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: gharauz@uoguelph.ca
*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 mmKCl pH 6.5), (b) a
mixture of trifluoroethanol (TFE-d
2
) and water (30 : 70% v ⁄v), and (c) a
dispersion of 100 mmdodecylphosphocholine (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, ahelix 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
ahelix 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 ª2006 The Authors Journal compilation ª2006 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
ahelix lying on the surface of the membrane at a 9tilt.
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
(http://www.biomsmedical.com) [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 mmKCl, 30% trifluoroethanol (TFE-d
2
by
volume in water) [4,30], and 100 mmdodecylphosphat-
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
`set al.
602 FEBS Journal 273 (2006) 601–614 ª2006 The Authors Journal compilation ª2006 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 mmKCl, 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 mmDPC-d
38
(Fig. 2E,F). The
TOCSY spectra exhibit the J-correlated iH
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 iand 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 mmKCl 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
¼5.4 p.p.m. versus F86)
and F10¢(DdH
N
¼0.46 p.p.m. versus F87), possibly
due to steric effects in the local environment. These
overall small deviations suggest similar Fand Yangles
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
`set al. Structure of MBP immunodominant epitope
FEBS Journal 273 (2006) 601–614 ª2006 The Authors Journal compilation ª2006 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 mMKCl, pH 6.5), (C, D) 30% TFE-d
2
,
(E, F) 100 mMDPC-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
`set al.
604 FEBS Journal 273 (2006) 601–614 ª2006 The Authors Journal compilation ª2006 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 ahelix 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 mmKCl, 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 mmDPC, 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 ahelix 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 mMDPC-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
`set al. Structure of MBP immunodominant epitope
FEBS Journal 273 (2006) 601–614 ª2006 The Authors Journal compilation ª2006 FEBS 605