Tài liệu Báo cáo khoa học: Solution NMR structure of an immunodominant epitope of myelin basic protein Conformational dependence on environment of an intrinsically unstructured protein doc

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

Cellular Biology, and Biophysics
Interdepartmental Group, University of
Guelph, 50 Stone Road East, Guelph,
Ontario, Canada, N1G 2W1
Fax: +1 519 837 2075
Tel: +1 519 824 4120, ext. 52535
E-mail:
*Present address
Max-Planck-Institut fu
¨
r Biophysikalische
Chemie, NMR-Based Structural Biology,
Go
¨
ttingen, Germany.
Christophe Fare
`
s and David S. Libich contri-
buted equally to this work.
(Received 19 October 2005, revised
1 December 2005, accepted 7 December
2005)
doi:10.1111/j.1742-4658.2005.05093.x
Using solution NMR spectroscopy, three-dimensional structures have been
obtained for an 18-residue synthetic polypeptide fragment of 18.5 kDa
myelin basic protein (MBP, human residues Q81–T98) under three condi-
tions emulating the protein’s natural environment in the myelin membrane
to varying degrees: (a) an aqueous solution (100 mm KCl pH 6.5), (b) a
mixture of trifluoroethanol (TFE-d
2

) 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

-CD
2
-OH); TSP, 3-(trimethylsilyl)-propionic acid.
FEBS Journal 273 (2006) 601–614 ª 2006 The Authors Journal compilation ª 2006 FEBS 601
(SDSL) and electron paramagnetic resonance (EPR)
spectroscopy to investigate the topology of MBP when
bound to lipid bilayers of composition mimicking that
of the cytoplasmic face of myelin [6,7]. In particular, the
segment P85-VVHFFKNIVT-P96 (human sequence
numbering, Fig. 1) was shown to be an amphipathic
a helix lying on the surface of the membrane at a 9° tilt.
The phenylalanyl residues in the middle of this segment
penetrated deeply (up to 12 A
˚
) into the bilayer, and the
lysyl residue was in an ideal position for snorkeling [7].
There had been several previous, contradictory predic-
tions of the kind of secondary structure of this segment
of MBP, due to the plethora of experimental conditions,
and the SDSL ⁄ EPR experiments demonstrated its
a-helicity in situ. More recent crystallographic structures
of an MBP polypeptide encompassing this segment, in
a complex with human major histocompatibility com-
plex (MHC) and autoimmune T-cell receptors [8,9],
revealed an extended conformation, due to the struc-
tural requirements for MHC II binding [5,10].
This segment of MBP is highly conserved in primary
structure (Fig. 1), and is of biological and medical
interest for several reasons. The human hMBP(P85–
P96) region is a minimal B-cell epitope for HLA DR2b

(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-

kinetics.
We have initiated solution NMR studies of
18.5 kDa rmMBP to probe its three-dimensional
conformation under structure-stabilizing conditions,
namely 100 mm KCl, 30% trifluoroethanol (TFE-d
2
by
volume in water) [4,30], and 100 mm dodecylphosphat-
idylcholine (DPC-d
38
). Direct application of solution
NMR to membrane-associated MBP is problematic
because of the reduced mobility of the protein in a
reconstituted protein–lipid assembly. The challenge is
to find sample preparation conditions that would allow
high-resolution NMR studies of MBP in an environ-
ment most closely mimicking the native myelin sheath.
Although there have been previous NMR studies of
other MBP-derived polypeptides [31–33], they could
not, at the time, be compared with other structural
analyses in environments representative of the in vivo
situation. Here, we describe a solution NMR and CD
spectroscopic investigation of a segment of MBP com-
prising the primary immunodominant epitope, to char-
acterize further its conformational dependence on
environment, and to complement and extend previous
structural analyses that used SDSL ⁄ EPR and X-ray
Fig. 1. Comparison of amino acid sequences of the primary immu-
nodominant epitope from various species. The
BLASTP ⁄ CLUSTALW

[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,

and by extension, tertiary structural elements.
Although there is no guarantee that the structure of
FF
2
will be representative of the intact protein, the
conditions used here will help define solution condi-
tions in which these criteria are met. Using chemical
shift index (CSI) analysis of the resonances of the
intact protein recorded in 30% TFE-d
2
, regions of sec-
ondary structure coincide very well with elements that
were either predicted or shown to be transient in
molecular dynamics simulations. Another major con-
cern in studying IUPs in solution is their inherent flexi-
bility and their extreme dependence on the global
environment (as demonstrated below), necessitating
novel NMR strategies [34,35]. A condition that creates
a homogeneous population in solution allows for a
‘snapshot’ of the protein to be taken using solution
NMR techniques. Thus, in addition to providing a
complete characterization of the peptide per se, this
work represents a step towards establishing and opti-
mizing physiologically relevant and experimentally
tractable solution NMR conditions that will eventually
be applied to structural studies of the intact protein.
Results and Discussion
NMR spectroscopy
Resonance assignment
Standard ‘through-bond’ and ‘through-space’

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

TOCSY spectra exhibit the J-correlated i H
N
to H
a
frequencies of all residues except for the N-terminus
and the two prolyl residues, whereas the NOESY spec-
tra show the cross-relaxation peaks with frequencies
corresponding to the H
N
of residue i and H
a
of residue
(i)1) in close proximity. Despite the small size of the
polypeptide, some degree of overlap was present, espe-
cially for the consecutive residues H8¢,F9¢, and F10¢
with similar spin systems (Fig. 2A,C,E), and additional
correlations from both experiments were needed to lift
the ambiguity. However, no secondary set of cross-
peaks was observed, which suggested that FF
2
formed
a single, dominant, fast-averaging structure in the three
solution conditions investigated. The complete reson-
ance assignments for the three conditions are given in
the Supplementary Material (Table S1).
To strengthen further the relevance of FF
2
as a
polypeptide model for the immunodominant epitope
of MBP, 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

2
) with definite peak identification (refer
to values described previously [30], Accession No. 6100
in the BioMagRes Bank database, http://www.
bmrb.wisc.edu), there generally is very good agreement
with the chemical shifts identified in FF
2
recorded
under the same conditions. The H
N
and C
a
atoms were
identified in 15 residues in the Q78–T95 sequence of
rmMBP and differ on average by 0.2 and 1.2 p.p.m.,
respectively, with the corresponding primed residues of
FF
2
. However, in each case there is one outlying larger
difference: residues F9¢ (DdC
a
¼ 5.4 p.p.m. versus F86)
and F10¢ (DdH
N
¼ 0.46 p.p.m. versus F87), possibly
due to steric effects in the local environment. These
overall small deviations suggest similar F and Y angles
in both structures throughout the central segment of
the polypeptide, with an exception perhaps in the
vicinity of the Phe–Phe pair. Observed differences in

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

Fig. 2. Results of NMR correlation experiments of the FF
2
polypeptide in (A, B) aqueous solution (100 mM KCl, pH 6.5), (C, D) 30% TFE-d
2
,
(E, F) 100 m
M DPC-d
38
micelles, pH 6.5. Panels present
1
H
N
–
1
H
a
fingerprint regions of (A, C, E) a two-dimensional TOCSY (DIPSI-2) spec-
trum with mixing time of 120 ms, and (B, D, F) a two-dimensional NOESY spectrum with mixing time of 300 ms. Labels were added show-
ing the relevant peak assignments, by residue number.
Structure of MBP immunodominant epitope C. Fare
`
s et al.
604 FEBS Journal 273 (2006) 601–614 ª 2006 The Authors Journal compilation ª 2006 FEBS
C
a
chemical shift difference (downfield displacement)
and a negative H
a
chemical shift difference (upfield
displacement) relative to the random coil chemical shift

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

polypeptide was
examined by CD spectroscopy under various condi-
tions (Fig. 4). In aqueous solution (pure water, and
100 mm KCl, pH 6.5), the spectra indicated that the
polypeptide had little or no regular secondary struc-
ture. In organic and membrane-mimetic conditions
(30% TFE and 20 mm DPC, respectively), the spec-
tra clearly indicated an a-helical conformation. These
results are consistent with previous CD spectroscopic
studies of MBP and MBP fragments [38–40] and
support the inclusion of loose dihedral angle
restraints in the structure calculations of FF
2
in
TFE-d
2
and DPC-d
38
(see below).
NOE analysis
The pattern and size of NOE connectivities extracted
from the NOESY experiment also provide an inde-
pendent indication of the secondary structure of FF
2
.
The diagrams in Fig. 3 show the classification of NOE
connectivities into either sequential (i, i+1) or medium
range (i, i+2) (i, i+3), and (i, i+4) categories. The
extremities of each line connect the cross-relaxing resi-
dues, whereas the thicknesses relate to the magnitude

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 of FF
2
dissolved in (A) aqueous solution (100 mM
KCl, pH 6.5), (B) 30% TFE-d
2
, and (C) 100 mM DPC-d
38
micelles,
pH 6.5. Thick, medium, and thin bars indicate strong, intermediate,
and weak NOE intensities, respectively, linking the residues
involved in sequential (d
aN
,d
bN
and d
NN
) and medium-range (d
ab
and d
aN
⁄ d
bN
) NOE connectivities. The
13
C
a
and
1
H
a

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.

Sufficient NOE cross-peaks were compiled, partially
assigned, and measured to calculate the structure of FF
2
in 100 mm KCl (pH 6.5), 30% TFE-d
2
, and in 100 mm
DPC-d
38
micelles. Although two-dimensional NOESY
spectra were measured for several mixing times (100,
200, and 300 ms) and were all used to assign connectivi-
ties, the magnitude of NOEs was based on the Gaus-
sian-function fitted volume of cross-peaks from the
two-dimensional spectrum recorded at 300 ms. For
the correlation time regime of FF
2
under all conditions,
the NOE build-up curves are expected to vary quasi-lin-
early over the time range covered by these mixing times.
The NOE cross-peaks with heavy overlap were fit using
the sum-over-box algorithm in the sparky package.
As described in Experimental Procedures, the
aria ⁄ cns calculations were provided with: (a) chemical
shift assignments; (b) a list of NOE cross-peak vol-
umes that were tentatively assigned; and (c) for the
TFE-d
2
and DPC-d
38
structures, loose initial backbone

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

and membrane-mimetic conditions, considering only
residues 5¢ to 16¢, these RMSD values are further
reduced by  0.5 A
˚
. This segment is a well-defined
helix, with F and Y torsion angle pairs falling
within the allowed a-helical region of the Ramachan-
dran plot [41]. Under aqueous conditions, deviations
from the allowed regions of the Ramachandran plot
are greater than observed under the other two
conditions, suggesting the incomplete formation of
an a-helical structure. It should be noted that the
majority of residues (81.4%) fall into the allowed or
generously allowed regions, which suggests that the
peptide adopts a structure (in the core region) sim-
ilar to a helix. There is extreme flexibility of the
polypeptide near the termini, particularly residues
D2¢,E3¢, R17¢ and T18¢ which have the largest devi-
ations from the most highly populated regions of the
Ramachandran plot, and which contribute to the
proportion of residues in the disallowed space.
In aqueous solution (100 mm KCl, pH 6.5), the
polypeptide forms a relatively stable core, and sug-
gests a weakly helical conformation in the most
highly conserved region (V6¢ to F10¢). These results
are consistent with the CD data (Fig. 4) and with
our recent molecular dynamics simulations that
Fig. 4. CD spectroscopy of the FF
2
polypeptide in various solution

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.

In the organic and membrane-mimetic environ-
ments, the helical segment stretches over 10 residues,
forms three loops, and exhibits little curvature. As
expected, the helix also delineates the discrete amphi-
pathic nature of the polypeptide. To illustrate this seg-
regation of hydrophobic ⁄ hydrophilic residues around
the helical conformation, Fig. 5 shows the electrostatic
surface charge of residues P5¢ to P16¢ of the proposed
structures of FF
2
from two orthogonal view angles.
The partitioning of charges onto opposing faces of the
helix further reinforces the amphipathic nature of this
peptide. A noticeable difference between the two struc-
tures is seen, however, in both N- and C-termini. In
TFE-d
2
, the ends bend abruptly at the site of the two
prolyl residues, and fold back towards the hydrophilic
side of the helix. In DPC-d
38
, the helix is more
elongated despite similar interruptions of the helix at
P5¢ and P16¢.
This important difference can be rationalized from
the nature of the solvent. Previously, Bates et al. [7]
performed molecular dynamics simulations of the cen-
tral immunodominant segment in water, with an added
chlorine (Cl
–

) 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

Total number of NOE restraints 182 266 199
Unambiguous 168 246 183
Intraresidue 122 116 107
Sequential 35 68 40
Short-range (long range) 11(0) 60(2) 36(0)
Dihedral angle 0 30 30
Restraint violations
Distance restraints ¼ 0.3 A
˚
(¼ 0.5 A
˚
) 0(0) 0(0.65) 0(0)
Dihedral angle restraints of ¼ 5 0 0.9 0
Deviations from idealized geometry
Bonds (A
˚
) 0.0015 ± 0.00009 0.00296 ± 0.00029 0.00185 ± 0.00007
Angles (°) 0.26 ± 0.01 0.46 ± 0.03 0.29 ± 0.01
Impropers (°) 0.13 ± 0.01 0.43 ± 0.15 0.12 ± 0.01
Dihedral (°) 44.23 ± 0.63 40.66 ± 0.70 39.63 ± 0.38
VdW (A
˚
) 12.07 ± 0.78 25.12 ± 2.71 10.92 ± 0.57
Energies (kcalÆmol
)1
)
NOE restraint energy 1.88 ± 0.87 9.69 ± 4.98 2.17 ± 0.39
Total energy ) 512.4 ± 35.5 ) 501.4 ± 39.9 ) 544.9 ± 28.8
Ramachandran statistics (%)
Most allowed 37.5 74.1 82.4

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

3
, which, respectively, partition
inside or outside the hydrophobic interior of the
micelles. These molecules act locally as strong signal-
relaxing agents, causing a broadening proportional to
the inverse of the average of the distance to the sixth
power (<r
)6
>), between the unpaired electron of the
paramagnetic agent and the interacting nucleus. Thus,
these agents can report the positioning of individual
residues, and on the orientation of the whole helix relat-
ive to the micellar core. The effects of these agents were
measured on an ensemble of cross-peaks belonging to
the same residue spin system in TOCSY spectra meas-
ured with a 40 ms mixing time, and are summarized in
Fig. 6. For the 5-DSA titration data, there are three
short regions of strong relaxation effect (V6¢–V7¢,
F9¢–F10¢–K11¢ and I13¢–V14¢), separated by regions of
lower effect (H8¢, N12¢). The termini of the polypeptide
are generally not affected by the presence of the 5-DSA
in the micelles. A reverse trend is apparent when the
experiment is repeated on the same FF
2
⁄ DPC-d
38
sam-
ple to which FeCl
3
was added, although the effect seems

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

used to convert the left structure to the right structure. The N-ter-
minus is at the left for every structure. The best-fit overlays of the
10 lowest overall energy structures obtained with the
ARIA protocol,
described in Experimental Procedures, are illustrated as a line-
model of the covalent bonds between heavy atoms, or as ribbons
(A only). In (B) and (C), the means of the 10 lowest energy struc-
tures are presented as schematic representations of a-helical struc-
ture, and as space-filling models. The surfaces in the latter
representations are colored with a red-to-white-to-blue gradient indi-
cating the electrostatic partial charge distribution (red ¼ positive,
white ¼ neutral, blue ¼ negative).
Structure of MBP immunodominant epitope C. Fare
`
s et al.
608 FEBS Journal 273 (2006) 601–614 ª 2006 The Authors Journal compilation ª 2006 FEBS
electrostatic interactions which are unfavorable in the
vicinity of the partially positively charged side chain of
histidine. These results demonstrate that the polypeptide
a helix forms distinct hydrophobic and electrostatic
contacts with the DPC micelles, and are in agreement
with the SDSL ⁄ EPR mapping and positioning of the
a-helical model of this epitope of MBP on the surface of
a lipid bilayer [6,7].
Biological significance
MBP is an ‘intrinsically unstructured’ (or ‘conforma-
tionally adaptive’) protein [4]. Such proteins constitute
roughly one third of the eukaryotic proteome, and are
generally involved in signaling and ⁄ or cytoskeletal
assembly [43,44]. Although seemingly unstructured in

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

site [27]. The segment QKSQRSQDENPV from the
guinea-pig sequence, corresponds to the 13-residue seg-
ment hMBP(Q74–V86) of the human sequence, which
is N-terminal to our 18-residue FF
2
polypeptide. Thus,
the overlap region between gpMBP(Q74–V85) and FF
2
is only six residues (QDENPV), of which QDE were
least well-defined conformationally in both studies, due
to being at the termini of both constructs. Similarly,
minimal direct comparison can be made with previous
studies of other MBP segments [31,33,46] or the cyclic
analogs [28].
The FF
2
sequence is highly conserved evolutionarily
compared with the rest of the protein (Fig. 1), and
there are several post-translational modifications
within it: Q1¢ can be deamidated, R17¢ can be deimi-
nated, and T15¢-and T18¢ can both be phosphorylated
by MAP kinases [4]. In all species except fish, this
sequence is followed by a triproline repeat
(P19¢P20¢P21¢) and comprises a potential SH3-ligand
(P16¢R17¢T18¢P19¢), which could be expected to form a
Fig. 6. Paramagnetic relaxation effects of 5-DSA, and of FeCl
3
,on
the FF
2

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-

ferent biophysical approaches, here and elsewhere [7].
This investigation serves to guide ongoing solution
NMR investigations of the full-length protein. The
problems faced here are similar to those in NMR
structural studies of other membrane-associated
and ⁄ or intrinsically unstructured proteins such as
a-synuclein [48–50], and similar strategies are thus
suggested to probe MBP’s conformational ensemble.
Whereas the study of Bates et al. [7] indicated that the
MBP segment PVVHFFKNIVTP was a-helical in situ
in a membrane, this high-resolution NMR structural
study proved its a-helicity in a stabilizing solution
environment, and supports the use of DPC-d
38
or
TFE-d
2
[30] as a structure-stabilizing condition for
solution NMR studies of the full-length protein.
Experimental procedures
Peptide synthesis
The 18-residue polypeptide hMBP(Q81–T98) (Q
1¢
DENPV-
VHFFKNIVTPRT
18¢
), encompassing the immunodominant
epitope region matching a membrane surface-interacting
a helix (V86 to T95), was synthesized via 9-fluorenylmeth-
oxycarbonyl (Fmoc) chemistry at the Advanced Protein

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,

pH 6.5, to a final concentration of 2 mm. The 550 lL
sample was transferred to a standard 5 mm high-precision
microcell tube (528 pp, Wilmad-Labglass, Buena, NJ). For
the measurements of natural abundance
13
C, the polypep-
tide concentration was increased to 20 mm. The sample
temperature was maintained at 298 K.
FF
2
⁄ 30% TFE-d
2
Homonuclear
1
H experiments were performed on a 600 lL
FF
2
NMR sample prepared by dissolving the polypeptide
to a concentration of 5 mm in 30% TFE-d
2
(Cambridge
Isotope Laboratories, Andover, MA) in H
2
O. As for the
aqueous solution, the sample was transferred to a standard
5 mm high-precision microcell tube. The polypeptide con-
centration was increased to 20 mm for experiments invol-
ving natural abundance
13
C. The sample temperature was

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

H,
13
C, and
15
N NMR spectra were
recorded on a Bruker Avance (Bruker BioSpin, Milton, ON,
USA), spectrometer operating at a field of 14.1 T (corres-
ponding to the resonance frequency of 600.1 MHz for
1
H)
and implemented with a triple resonance gradient inverse
probe. The 90° pulses were typically 12 and 15 l s, and the
spectral widths were set to 12 and 165 p.p.m. for
1
H and
13
C,
respectively. Solvent (water) signal purging was achieved
using a 2 s presaturation pulse with the carrier frequency set
on the water
1
H signal. The phase-sensitive two-dimensional
TOCSY [36] (with DIPSI-2 [51] isotropic mixing times:
50–120 ms) and two-dimensional NOESY [52] (mixing times:
100–300 ms) experiments were typically acquired using a
recycling delay of 2 s, 128 increments, and 96 scans per
increment, for a total experimental time of  5.12 h. The nat-
ural abundance
1
H–

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,

and the connectivities were identified mainly from
1
H
N
–
1
H
N
and
1
H
N
–
1
H
a
fingerprint regions. The
1
H–
1
H NOE cross-
peaks were fit to a Gaussian curve and integrated using
sparky. The peak volumes and chemical shifts were used as
the distance restraint input for aria, along with their most
probable assignment. Stereochemical and overlap assignment
ambiguities were automatically resolved by the aria proto-
col. Additional restraining input parameters included loose
H-bonding (C ¼ O
i
HN

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

2
(0.2 mgÆmL
)1
) in aque-
ous solution (100 mm KCl, pH 6.5), in 30% TFE, and in
20 mm DPC (pH 6.5), was performed using a Jasco J600
spectrapolarimeter (Japan Scientific, Tokyo, Japan). Sam-
ples had volumes of 1 mL, and were measured in a 0.1 cm
path-length quartz cuvette. Measurements were taken at a
100 nmÆmin
)1
rate, at 0.1 nm intervals, over a range of 190
to 250 nm. All measurements were recorded at ambient
room temperature. Four successive scans were recorded, the
sample blank was subtracted, and the scans were averaged.
The data averaging and smoothing (using an inverse square
algorithm) operations were accomplished with the sigma-
plot (SPSS, Chicago, IL) computer program.
Acknowledgements
This work was supported by the Canadian Institutes
of Health Research (Operating Grant MOP 43982 to
GH), and by the Natural Sciences and Engineering
Research Council of Canada (to GH). DSL was
the recipient of an Ontario Graduate Scholarship. The
authors are grateful to Dr Nam-Chiang Wang of the
Peptide Synthesis Facility (Hospital for Sick Children,
Toronto) for synthesizing the FF
2
polypeptide, and to
Ms Valerie Robertson, Dr Martine Monette, and Dr

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

The following supplementary material is available
online:
Table S1. The
1
H and
13
C chemical shifts of of the
FF
2
polypeptide, referenced to TSP, under various
solution conditions: (A) 100 mm KCl, pH 6.5; (B) 30%
(vol) TFE-d
2
; (C) 100 mm DPC-d
38
, pH 6.5. These data
have been deposited in the BioMagResBank (http://
www.bmrb.wisc.edu) with accession number 6857.
This material is available as part of the online article
from
Structure of MBP immunodominant epitope C. Fare
`
s et al.
614 FEBS Journal 273 (2006) 601–614 ª 2006 The Authors Journal compilation ª 2006 FEBS