two distinct structures of α-conotoxin gi in aqueous solution

Eur. J. Biochem.254, 2382247 (1998) FEBS1998

Two distinct structures of B-conotoxin GI in aqueous solution

Innokenty V. MASLENNIKOV, Alexander G. SOBOL, Konstantin V. GLADKY, Alexey A. LUGOVSKOY, Andrey G. OSTROVSKY,Victor I. TSETLIN, Vadim T. IVANOV and Alexander S. ARSENIEV

Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia

(Received 30 January1998) 2 EJB 98 0146/3

The detailed analysis of conformational space ofA-conotoxin GI in aqueous solution has been per-formed on the basis of two-dimensional NMR spectroscopy data using multiconformational approach. Asthe result, two topologically distinct interconvertible sets of GI conformations (populations of 78% and22%) have been found. A common feature of the two sets is the Asn4-Cys7β-turn. The Gly8 to Tyr11region has a structure of right-handed helical turn in the major set and two sequential bends in the minorone. N-terminus and C-terminus also have different orientations, anti-parallel in the major conformationalset and parallel in the minor one. An average pairwise rmsd for backbone heavy atoms is 0.56 A˚ in themajor set, 0.23 A˚ in the minor, and1.85 A between the structures of the two sets. The X-ray structure ofGI [Guddat, L. W., Martin, J. A., Shan, L., Edmundson, A. B. & Gray, W. R. (1996) Biochemistry 35,11329211335] has the same folding pattern as the major NMR set, the average backbone rmsd betweenthe two structures being 0.77 A˚ .

Keywords:conotoxin ; nicotinic acetylcholine receptor; NMR; conformational analysis.

The venom of marine snails of theConusgenus contains structure, dynamics, and their relationship with the biologicalfunction of peptides. However, in practice high-resolution X-rayoligopeptide neurotoxins which are sophisticated tools for study-

ing a wide range of receptors and ion channels:A-conotoxins structures of small flexible peptides are often biased by crystalpacking forces and may differ from a biologically active confor-block nicotinic acetylcholine receptors (nAChR),µ-conotoxins

and ω-conotoxins act on voltage-gated sodium and calcium mation. In turn, NMR spectroscopy faces technical difficultieswhen dealing with flexible molecules. Quite often it is statedchannels, respectively, while the targets of the contatokins are

the N-methyl-D-aspartate receptors (see review [1]). The most that a number of different conformations in fast exchange arepresent in solution, among which one can expect a biologicallynumerous family is that ofA-conotoxins consisting of over10

members whose primary structure has been established and bio- active conformation. Several techniques were proposed foranalysis of NMR data on flexible molecules [11218], but thelogical activity analysed. The efficiency of the interaction be-

tween nAChR andA-conotoxins depends on the species and sub- problem is still far from being solved. Here we introduce a newapproach for the analysis of NMR data of flexible peptides andtype of the nAChR. Most ofA-conotoxins block the neuromus-

cular nAChR. However, several recently discoveredA-conotox- show thatA-conotoxin GI in aqueous solution is represented bytwo topologically distinct sets of interconvertible conformations.ins act on neuronal nAChR [2]. Besides the above-mentioned

species-selectivities and subtype-selectivities, recent data de-scribe the affinity and binding selectivity to theTorpedomem-branes or to AChR in the mouse cell line for the naturally occur-

MATERIALS AND METHODSring A-conotoxins GI, M1 and S1 [325] as well as for severalsynthetic analogs of GI and S1 [6, 7]. It was shown that M1 and Synthesis and purification ofB-conotoxin GI. A-ConotoxinGI bind more efficiently than S1 to the high-affinity site at the GI was synthesised in solution using a block condensationA/γ subunit interface of theTorpedoAChR, whereas these three51(315). In protecting the carboxyls of glycine and proline res-toxins bind with the same potency to the low-affinityA/δ site of idues, benzyl, methyl or 9-fluorenylmethyl esters were tested.the receptor [5, 6]. The latter were found most preferable in terms of yields and

Spatial structure ofA-conotoxin GI has been recently solvedpurity of final products.A-Amino groups were temporarily pro-to 1.2-A resolution by X-ray crystallography [8]. There are over-tected by butyloxycarbonyl groups, whereas side-chain func-all similarities between the X-ray structure and those derived attional groups were blocked by benzyl-type groupings. The SH-relatively low resolution by NMR spectroscopy in water [9] andgroups of cysteine residues were protected in the following man-(2H6)dimethylsulfoxide [10] solutions. ner: 4-methylbenzyl groups for Cys3 and Cys13, and

It is well known that X-ray crystallography and NMR acetamidomethyl groups for Cys2 and Cys7. Fragment conden-spectroscopy should provide complementary data on the spatialsation was performed with the aid ofN-hydroxy-5-norboren-2,3-

dicarboxyimide. The protecting groups were removed by liquidCorrespondence toA. S. Arseniev, Shemyakin-Ovchinnikov Insti-

HF in the presence ofp-cresol andp-thiocresol. The disulfidetute,16/10 Miklukho-Maklaya,117871 Moscow, Russiabridge between Cys3 and Cys13 was closed by air oxidation atFax: 17 95 335 50 33.pH 8.5 (room temperature, 24 h). After this, acetamidomethylE-mail : [email protected] were removed by iodine in the dimethylformamide/aceticAbbreviations. AChR, acetylcholine receptor ; nAChR, nicotinic

AChR; DQF-COSY, double-quantum-filtered COSY. acid mixture (9:1) at 4°C in 15 min with simultaneous closure

239Maslennikov et al. (Eur. J. Biochem. 254)

(a) extraction of distance (1/r6-approximation) and torsionof the disulfide bridge Cys2-Cys7. Purification of the intermedi-ate product with one disulfide bridge, as well as of the target angle constraints, proton stereospecific assignments;

(b) generation of initial structure set by the DIANA pro-product was achieved by reverse-phase HPLC on a Nucleosil7C18 column (6 mm3250 mm) in a linear gradient of acetoni- gram;

(c) calculation of refined upper and generation of lower dis-trile (from 5% to 35% in 30 min). The yield of the final product,calculated for the crude product obtained after HF treatment, tance constraints by the MARDIGRAS program on the basis of

NOE cross-peak volumes and initial structure set;was 26%.The identity and purity of the synthetic peptide GI was (d) hydrogen bond detection and introduction of upper

(NH. ..O 2.3 A, N.. .O 3.3 A) and lower (NH.. .O1.8 A, N. ..Ochecked by matrix-assisted laser-desorption ionisation massspectrometry. NMR spectra obtained under the conditions de- 2.8 A˚ ) distance constraints to meet the hydrogen bond length

and angle criteria;scribed in [9] demonstrated absence of impurities or disulfideisomers in the GI sample. (e) eight additional lower distance constraints (no-NOE),

based on cross-peaks expected according to the structures ob-NMR spectroscopy.To facilitate water signal suppression,all NMR experiments were performed in 5-mm heavy-wall tained after step (b) but not present in NOESY spectra were

introduced. The no-NOE constraints were introduced as lowerNMR tube (sample volume150 µl). 4.5 mg of GI were dissolvedin 150µl of H2O (10% D2O) or D2O (100% deuterium, SIL). bound distance constraints of 3.0 A˚ . The value of 3.0 A˚ is quite

cautious, as the smallest of observed NOE corresponds to 5.3 A˚1H-NMR spectra were obtained using a 600 MHz (Varian Unity600) spectrometer at 5°C unless otherwise specified. That tem- upper distance constraint.

(f) the final standard set of18 GI structures was obtainedperature proved optimal for obtaining NOEs in GI.Double-quantum-filtered (DQF)-COSY [19], TOCSY [20] using all upper and lower distance and torsion angle constraints.

The criterion of the final structure selection was the DIANAwith a mixing time of 50 ms, and NOESY [21] with mixingtimes of100, 200 and 400 ms, were recorded in the pure phase- penalty function.

The multiconformational approach to spatial structure calcu-absorption mode by collecting hypercomplex data [22]. Relax-ation delay of1.6 s was used throughout. To detect amide pro- lation consists of the following steps:

(a) analysis of experimental data and discrimination of thetons with slow hydrogen-deuterium exchange rates, one-dimen-sional spectra were recorded starting at 5 min on GI solution in subsets of non-averaged (static) and ensemble-averaged NOE

and spin-spin coupling data. For this purpose we tested the con-D2O.NMR data was processed with VNMR, Varian software, and sistency of experimental data set for each residue by comparing

spin-spin coupling constant values with local distance con-analysis of the data was performed with the XEASY program[23]. Complete proton resonance assignment was done using a straints and calculating the local DIANA penalty function and

Rx-factor;previously published assignment [9] with the XEASY program.Proton spin-spin coupling constants (12 H-NCA-H and 20 H- (b) generation of structure set by using standard approach

(see above) on the basis of only static data (NOEs, torsion-angleCACβ-H) were measured in one-dimensional spectra. Volumes of316 unambiguously assigned NOE cross-peaks were measured constraints and stereospecific assignments). The generation was

stopped when maximal rmsd between structures in the set ceasedin 100 ms and 200 ms NOESY spectra with the programXEASY based on non-linear approximation algorithm for the to grow. So the set covers all possible (according to the static

constraints) conformational space. Each structure from this setcross-peaks line shapes in both directions of two-dimensionalspectrum and on least-squares method. did not show violation of the applied constraints greater than

0.2 A for distance, 0.1 A for van-der-waals, and 2 degrees forExperimental constraints. Distance constraints were de-duced from the NOE cross-peaks volumes measured in 200 ms angle constraints;

(c) clustering of the conformations obtained in step (b) byNOESY spectra. The1/r6 approximation was used for evaluationof initial upper constraints (program CALIBA, [24]). Refined the hierarchical method based on average linkage algorithm. The

NMRCLUST program [26] was used here to reduce the com-upper and lower constraints were derived by complete relaxationmatrix approach (program MARDIGRAS, [25]) at the final plexity of the structure set and to minimise the loss of informa-

tion. The conformations were grouped together using rmsd ofstages of structure calculation. Rotational correlation time wasestimated as 2.0 ns in relaxation matrix calculations according Cys2-Cys13 region backbone atom coordinates as a measure of

conformational similarity. The structure with minimal rmsd toto the size of GI and viscosity of the sample. Additional sixupper and six lower distance constraints were introduced for two geometrical averaged pseudo-structure in each cluster was se-

lected as a representative one;disulfide bonds between residues Cys2-Cys7 and Cys3-Cys13.Stereospecific assignments ofβ-methylene protons were ob- (d) calculation of theoretical NOE cross-peak volumes for

each representative structure by complete relaxation matrixtained with the use of the GLOMSA program [24] based oncombination of the available spin-spin coupling constants and analysis program CORMA [27] ;

(e) evaluation of relative populations of representative struc-NOE-derived distance constraints. Torsion angle constraintswere derived from proton spin-spin coupling constants H-NCA-H tures as a solution of the linear system:(f angle) and H-CACβ-H (χ1 angle), and obtained stereospecificassignments.

Spatial structure calculation. Spatial structure calculation HoN

i 51ai

jxi 5 bj

oN

i51xi 5 1

was performed with the use of standard (distance-geometry algo-rithm) and multiconformational approaches.

The standard approach was based on the distance-geometryalgorithm program DIANA [24]. As input data we used the com-with additional inequality constraints accounting for no-NOEs:plete set of distance constraints and torsion-angle constraints, as

oN

i51ai

jxi < n .well as information on stereospecific assignment of prochiralgroups (see experimental constraints above). A standard pro-cedure of spatial structure calculation involves the following Theai

j designates the volume ofj-th (1< j < M) theoreticalNOE calculated fori-th (1 < i < N) structure (see step d),bj ismain steps:

240 Maslennikov et al. (Eur. J. Biochem. 254)

the volume ofj-th experimental NOE,xi is the vector of relativepopulations,N is the number of representative structures.M isthe number of experimental NOE cross-peaks. Inequality con-straints for no-NOE intensities are introduced when interprotondistance was found less than 4.0 A˚ in one or several representa-tive structures but corresponding NOE cross-peak was not pre-sent in the NOESY spectrum.n is the noise level in the NOESYspectrum. The linearly constrained least-squares approach [28]has been implemented for solution of the system.

Structure analysis and visualisation.DIANA penalty func-tion was calculated according to the standard procedure [24].Populations of different rotamers ofχ1 torsion angle were calcu-lated based on spin-spin coupling constants [29], which weretested for consistency with theoretical values of coupling con-stants calculated for multiconformational structure set.

Rx-factor values assessing the conformity of theoretical andexperimental NOE cross-peak volumes were calculated by equa-tion:

Rx 5oN

i 51|6!Ii

O 2 6!IiC|

oN

i 51

6!I iO

,

whereN designates the number of unambiguously assigned NOEcross-peaks,IO and IC are the observed and calculated volumesof NOE cross-peaks respectively.

The local DIANA penalty function andRx-factors were cal-culated as shown above, accounting for all intraresidue con-straints and NOE cross-peak volumes between protons of resi-due i and amide proton NH of residuei 11.

To reveal mismatches between the calculated structure andeach individual NOE cross-peak volume we introduceDx-factor,calculated by equation:

Dx 5|6!IO 2 6!IC|

6!IO

, Fig.1. Diagonal plots representing the survey of NOE contacts.(a)Experimental and (b) calculated for the best standard (lower left) andmulticonformational set (upper right) of GI structures. A square in thewhereIO is the experimental NOE cross-peak volume andIC isposition (i, j) of the (a) and (b) plots indicates that betweeni and j

the calculated volume (standard approach) or the weighted sumresidues one or several NOE contacts were observed and calculated,of calculated volumes (multiconformational approach) of therespectively. Darkness of the squares correspond to the number of NOEs.corresponding NOE cross-peak. The maximalDx-factor for NOE (c) Diagonal plot of maximalDx-factors, calculated for the best standardcross-peaks between protons of each pair of residues was used(lower left) and multiconformational set (upper right) of GI structures.

A square or a circle in the position (i, j) of the plot indicates that theto demonstrate the discrepancies between theoretical and experi-maximalDx-factor corresponds to experimentally observed NOE or no-mental NOEs.NOE between protons of residuesi and j, respectively. Darkness of theVisual analysis of the structures obtained and preparation ofsquares and circles corresponds to the maximalDx-factor value as shownfigures was performed using MOLMOL program [30].at the right-hand side.

RESULTS AND DISCUSSIONtions of 0.0620.18 ppm from those reported by Pardi et al. [9].The absence of other significant (more than 0.05 ppm) devia-NMR structures ofA-conotoxin GI in aqueous [9] and

dimethylsulfoxide solution [10] have been previously studied. tions of chemical shifts testifies to the identity of the samples.The observed NOEs are presented in the form of diagonalThe data of [9] contain only 49 distance constraints (Table 3 in

[9]) and do not include spin-spin coupling constants. As a result, plot in Fig.1a. The H-NCA-H and H-CACβ-H coupling constantsand obtained stereospecific assignments ofβ-methylene protonsthe backbone conformation of GI was defined with the average

rmsd 0.98 A, but the side-chain conformations were generally of GI are tabulated in Table1. The obtained experimental dataand the derived constraints are summarised in Table 2.undefined (the average rmsd calculated for all heavy atoms is

1.62 A, [9]). Recent progress in NMR spectroscopy techniques Only one amide proton (NH Cys7) was found in spectra re-corded 5 min after dissolving GI in D2O. Relatively slow hy-allows us to obtain an expanded and more precise data set. We

believed that the larger experimental data set would allow us to drogen-deuterium exchange rate of NH Cys7 correlates with alow temperature coefficient of its chemical shift (0.8 ppb/°C)refine the spatial structure of GI in aqueous solution signifi-

cantly. and confirms its participation in hydrogen bonding.The complete resonance assignment was carried out by using

a standard procedure [31] on the basis on DQF-COSY, TOCSY Standard approach to spatial structure calculation. The setof 18 GI structures was obtained on the basis of all availableand NOESY spectra and the previously reported [9] resonance

assignment for GI in aqueous solution. Only CβH2, CγH2, and experimental distance and torsion angle constraints, stereo-specific assignments, hydrogen bond and no-NOE constraintsCδH2 protons of Arg9 residue demonstrate chemical shift devia-


Fig. 2. Stereoview of 18 GI structures obtained by the standard approach and superimposed at the backbone heavy atoms of Cys2-Cys13region. All heavy atoms are shown. All side chains are shown in grey except for cysteines and proline. Corner residues (Glu1, Pro5 and Arg9) aremarked by the numbers.

Table 1. Experimental (exp.) and calculated for ensemble of structures (calc.) spin-spin coupling constants, stereospecific assignments, andunambiguously determined ranges ofχ1 torsion angles for B-conotoxin GI. Coupling constants indicating a static angle are denoted by bold.

Residue 3J-coupling constants χ1, 630

HN-CAH HACCβ2H HACCβ3H HACCβ2H HACCβ3H

exp. calc. exp. calc.

Hz deg.

Glu1 2 8.1, 6.1 5.4, 4.9Cys2 6.5 6.5 2.0 10.1 2.5 11.0 180Cys3 8.7 8.6 4.0, 8.4 3.9, 8.7Asn4 7.4 7.5 5.5, 6.9 4.0, 6.4Ala6 5.4 5.3Cys7 #3 3.5 11.0 3.0 11.7 3.6 260Gly8 9.2; 3.8Arg9 5.7 6.3 10.2 4.0 10.4 2.2 260His10 7.9 7.8 3.5 11.0 3.2 11.1 180Tyr11 3.3 4.0 5.3 9.9 5.2 10.2Ser12 6.0 6.3 4.5, 8.8 5.7, 7.4Cys13 7.7 7.0 5.3, 7.0 4.8, 8.9

(Table 2). The GI structures have the backbone fold similar to Low values of the local DIANA penalty function (Fp) andthe local Rx-factor (Fig. 3a) testify that the conformation ofthat found in [9], consisting of twoβ-turns: type I, formed by

Asn4-Cys7 residues, and type II′, formed by Gly8-Tyr11 resi- Cys2-Cys7 loop was accurately determined by standard ap-proach. This region is stabilised by the Cys2-Cys7 disulfidedues (Fig. 2). The structures are characterized by average pair-

wise backbone and all heavy atoms rmsd of 0.5160.23 A and bridge and the NH(Cys7)-CO(Asn4) hydrogen bond.A number of significant inconsistencies between experimen-1.1960.39 A, respectively. A single rotamer ofχ1 torsion angle

is obtained for four residues. Thus, utilization by standard ap- tal and theoretical NOEs, calculated for standard set of struc-tures, has been observed. Our attempts to find better calibrationproach of a more extensive data set than the one used by Pardi

et al. [9], allowed us to lower rmsd values and to fix someχ1 of the constraints by reasonable variation of the MARDIGRASparameters (such as a rotational correlation time, noise leveltorsion angles.


Table 2. Experimental data and results of spatial structure calcula-tion using the standard protocol and the multiconformational(MCA) approach for B-conotoxin GI.

Parameter Quantity U Standard MCA

Experimental data set NOE intensities 316 3163JHNCAH 12 123JHACCβH 20 20

Number of constraints distancea 163 163backbone angles 4 4side chain angles 4 4

Data set used forstructure calculations NOE 163 163 (73)b

overlapped NOE 0 12no-NOE 8 192disulfide bridges 2 2H-bond 1 1coupling constants 12 12

DIANA target function minimal A2 14.2 0.02c

average in the set A˚ 2 18.9 0.07c

Dx-factor number.0.2 11 0maximal 0.32 0.14

Rx-factor 0.20d 0.16Average rmsd backbone atoms A˚ 0.51 0.56/0.23e

for the region 2213 all heavy atoms A˚ 1.39 1.61/0.64e

a The number of pairs (upper and lower) of NOE-derived distanceconstraints is presented.

b Static data used to generate available conformational space (seeMaterials and Methods) are shown in parentheses.

c The values corresponding to the structures calculated with staticFig.3. Histogram of localRx factor and DIANA penalty function ( Fp)data set only.values calculated for GI structures obtained by standard (a) andd Average value in the set of structures is presented.multiconformational (b) approaches.e The values for major/minor sets are presented.

etc.) or calculations using NOESY spectra with mixing time ofrestrained by the disulfide bridge Cys3-Cys13. However, the100 ms have failed. Violations greater than 0.5 A˚ occur both for HAC-CβH coupling constants of Cys3 and Cys13 (Table1) areshort [between adjacent residues, e.g. CAH(Ser12)-NH(Cys13), indicative of two or threeχ1 rotamers, so several configurationsCβH(Ser12)-NH(Cys13)] and long-range distance constraintsof the disulfide bridge could exist in solution. All this led us to[between sequentially distant residues, e.g. NδH(Asn4)- conclusion that observed inconsistencies in the constraints areNH(Tyr11), CδH(Pro5)-CδH(Tyr11), CA1H(Gly8)-NH2(C-termi- caused by presence of several distinct GI conformations.nus)]. Comparison of experimental (Fig.1a) and calculatedbased on the standard structure set (below diagonal on Fig.1b) Multiconformational approach. Peptides and proteins in solu-

tions are often in an exchange between two or more differentNOEs maps shows the disagreement between GI standard struc-tures and long-range NOE data. Analysis of the mismatch, cal- conformations. Interpretation of experimental NMR parameters,

such as chemical shifts, NOEs and coupling constants, measuredculated as a sum ofDx-factors for each pair of residues (belowdiagonal on Fig.1c), shows the problems of the structure calcu- for a flexible molecule, depends on exchange rate or, in other

words, the time range of the exchange process. A conforma-lation within the Gly8-Tyr11 region and in orientation of N-terminus and C-terminus of GI (Glu1-Cys3 and Ser12-Cys13 tional exchange is defined as fast, intermediate or slow when

the exchange rate is considerably higher, close to, or much lessregions). The contradictions of the experimental data result alsoin eight van-der-waals and five lower distance-constraint viola- than the frequency difference between the values of a given

NMR parameter in distinct conformations. Conformational in-tions greater than 0.3 A˚ , and 3 torsion-angle constraint violationsgreater than 8°. terconvertions that are fast on a chemical shift time-scale (life-

times of the conformations of less than a few milliseconds) leadBy this means the experimental data themselves are incom-patible; that is no single conformation, simultaneously satisfying to a single set of signals in NMR spectra, that corresponds to a

set of conformationally averaged NOEs [11]. Obviously, an at-all of them can be found. An analysis of the experimental dataproves the lability of the GI molecule. The degenerated chemical tempt to interpret the ensemble-averaged NMR data in the

frames of a single structure would give unrealistic conforma-shift values of Glu1 CβH2 and CγH2 protons indicate the highmobility of its side chain. The3JHNCAH value of Cys2 (6.5 Hz, tions.

A number of investigations has been done aimed at a properTable1) suggests a non-fixed torsion anglef of the Cys2 whichleads to unconstrained mobility of Glu1. The HN-CAH spin-spin accounting of conformationally averaged NMR experimental

data. A time-averaged model was used in distance-based refine-coupling constants for Arg9, His10, Ser12 and Cys13 residuesare close to the values typical for random coil peptides (Table ment [12, 13] or in refinement directly against the NOE cross-

peak volumes [15]. An alternative approach treats experimental1) and do not allow us to restrain the correspondingf torsionangles. The conformational mobility of the Gly8-Cys13 loop is data as ensemble averaged [16218].


Fig. 4. Stereoviews of conformational space available to G1 according to the static subset of NMR data.(a) 973 structures and (b) 71representative structures superimposed at the backbone heavy atoms of Cys2-Cys13 region. Side chains of Pro5 (black) along with cysteine andArg9 residues (grey) are shown. Hydrogen bond NH(Cys7)-CO(Asn4) is shown by three dots.

A complete search for an ensemble is very time consuming. cal DIANA penalty function stand for impossibility to satisfyNOEs, J-couplings, disulfide bridges and van-der-waals con-However, different regions of GI differ in conformational lability

(see above). Using experimental constraints that correspond to straints within a single conformation. High values of the localRx-factor show that the obtained structure is inadequate to exper-relatively rigid regions of the molecule, one can considerably

restrict the possible conformational space of the molecule. imental NOE data, i.e. complete set of local NOE constraintscould not be satisfied simultaneously.Therefore, objective criteria of non-averaged experimental

data selection have to be formulated. While an ensemble averag- Fig. 3 illustrates the sequence dependencies of the localDIANA penalty function (Fp) and localRx-factor. Low Fp anding of spin-spin coupling constants could be easily detected at

the stage of angle constraints generation [29], an ensemble aver-Rx values indicate that Cys2 and Pro5-Cys7 residues are confor-mationally rigid. The experimental data for the rest of the mole-aging of NOE cross-peak volumes usually shows up only during

structure refinement by an increase in penalty function and sys- cule, including residues Glu1, Gly8-Cys13 (Fig. 3a), and sidechains of Cys3 and Asn4 (Table1), are incompatible in thetematic distance-constraint violations. For the analysis of experi-

mental data mismatch, we used two criteria that are local frames of a single-structure approach. However, H-NCA-H cou-pling constants of Cys3 and Tyr11 correspond to a single rangeDIANA penalty function and localRx-factor. High values of lo-


Fig. 5. Stereoviews of major NMR set (black) and X-ray crystallography structure (red), superimposed at the backbone heavy atoms ofCys2-Cys13 region (a) and stereoview of minor NMR structure (b).Side chains of cysteines, Pro5, Arg9 and His10 are shown. Hydrogen bondNH(Cys7)-CO(Asn4) is shown by three dots.

of f torsion angles (Table1). Some of the H-CACβ-H coupling als and Methods) with the optimal threshold of 0.31 A. A repre-sentative structure was selected in each of the 71 obtained clus-constants and NOEs at the labile region of GI permit us to obtain

stereospecific assignments and therefore to fix side-chain rotam- ters. Sets of the superimposed 973 structures and the 71 repre-sentative structures are shown in Fig. 4. The pairwise rmsd ofers of several residues (Table1). So, not all of the experimental

data are inconsistent and ensemble averaged. Using the above- Cys2-Cys13 region backbone atoms in the set of 71 representa-tive structures varied from 0.29 A˚ to 2.42 A. Thus the numbermentioned local characteristics of ‘spatial structure-NMR data’

compatibility, we revealed the region of static conformation, of structures describing the conformational space was reducedfrom 973 to 71 without narrowing of the favourable conforma-which included Cys2, Pro5-Cys7 residues and backbone of

Cys3, and proposed that the experimental constraints within this tional space. We expect that a combination of representativestructures should form the required ensemble.region are static. Constraints obtained for the torsion angles of

side chains with the stereospecific assignments (Table1) were In general, the formalism of ensemble-averaged NOEdata computation depends on the rate of conformational ex-considered as static too.

Thus, the complete set of NMR data was divided into the change. Conformational exchange with the rate slower orequal to the rate of molecule overall tumbling, lead to the aver-static and ensemble-averaged subsets. The static subset consists

of 73 NOE cross-peak volumes, four3JHNCAH and eight3JHACCβH aging of the NOE intensities or the cross-relaxation rates, re-spectively; in both cases the results are quite similar [32]. More-coupling constants. The ensemble-averaged subset consists of

90 NOE cross-peak volumes, 63JHNCAH and12 3JHACCβH coupling over, relatively small spin diffusion allows us to accept theintensity averaging model that requires less calculation time.constants. Using the static experimental data subset, 973 struc-

tures were generated by the DIANA program with the range of Theoretical NOE cross-peak volumes were calculated for eachof the obtained 71 representative structures. The experimentalpairwise rmsd of Cys2-Cys13 region backbone atoms from

0.001 A to 2.42 A. Structure generating was terminated when NOE cross-peak volume was fitted to weighted sum of theoreti-cal NOE cross-peak volumes calculated for each representativemaximal pairwise rmsd in the set ceased to grow and the average

pairwise rmsd began to decline monotonously. structure in the ensemble. The weighting coefficients in this sumare the population weights of respective structures in the en-Thus we obtained the conformational space of GI consistent

with the static experimental constraints. To exclude similar semble.Population weights were determined by solving the linearlystructures and to reduce their number, the clustering of the pos-

sible structures was done. Hierarchical clusterng of 973 struc- constrained least-squares problem formulated in Materials andMethods. Population weights of the 71 theoretical spectra ob-tures was performed by the NMRCLUST program (see Materi-


Fig. 6. Stereoview of van-der-waals surface models of the most populated major (a) and minor (b) representative structures.Orientations ofthe static Cys2-Cys7 regions (orange) are the same. Flexible regions (Glu1 and Gly8-Cys13) are shown in grey. N-terminal NH1

3 group and guanidogroup of Arg9 are coloured in dark blue and carboxyl group of Glu1 is shown in magenta.

tained from the 71 representative structures were found as a so-1.8560.12 A for backbone and 3.7260.17 A for all heavyatoms. Distribution off and ψ torsion angles (Fig. 7) showslution of the overdetermined system containing 367 (163 for re-

solved and12 for overlapped experimental NOEs,192 for no- similar conformations in Cys2-Cys7 region for all structures[type I β-turn, stabilised by NH(Cys7)-CO(Asn4) hydrogenNOEs) of approximately satisfied (in the least-squares sense)

equations, and one exactly satisfied equation (the sum of popula- bond]. An average pairwise rmsd for Cys2-Cys7 region of allstructures is equal to 0.1960.07 A for backbone andtion weights equal to unity).

The applied procedure produced non-zero population 0.7060.27 A for all heavy atoms. The average pairwise rmsdfor backbone atoms of Gly8-Cys13 region is 0.9660.83 A forweights for 8 representative structures out of 71. Theoretical

NOEs, calculated according with the obtained weights of struc- all structures, while for the structures of the major set it is0.5360.17 A.tures, as well as observed mismatches between them and experi-

mental NOEs are summarised in the Fig.1. Two principally dif- The helical turn in the major set is stabilised by NH(His10)-CO(Cys7) and/or NH(Tyr11)-CO(Gly8) hydrogen bonds. Theferent backbone topologies have been discerned, basing on back-

bone pairwise rmsd values and visual inspection of the eight antiparallel orientation of GI termini is braced by hydrogen bondNH(Cys2)-CO(Ser12), suggesting that theβ-bridge is formed byrepresentative structures having non zero population weights

(Fig. 5). The major topology consists of seven clusters compris- Cys2 and Ser12 residues. In the minor set NH(Cys2)-OγH(Ser12) hydrogen bond stabilises the parallel N-terminus toing 78% of the total population and has the average pairwise

rmsd 0.5660.14 A for backbone and1.6160.26 A for all heavy C-terminus orientation. However, amide protons of Cys2, His10or Tyr11 demonstrate fast exchange with water deuterons. Theatoms. In the major set the C-terminus and N-terminus of GI

have anti-parallel orientation and Gly8-Cys13 loop is described possible reason of the fast exchange of the NH protons involvedin hydrogen bonds could be the proximity of a positivelyas having a helical turn Gly8-Tyr11 (Fig. 5a). The minor topol-

ogy (22% population, average rmsd 0.2360.06 A for backbone charged group. Three groups have a positive charge at the usedpH 5.3: A-amino group of Glu1 (nearby the Cys2 amide group),and 0.6460.19 A for all heavy atoms) represents the remaining

cluster. In the minor set the C-terminus and N-terminus have guanido group of Arg9 and imidazol ring of His10 (close toHis10 and Tyr11 amide groups). Another reason for the fast ex-parallel orientation and Gly8-Cys13 loop comprises two consec-

utive bends, Gly8-Arg9 and His10-Tyr11 (Fig. 5b). The average change is the exchange between the hydrogen-bonded (helicalturn) and non-bonded (two bends) conformations of the Gly8-rmsd between the structures of the major and the minor sets is


the general fold of Cys2-Cys7 loop in the NMR and X-ray struc-tures is similar. However, peptide bonds Cys3-Asn4 and Pro5-Ala6, are oriented differently in the NMR and X-ray structures.This difference could be due to the van-der-waals contacts ofCys3 and Pro5 backbone atoms with neighbouring molecule, aswell as the intermolecular hydrogen bond formed by Cys3 COand C-terminal amide group in the crystal [8]. The mobility ofN-terminal Glu1 residue is restricted in the crystal by anintermolecular hydrogen bond between the carboxylate group ofGlu1 and the phenolic OεH-group of Tyr11 as well as by theintramolecular CγO(Glu1)-NH(Ser12) hydrogen bond; these re-strictions do not operate in the solution.

Several side chains of GI have different conformations in theX-ray and NMR structures. NMR data revealed that Cys3 andTyr11 residues have twoχ1 rotamers, while for Glu1, Asn4,Ser12 and Cys13 residues all threeχ1 rotamers are allowed. Inthe crystal, CγO-group of Asn4 forms an intermolecular hy-drogen bond with the imidazole Nε2H of the His10 of the neigh-bouring molecule, thus stabilising the conformation of the Asn4side chain. The Cys3-Cys13 disulfide bridge configuration in theX-ray structure is also well defined. The tight crystal packing ofthree water molecules associated with the hydroxyl group ofFig. 7. Scatter plot of thef, ψ, χ1 angles calculated for eight majorSer12 restricts the mobility of the Ser12 side chain in the X-(+) and one minor (x) NMR structures presented together with theray structure. Side-chain conformations of Arg9 (χ19<260°) andangles of the X-ray (s) structure of B-conotoxin GI.His10 (χ110<180°) in the NMR structures differ from those deter-mined by X-ray crystallography (χ19 < 160° and χ110 <260°). The Arg9 and His10 side chains are closely packed inCys13 loop. Rearrangement of the C-terminus relative to the N-the crystal, even though both chains are positively charged atterminus in GI from antiparallel to parallel fashion leads to thethe pH 5.2 used by Guddat et al. [8]. This is explained [8] bytemporal character of hydrogen bonds formed by the Cys2 NHsymmetry contacts in the crystal, such as the intermolecular hy-and its fast exchange rate.drogen bond CγO(Asn4)-Nε2H(His10) and the tight packing ofTo compare the consistency of a structure or structural setthe Arg9 side chain with the Tyr11 side chain of the symmetry-with the experimental data we usedRx andDx-factors (see Mate-related molecule.rials and Methods). AnRx-factor value calculated for any of the

Thus all differences between the X-ray structure and the ma-individual structures obtained by the standard approach does notjor NMR structure of GI can be explained by crystal-packingfall below 0.19. The Rx-factor calculated for a single structureforces, which distort side-chain and backbone conformations.obtained by multiconformational approach was within the 0.192Clearly the same forces select for crystallisation of the major0.31 range, however, when calculated for the ensemble of struc-structure of GI out of the two coexisting in solution.tures it went down up to 0.16. Moreover, all of the structures

Thus, two distinct GI spatial structures are present in aque-obtained by the standard approach show11 Dx-factor values inous solution. Most likely, the observed conformational labilitythe range of 0.2020.32, while the maximalDx-factor calculated(or diversity) of GI is a feature of the most of the conotoxinsfor the ensemble was 0.14. Comparison of experimental and cal-(see discussion in [9,10, 33] where NMR spectroscopy was usedculated NOEs (Fig.1) as well asRx-factor andFp values (Fig. 3)to characterise the spatial structures of conotoxins in solution).presents further evidence of more adequate interpretation of theThe question arises, what is the biological significance, if any,experimental data by multiconformational approach.of this conformational diversity? It was shown [6, 7] that Arg9forms one of the determinants important for site-dependent se-Comparison of the X-ray and major NMR structures. GI so-

lution structures from the major set are very similar to the re- lectivity of GI action. Our results indicate that Arg9 is locatedat the labile region and orientation of its side chain relative tocently reported [8] X-ray structure (Fig. 5). Average rmsd values

between the X-ray structure and structures from the major set Cys2-Cys7 region differs significantly in two distinct structures(Fig. 6). At present we cannot exclude that only one of the twoare 0.7760.19 A for backbone and 2.2160.23 A for all heavy

atoms of Cys2-Cys13 residues. distinct structures of GI is responsible for its biological activity.However, the conformational diversity of conotoxins might beIn the major set the backbone is stabilised by one constant

[NH(Cys7)-CO(Asn4)] and two temporary [NH(His10) an essential factor facilitating their binding to a variety of loci,e.g. to different receptors or distinct sites of the same receptor.-CO(Cys7) and/or NH(Tyr11)-CO(Gly8)] hydrogen bonds. The

three hydrogen bonds are present in the X-ray structure [8]. This conformational diversity could be used by cone snails asan addition to the combinatorial peptide library strategy [34] toWhile Guddat and co-authors [8] proposed that the 35 intermo-

lecular contacts involving 8 backbone atoms of the symmetry- produce peptides active against a variety of targets.Subsequent NMR and X-ray studies of different conotoxins,related molecules were too weak to effect substantially the back-

bone conformation in the crystal, the NMR structures demon- along with site-directed modifications and detailed studies ofinteractions ofA-conotoxins with different subclasses of well-strated a noticeable difference from the X-ray structure (Figs 5

and 7). The backbone torsion angles of Cys3 (ψ), Asn4 (f), characterised receptors, could help to elucidate sequence, spatialstructure and dynamics features of conotoxins that underlie theirPro5 (ψ) and Ala6 (f) in the X-ray structure fall outside the

NMR-derived angle intervals (Fig. 7). The largest backbone an- unique selectivity.gle differences (about100°) were found for Cys3ψ and Asn4 This work was supported by grants from Russian Basic Researchf angles. The differences have opposite signs for sequentialψi Foundation (project number 96-04-50375) and the Ministry of Science

and Technical Politics (96-03-08).and fi11 torsion angles and compensate each other. Therefore,


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two distinct structures of α-conotoxin gi in aqueous solution

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