Solution NMR structure of an immunodominant epitope
of myelin basic protein
Conformational dependence on environment of an intrinsically
unstructured protein
Christophe Fare
`
s
1,
*, David S. Libich
1
and George Harauz
1
1 Department of Molecular and Cellular Biology, and Biophysics Interdepartmental Group, University of Guelph, Canada
Multiple sclerosis is characterized by chronic inflamma-
tion of the myelin in the central nervous system (CNS),
and major variants of the illness are considered to be
primarily autoimmune in nature [1]. The 18.5 kDa
isoform of myelin basic protein (MBP) is one of the
most abundant proteins in CNS myelin; MBP maintains
the compaction of the sheath by anchoring the cytoplas-
mic faces of the oligodendrocyte membranes [2], and is a
candidate antigen for T cells and autoantibodies in
multiple sclerosis [3]. The three-dimensional structure of
MBP has not yet been elucidated to high resolution
[4,5]. We recently used site-directed spin-labeling
Keywords
correlation spectroscopy; multiple sclerosis;
myelin basic protein; immunodominant
epitope; solution NMR
Correspondence
G. Harauz, Department of Molecular and

) and water (30 : 70% v ⁄ v), and (c) a
dispersion of 100 mm dodecylphosphocholine (DPC-d
38
, 1 : 100 pro-
tein ⁄ lipid molar ratio) micelles. This polypeptide sequence is highly con-
served in MBP from mammals, amphibians, and birds, and comprises a
major immunodominant epitope (human residues N83–T92) in the auto-
immune disease multiple sclerosis. In the polypeptide fragment, this epitope
forms a stable, amphipathic, a helix under organic and membrane-mimetic
conditions, but has only a partially helical conformation in aqueous solu-
tion. These results are consistent with recent molecular dynamics simula-
tions that showed this segment to have a propensity to form a transient
a helix in aqueous solution, and with electron paramagnetic resonance
(EPR) experiments that suggested a a-helical structure when bound to a
membrane [I. R. Bates, J. B. Feix, J. M. Boggs & G. Harauz (2004) J Biol
Chem, 279, 5757–5764]. The high sensitivity of the epitope structure to its
environment is characteristic of intrinsically unstructured proteins, like
MBP, and reflects its association with diverse ligands such as lipids and
other proteins.
Abbreviations
CNS, central nervous system; CSI, chemical shift index; DIPSI, decoupling in the presence of scalar interactions; DPC-d
38
, perdeuterated
dodecylphosphatidylcholine; DSA, doxylstearic acid; EPR, electron paramagnetic resonance; Fmoc, 9-fluorenylmethoxycarbonyl; gpMBP,
guinea pig myelin basic protein; hMBP, human myelin basic protein; MAP, mitogen-activated protein; MBP, myelin basic protein; MHC,
major histocompatibility complex; rmMBP, recombinant murine; RMSD, root mean squared deviation; SDSL, site-directed spin-labeling; SH3,
Src homology domain 3; TFE-d
2
, deuterated 2,2,2-trifluoroethanol (CF
3

(DRB1*1501)-restricted T cells [3,11], and overlaps
the DR2a-restricted epitope for T cells reactive to
hMBP(V87–G106) [12]. There is evidence that segment
hMBP(V86–P96) contributes to autoantibody binding,
and also contains the T-cell receptor and MHC con-
tact points [11,13]. Moreover, this portion of MBP is
also a potential Ca
2+
–calmodulin binding site [14],
and borders a potential SH3-ligand and two known
mitogen activated protein (MAP) kinase sites [4].
Experimental treatments for multiple sclerosis based
on polypeptide mimetics of MBP have focused on this
and neighboring regions of the protein [11,13,15–28].
Several linear and cyclic analogs of hMBP(V87–P99)
have been designed, analyzed structurally using NMR
and molecular modeling, and evaluated for their ability
to induce and ⁄ or inhibit experimental autoimmune
encephalomyelitis in rats [22,23,25,28]. The cyclic ana-
logs, in particular, showed promise as potential antag-
onist mimetics for treating multiple sclerosis as
artificial regulators of the immune response. The linear
polypeptide D82-ENPVVHFFKNIVTPR-T98 (human
numbering) has been used to induce immunologic tol-
erance in patients with progressive multiple sclerosis
[20], and clinical efficacy is under evaluation in a phase
II ⁄ III clinical trial that is currently enrolling patients
() [29]. Thus, comparison
of the tertiary structures of this epitope under various
conditions is of interest to understand its pharmaco-

[56,57] alignment of sequences of 18.5 kDa MBP from mouse
(Mus musculus), rat (Rattus norvegicus), chimpanzee (Pan troglo-
dytes), human (Homo sapiens), bovine (Bos taurus), pig (Sus
scrofa), horse (Equus caballus), rabbit (Oryctolagus cuniculus), gui-
nea pig (Cavia porcellus), chicken (Gallus gallus), African clawed
frog (Xenopus laevis), little skate (Raja erinacea), spiny dogfish
(Squalus acanthias), and horn shark (Heterodontus francisci). Sym-
bols mean that residues in that column are (*) identical in all
sequences, (:) substitutions are conservative, and (.) substitutions
are semiconservative. The sequence has been numbered 1¢ to 18¢ ,
where 1¢ corresponds to residues 81 and 78 in human and murine
full-length 18.5 kDa MBPs, respectively. There is a high degree of
conservation in this epitope, particularly in residues V6¢ to F10¢.
Structure of MBP immunodominant epitope C. Fare
`
s et al.
602 FEBS Journal 273 (2006) 601–614 ª 2006 The Authors Journal compilation ª 2006 FEBS
crystallographic techniques. The 18-residue polypeptide
Q
1¢
DENPVVHFFKNIVTPRT
18¢
, was synthesized and
is referred to here as FF
2
, because it comprises the sec-
ond Phe–Phe pair (viz. F9¢–F10¢) within the classic
18.5 kDa MBP isoform.
A key consideration for solution NMR experiments
on full-length MBP is the stabilization of secondary,

1
H–
1
H
homonuclear correlation experiments were employed
to assign the resonances of the polypeptide FF
2
, and
ultimately to provide the semiquantitative distance
restraints for the calculation of its structure in aqueous
(100 mm KCl, pH 6.5), organic (30% TFE-d
2
), and
membrane-mimetic (DPC-d
38
micelles, 1 : 100 polypep-
tide ⁄ lipid molar ratio) environments. The
1
H spin sys-
tems for all of the 18 residues were revealed as
frequency-connected peak families created by the iso-
tropic mixing of the TOCSY experiments [36]. The
sequence-specific assignment of these spin systems was
deduced from the ‘fingerprint’ regions of the TOCSY
and NOESY experiments, shown in Fig. 2 for all three
conditions: aqueous solution (Fig. 2A,B), 30% TFE-d
2
(Fig. 2C,D), and 100 mm DPC-d
38
(Fig. 2E,F). The

13
C frequencies of the backbone spins of
FF
2
were also assigned and compared with those pre-
viously published for full-length MBP under the same
30% TFE-d
2
conditions [30]. Assignments were carried
out on the standard heteronuclear single-quantum
(HSQC) experiment and were based on the
1
H assign-
ment presented above. Because of the low abundance
of the
13
C nuclei, the sample concentration was raised
to 20 mm, for which excellent solubility was still
achievable in 100 mm KCl and 30% TFE-d
2
. At this
concentration, only minor
1
H chemical shift differences
were observed relative to the low concentration sam-
ples (data not shown), which implied that polypeptide
aggregation was minimal.
Secondary structure analysis
For those residues of the full-length rmMBP (recorded
in 30% TFE-d

the C
a
chemical shifts may be due to changes in local
environment because of tertiary interactions present in
C. Fare
`
s et al. Structure of MBP immunodominant epitope
FEBS Journal 273 (2006) 601–614 ª 2006 The Authors Journal compilation ª 2006 FEBS 603
the intact protein and absent in FF
2
. Small deviations
in the pH of the two samples may also account for the
chemical shift differences.
The secondary fold of FF
2
in all three conditions
was assessed using the chemical shifts of the H
a
and
C
a
atoms. A database of chemical shift indices was
compiled by Wishart et al. [37] to identify residues
involved in ordered secondary structures. Typically,
a-helical structures are identified by an uninterrupted
segment of four or more residues that have a positive
AB
D
FE
C

values for the same residue dissolved in water [37]. The
CSI analyses of our assignments, shown in Fig. 3, indi-
cate a noticeable tendency of a central 10-residue
segment of the polypeptide to adopt a helical
conformation from residues 5¢ to 14¢, for samples in
TFE-d
2
(Fig. 3B) and in DPC-d
38
(Fig. 3C), but not in
KCl (Fig. 3A). This tendency is shown by the uninter-
rupted downfield C
a
and upfield H
a
shifts for that
stretch of amino acids. Based on the CSI of FF
2
in
KCl, there is conflicting evidence of secondary struc-
ture formation (Fig. 3A). The H
a
shifts seem to indi-
cate weak a helix formation, which is unsubstantiated
by the C
a
chemical shifts.
In order to explain this apparent ambiguity, the
global conformation of the FF
2

of the interaction (weak, medium, strong). The charac-
teristic types of NOE connectivities for an a helix were
observed throughout the sequence, but were partic-
ularly consistent for a segment of residues between
positions 5¢ and 15¢. These included the sequential
d
NN
(i, i+1) and d
aN
(i, i+1), and medium-range d
ab
(i,
i+3), d
aN
(i, i+2), d
bN
(i, i+2), d
aN
(i, i+3), and d
bN
(i,
i+3). Numerous other (i, i+3) and (i, i+4) connectivi-
ties were also observed between side-chain protons
over this same sequence. This pattern reinforces the
a-helical model for the stretch of residues between P5¢
and P16¢.
Fig. 3. Amino acid sequence of the FF
2
polypeptide, and survey of
sequential and medium-range NOEs, and conformation-dependent

chemical shifts
are plotted relative to the random coil values available from Wishart
et al. [37], calibrated to TSP.
C. Fare
`
s et al. Structure of MBP immunodominant epitope
FEBS Journal 273 (2006) 601–614 ª 2006 The Authors Journal compilation ª 2006 FEBS 605
The structures of the FF
2
polypeptide presented here
are largely based on intramolecular NOE connectivi-
ties. The monomeric medium-sized FF
2
(2.2 kDa) is
predicted to have a rotational correlation time just
above the critical value for which NOE cross-peaks
vanish, owing to the equal contribution of the cross-
relaxation through the zero- and double-quantum tran-
sitions. Correspondingly, in the 100 mm KCl and 30%
TFE-d
2
samples, the NOESY cross-peaks are small
but have the same sign as the diagonal peak. In the
100 mm DPC-d
38
sample, cross-peaks are larger
because the FF
2
polypeptides in association with the
micelles have a longer correlation time.

dihedral restraints ()180° < F <0,)90° < Y <30°).
Additional loose H-bond distance restraints (2.5 < O
i
N
i+4
< 3.5) did not improve the quality of the
10 best structures, but reduced the occurrence of
NOE-violated structures over the ensemble of 100
structures. Approximately 200 NOE distance res-
traints were used for each condition, of which  50%
were interresidual (Table 1). These NOE connectivities
were either sequential, and⁄ or short-ranged (connect-
ing
1
H separated by 2–4 residues in the primary
sequence).
For each solution condition, the 10 lowest energy
structures were overlaid and represented from two
different orthogonal perspectives as line-connected
heavy atoms (backbone), as secondary structure sche-
matics (ribbons), and as space-filling models (Fig. 5).
As summarized in Table 1, these structures have low
energies (both for the restraint potentials and overall
potentials), small distance and angular deviations
from idealized molecular geometries, and few NOE
violations. The root mean square deviations
(RMSD), calculated from atom positions of the 10
best structures relative to the mean structure, are
reasonably low for all heavy nuclei (i.e. excluding
hydrogens) and for backbone nuclei. For the organic

conditions. The solid line represents FF
2
in 100 mM KCl, pH 6.5;
the dotted line represents FF
2
in 20 mM DPC; the dashed line rep-
resents FF
2
in 30% TFE; the dot-dash line represents FF
2
in water.
The spectra of FF
2
in TFE and DPC show the characteristic double
minima at 207 nm and 222 nm of an a helix. In contrast, the spec-
tra of FF
2
in 100 mM KCl and pure H
2
O are indicative of a primarily
random coil conformation.
Structure of MBP immunodominant epitope C. Fare
`
s et al.
606 FEBS Journal 273 (2006) 601–614 ª 2006 The Authors Journal compilation ª 2006 FEBS
showed this segment to have a propensity to form
transient a helices in aqueous solution [7]. The NMR
structures obtained under such conditions would thus
be consistent with a compendium of conformers in
fast exchange.

) counterion, and demonstrated that it
had a propensity to form an a helix. However, this
structure was transient in the absence of stabilizing
factors. In general, the organic solvent TFE is electric-
ally neutral and preferentially aggregates around the
polypeptide, displacing water, and thereby forming a
low dielectric environment that favors the formation of
intrapeptide hydrogen bonds [42]. Hence, in this
instance, the terminal and side chain charges must
come into close contact at the expense of bending
energies. The zwitterionic DPC, by contrast, provides
not only a hydrophobic surface from its acyl chain,
but both positive- and negative-charge contacts to the
polypeptide chain, allowing it to adopt a much more
relaxed conformation. The notion that the solvent
Table 1. Structural statistics of the FF
2
polypeptide structures under various solution conditions: 100 mM KCl, pH 6.5; 30% (vol) TFE-d
2
;
100 m
M DPC-d
38
, pH 6.5.
100 m
M KCl 30% TFE-d
2
100 mM DPC-d
38
Restraint for calculation

Additionally allowed 43.9 13.5 10.0
Generously allowed 9.3 5.9 1.2
Disallowed 9.3 6.4 6.4
RMSD from mean structure
Backbone atoms (overall) 3.69 ± 1.25 1.07 ± 0.26 0.86 ± 0.37
All heavy atoms (overall) 4.34 ± 1.28 1.55 ± 0.31 1.41 ± 0.41
Backbone atoms (2° structure) 0.29 ± 0.12 0.67 ± 0.18 0.42 ± 0.17
All heavy atoms (2° structure) 0.84 ± 0.35 1.09 ± 0.27 0.92 ± 0.19
C. Fare
`
s et al. Structure of MBP immunodominant epitope
FEBS Journal 273 (2006) 601–614 ª 2006 The Authors Journal compilation ª 2006 FEBS 607
environment can elicit structural changes in this
polypeptide, and by extension to the whole rmMBP
structure, is a major concern in the choice of mem-
brane-mimetic environment [4]. However, despite the
slight bend in the termini, the overall secondary struc-
ture is preserved by the presence of TFE-d
2
, while
avoiding possible aggregation and precipitation at the
high concentrations necessary for NMR.
Paramagnetic relaxation effects
The position of FF
2
in DPC-d
38
micelles was also inves-
tigated using two paramagnetic agents, 5-doxylstearic
acid (5-DSA) and FeCl

less pronounced. Here, the regions of larger broadening
are located near positions V7¢ and N12¢, as well as in the
vicinity of the C-terminus. However, the regions of high
relaxation with 5-DSA have relatively lower relaxation
because of the presence of Fe
3+
. The apparent fast
relaxation of V7¢ in the presence of both paramagnetic
agents suggests that the residue may lie at the micellar
interface where it would be exposed to both Fe
3+
and
5-DSA. A residue that shows slow relaxation under
both conditions is H8¢, although this may be due to
unfavorable electrostatic interaction between its side
chain and the Fe
3+
ions. Although the Fe
3+
ion is sol-
uble in aqueous solution, its location is also dictated by
Fig. 5. Structure of the FF
2
polypeptide in (A) 100 mM KCl, pH 6.5,
(B) 30% TFE-d
2
, (C) DPC-d
38
micelles, pH 6.5. To provide two dif-
ferent perspectives, a 90° rotation along the horizontal axis was

isolation, their large effective volume facilitates rapid
and specific interaction with a variety of ligands, the
association of which, in turn, effects a conformational
change. Often, defined segments of these proteins have
a propensity to form an a helix, and represent a bind-
ing target for some other protein [44]. The classic
18.5 kDa MBP isoform fits well into this paradigm,
because it is membrane-associated in vivo, but also
interacts with a plethora of other proteins, such as cal-
modulin, actin, tubulin, clathrin, and SH3-domain
containing proteins [4]. Here, we focused on a con-
served segment of MBP which is known to be a-helical
when bound to a membrane, is a potential calmodulin-
binding site, and also a primary immunodominant epi-
tope in multiple sclerosis. The helicity of this epitope
when associated with calmodulin is probable but not
yet proven [14], but it is extended when bound to the
MHC [8,9]. Thus, it exhibits a conformational adap-
tability depending on its environment and binding
partners.
Numerous epitopes of MBP have antigenic proper-
ties (13–32, 83–99, 111–129, 145–170, human sequence
numbering) [45]. Their structural characterization is
necessary to gain insight into their behavior as thera-
peutic agents, conditions under which a large variety
of environments are encountered. Recently, Tzakos
et al. determined the structure of the guinea pig myelin
basic polypeptide gpMBP(Q74–V85), using solution
NMR of the polypeptide dissolved in dimethylsulfox-
ide, and modeled its interaction with an MHC receptor

polypeptide in DPC-d
38
micelles. Normalized signal ampli-
tude of TOCSY (mixing time ¼ 40 ms) spin system cross-peaks is
displayed as a function of residue position for FF
2
dispersed in
DPC-d
38
micelles for each step of the titration of (A) 5-DSA (0.5–
2m
M), and (B) FeCl
3
(0–1.5 mM). The residual amplitudes were
measured for the ensemble of resolvable peaks of each spin sys-
tem at the x2 frequency of the H
N
.
C. Fare
`
s et al. Structure of MBP immunodominant epitope
FEBS Journal 273 (2006) 601–614 ª 2006 The Authors Journal compilation ª 2006 FEBS 609
polyproline type II helix [47]. Thus, the MBP segment
that we have studied may be critical in the protein’s
interaction with the myelin membrane, potentially in
proper positioning of this putative SH3-ligand and the
two known MAP kinase sites for functional roles
beyond membrane adhesion.
The structures of this segment have been well-char-
acterized under a variety of conditions and using dif-

Technology Centre (Hospital for Sick Children, Toronto,
Canada). The polypeptide was purified by reversed-phase
HPLC on a C
18
column (7.8 · 300 mm, Phenomenex, Tor-
rance, CA). As determined spectroscopically at 230 nm, the
polypeptide eluted after 30 min from a linear gradient bin-
ary solvent system (0–60% CH
3
CN in H
2
O with 0.1% tri-
fluoroacetic acid, in 60 min) at a flow rate of 1 mLÆmin
)1
.
This method yielded 200 mg of polypeptide; purity and
identity were confirmed by ESI-MS (not shown). The poly-
peptide, here referred to as FF
2
(because it comprises the
second of two Phe–Phe pairs within 18.5 kDa MBP, viz.,
F9¢–F10¢), required no further purification and was used
directly.
Sample preparation for NMR spectroscopy
FF
2
⁄ KCl
The FF
2
polypeptide was dissolved in 100 mm KCl,

maintained at 300 K.
FF
2
⁄ DPC-d
38
All experiments were performed on a 550 lL sample com-
prising 1 mm FF
2
polypeptide and 100 mm perdeuterated
DPC-d
38
(Cambridge Isotope Laboratories) in a 50 mm
phosphate buffer, adjusted to pH 6.5 and containing 10%
D
2
O. After dissolving the detergent and the polypeptide in
the buffer, the sample was transferred to a standard 5 mm
high-precision microcell tube and left to anneal for 30 min
at 60 °C before use. The sample temperature was main-
tained at 318 K during measurements. This sample was also
titrated with 5-DSA (55 mm solution in CD
3
OH) to obtain
final concentrations in the range of 0–2 mm, and FeCl
3
(55 mm aqueous solution) to obtain final concentrations
ranging from 0 to 1.5 mm.
Solution NMR spectroscopy
The high-resolution
1

13
C HSQC [53] spectra were acquired
using gradient pulses for coherence selection recording: 112
increments · 1024 scans, and 144 increments · 160 scans,
respectively. The
1
H and
13
C chemical shifts were referenced
Structure of MBP immunodominant epitope C. Fare
`
s et al.
610 FEBS Journal 273 (2006) 601–614 ª 2006 The Authors Journal compilation ª 2006 FEBS
indirectly to 3-(trimethylsilyl)-propionic acid (TSP). The
resonance assignments are reported in Table S1 (Supple-
mentary Material). All spectra were processed using the
xwinnmr package (Bruker BioSpin), and analyzed using
sparky 3 (TD Goddard & DG Kneller, University of
California, San Francisco).
Structure calculation and molecular modeling
All interhydrogen distance restraints were derived from the
NOE cross-peak volumes measured on the 300 ms two-
dimensional NOESY spectra and were used towards calcula-
ting a family of structures determined using cns v1.1 [54],
operating under aria v2.0 [55] for partially automated NOE
assignments; both programs were installed on a personal
computer running the Intel ⁄ Linux operating system. The
chemical shift assignment was based on the identification of
1
H spin systems on the two-dimensional TOCSY spectra,

i+4
) and backbone torsion angles
()180° < F <0,)90° < Y <30°), based on the CD and
chemical shift analyses (see Results and Discussion). One
hundred structures were generated at the end of 14 iterated
simulated annealing steps, with gradual decreases in the
NOE violation tolerance and in the partial assignment
threshold. In each simulated annealing step, the polypeptide
underwent molecular dynamics simulations with 3 fs time
steps, during which it was submitted to 10 000 heating steps
from 0 to 2000 K, and 16 000 cooling steps back to 50 K.
Each step used square-well distance and torsion restraints,
and the standard protein topology and parameters defining
the bonded and nonbonded geometrical energy functions
provided by the cns package [54]. Structural analyses and
generation of structure figures were carried out using mol-
mol 2.6 (ETH, Zu
¨
rich, Switzerland), also running on an
Intel ⁄ Linux operating system.
Data deposition
The
1
H and
13
C chemical shifts (Supplementary Material,
Table S1) were deposited in the BioMagResBank (http://
www.bmrb.wisc.edu) with Accession No. 6857.
CD spectroscopy
CD spectroscopy of samples of FF

Vladimir Ladizhansky for insightful discussion and
advice with the NMR experiments.
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Supplementary material