structural
features of a protein without specifying the positions of atoms. A more detailed representation
could, for example, include the positions of atoms.
In some embodiments, a representation can include information in addition to the
structural coordinates of the atoms in the human LINGO-1 polypeptide. For example, a
representation can provide information regarding the shape of a solvent accessible surface, the
van der Waals radii of the atoms of the model, and the van der Waals radius of a solvent (e.g.,
water). Other features that can be derived from a representation include, for example,
electrostatic potential, the location of voids or pockets within a macromolecular structure, and
the location of hydrogen bonds and salt bridges.
An agent that interacts with (e.g., binds) the human LINGO-1 polypeptide can be
identified or designed by a method that includes using a representation of the polypeptide or a
fragment of the polypeptide. Exemplary types of representations include the representations
discussed above. In some embodiments, the representation can be of an analog polypeptide or
polypeptide fragment. A candidate agent that interacts with the representation can be designed
or identified by performing computer fitting analysis of the candidate agent with the
representation. In general, an agent is a molecule. Examples of agents include polypeptides,
nucleic acids (including DNA or RNA), steroids and non-steroidal organic compounds. An
agent that interacts with a polypeptide (e.g., a human LINGO-1 polypeptide) can interact
transiently or stably with the polypeptide. The interaction can be mediated by any of the forces

noted herein, including, for example, hydrogen, bonding, electrostatic forces, hydrophobic
interactions, and van der Waals interactions.
A.s noted above, X-ray crystallography can be used to obtain structural coordinates of the
human LINGO-1 polypeptide. However, such structural coordinates can be obtained using other
techniques including NMR techniques. Additional structural information can be obtained from
spectral techniques (e.g., optical rotary dispersion (ORD), circular dichroism (CD)), homology
modeling, and computational methods (e.g., computational methods that can include data from
molecular mechanics, computational methods that include data from dynamics assays).
In some embodiments, the X-ray diffraction data can be used to construct an electron
density map of the human LINGO-1 polypeptide or a fragment of the polypeptide, and the
electron density map can be used to derive a representation (e.g., a two dimensional
representation, a three dimensional representation) of the human LINGO-1 polypeptide.
Creation of an electron density map typically involves using information regarding the phase of
the X-ray scatter. Phase information can be extracted, for example, either from the diffraction
data or from supplementing diffraction experiments to complete the construction of the electron
density map. Methods for calculating phase from X-ray diffraction data include, for example,
multiwavelength anomalous dispersion (MAD), multiple isomorphous replacement (MIR),
multiple isomorphous replacement with anomalous scattering (MIRAS), single isomorphous
replacement with anomalous scattering (SIRAS), reciprocal space solvent flattening, molecular
replacement, or any combination thereof. These methods generate phase information by making
isomorphous structural modifications to the native protein, such as by including a heavy atom or
changing the scattering strength of a heavy atom already present, and then measuring the
diffractioa amplitudes for the native protein and each of the modified cases. If the position of the
additional heavy atom or the change in its scattering strength is known, then the phase of each
diffracted X-ray can be determined by solving a set of simultaneous phase equations. The
location of heavy atom sites can be identified using a computer program, such as SHELXD
(Bruker-AXS, Madison, WI) or SHELXS (Sheldrick, Institut Anorg. Chemie, Gottingen,
Germany), and XPREP (Bruker-AXS, Madison, WI); and diffraction data can be processed using
computer programs such as MOSFLM, SCALA, SOLOMON, and SHARP ("The CCP4 Suite:
Programs for Protein Crystallography," Ada Crystallogr. Sect. D. 54:905-921.1997; deLa
Fortelle and Brigogne, Meth. Enzym. 276:472-494,1997). The phase of X-ray scatter for a

crystalline human LINGO-1 polypeptide, for example, can be determined by SIRAS using
crystals of a platinum derivative of the LINGO-1 polypeptide. To create a platinum derivative of
a crystalline LINGO-1 polypeptide, the crystalline LINGO-1 polypeptide can be soaked in a
solution containing platinum. Phases obtained by SIRAS from the platinum derivative can then
be refined using, for example, non-crystallographic symmetry (NCS) averaging and phase
extension in a computer program such as DM (Cowtan and Main, Ada Cryst. D49:148-157,
1993). The resulting model can be further derived by molecular replacement with a second data
set. For example, a model derived from a crystalline LINGO-1 polypeptide having space group
1222 can be refined using molecule replacement with a data set from a crystalline LINGO-1
polypeptide having space group P2]2i2. Phases obtained by SIRAS from crystals of the native
crystalline polypeptide and the platinum derivative can then be used to create an electron density
map of the LINGO-1 polypeptide.
The electron density map can be used to derive a representation of a polypeptide or a
fragment of a polypeptide by aligning a three-dimensional model of a polypeptide with the
electron density map. For example, the electron density map corresponding to the native
crystalline human LINGO-1 polypeptide can be aligned with the electron density map
corresponding to the platinum derivative of the crystalline human LINGO-1 polypeptide
complex derived by an isomorphous replacement method.
The alignment process results in a comparative model that shows the degree to which the
calculated electron density map varies from the model of the previously known polypeptide or
the previously known complex. The comparative model is then refined over one or more cycles
(e.g., two cycles, three cycles, four cycles, five cycles, six cycles, seven cycles, eight cycles, nine
cycles, ten cycles) to generate a better fit with the electron density map. Software programs such
as CNS (Brunger et al, Ada Crystallogr. D54:905-921, 1998) and REFMAC (Collaborative
Computational Project, Number 4. The CCP4 suite: Programs for Protein Crystallography, Ada
Crystallogr. D50:760-776, 1994) can be used to refine the model. The quality of fit in the
comparative model can be measured by, for example, an Rfactor or Rfree value. A smaller value of
Rfactor or Rfree generally indicates a better fit. Misalignments in the comparative model can be
adjusted to provide a modified comparative model and a lower Rfactor or Rrree value. The
adjustments can be based on information relating to variations of the human LINGO-1
polypeptide (e.g., sequence variation information, alternative structure information, heavy atom

derivative information) as appropriate. As an example, in embodiments in which a model of a
heavy atom derivative of a crystalline LINGO-1 polypeptide is used, an adjustment can include
fitting an approximate model of the native LINGO-1 polypeptide over the model of the heavy
atom derivative. As another example, in certain embodiments, an adjustment can include
replacing an amino acid in the previously known LINGO-1 polypeptide with an amino acid
having a similar structure (a conservative amino acid change). When adjustments to the
modified comparative model satisfy a best fit to the electron density map, the resulting model is
that which is determined to describe the polypeptide or complex from which the X-ray data was
derived. Methods of such processes are disclosed, for example, in Carter and Sweet, eds.,
"Macromolecular Crystallography" in Methods in Enzymology. Vol. 277, Part B, New York:
Academic Press, 1997, and articles therein, e.g., Jones and Kjeldgaard, "Electron-Density Map
Interpretation," p. 173, and Kleywegt and Jones, "Model Building and Refinement Practice,"
p. 208.
A machine, such as a computer, can be programmed in memory with the structural
coordinates of the human LINGO-1 polypeptide together with a program capable of generating a
graphical representation of the structural coordinates on a display connected to the machine. A
software system can also be designed and/or utilized to accept and store the structural
coordinates. The software system can be capable of generating a graphical representation of the
structural coordinates. The software system can also be capable of accessing external databases
to identify one or more candidate agents likely to interact with the human LINGO-1 polypeptide.
A machine having a memory containing structure data or a software system containing
such data can aid in the rational design or selection of a human LINGO-1 polypeptide agonist of
a human LINGO-1 polypeptide antagonist. For example, such a machine or software system can
aid in the evaluation of the ability of an agent to associate with the human LINGO-1 polypeptide,
or can aid in the modeling of compounds or proteins related by structural or sequence homology
to the human LINGO-1 polypeptide. As used herein, an agonist refers to a compound that
enhances at least one activity of the human LINGO-1 polypeptide, and an antagonist refers to a
compound that inhibits or counteracts at least one activity of the human LINGO-1 polypeptide.
For example, a compound may function as an antagonist of the human LINGO-1 polypeptide by,
for example, increasing the rate of myelin production by a neural cell, or by inhibiting interaction
of the human LINGO-1 polypeptide with the Nogo receptor complex.

The machine can produce a representation (e.g., a two dimensional representation, a three
dimensional representation) of the human LINGO-1 polypeptide or a fragment of the
polypeptide. A software system, for example, can cause the machine to produce such
information. The machine can include a machine-readable data storage medium including a data
storage material encoded with machine-readable data. The machine-readable data can include
structural coordinates of atoms of the human LINGO-1 polypeptide. Machine-readable storage
media (e.g., data storage material) include, for example, conventional computer hard drives,
floppy disks, DAT tape, CD-ROM, DVD, and other magnetic, magneto-optical, optical, and
other media which may be adapted for use with a machine (e.g., a computer). The machine can
also have a working memory for storing instructions for processing the machine-readable data, as
well as a central processing unit (CPU) coupled to the working memory and to the machine-
readable data storage medium for the purpose of processing the machine-readable data into the
desired three-dimensional representation. A display can be connected to the CPU so that the
three-dimensional representation can be visualized by the user. Accordingly, when used with a
machine programmed with instructions for using the data (e.g., a computer loaded with one or
more programs of the sort described herein) the machine is capable of displaying a graphical
representation (e.g., a two dimensional graphical representation, a three-dimensional graphical
representation) of any of the polypeptides, polypeptide fragments, complexes, or complex
fragments described herein.
A display (e.g., a computer display) can show a representation of the human LINGO-1
polypeptide or a fragment of the human LINGO-1 polypeptide. The user can inspect the
representation and, using information gained from the representation, generate a model of the
human LINGO-1 polypeptide or polypeptide fragment bound to a ligand. The model can be
generated, for example, by altering a previously existing representation of the human LINGO-1
polypeptide. Optionally, the user can superimpose a three-dimensional model of an agent on the
representation of the human LINGO-1 polypeptide. The agent can be an agonist (e.g., a
candidate agonist) of the human LINGO-1 polypeptide. In some embodiments, the agent can be
a known compound or a fragment of a known compound. In certain embodiments, the agent can
be a previously unknown compound, or a fragment of a previously unknown compound.
It can be desirable for the agent to have a shape that complements the shape of the active
site. There can be a preferred distance, or range of distances, between atoms of the agent and

atoms of the human LINGO-1 polypeptide. Distances longer than a preferred distance may be
associated with a weak interaction between the agent and active site (e.g., the active site of the
human LINGO-1 polypeptide). Distances shorter than a preferred distance may be associated
with repulsive forces that can weaken the interaction between the agent and the polypeptide. A
steric clash can occur when distances between atoms are too short. A steric clash occurs when
the locations of two atoms are unreasonably close together, for example, when two atoms are
separated by a distance less than the sum of their van der Waals radii. If a steric clash exists, the
user can adjust the position of the agent relative to the human LINGO-1 polypeptide (e.g., a rigid
body translation or rotation of the agent) until the steric clash is relieved. The user can adjust the
conformation of the agent or of the human LINGO-1 polypeptide in the vicinity of the agent in
order to relieve a steric clash. Steric clashes can also be removed by altering the structure of the
agent, for example, by changing a "bulky group," such as an aromatic ring, to a smaller group,
such as to a methyl or hydroxyl group, or by changing a rigid group to a flexible group that can
accommodate a conformation that does not produce a steric clash. Electrostatic forces can also
influence an interaction between an agent and a ligand-binding domain. For example,
electrostatic properties can be associated with repulsive forces that can weaken the interaction
between the agent and the human LINGO-1 polypeptide. Electrostatic repulsion can be relieved
by altering the charge of the agent, e.g., by replacing a positively charged group with a neutral
group.
Forces that influence binding strength between a candidate agent and the human LINGO-
1 polypeptide, respectively, can be evaluated in the polypeptide/agent model. These can include,
for example, hydrogen bonding, electrostatic forces, hydrophobic interactions, van der Waals
interactions, dipole-dipole interactions, 7r-stacking forces, and cation-7t interactions. The user
can evaluate these forces visually, for example by noting a hydrogen bond donor/acceptor pair
arranged with a distance and angle suitable for a hydrogen bond. Based on the evaluation, the
user can alter the model to find a more favorable interaction between the human LINGO-1
polypeptide and the agent. Altering the model can include changing the three-dimensional
structure of the polypeptide without altering its chemical structure, for example by altering the
conformation of amino acid side chains or backbone dihedral angles. Altering the model can
include altering the position or conformation of the agent, as described above. Altering the
model can also include altering the chemical structure of the agent, for example by substituting,

adding, or removing groups. For example, if a hydrogen bond donor on the human LINGO-1
polypeptide is located near a hydrogen bond donor on the agent, the user can replace the
hydrogen bond donor on the agent with a hydrogen bond acceptor.
The relative locations of an agent and the human LINGO-1 polypeptide, or their
conformations, can be adjusted to find an optimized binding geometry for a particular agent to
the human LINGO-1 polypeptide. An optimized binding geometry is characterized by, for
example, favorable hydrogen bond distances and angles, maximal electrostatic attractions,
minimal electrostatic repulsions, the sequestration of hydrophobic moieties away from an
aqueous environment, and the absence of steric clashes. The optimized geometry can have the
lowest calculated energy of a family of possible geometries for the human LINGO-1
polypeptide/agent complex. An optimized geometry can be determined, for example, through
molecular mechanics or molecular dynamics calculations.-
A series of representations of the human LINGO-1 polypeptide having different bound
agents can be generated. A score can be calculated for each representation. The score can
describe, for example, an expected strength of interaction between the human LINGO-1
polypeptide and the agent. The score can reflect: one of the factors described above that
influence binding strength. The score can be an aggregate score that reflects more than one of
the factors. The different agents can be ranked according to their scores.
Steps in the design of the agent can be carried out in an automated fashion by a machine.
For example, a representation of the human LINGO-1 polypeptide can be programmed in the
machine, along with representations of candidate agents. The machine can find an optimized
binding geometry for each of the candidate agents to an active site, and calculate a score to
determine which of the agents in the series is likely to interact most strongly with the human
LINGO-1 polypeptide.
A software system can be designed and/or implemented to facilitate these steps.
Software systems (e.g., computer programs) used to generate representations or perform the
fitting analyses include, for example: MCSS, Ludi, QUANTA® (macromolecular X-ray
crystallography software), Insight II® (biologicall compound modeling and simulation software),
Cerius2® (modeling and simulation software) , CHARMm® (software for simulation of
biological macromolecules) CHARMm® (software for simulation of biological

macromolecules), and Modeler from Accelrys, Inc. (San Diego, CA); SYBYL® (molecular
modeling software), Unity, FleXX, and LEAPFROG from TRIPOS, Inc. (St. Louis, MO);
AUTODOCK (Scripps Research Institute, La Jolla, CA); GRID (Oxford University, Oxford,
UK); DOCK (University of California, San Francisco, CA); and Flo+ and Flo99 (Thistlesoft,
Morris Township, NJ). Other useful programs include ROCS, ZAP, FRED, Vida, and Szybki
from Openeye Scientific Software (Santa Fe, NM); Maestro, Macromodel, and Glide from
Schrodinger, LLC (Portland, OR); MOE (Chemical Computing Group, Montreal, Quebec),
Allegrow (Boston De Novo, Boston, MA), CNS (Brunger, et al, Acta Crystall. Sect. D 54:905-
921, 1997) and GOLD (Jones et al, J. Mol. Biol 245:43-53, 1995). The structural coordinates
can also be used to visualize the three-dimensional structure of the human LINGO-1 polypeptide
using MOLSCRIPT, RASTER3D, or PYMOL (Kraulis, J. Appl. Crystallogr. 24: 946-950, 1991;
Bacon and Anderson, J. Mol. Graph. 6: 219-220, 1998; DeLano, The PYMOL Molecular
Graphics System (2002) DeLano Scientific, San Carlos, CA).
The agent can, for example, be selected by screening an appropriate database, can be
designed de novo by analyzing the steric configurations and charge potentials of a human
LINGO-1 polypeptide in conjunction with the appropriate software systems, and/or can be
designed using characteristics of known ligands, such as the neurotrophin receptor p75 and the
Nogo receptor (NogoR). The method can be used to design or select agonists or antagonists of
the human LINGO-1 polypeptide. A software system can be designed and/or implemented to
facilitate database searching, and/or agent selection and design. '
Once an agent has been designed or identified, it can be obtained or synthesized and
further evaluated for its effect on the activity of the human LINGO-1 polypeptide. For example,
the agent can be evaluated by contacting it with the human LINGO-1 polypeptide and measuring
the effect of the agent on polypeptide activity. A method for evaluating the agent can include an
activity assay performed in vitro or in vivo.
An activity assay can be a cell-based assay, for example. A cell based assay can include
monitoring the effect of a candidate agent on myelin production. Such assays for human
LINGO-1 polypeptide inhibitors may involve contacting a candidate inhibitor with cells
expressing the human LINGO-1 polypeptide and assaying for an effect on cell morphology. For
example, a neural cell line can be contacted with a candidate agent and the cells monitored for an

effect on oligodendrocyte differentiation. Decreases in LINGO-1 activity have been shown to
increase the differentiation of cells into mature oligodendrocytes, as evidenced by increases in
the length of cell processes (Mi et al., Nat. Neuroscience 8:745-751,2005). LINGO-1 activity
can also be assayed by measuring levels of proteins involved in myelin production, such as
myelin-associated glycoprotein (MAG), a protein expressed at the onset of myelination; myelin
basic protein (MBP), the major protein component of myelin; and other myelin components
including oligodendrocyte-myelin glycoprotein (OMpg), myelin oligodendrocyte glycoprotein
(MOG) and cyclic nucleotide phosphodiesterase (CNPase). LINGO-1 inhibitors have been
shown to promote the upregulation of expression of each of these proteins (Mi et al., Nat.
Neuroscience 8:745-751, 2005). Protein levels can be assayed by standard protein detection
techniques, such as immunohistochemistry by Western blot analysis or in situ hybridization in
cultured cells or whole tissue sections.
Depending upon the action of the agent on the human LINGO-1 polypeptide, the agent
can act either as an agonist or antagonist of human LINGO-1 polypeptide activity. An agonist,
for example, may decrease the rate of myelin production, or increase the binding affinity of the
human LINGO-1 polypeptide to the Nogo receptor complex. Conversely, an antagonist may
increase the rate of myelin production or decrease the binding affinity of the human LINGO-1
polypeptide to the Nogo receptor complex. The agent can be contacted with the human LINGO-
1 polypeptide in the presence of one or more components of the Nogo receptor complex (e.g.,
p75 or Nogo-66) in order to determine whether or not the agent inhibits binding of the human
LINGO-1 polypeptide to the Nogo receptor complex. A crystal containing the human LINGO-1
polypeptide bound to the identified agent can be grown and the structure determined by X-ray
crystallography. A second agent can be designed or identified based on the interaction of the
first agent with the human LINGO-1 polypeptide.
Various molecular analysis and rational drug design techniques are further disclosed in,
for example, U.S. Patent Nos. 5,834,228, 5,939,528 and 5,856,116, as well as in PCT
Application No. PCT7US98/16879, published as WO 99/09148.
While certain embodiments have been described, other embodiments are also
contemplated.

As an example, while embodiments involving the human LINGO-1 polypeptide have
been described, the description herein is more generally directed to any LINGO-1 polypeptide.
A LINGO-1 polypeptide can be a full-length, mature polypeptide, including the full-
length amino acid sequence of any isoform of a LINGO-1 polypeptide. An isoform is any of
several multiple forms of a protein that differ in their primary structure. The full length amino
acid and nucleotide sequences of human LINGO-1 are disclosed in FIGs. 8A-8B.
A LINGO-1 polypeptide can be a fragment of a human LINGO-1 polypeptide, such as a
signal sequence, an LRR-type domain, an Ig-like domain, a transmembrame domain, a
cytoplasmic domain, or a combination thereof. A fragment of a LINGO-1 polypeptide can
include more than 40%, 50%, 60%, 80%, 90% or more of a LINGO-1 polypeptide sequence
{e.g., SEQ ID NO.l). For example, a LINGO-1 polypeptide can include one or more of the
following domains: an LRR-type domain {e.g., about amino acids 1-382 of SEQ ID NO:1), or a
fragment or selected residue thereof {e.g., one or more of amino acids Asp 13, Arg20, Arg22,
Arg43, Glu60, Glu62, Leu94, Leul20, Metl23, Hisl76, Tyrl42, Hisl45, Hisl85, His209,
His233, Ala238, Trp346, Arg347, Asn349, Asn351, Arg352, and/or Gln353, of SEQ ID NO:1);
an Ig-like domain {e.g., about amino acids 383-477 of SEQ ID NO:1), or a fragment or selected
residue thereof {e.g., one or more of amino acids Phe396, Arg408, Leu420, Leu426, Phe438,
Asp440, Arg446, Tyr447 and/or Ile459 of SEQ ID NO:1); and/or a stalk region {e.g., about
amino acids 478-516 of SEQ ID NO:1), or a fragment or selected residue thereof. In other
embodiments, the LINGO-1 polypeptide can contain one or more of the active sites and/or
structural folds, as described herein. The LINGO-1 polypeptide may contain one or more
conservative amino acid substitutions that yield a similar three-dimensional structure, as
described herein. For example, in some embodiments, the LINGO-1 polypeptide (or fragment
thereof) includes up to 5, 10, 20, 30, 50, 75, 100, 150, or 200 or more conservative substitutions
from a predetermined sequence, e.g., a human LINGO-1 amino acid sequence (e.g., SEQ ID
NO: I).
A LINGO-1 polypeptide can have an active site. For example, the LRR-like domain
includes a likely active site of a LINGO-1 polypeptide. In general, an active site can include a
site of ligand binding, or a site of phosphorylation, glycosylation, alkylation, acylation, or other
covalent modification. A site of ligand binding can be a site of a site of binding of a component
of the Nogo receptor complex, or an agonist or antagonist of LINGO-1 activity. An active site

can include a glycosylation site. A ligand binding site can include accessory binding sites
adjacent to or proximal to the actual site of binding that may affect activity upon interaction with
the ligand. Candidate active sites of the human LINGO-1 polypeptide can be identified by
comparing the structure of the LINGO-1 polypeptide with homologous structures of other known
polypeptides. For example, one or more active sites of the human LINGO-1 polypeptide can be
identified by comparing the structure of the LINGO-1 polypeptide with the structure of the
platelet membrane glycoprotein Gplbα A candidate active site on LINGO-1 including Trp346
and Arg352 was identified by its similarity to the structure of a ligand-binding site on Gplbα.
Cavities located on the surface of the human LMGO-1 polypeptide also represent candidate
ligand-binding sites.
An active site of the human LINGO-1 polypeptide can include amino acids of SEQ ID
NO:1 (PIG. 1). For example, an active site of the human LINGO-1 polypeptide can include one
or moreof amino acids Aspl3, Arg20, Arg22, Arg43, Glu60, Glu62, Leu94, Leul20, Metl23,
Hisl76,Tyrl42, Hisl45, Hisl85, His209, His233, Ala238, Trp346, Arg347, Asn349, Asn351,
Arg352,Gln353, Phe396, Arg408, Leu420, Leu426, Phe438, Asp440, Arg446, Tyr447 and
He459 as set forth in the amino acid positions of SEQ ID NO:1. An active site can include a
subset of amino acids located at a particular region of the human LINGO-1 polypeptide. For
example, an active site can be located on the concave surface of the LRR-domain and include
Trp346 and Arg352; His 185, His209, and His233; Aspl3; Glu60 and Glu62; or Arg20, Arg22,
and Arg43 of SEQ ED NO:1. In another example, an active site can be located on the convex
surface of the LRR-domain and include Tyrl42 and Hisl45; Leu94, Leul20, and Metl23;
Hisl76; or Ala238 of SEQ ID NO:1. In another example, an active site can be located on the Ig-
domain and include Arg446 and Tyr447; Arg408, Phe438, and Asp440; Phe396; or Leu420,
Leu426, and IIe459 of SEQ ID NO:1.
The numbering of the amino acids of the human LINGO-1 polypeptide may be different
than that set forth herein, and the sequence of the human LINGO-1 polypeptide may contain
certain conservative amino acid substitutions that yield the same three-dimensional structure.
For example, the numbering of the human LINGO-1 polypeptide may be different than that set
forth in FIG. 1, and the sequence of the human LINGO-1 polypeptide may contain conservative
amino acid substitutions but yield the same structure as that defined by the coordinates of Table
2 and illustrated in FIGs. 2-4 and FIGs. 5A-5C. Corresponding amino acids and conservative

substitutions in other isoforms or analogs are easily identified by visual inspection of the relevant
amino acid sequences or by using commercially available homology software programs (e.g.,
MODELLAR, MSI Management Simulations, Inc., San Diego, CA). The crystal structure
coordinates shown in Table 2 were determined from a crystal having space group P2J2J2, but the
same coordinates +/- a root mean square deviation for alpha carbon atoms of not more than 1.5A,
can correspond to a crystalline LINGO-1 polypeptide having a different space group, such as
1222.
An analog is a polypeptide having conservative amino acid substitutions. A conservative
substitution can include switching one amino acid for another with similar polarity, steric
arrangement, or of the same class (e.g., hydrophobic, acidic or basic), and includes substitutions
having an inconsequential effect on the three-dimensional structure of the human LINGO-1
polypeptide with respect to identification and design of agents that interact with the polypeptide,
as well as for molecular replacement analyses and/or for homology modeling.
Variants or mutants of a LINGO-1 polypeptide can be used in the methods, compositions,
and three-dimensional models disclosed herein. A variant or mutant of a LINGO-1 polypeptide,
or fragment thereof, includes chimeric or fusion proteins, labeled proteins (e.g., radiolabeled
proteins), fusion proteins, mutant proteins, proteins having similar (e.g., substantially similar)
sequences (e.g., proteins having amino acid substitutions (e.g., conserved amino acid
substitutions), deletions, insertions), protein fragments, mimetics, so long as the variant has at
least a portion of an amino acid sequence of a native protein, or at least a portion of an amino
acid sequence of substantial sequence identity to the native protein. In embodiments, the mutant
LINGO-1 differs from the amino acid sequence of SEQ ID NO:1 or a fragment thereof by up to
5, 10, 20, 30, 50, 75, 100, 150, or 200 or more amino acid residues. In other embodiments, the
mutant LINGO-1 is at least about 70%, 80%, 90%, 95% or more identical to a LINGO-1
polypeptide sequence, e.g., a human LINGO-1 polypeptide (e.g., SEQ ID NO:1).
A "chimeric protein"or "fusion protein" is a fusion of a first amino acid sequence
encoding a polypeptide with a second amino acid sequence, wherein the first and second amino
acid sequences do not occur naturally as part of a single polypeptide chain.
As used herein, the term "substantially similar" (or "substantially" or "sufficiently"
"homologous" or "identical") is used herein to refer to a first amino acid or nucleotide sequence
that contains a sufficient number of identical or equivalent (e.g., with a similar side chain, e.g.,

conserved amino acid substitutions) amino acid residues or nucleotides to a second amino acid or
nucleotide sequence such that the first and second amino acid or nucleotide sequences have
similar activities. Sequences similar or homologous (e.g., at least about 85% sequence identity)
to the sequences disclosed herein are also part of this application. In some embodiments, the
sequence identity can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
higher.
Calculations of "homology" or "sequence identity" between two sequences are performed
as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal
alignment and non- homologous sequences can be disregarded for comparison purposes).
Typically, the length of a reference sequence aligned for comparison purposes is at least 30%,
preferably at least 40%, more preferably at least 50%, even more preferably at lo least 60%, and
even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide
positions are then compared. When a position in the first sequence is occupied by the same
amino acid residue or nucleotide as the corresponding position in the second sequence, then the
molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is
equivalent to amino acid or nucleic acid "homology"). The percent identity between the two
sequences is a function of the number of identical, positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, which need to be introduced for optimal
alignment of the two sequences.
The comparison of sequences and determination of percent identity between two
sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent
identity between two amino acid sequences is determined using the Needlernan and Wunsch
((1970) /. Mol. Biol. 48:444-453) algorithm which has been incorporated into the commercially
available GAP program in the GCG software package, using either a Blossum 62 matrix or a
PAM250 matrix, and a gap weight of 16, 14,12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5,
or 6. In yet another embodiment, the percent identity between two nucleotide sequences is
determined using the commercially available GAP program in the GCG software package, using
a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 30 70, or 80 and a length weight of 1,
2, 3, 4, 5, or 6. Parameters typically used to determine percent homology are a Blossum 62

scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty
of 5. The percent identity between two amino acid or nucleotide sequences can also be
determined using the s algorithm of E. Meyers and W. Miller ((1989) CABIOSA-A1-17) which
has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4.
A LINGO-1 polypeptide can originate from a nonmammalian or mammalian species. A
mammalian LINGO-1 polypeptide can originate from a human, for example. Exemplary
nonhuman mammals include a nonhuman primate (such as a monkey or ape), a mouse, rat, goat,
cow, bull, pig, horse, sheep, wild boar, sea otter, cat, and dog. Exemplary nonmammalian
species include chicken, turkey, shrimp, alligator, and fish. The LINGO-1 amino acid and
nucleotide sequences from several mammalian and nonmammalian species (including, for
example, human, mouse, rat, dog, chimpanzee and chicken) are known in the art {see e.g.y Mi et
al. (2005) Nat. Neuroscience 8:745-751; Carim-Todd, L. et al. (2003) Eur. J. Neurosci.
18(12):3167-3182; and Okafuji et .al. (2005) Gene Expr. Patterns 6(1): 57-62).
An. agent can be, for example, a chemical, compound {e.g., a polypeptide, nucleic acid,
peptidomimetic). A peptidomimetic is a chemical compound that can mimic the ability of a
peptide to recognize certain physiological molecules, such as proteins and nucleic acids. In some
instances, the peptidomimetic includes non-peptidic structural elements that are capable of
mimicking or antagonizing the biological action(s) of a natural parent peptide. For example,
scissile peptide bonds can be replaced with one or more non-scissile dipeptide isosteres. .
The following example is illustrative and not intended as limiting.
EXAMPLE
LINGO-1 was crystallized and its structure determined. The human LINGO-1 is a 614
amino acid protein containing a signal sequence, a leucine-rich repeat-like domain, an
immunoglobulin-like domain, a stalk domain, a transmernbrane domain and a short cytoplasmic
tail (Mi et al.r Nat. Neurosci. 7:221-228, 2004). The extracellular region of human LINGO-1
(including the LRR motifs, the Ig-like domain and the stalk region, and not including the signal
sequence) was fused at its C-terminus to a hexahistidine tag (FIG. 1). The resulting LINGO-
lK549his6 construct was subcloned into a pSMEG vector (SEQ ID NO:2) (FIGs. 6, and 7A-1 to
7A-3) behind a murine cytomegalovirus (CMV) promoter (K549 refers to the lysine that occurs

at amino acid position 249 of the full-length human LINGO-1 construct, including the 33 amino
acid signal sequence. The signal sequence was not present in the LINGO-lK549hiS6
polypeptide.). The construct was verified by sequencing. Mammalian Chinese hamster ovary
(CHO) cells were grown and maintained, in a humidified incubator with 5% CO2 at 37°C. Cell
culture and DNA transfection for Lec3.2.8.1 cells are described in Zhong et .al. (Biochim.
Biophys. Ada 1723:143-150.2005). A stable clone for LINGO-lK549his6 construct in Lec3.2.8.1
(1042-5) was established by screening with anti-his4 antibody. Procedures for large-scale
production of conditioned media for LINGO- lK549hiS6 were as described previously (Zhong et
al, Biochim. Biophys. Acta 1723:143-150. 2005).
To purify LINGO-lK549h,s6, conditioned CHO media expressing the fusion protein
(FIG 1) was exchanged for a buffer of 50 mM Tris at pH 8.0, 100 mM NaCl, to which a cocktail
of protease inhibitors was added (complete inhibitors from Roche, Nutley, NJ). To capture the
fusion protein, Ni-NTA resin was equilibrated in 50 mM Tris, pH 8.0, 100 mM NaCl and then
added to the cultured media for batch binding (end-over-end) for 2 hours. Once the cultured
media was removed, the Ni-NTA resin was packed into a column. The column was connected to
an AKTA. Explorer (HPLC) and was washed with 4-5 column volumes of 50 mM Tris, pH 8.0;
100 mM NaCl. The protein was eluted with a 12-column volume gradient from 0% to 100%
50 mM Tris, pH 8.0; 100 mM NaCl, 300 mM imidazole. The collected protein fractions were
dialyzed into 50 mM Tris, pH 8.0; 500 mM NaCl. For crystallization, the protein was incubated
with Carboxypeptidase A (Sigma, St. Louis, MO) for 14-16 hours at 25°C, and then dialyzed into
50 mMMES, pH 6.1; 50 mM NaCl. After dialysis, the protein was loaded onto an SP sepharose
column (GE Healthcare) and washed with 4-5 column volumes of 50 mM MES, pH 6.1; 50 mM
NaCl. The protein was eluted with a 12-column volume gradient from 0% to 100% 50 mM
MES, pH 6.1; 750 mM NaCl. The protein peak fractions were collected, concentrated to 5-8
mg/ml and then loaded onto the gel filtration Superdex 200 column equilibrated with TBS. The
protein peak fractions were pooled together and concentrated to 4-5 mg/ml for crystallization.
The purified protein was fully glycosylated.
LINGO-lK549his6 crystals were obtained using the hanging drop vapor diffusion method
at 18°C. Diffraction quality crystals were grown from protein solutions with 4 mg/mL of protein
and precipitating solutions of 1.2 -1.4 M (NILO2SO4, 0.1 M Na-Citrate, pH 5.0. The protein
crystallized in two forms under identical conditions, with both forms found in the same

crystallization droplets: 1222, with two molecules per asymmetric unit and 74% solvent content,
and P21212, with four molecules per asymmetric unit and 73% solvent content. For data
• collection, crystals were gradually transferred from the mother liquor to the stabilizing-
cryoprotecting solution containing 2.9 M sodium malonate, pH 5.2 (Holyoak et ah, Biological
Crystallography D59:2356-2358, 2003). This solution, in which crystals were found to be stable
over the period of several days and over the pH range 5-7, was used for crystal derivatization. A
single derivative that allowed structure determination by the method of single isomorphous
replacement with anomalous scattering (SIRAS) was obtained from a platinum derivative,
created by soaking the crystals in 50 mM K2PtCl6 and 2.9 M sodium malonate at pH 7.0 for 24
hours. Prior to data collection, all crystals were flush cooled under a nitrogen stream at 100 K.
Two data sets obtained from crystals of space group 1222 were used for phase
determination: the 3.5 A native data set and the 3.6 A data set for the platinum derivative, both
measured in-house with a Satum92 CCD mounted on an FR-E Cu Kor rotating anode source
(Rigaku, Japan). The higher resolution native data set was collected to 2.7 A at Advanced
Photon Source (APS) (Argonne, Illinois) with beamline 22-ID of SER-CAT from a crystal that
belongs to the P21212 space group. All data were integrated and scaled with HKL2000
(Otwinowski and Minor, Methods Enzymol. 276:307-326, 1997).
The initial positions of the platinum (Pt) atoms in the derivative crystal were located with
SHELXD (2001, Bruker-AXS, XM, Ver. 6.12; Bruker-AXS, Madison, WI) using anomalous
differences of Pt atoms at the Cu Kar-edge. The input SAS coefficients were prepared with
XPREP (2001 Bruker-AXS, Ver. 6.12; Bruker-AXS, Madison, WI). Refinement of heavy-atom
parameters, phase calculation and density modification by SOLOMON (Abrahams and Leslie,
Acta Cryst. D52: 30-42, 1996) were performed with SHARP (de La Fortelle and Bricogne,
Methods Enzymol. 276:472-494, 1997) at 20-3.6 A resolution, using both anomalous and
isomorphous differences from the native and derivative data sets. The final 3.6 A single
isomorphous replacement with anomalous scattering (SIRAS) maps produced with SHARP were
of interpretable quality and revealed two LINGO-1 molecules in the asymmetric unit. SHARP
phases were further improved by two-fold non-crystallographic symmetry (NCS) averaging and
phase extension to 3.5 A in DM (Cowtan and Main, Acta Cryst. D49:148-157, 1993). The
resulting maps allowed us to build an initial, 90% complete (-855 residues) model with
QUANTA (Accelrys, Inc., San Diego, CA). This model was then used for molecular

replacement with the P21212 data set to utilize the higher 2.7 A resolution data. A clear solution
for four molecules in the asymmetric unit was identified with PHASER (McCoy et ah, Ada
Cryst. 061:458-464,2005). The 1222 and P21212 crystal forms share the same tetrameric
packing, in which the tetramer can be built bj' replicating a dimer around the two-fold axis.
Subsequent rounds of rebuilding and refinement against the 2.7 A data set were done with the
(Crystallographic Object)-Oriented Toolkit (COOT) (Emsley and Cowtan, Ada Crys. D
60:2126-2132, 2004) and REFMAC (Murshudov et ah, Ada Crystallogr. D 53:240-255, 1997).
The final model contains four protein molecules (residues A1-477, B3-475, C2-477, and
D3-476), 39 N-acetylglucosamine and 12 mannose residues, and 310 water molecules.
Residues 1-2 at the N-termini of B, C and D; 476-477 at the C-termini of B and D; and residues
D32-34 were not modeled into the structure due to the lack of adequate electron density,
presumably because of disordering. Geometric analysis of the final refined structure performed
with MolProbity places 94% of all residues in favored regions and 0.16% as outliers. Statistics
for data collection, phasing and refinement are summarized in Table 1.
Table 2 lists the crystal structure coordinates as deduced from the crystal of space group
P21212 (see Table 1). The native and derivative structures having space group 1222 revealed
essentially the same structure and were used to help with phasing. In Table 2, the "#" column
assigns an index to each atom for which coordinates are given. The "name" column indicates
what type of atom, and the "res" column indicates what type of residue the atom belongs to. The
"chain" indicates which polypeptide the atom belongs to. "Res #" gives the residue number for
the atom. For example, atom number 1 (the first row in Table 2) is the Nitrogen (N) of Thrl
(according to the sequence set forth in SEQ ED NO:1). Its x, y, and z structural coordinates are
given in the X, Y, and Z columns, respectively. The column headed "occ" describes the
occupancy assigned to the atom (1.00 = full occupancy), and the "B" column provides B factors
(or temperature factors) in units of A2. The column labeled "element" lists the. atom's element
symbol. Water is denoted by "HOH." "NAG" and "MAN" indicate N-acetylglucosamine and
mannose residues, respectively.

Table 1. Statistics for data collection, phasing and refinement

"Rsym = Σ Σ I I(h)i - | / Σ Σ I(h)i, where is the mean intensity. Numbers in
parentheses reflect statistics for the highest resolution shells.
bRiso= I I Fnat(h) - Fpt(h) | / Σ Fnat(h) and Rano is calculated for the amplitudes of the
positive and negative counterparts of the Bijvoet pairs.
cPhasing Power is defined by <|Fn|>/<(lack-of-closure)>, where 'H' represents heavy-atom.
dMean figure of merit is the estimated mean cosine of the phase error.
*Rfree is calculated with 5% of the data.

Both crystal forms of LINGO-1 (1222 and P21212) revealed a tetrameric structure,
consistent with gel filtration data and dynamic light scattering measurements, showing that
LINGO-1 exists as a tetramer in solution with a molecular weight of-240 kDa. FIG 2 shows a
fragment of the electron density for the refined 2.7 A resolution structure.
An overall view of the monomeric LINGO-1 structure is shown in FIG 3. Each
monomer is formed from two distinct domains: residues 1-382 constitute the N-terminal LRR-
domainand residues 383-477 form the C-terrninal Ig-like domain. The relative orientation of
the two constituent domains gives the monomeric structure a question mark shape.
The LRR-domain (residues 1-382) adopts an elongated arc shape with a parallel 15-
stranded B-sheet on the concave inner face and with highly irregular secondary structures on the
convex outer face (FIG 3). In total, there are twelve LRRs, 23 to 25 residues each, that together
create a continuous right-handed super-helical assembly. Each LRR begins with a J3-strand and
has the consensus 24-residue sequence repeat motif:
xL2xxL5xL7xxN10xL12xxL15xxxxF20xxL23x (SEQ ID NO:9), where x can be any amino acid; L
is a hydrophobic residue, preferentially Leu, but: also IIe, Val, Met, Phe or Thr; N are less
conserved in nature and includes mostly Asn, but also Cys, Asp, Leu or Trp; and F represents
the hydrophobic residue Phe or Leu. The consensus residues at the indicated positions make up
the interior of the LRR-domain. As in several other LRR proteins, the N- and C-termini of the
LRR hydrophobic core are shielded from the solvent by two LRR caps: 'Neap' and 'Ccap' (FIG
3). The 'Neap' has two anti-parallel ß-strands with two disulfide bridges at the base (Cys3-Cys9
and Cys7-Cysl8), an arrangement that is very close in structure to NogoR (He, et al., Neuron
38:177-185, 2003). The 'Ccap' is also supported by two disulfide bridges (Cys334-Cys357 and
Cys336-Cys382) and, like NogoR, has a characteristic motif consisting of one α-helix and three
short 3|0-helices. Unlike in the NogoR, however, the interior of the 'Ccap' in LINGO-1 is filled
almost entirely with a phenylalanine cluster (six aromatic rings from Phe342, Phe350, Phe362,
Phe368, Phe371, Phe380 and one aromatic ring from Tyr379). In addition, a short loop segment
(residues 349-353) following the a-helix is integrated into the canonical LRR structure in a
slightly different way than in NogoR. The result is such that the top of the 5-residue loop
bulges away from the 6-sheet into the concave space by at least 7.5 A, therefore showing a more
protruding character than the corresponding segment of the same length in NogoR. The
position of the bulge loop (FIG 3), which corresponds to the ligand binding 6-switch loop in the

platelet membrane glycoprotein Gplbα (Huizinga et al, Science 297:1176-1179, 2002),
combined with a character of the surface-exposed and protruding side chains (Trp346, Arg347,
and Asn349, Asn351, Gln353, and Arg352 at the tip of the loop), suggests a likely candidate for
interaction with a ligand. The electrostatic surface of the molecule (FIG 4) reveals a
pronounced set of electropositive and electronegative patches on all sides of the LRR-domain,
except one relatively large hydrophobic patch found on the N-terminal convex side.
Based on the structure of the tetrameric complex, five subsets of amino acids located on
the concave surface of the LRR domain were determined to be candidate ligand binding sites:
(1) Trp346 and Arg352; (2) Hisl85, His209, and His233; (3) Aspl3; (4) Glu60 and Glu62; and
(5) Arg20, Arg22, and Arg43. Likewise, four subsets of amino acids located on the convex
surface of the LRR domain were determined to be candidate ligand binding sites: (1) Tyrl42
and Hisl45; (2) Leu94, Leul20, and Metl23; (8) Hisl76; and (9) Ala238.
Alter completing the LRR-domain, the polypeptide chain enters the compact Ig-like fold
(residues 383-477) which represents the classical ß-sandwich of two ß-sheets cross-linked by
one disulfide bond (Cys407-Cys458). Four strands (A, B, D and E) form one 8-sheet and five
strands (A', C, C\ F and G) make up the second ß-sheet. There is one 310 α-helical turn that
connects the E and F strands. Comparison with other Ig-like structures indicates that the closest
structural analog of the LINGO-1 Ig-like domain is the Ig3-module of the neural cell adhesion
molecule, NCAM (Soroka et al.. Structure 10:1291-1301,2003), with the calculated identity
between the two sequences of-30%. Not surprisingly, the majority of the amino acid
replacements occurs at the Ig-domain surfaces altering the side chain volumes and/or surface
polarity, and most of the conserved amino acids map to the core of the structure and have
equivalent conformations to satisfy the fold. The surface electrostatic potential is a combination
of negative and positive patches on the ABDE-surface. On the contrary, the opposite A'CC'FG-
surface is highly enriched in hydrophobic residues (Ala4l6, Leu418, Leu420, Leu426, Leu457,
Ile459, Ala461, AIa463) surrounded by a number of polar and positively charged residues.
The Ig-domain orientation is such that it projects away from the convex LRR arc
creating a wide solvent-exposed cleft at the back of the LRR-domain, with an elbow angle of
~90 degrees (FIG 4). The walls of the cleft are formed by the C-terminal convex surface of
LRR (repeats 10-12 and the Ccap), on one side, and by the A'CC'FG ß-sheet of the Ig-domain
on the other. The opposing surfaces are relatively far apart and have different properties. The

convex surface is made up of mostly polar and charged residues, while the opposing Ig-surface
is rather flat and is lined up with the amino acids that are predominantly hydrophobia (as
indicated above). Small interdomain interactions occur at the tip of the elbow and involve van
der Waals contacts between residues on the FG-loop (Ala463, Gly464) and on the C-terminal
portion of LRR (Cys336, Arg337 and Cys382). Of note are the disulfide bond at the tip of the
elbow, Cys336-Cys382, and the absence of an interdomain linker (as Arg383 is the first residue
of the first ß-strand and a component of the ABDE C-sheet). Such linkage between the domains
constrains the positioning of the two relative to each other.
Based on the structure of the tetrarneric complex, five subsets of amino acids on the Ig
domain were determined to be candidate ligand binding sites: (1) Arg446 and Tyr447;
(2) Arg408, Phe438, and Asp440; (3) Phe396; and (4) Leu420, Leu426, and Ile459.
The glycoform of LINGO-1 in CHO restricted cells is expected to be predominantly
high-mannose type glycans such as MansGlcNAc2, which is consistent with our mass
spectroraetry data. The crystallized glycoprotein contains eight typical N-glycosylation
consensus sites. Six of them are located in the LRR-domain (Asnl05, Asnl63, Asn225,
Asn235, A.sn254, and Asn302) and two in the Ig-like domain (Asn466 and Asn453). All of
these sites are positioned at the domain surfaces. Electron density can be assigned for
carbohydrate moieties at all or these locations, except for the Asn466 site. The latter, although
located at the surface, is wrapped up closely with a symmetry-related LINGO-1 partner, as
described below and as shown in FIG 5B. Since the protein packing leaves no room for
glycosylation at Asn466, the N-glycan linked to Asn466 would prevent the LINGO-1
tetramerization observed in the crystals. The oligosacchai ides at the remaining seven sites are
spread over the structure as shown by the numbered residues in FIG. 4, where #1 corresponds to
Asn 105, #2 to Asnl63, #3 to Asn 225, #4 to Asn 235, #5 :o Asn 254, #6 to Asn 302, and #7 to
Asn 453. Out of four faces of the LRR-domain, only the convex outer surface is completely
bare of carbohydrates, a finding consistent with its role in self-association. The concave and the
two side surfaces, each bearing two N-glycan binding site;, are largely covered by
carbohydrate. The last carbohydrate moiety maps to the Ig ;-face A'CC'FG, proximal to the C-
terminus and remote from the intermolecular interface (FIG 4).
In general, the presence of extensive glycosylation on glycoprotein surfaces limits their
accessibility for interaction with ligands, and in most cases the surfaces that are not covered by

sugars are where the ligands are predicted to bind (Rudd et al, JMB 293:351-366, 1999).
Therefore, it seems unlikely that the concave LRR surface of LENGO-1, with two sugar
moieties right at the arc centre, can play a major role in accommodating such large protein
partners as NogoR or p75. This would be contrary to the other LRR-containing receptors that
frequently use the concave surfaces for interaction with their ligands (Bell et al. Trends
Immunol. 24:528, 2003). Since there is no evidence to suggest that the Asn-linked N-glycans are
directly associated with ligand binding, it is also possible that the LINGO-1 glycosylation helps
prevent non-specific protein-protein interactions or helps maintain the proper scaffold for
subsequent ligand binding.
Crystallization, size-exclusion chromatography and dynamic light scattering all indicate
that LINGO-1 adopts a tetrameric form both in crystals and in solution. In the crystal, the four
independent copies of LINGO-1 interact to constitute a condensed tetrameric structure. A
pseudo four-fold axis of rotation relates the four molecules in a ring-like structure with a large
hole at the center (~40 A in diameter; outer ring diameter -95 A; FIG 5A). This arrangement
places the N-terminal convex side of each LRR-domain at the elbow between the domains of
the adjacent molecule (FIG 5B), reminiscent of a cytokine/receptor binding mode. As in the
latter case, the attachment point between two molecules is provided by three surfaces: one
formed by the N-terminal curved repeats 1-6 and two elbow surfaces formed, as described
above, by the C-terminal curved repeats 11-12, the 'C-cap' and the A'CC'FG B-strands,
respectively. Hence, the four LRR-domains interlock the ring in a head-to-tail fashion, and the
corresponding Ig-like modules extend from the perimeter of the ring in a landing gear fashion
(see FIG. 5C). If we apply this geometry relative to a cell surface, then the protein rotation axis
will lie approximately normal to the surface with the membrane-proximal C-terminal ends
pointing in the same direction, i.e., towards the cell membrane.
Each intermolecular interface reveals a tight geometric match between the binding
surfaces and the precise stereochemical complementarity of the interaction. The solvent-
accessible surface area buried at each intermolecular interface is relatively large, ~2400 A2 total
from the three individual components, which suggests a strong association. The above evidence
combined with the multivalent character of interaction suggest that the contacts in the tetramer
are not just mere crystal contacts.

WHAT IS CLAIMED IS:
1. A crystallized LINGO-1 polypeptide.
2. The crystallized LINGO-1 polypeptide of claim 1 or 2, wherein the LINGO-1 polypeptide
comprises a leucine-rich repeat (LRR) domain.
3. The crystallized LINGO-1 polypeptide of claim 2, wherein the LINGO-1 polypeptide
comprises an immunoglobulin-like (Ig-like) domain.
4. The crystallized LINGO-1 polypeptide of claim 1, wherein the LINGO-1 polypeptide
consists essentially of a LKR-like domain, an Ig-like domain, and a stalk domain.
5. The crystallized LINGO-1 polypeptide of claim 1, wherein the LINGO-1 polypeptide
comprises the amino acid sequence of SEQ ID NO:1.
6. The crystallized LINGO-1 polypeptide of any of claims 1-5, wherein the crystallized
LINGO-1 polypeptide has space group P21212.
7. The crystallized LINGO-1 polypeptide of claim 6, wherein the crystallized LINGO-1
polypeptide has unit cell dimensions a=201.5 A, b=149.7 A, c=157.5 A, and α=β=γ=90o
8. The crystallized LINGO-1 polypeptide of any of claims 1-5, wherein the crystallized
LINGO-1 polypeptide has space group 1222.
9. The crystallized LINGO-1 polypeptide of claim 8, wherein the crystallized LINGO-1
polypeptide has unit cell dimensions a=148.7 A, b=158.6 A, c=200.0 A, and α=β=γ= 90°.
10. The crystallized LINGO-1 polypeptide of claim 8, wherein the crystallized LINGO-1
polypeptide has unit cell dimensions a=149.6 A, b=157.3 A, c=200.3 A, and α=β=γ= 90°.

11. The crystallized LINGO-1 polypeptide of any of claims 1 or 6-10, wherein the LINGO-1
polypeptide is from a mammalian species.
12. The crystallized LINGO-1 polypeptide of any of claims 1 or 6-10, wherein the LINGO-1
polypeptide is from a nonmammalian species.
13. The crystallized LINGO-1 polypeptide of any of claims 1 or 6-10, wherein the LINGO-1
polypeptide is from a human.
14. The crystallized LINGO-1 polypeptide of claim 1, wherein the crystallized polypeptide is
capable of diffracting X-rays to a resolution of at least about 3.7 A.
15. The crystallized LINGO-1 polypeptide of any of claims 1 or 6-10, wherein the
crystallized polypeptide comprises the structural coordinates of Table 2, +/- a root mean square
deviation for alpha carbon atoms of not more than 1.5 A.
16. The crystallized LINGO-1 polypeptide of any of claims 1 or 6-10, wherein the
crystallized polypeptide comprises structural coordinates of an atom selected from the group
consisting of atoms of amino acids Asp 13, Arg20, Arg22, Arg43, Glu60, Glu62, Leu94, Leul20,
Metl23, Hisl75, Tyrl42, Hisl45, Hisl85,His209, His233, Ala238, Trp346, Arg347, Asn349,
Asn351, Arg352, Gln353, Phe396, Arg408, Lcu420, Leu426, Phe438, Asp440, Arg446, Tyr447 and
Ile459 of the LINGO-1 polypeptide as set forth in SEQ ID NO:1.
17. A crystallized polypeptide-ligand complex comprising:
a LINGO-1 polypeptide; and
a ligand that is an agonist of the LINGO-1 polypeptide or an antagonist of the LINGO-1
polypeptide.
18. The crystallized polypeptide-ligand complex of claim 17, wherein the LINGO-1
)olypeptide comprises a leucine-rich repeat (LRR) domain.

19. The crystallized polypeptide-ligand complex of claim 17 or 18, wherein the LINGO-1
polypeptide comprises an immunoglobulin-like (Ig-like) domain.
20. The crystallized polypeptide-ligand complex of claim 17, wherein the LINGO-1
polypeptide consists essentially of a LRR-like domain, an Ig-like domain, and a stalk domain.
21. The crystallized polypeptide-ligand complex of claim 17, wherein the LINGO-1
polypeptide comprises the amino acid sequence of SEQ ID NO:1.
22. A method comprising:
using a three-dimensional model of a LINGO-1 polypeptide to design an agent that binds the
LINGO-1 polypeptide.
23. The method of claim 22, wherein the three-dimensional model comprises an LRR
domain of the LINGO-1 polypeptide.
24. The method of claim 22 or 23, wherein the three-dimensional model comprises an Ig-
like domain of the LINGO-1 polypeptide.
25. The method of claim 22, wherein the three-dimensional model consists essentially of
an LRR domain, an Ig-like domain, and a stalk domain of the LINGO-1 polypeptide.
26. The method of claim 22, wherein the agent inhibits LINGO-1 activity.
27. The method of any of claims 22-26, wherein the three-dimensional model comprises
structural coordinates of atoms of the LINGO-1 polypeptide.
28. The method of claim 27, wherein the structural coordinates are experimentally
determined coordinates.

29. The method of claim 28, wherein the structural coordinates are according to Table 2,
+/- a root mean square deviation for alpha carbon atoms of not more than 1.5 A.
30. The method of any of claims 22-29, wherein the three-dimensional model comprises
structural coordinates of an atom selected from the group consisting of atoms of amino acids Asp 13,
Arg2O, Arg22, Arg43, Glu60, Glu62, Leu94, Leul20, Metl23, Hisl76, Tyrl42, Hisl45, Hisl85,
His209, His233, Ala238, Trp346, Arg347, Asn349, Asn351, Arg352, Gln353, Phe396, Arg408,
Leu420, Leu426, Phe438, Asp440, Arg446, Tyr447 and Ile459 of the LINGO-1 polypeptide as set
forth in SEQ lD NO:l.
31. The method of any of claims 22-29, further comprising calculating a distance between
an atom of the LINGO-1 polypeptide and an atom of the agent.
32. The method of any of claims 22-29, further comprising providing a composition
comprising the LINGO-1 polypeptide.
33. The method of claim 32, wherein the composition includes the agent designed to bind
the LINGO-1 polypeptide.
34. The method of any of claims 22-29, further comprising experimentally determining
the interaction of the agent with the LINGO-1 polypeptide.
35. The method of any of claims 22-29, further comprising comparing the interaction of
the agent with the LINGO-1 polypeptide to an interaction of a second agent with the LINGO-1
polypeptide.
36. A method comprising:
using a three-dimensional model of a LINGO-1 polypeptide-ligand complex to design an
agent that binds the LINGO-1 polypeptide.

37. The method of claim 36, wherein the three-dimensional model comprises an LRR
domain of the LINGO-1 polypeptide.
38. The method of claim 36 or 37, wherein the three-dimensional model comprises an Ig-
like domain of the LINGO1 polypeptide.
39. The method of claim 36, wherein the three-dimensional model consists essentially of
an LRR domain, an Ig-like domain, and a stalk domain of the LINGO-1 polypeptide.
40. The method of claim 36, wherein the agent inhibits LINGO-1 activity.
41. The method of any of claims 36-40, further comprising experimentally determining
the interaction of the agent with the LINGO-1 polypeptide.
42. A method, comprising:
using a three-dimensional model of a complex comprising a LINGO-1 polypeptide to design
an agent that binds the LINGO-1 polypeptide.
43. The method of claim 42, wherein the three-dimensional model comprises an LRR
domain of the LINGO-1 polypeptide.
44. The method of claim 42, wherein the three-dimensional model comprises an Ig-like
domain of the LINGO-1 polypeptide.
45. The method of claim 42 or 43, wherein the three-dimensional model consists
essentially of an LRR domain, an Ig-like domain and a stalk domain of the LINGO-1 polypeptide.
46. The method of claim 42, wherein the agent inhibits LINGO-1 activity.
47. The method of any of claims 42-46, wherein the three-dimensional model comprises
structural coordinates of atoms of the LINGO-1 polypeptide.

48. The method of claim 47, wherein the structural coordinates are experimentally
determined coordinates.
49. The method of claim 48, wherein the structural coordinates are according to Table 2,
+/- a root mean square deviation for alpha carbon atoms of not more than 1.5 A.
50. The method of any of claims 42-49, wherein the three-dimensional model comprises
structural coordinates of an atom selected from the group consisting of atoms of amino acids Aspl3,
Arg20, Arg22, Arg43, Glu60, Glu62, Leu94, Leul20, Metl23, Hisl76, Tyrl42, Hisl45, Hisl85,
His209, His233, Ala238, Trp346, Axg347, Asn349, Asn351, Arg352, Gln353, Phe396, Arg408,
Leu420, Leu426, Phe438, Asp440, Arg446, Tyr447 and Ile459 of the LESTGO-l polypeptide as set
forth in SEQ ID NO:l.
51. The method of any of claims 42-49, further comprising calculating a distance between
an atom of the LINGO-1 polypeptide and an atom of the agent.
52. The method of any of claims 42-49, further comprising providing a composition
comprising the LINGO-1 polypeptide.
53. The method of claim 52, wherein the composition includes the agent designed to bind
the LINGO-1 polypeptide.
54. The method of any of claim 42-49, further comprising experimentally determining the
interaction of the agent with the LINGO-1 polypeptide.
55. The method of claim 54, further comprising comparing the interaction of the agent
with the LINGO-1 polypeptide to an interaction of a second agent with the LINGO-1 polypeptide.
56. A method, comprising:

selecting an agent by performing rational drug design with a three-dimensional structure of a
LINGO-1 polypeptide;
contacting the agent with the LINGO-1 polypeptide; and
detecting an ability of the agent to bind the LINGO-1 polypeptide.
57. The method of claim 56, wherein the agent is selected via computer modeling.
58. The method of claim 56 or 57, further comprising synthesizing the agent.
59. The method of claim 58, further comprising detecting an ability of the agent to inhibit
LINGO-1 activity.
60. The method of claim 58, further comprising detecting an ability of the agent to
increase myelin formation in vitro or in vivo.
61. The method of any of claims 56-60, further comprising:
obtaining a supplemental crystalline complex comprising the LINGO-1 polypeptide and the
agent;
determining the three-dimensional structure of the supplemental crystalline complex;
selecting a second agent by performing rational drug design with the three-dimensional
structure of the supplemental crystalline complex;
contacting the second agent with the LINGO-1 polypeptide; and
detecting the ability of the second agent to bind the LINGO-1 polypeptide.
62. The method of claim 61, wherein the second agent is selected via computer modeling.
63. The method of claim 61, further comprising synthesizing the second agent.
64. The method of claim 61, further comprising detecting an ability of the second agent to
inhibit LINGO-1 activity.

65. The method of claim 61, further comprising detecting an ability of the second agent to
increase myelin, levels in vitro or in vivo.
66. A method, comprising:
selecting an agent by performing rational drug design with a three-dimensional structure of a
LINGO-1 polypeptide-ligand complex;
contacting the agent with the LINGO-1 polypeptide; and
detecting an ability of the agent to bind the LINGO-1 polypeptide.
67. The method of claim 66, wherein the agent is selected via computer modeling.
68. The method of any of claim 66-67, further comprising synthesizing the agent.
69. The method of claim 68, further comprising detecting an ability of the agent to inhibit
LINGO-1 activity.
70. The method of claim 68, further comprising detecting an ability of the agent to
increase myelin formation in vitro or in vivo.
71. The method of any of claims 66-70, further comprising:
obtaining a supplemental crystalline complex comprising the LINGO-1 polypeptide and the
agent;
determining the three-dimensional structure of the supplemental crystalline complex;
selecting a second agent by performing rational drug design with the three-dimensional
structure of the supplemental crystalline complex;
contacting the second agent with the LENGO-1 polypeptide; and
detecting the ability of the second agent to bind the LINGO-1 polypeptide.
72. The method of claim 71, wherein the second agent is selected via computer modeling.
73. The method of claim 71, further comprising synthesizing the second agent.

74. The method of claim 71, further comprising detecting an ability of the second agent to
inhibit LINGO-1 activity.
75. The method of claim 71, further comprising detecting an ability of the second agent to
increase myelin levels in vitro or in vivo.
76. A method, comprising:
providing a composition comprising a LINGO-1 polypeptide; and
crystallizing the composition to form a crystalline complex comprising the LINGO-1
polypeptide,
wherein the crystalline complex can diffract X-rays to a resolution of at least about 3.7 A.
77. The method of claim 76, wherein the method includes using hanging drop vapor
diffusion.
78. A method, comprising:
providing a composition comprising a LINGO-1 polypeptide-ligand complex; and
crystallizing the composition to form a crystal comprising the LINGO-1 polypeptide-ligand
complex,
wherein the crystal can diffract X-rays to a resolution of at least about 3.7 A.
79. The method of claim 78, wherein the method includes using hanging drop vapor
diffusion.
80. A software system, comprising instructions for causing a computer system to:
accept information relating to the structure of a LINGO-1 polypeptide;
accept information relating to a candidate agent; and
determine binding characteristics of the candidate agent to the LINGO-1 polypeptide,
wherein the determination is based on the information relating to the structure of the LINGO-
1 polypeptide, and the information relating to the candidate agent.

81. A computer program residing on a computer readable medium having a plurality of
instructions stored thereon, which, when executed by one or more processors, cause the one or more
processors to:
accept information relating to the structure of a complex comprising a LINGO-1 polypeptide;
accept information relating to a candidate agent; and
determine binding characteristics of the candidate agent to the LINGO-1 polypeptide,
wherein the determination is based on the information relating to the structure of the LINGO-
1 polypeptide and the information relating to the candidate agent.
82. A method, comprising:
accepting information relating to the structure of a LINGO-1 polypeptide; and
modeling the binding characteristics of the LINGO-1 polypeptide with a candidate agent,
wherein the method is implemented by a software system.
83. A computer program residing on a computer readable medium having a plurality of
instructions stored thereon, which, when executed by one or more processors, cause the one or more
processors to:
accept information relating to a structure of a LINGO-1 polypeptide; and
model the binding characteristics of the LINGO-1 polypeptide with a candidate agent.
84. A software system, comprising instructions for causing a computer system to:
accept information relating to a structure of a LINGO-1 polypeptide; and
model the binding characteristics of the LTNGO-l polypeptide with a candidate agent.
85. A method of modulating LINGO-1 activity in a subject, comprising:
usingrational drug design to select an agent that is capable of modulating LINGO-1 activity;
and
administering a therapeutically effective amount of the agent to the subject.

86. The method of claim 85, wherein the agent is capable of increasing myelin levels in
vivo.
87. The method of claim 85 or 86, wherein the rational drug design includes using a three-
dimensional structure of a crystalline complex that comprises a LINGO-1 polypeptide.
88. The method of claim 87, wherein the crystalline complex further comprises a ligand.
89. A method of treating a subject having a condition associated with LINGO-1 activity,
comprising:
using rational drug design to select an agent that is capable of affecting LINGO-1 activity; and
administering a therapeutically effective amount of the agent to a subject in need thereof.
90. The method of claim 89, wherein the agent is capable of inhibiting LINGO-1 activity.
91. The method of claim 89 or 90, wherein the agent is capable of increasing myelin levels
in vivo.
92. The method of any of claims 89-91, wherein the condition is a demyelinating disease.
93. The method of any of claims 89-91, wherein the condition is multiple sclerosis.
94. A method of prophylactically treating a subject susceptible to a condition associated
with LINGO-1 activity, comprising:
determining that the subject is susceptible to the condition associated with LINGO-1 activity;
using rational drug design to select an agent that is capable of effecting LINGO-1 activity; and
administering a therapeutically effective amount of the agent to the subject.
95. The method of claim 94 or 95, wherein the agent is capable of inhibiting LINGO-1
activity.

96. The method of any of claims 94-95, wherein the agent is capable of increasing myelin
levels in vivo.
97. The method of any of claims 94-96, wherein the condition is a demyelinating disease.
98. The method of any of claims 94-97, wherein the condition is multiple sclerosis.
99. Use of an agent designed or selected according to any of claims 22-75 in the
manufacture of a medicament for the prophylaxis or treatment of a condition associated with LINGO-
1 activity.
100. The use according to claim 99, wherein the agent is capable of inhibiting LINGO-1
activity.
101. The use according to claim 99 or 100, wherein the condition is a demyelinating
disease.
102. The use according to any of claims 99-101, wherein the condition is multiple sclerosis.
103. The use according to any of claims 99-102, wherein the agent binds to LINGO-1 at a
ligand binding site.
104. The use according to claim 103, wherein the ligand binding site is located on a
concave surface of an LRR domain.
105. The use according to claim 104, wherein the concave surface of the LRR domain
comprises one or more of Trp346 and Arg352; Hisl85, His209 and His233; Aspl3; Glu60 and Glu62;
or Axg20, Arg22 and Arg43.
106. The use according to claim 103, wherein the ligand binding site is located on a convex
surface of an LRR domain.

107. The use according to claim 106, wherein the convex surface of the LRR domain
comprises one or more of Tyrl42 and Hisl45; Leu94, Leul20, and Metl23; Hisl76; or Ala238.
108. The use according to claim 103, wherein the ligand binding site is located on an Ig
domain.
109. The use according to claim 108, wherein the Ig domain comprises one or more of
Arg446 and Tyr447; Arg408, Phe438 and Asp440; Phe396; or Leu420, Leu426 and Ile459.
110. An agent designed or selected according to any of claims 22-75 for use in the
prophylaxis or treatment of a condition associated with LINGO-1 activity.
111. The agent according to claim 110, wherein the agent is capable of inhibiting LINGO-1
activity.
112. The agent according to claim 110 or 111, wherein the condition is a demyelinating
disease.
113. The agent according to any of claims 110-112, wherein the condition is multiple
sclerosis.
114. The agent according to any of claims 110-113, wherein the agent binds to LINGO-1 at
a ligand binding site.
115. The use according to claim 114, wherein the ligand binding site is located on a
concave surface of an LRR domain.
116. The use according to claim 115, wherein the concave surface of the LRR domain
comprises one or more of Trp346 and Arg352; Hisl85, His209 and His233; Aspl3; Glu60 and GIu62;
or Arg20, Arg22 and Arg43.

117. The use according to claim 114, wherein the ligand binding site is located on a convex
surface of an LRR domain.
118. The use according to claim 117, wherein the convex surface of the LRR domain
comprises one or more of Tyrl42 and Hisl45; Leu94, Leul20, and Metl23; Hisl76; or Ala238.
119. The use according to claim 114, wherein the ligand binding site is located on an Ig
domain.
120. The use according to claim 119, wherein the Ig domain comprises one or more of
Arg446 and Tyr447; Arg408, Phe438 and Asp440; Phe396; or Leu420, Leu426 and Ile459.

This disclosure relates to LINGO-1 polypeptides,
LINGO-1 polypeptide/ligand complexes, crystals of LINGO-1
polypeptides, crystals of LINGO-1 polypeptide/ligand complexes,
and related methods and software systems.