We recently used site-directed spin-labeling Keywords correlation spectroscopy; multiple sclerosis; myelin basic protein; immunodominant epitope; solution NMR Correspondence G.. This pol
Trang 1of myelin basic protein
Conformational dependence on environment of an intrinsically
unstructured protein
Christophe Fare`s1,*, David S Libich1and George Harauz1
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 mm KCl pH 6.5), (b) a mixture of trifluoroethanol (TFE-d2) and water (30 : 70% v⁄ v), and (c) a dispersion of 100 mm dodecylphosphocholine (DPC-d38, 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-d38, 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.
Trang 2(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 Ca2+–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 mm KCl, 30% trifluoroethanol (TFE-d2by volume in water) [4,30], and 100 mm dodecylphosphat-idylcholine (DPC-d38) 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¢.
Trang 3crystallographic techniques The 18-residue polypeptide
Q1¢DENPVVHFFKNIVTPRT18¢, was synthesized and
is referred to here as FF2, 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
FF2 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-d2, 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’ 1H–1H
homonuclear correlation experiments were employed
to assign the resonances of the polypeptide FF2, 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-d2), and
membrane-mimetic (DPC-d38micelles, 1 : 100
polypep-tide⁄ lipid molar ratio) environments The 1H 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-d2
(Fig 2C,D), and 100 mm DPC-d38 (Fig 2E,F) The
TOCSY spectra exhibit the J-correlated i HN to Ha
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 HNof residue i and Haof 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 FF2 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 FF2 as a polypeptide model for the immunodominant epitope
of MBP, the 13C frequencies of the backbone spins of
FF2 were also assigned and compared with those pre-viously published for full-length MBP under the same 30% TFE-d2conditions [30] Assignments were carried out on the standard heteronuclear single-quantum (HSQC) experiment and were based on the 1H assign-ment presented above Because of the low abundance
of the 13C nuclei, the sample concentration was raised
to 20 mm, for which excellent solubility was still achievable in 100 mm KCl and 30% TFE-d2 At this concentration, only minor1H 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-d2) 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 FF2 recorded under the same conditions The HNand Caatoms 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
FF2 However, in each case there is one outlying larger difference: residues F9¢ (DdCa¼ 5.4 p.p.m versus F86) and F10¢ (DdHN¼ 0.46 p.p.m versus F87), possibly due to steric effects in the local environment These overall small deviations suggest similarF 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 Ca chemical shifts may be due to changes in local environment because of tertiary interactions present in
Trang 4the intact protein and absent in FF2 Small deviations
in the pH of the two samples may also account for the
chemical shift differences
The secondary fold of FF2 in all three conditions
was assessed using the chemical shifts of the Ha and
Ca 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
D
F E
C
Fig 2 Results of NMR correlation experiments of the FF 2 polypeptide in (A, B) aqueous solution (100 m M KCl, pH 6.5), (C, D) 30% TFE-d2, (E, F) 100 m M DPC-d38micelles, pH 6.5 Panels present 1 HN– 1 Hafingerprint 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 specspec-trum with mixing time of 300 ms Labels were added show-ing the relevant peak assignments, by residue number.
Trang 5Ca chemical shift difference (downfield displacement)
and a negative Ha 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-d2(Fig 3B) and in DPC-d38(Fig 3C), but not in
KCl (Fig 3A) This tendency is shown by the
uninter-rupted downfield Ca and upfield Ha shifts for that
stretch of amino acids Based on the CSI of FF2 in
KCl, there is conflicting evidence of secondary
struc-ture formation (Fig 3A) The Ha shifts seem to
indi-cate weak a helix formation, which is unsubstantiated
by the Cachemical shifts
In order to explain this apparent ambiguity, the
global conformation of the FF2 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 FF2 in
TFE-d2 and DPC-d38 (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 FF2
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
dNN(i, i+1) and daN(i, i+1), and medium-range dab(i,
i+3), daN(i, i+2), dbN(i, i+2), daN(i, i+3), and dbN(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 FF2dissolved in (A) aqueous solution (100 m M
KCl, pH 6.5), (B) 30% TFE-d2, and (C) 100 m M DPC-d38 micelles,
pH 6.5 Thick, medium, and thin bars indicate strong, intermediate, and weak NOE intensities, respectively, linking the residues involved in sequential (daN, dbN and dNN) and medium-range (dab and daN⁄ d bN ) NOE connectivities The 13 Caand 1 Hachemical shifts are plotted relative to the random coil values available from Wishart
et al [37], calibrated to TSP.
Trang 6The structures of the FF2polypeptide presented here
are largely based on intramolecular NOE
connectivi-ties The monomeric medium-sized FF2 (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-d2 samples, the NOESY cross-peaks are small
but have the same sign as the diagonal peak In the
100 mm DPC-d38 sample, cross-peaks are larger
because the FF2 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 FF2
in 100 mm KCl (pH 6.5), 30% TFE-d2, and in 100 mm
DPC-d38 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 FF2under 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-d2 and DPC-d38structures, loose initial backbone dihedral restraints ()180 < F < 0, )90 < Y < 30) Additional loose H-bond distance restraints (2.5 < Oi Ni+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 1H 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 m M KCl, pH 6.5;
the dotted line represents FF2in 20 m M 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 m M KCl and pure H2O are indicative of a primarily
random coil conformation.
Trang 7showed 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 FF2 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-d2, the ends bend abruptly at the site of the two
prolyl residues, and fold back towards the hydrophilic
side of the helix In DPC-d38, 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 preferentielectric-ally 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 m M KCl, pH 6.5; 30% (vol) TFE-d2;
100 m M DPC-d38, pH 6.5.
Restraint for calculation
Restraint violations
Deviations from idealized geometry
Energies (kcalÆmol)1)
Ramachandran statistics (%)
RMSD from mean structure
Trang 8environment 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-d2, while
avoiding possible aggregation and precipitation at the
high concentrations necessary for NMR
Paramagnetic relaxation effects The position of FF2in DPC-d38micelles was also inves-tigated using two paramagnetic agents, 5-doxylstearic acid (5-DSA) and FeCl3, 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 FF2⁄ DPC-d38 sam-ple to which FeCl3was 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 Fe3+ 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 Fe3+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 Fe3+ions Although the Fe3+ion is sol-uble in aqueous solution, its location is also dictated by
Fig 5 Structure of the FF 2 polypeptide in (A) 100 m M KCl, pH 6.5, (B) 30% TFE-d2, (C) DPC-d38micelles, 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 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).
Trang 9electrostatic 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 epiepi-tope 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 FF2 polypeptide Thus, the overlap region between gpMBP(Q74–V85) and FF2
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 FF2 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 FF2polypeptide 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–
2 m M ), and (B) FeCl3 (0–1.5 m M ) The residual amplitudes were
measured for the ensemble of resolvable peaks of each spin
sys-tem at the x2 frequency of the H N
Trang 10polyproline 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-d38 or
TFE-d2 [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) (Q1¢
DENPV-VHFFKNIVTPRT18¢), 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 C18column (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% CH3CN in H2O 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 FF2 (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
FF2⁄ KCl
The FF2 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 13C, the polypep-tide concentration was increased to 20 mm The sample temperature was maintained at 298 K
FF2⁄ 30% TFE-d2
Homonuclear 1H experiments were performed on a 600 lL
FF2 NMR sample prepared by dissolving the polypeptide
to a concentration of 5 mm in 30% TFE-d2 (Cambridge Isotope Laboratories, Andover, MA) in H2O 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 13C The sample temperature was maintained at 300 K
FF2⁄ DPC-d38
All experiments were performed on a 550 lL sample com-prising 1 mm FF2 polypeptide and 100 mm perdeuterated DPC-d38 (Cambridge Isotope Laboratories) in a 50 mm phosphate buffer, adjusted to pH 6.5 and containing 10%
D2O 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 60C 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 CD3OH) to obtain final concentrations in the range of 0–2 mm, and FeCl3 (55 mm aqueous solution) to obtain final concentrations ranging from 0 to 1.5 mm
Solution NMR spectroscopy The high-resolution 1H, 13C, and 15N 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 1H) and implemented with a triple resonance gradient inverse probe The 90 pulses were typically 12 and 15 ls, and the spectral widths were set to 12 and 165 p.p.m for1H and13C, respectively Solvent (water) signal purging was achieved using a 2 s presaturation pulse with the carrier frequency set
on the water1H 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 1H–13C HSQC [53] spectra were acquired using gradient pulses for coherence selection recording: 112 increments· 1024 scans, and 144 increments · 160 scans, respectively The1H and13C chemical shifts were referenced