Long-range effects in protein–ligand interactions mediatepeptide specificity in the human major histocompatibilityantigen HLA-B27~B*2701!

STEFAN KREBS,1 DIDIER ROGNAN,2 and JOSÉ A. LÓPEZ DE CASTRO11Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas~CSIC! andUniversidad Autónoma de Madrid, Facultad de Ciencias, Cantoblanco, 28049 Madrid, Spain

2Department of Pharmacy, ETH Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland

~Received January 19, 1999;Accepted March 18, 1999!

Abstract

B*2701 differs from all other HLA-B27 subtypes of known peptide specificity in that, among its natural peptide ligands,arginine is not the only allowed residue at peptide position 2. Indeed, B*2701 is unique in binding many peptides withGln2 in vivo. However, the mutation~Asp74Tyr! responsible for altered selectivity is far away from the B pocket of thepeptide binding site to which Gln0Arg2 binds. Here, we present a model that explains this effect. It is proposed that anew rotameric state of the conserved Lys70 is responsible for the unique B*2701 binding motif. This side chain shouldbe either kept away from pocket B through its interaction with Asp74 in most HLA-B27 subtypes, or switched to thispocket if residue 74 is Tyr as in B*2701. Involvement of Lys70 in pocket B would thus allow binding of peptides withGln2. Binding of Arg2-containing peptides to B*2701 is also possible because Lys70 could adopt another conformation,H-bonded to Asn97, which preserves the same binding mode of Arg2 as in B*2705. This model was experimentallyvalidated by mutating Lys70 into Ala in B*2701. Edman sequencing of the B*2701~K70A! peptide pool showed onlyArg2, characteristic of HLA-B27-bound peptides, and no evidence for Gln2. This supports the computational model anddemonstrates that allowance of B*2701 for peptides with Gln2 is due to the long-range effect of the polymorphic residue74 of HLA-B27, by inducing a conformational switch of the conserved Lys70.

Keywords: HLA-B27; molecular modeling; peptide specificity; pool sequencing; protein–ligand interactions

The human major histocompatibility antigen HLA-B27 is stronglyassociated to ankylosing spondylitis and reactive arthritis~Brew-erton et al., 1973; Kingsley & Sieper, 1993!. That presentation ofself-derived and bacterial peptides, and their recognition by HLA-B27-restricted T-cells, might be crucial in the pathogenesis of thesediseases~Benjamin & Parham, 1990! has gained support from theestablishment of disease models with HLA-B27 transgenic rats~Hammer et al., 1990; Zhou et al., 1998!, the isolation of B27-restricted autoreactive and bacteria-specific CTL from affected pa-tients ~Hermann et al., 1993; Ugrinovic et al., 1997!, and thedifferential association of B27 subtypes with ankylosing spondy-litis ~D’Amato et al., 1995; López-Larrea et al., 1995; Gonzalez-Roces et al., 1997; Nasution et al., 1997; Ren et al., 1997!. HowHLA-B27 polymorphism affects the peptide repertoire and mod-ulates disease susceptibility still cannot be explained. Therefore,the molecular mechanisms that select the peptide repertoire pre-sented by each subtype need to be better understood.

Class I MHC molecules present a broad repertoire of endog-enous peptides, whose nature is modulated by MHC polymor-

phism. This dictates the functional properties of HLA class I proteinsas antigen-presenting molecules for T-cells. Therefore, the rela-tionship between the structure of a given class I protein and thetype of peptides that it can bind is crucial to understanding HLAclass I function. Peptides bind to MHC molecules through non-specific main-chain contacts and more selective side-chain inter-actions~Madden, 1995; Batalia & Collins, 1997!. These restrictbinding to one or a few residues at the so-called main anchorpositions of the peptide~Falk et al., 1991!. The specificity forgiven anchor residues is controlled by HLA polymorphism, muchof which affects the structure of the corresponding binding pockets~Garrett et al., 1989; Saper et al., 1991!. The main anchor residuesof HLA-B27-binding peptides are Arg at position 2 and theC-terminal one. This is either a hydrophobic or basic residue, withsubtype-specific fine-tuning~Jardetzky et al., 1991; Rojo et al.,1993; Rötzschke et al., 1994; Boisgérault et al., 1996; Fiorilloet al., 1997; García et al., 1997a, 1997b, 1998; Tieng et al., 1997!.Auxiliary anchors are found at positions 1, 3, and 7~Madden et al.,1992; Colbert et al., 1994; Rovero et al., 1994!. In HLA-B*2705,the binding site for Arg2, termed B pocket, is formed by multipleresidues, including His9, Thr24, Glu45, Cys67, and Tyr99~Mad-den et al., 1992!. Three direct and two water-mediated hydrogenbonds are engaged between Arg2 and pocket B residues. Thus, theB pocket in HLA-B27 is optimally designed for interacting with an

Reprint requests to: Dr. José A. López de Castro, Centro de BiologíaMolecular Severo Ochoa, Universidad Autónoma de Madrid, Facultadde Ciencias, Cantoblanco, 28049 Madrid, Spain; e-mail: aldecastro@cbm.uam.es.

Protein Science~1999!, 8:1393–1399. Cambridge University Press. Printed in the USA.Copyright © 1999 The Protein Society

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Arg side chain. A remarkable feature of this interaction is thepresence of a bound water molecule in pocket B that enhances thiselectrostatic complementarity~Madden et al., 1992!. Glu45 is crit-ical by serving as a countercharge for the peptidic Arg2 residue,which is present in almost all naturally presented peptides identi-fied so far. Nevertheless peptides containing Gln at position 2 bindin vitro with reduced affinity to B*2705, B*2702, and other sub-types~Parker et al., 1994; Villadangos et al., 1995; Galocha et al.,1996; Raghavan et al., 1996! and can act as antigens in vivo~Simmons et al., 1997!. In B*2701, which differs from B*2705 inthree residues, Asp74r Tyr, Asp77r Asn, and Leu81r Ala,Gln2 is, besides Arg2, a peptide motif found among natural ligands~García et al., 1997a!. The Asp74r Tyr change, which is uniqueamong B27 subtypes, is largely responsible for this feature~Garcíaet al., 1997a!. A direct interaction of Gln2 with the Tyr74 residue,however, has to be excluded on the basis of the B*2705 crystalstructure~Madden et al., 1992!. Hence, Asp74 is located far out-side the B pocket, but forms a salt bridge with Lys70. In B*2705,the Lys70 side chain, although located at the rim of the B pocket,is kept away from it, and does not interact with Arg2 in the X-raystructure~Madden et al., 1992! or with Gln2 in a simulated B*2705peptide complex~Rognan et al., 1997!. Lys70 is only found inHLA-B27 and HLA-B73 among class I MHC proteins and is presentin most of the B27 subtypes.

Here we present a model explaining how B*2701 is able to bindpeptides with either Arg2 or Gln2 with high affinity, by adoptingdifferent patterns of interaction in the B pocket via rotation of theLys70 side chain. To confirm this computational model and thecritical role of Lys70 in B pocket specificity, this residue wasmutated to Ala with the expectation that the natural peptide pool ofthis mutant would lack the Gln2 motif, as a consequence of spe-cifically disrupting those interactions involving Lys70 that favorbinding of such peptides.

Results

Lys70 in B*2701 adopts different conformationswith Gln2- or Arg2-containing peptides

A full systematic search of all Lys70 rotameric states in B*2701was undertaken to check whether some preferred conformationalstates might be dependent on position 2 of the bound peptide. Torestrict the conformational scan, a Gln2-containing virtual peptidepool was defined from 28 class I MHC-bound peptides~see Materialsand methods!. In the presence of Gln2-containing peptides, mostenergy minima adopt similar conformations in which Lys70 islocated within H-bond distances of the Gln2 side chain~Fig. 1A!.The bulky Tyr74 prevents a “B*2705-like” orientation of the Lys70side chain toward residue 74. To test if this new interaction wasstable, B*2701 was modeled with a natural B*2701 ligand bearingGln2: GQAPGYSY~García et al., 1997a!. After 200 ps MD sim-ulation in water, the whole complex as well as the Lys70–Gln2H-bond were shown to be very stable~Fig. 2A!. The latter inter-action occurred in 72% of the 400 MD conformers. From thissimulation is derived a general model in which Lys70 is preferen-tially H-bonded to two MHC side chains~His9, Tyr99! and to Gln2of the bound peptide~Fig. 3A! . The bound water molecule in theB pocket of B*2705~Madden et al., 1992! has been expelled fromthe subsite to allow a proper contact of Lys70 with the peptidicGln2.

Sequencing of natural B*2701 ligands and in vitro binding as-says indicate that Arg remains a strong anchor at P2~García et al.,1997a!. This suggests that Lys70 should adopt a different confor-mation when B*2701 is bound to Arg2-containing peptides. Thus,a new conformational search of the Lys70 side chain was under-taken in the presence of a second virtual peptide pool containingArg2. If P2 is Arg, Lys70 was predicted to fold toward pocket Cand to be preferentially H-bonded to Asn97~Fig. 1B!. A naturalB*2701 ligand with Arg2~RRYQKSTEL! ~García et al., 1997a!was simulated in the binding groove of B*2701 to study the dy-namics of the interaction. It should be stated here that using ref-erence peptides of different lengths~octamer when P25 Gln,nonamer when P25 Arg! should not have significant conse-quences because the starting position~see above! of the Lys70 sidechain in both allotypes was determined in the presence of a virtualpeptide pool made of octa, nona, and decamers~see Materials andmethods!. The Lys70 side chain was predicted to be mainlyH-bonded to Asn97 and, less frequently, to the Tyr74 hydroxylegroup ~Fig. 2B!. Again, a general model can be depicted for the

A

B

Fig. 1. Peptide-restricted conformational analysis of Lys70 side chain ofB*2701 in the context of two virtual peptide pools.A: Gln2-containingpeptides.B: Arg2-containing peptides. H-bonding distances~Å! betweenLys70 ~NZ atom! and the major H-bond acceptor~P2 Gln or Asn97! areindicated vs. internal energy of B*2701.

1394 S. Krebs et al.

interaction of B*2701 with Arg2-containing peptides~Fig. 3B!. Inthis model Arg2 is bound to the B pocket of B*2701 as in thecrystal structure of B*2705~Fig. 3C! with the participation of abound water molecule. However, the main difference with B*2705is that in B*2701 the side chain of Lys70 stacks between His9,Asn97, and Tyr74. Thus, dual specificity of B*2701 for peptideswith either Arg2 or Gln2 is explained because Lys70 is predictedto adopt different rotameric states and contacts depending on thepeptidic P2 residue.

Peptides eluted from the B*2701 mutant K70Alack the Gln2 motif

Cells transfected with this mutant where recognized by the mAbME1 ~see Materials and methods!. The expression level of theselected transfectant clone was 124% relative to the B*2705 con-trol, indicating normal surface expression~Fig. 4!. The amount ofpeptides eluted from the mutant was also comparable to B*2705.

Edman degradation of the peptide pool eluted from the immu-nopurified K70A mutant of B*2701~Fig. 5! showed only pth-Arg

in the second cycle. Neither Gln nor Glu or any other residue wereincreased relative to the previous cycle. This result demonstratesthat the allowance of Gln2 in peptides bound to B*2701 criticallydepends on Lys70 and that removal of this side chain has noadditional effect on P2 residue selection other than restoring thespecificity of the B pocket for Arg2. In the third cycle polar~Asn,Thr!, aliphatic~Met, Val, Ile, Leu!, aromatic~Tyr, Phe!, and basic~His, Lys! amino acids were present. This is in contrast to wild-type B*2701, where basic residues at this position were absent~García et al., 1997a!. At position 9, which is the C-terminal onefor the majority of class I-bound peptides, only Tyr and Phe weredetected. However, since pool sequence analysis usually under-estimates the allowed C-terminal motifs, this does not rule out thatpeptides with nonpolar aliphatic residues may also be present inthe peptide pool.

Discussion

Gln at position 2 is a suboptimal residue for peptide binding toHLA-B*2705 ~Parker et al., 1994; Villadangos et al., 1995; Ga-locha et al., 1996!. Gln2 is also suboptimal for B*2702, althoughfor this subtype the binding differences in vitro between Arg2-containing peptides and their Gln2 analogues are smaller~Ragha-van et al., 1996; Galocha et al., 1996!. This is probably due tostronger anchoring of C-terminal hydrophobic residues to this sub-type, which could partially compensate for the weaker binding ofGln2. Peptides with Gln2 have not been isolated from B*2705 orB*2702 expressed on human cells, although one such peptide wasisolated from B*2705 in transgenic rats~Simmons et al., 1997!. Incontrast, a significant presence of Gln2 among B*2701-bound pep-tides has been confirmed by pool sequencing and identification ofindividual ligands carrying this residue: two of eight B*2701 li-gands of known sequence have Gln2, and the molar ratio of pth-Gln2:pth-Arg2 upon pool sequencing was 1:3~García et al., 1997a!.This unique feature could be assigned to a polymorphism at posi-tion 74, as the B*2705 mutant D74Y was able to mimic the B*2701motif ~García et al., 1997a!. Comparative modeling of B*2705 andB*2701 with Arg2- and Gln2-biased peptide pools suggested thatLys70 can directly contact Gln2 of B*2701-bound peptides. Thiswas a consequence of the conformational freedom of the Lys70side chain in B*2701, due to absence of the salt bridge that inB*2705 is established between Lys70 and Asp74~Madden et al.,1992!. To probe this model, we examined the peptide motif of theB*2701 K70A mutant. We reasoned that if Lys70 was critical forallowing Gln2 to bind in the B pocket, upon removal of Lys70, thepeptide pool of the B*2701 mutant should only contain peptideswith Arg2, despite the presence of Y74.

The absence of a Gln signal in the second cycle of the Edmandegradation of the peptide pool eluted from the B*2701 K70Amutant was in agreement with the proposed binding mode of Gln2-containing peptides, which cannot be accommodated in an ener-getically favored manner without Lys70 as a binding partner. Mostsignificant, besides the lack of Gln2, was the presence of basicresidues~Lys and His! in cycle three, which are absent in B*2701~García et al., 1997a!. Possibly the removal of both the positivecharge and the bulky side chain by the K70A mutation can facil-itate accommodation of such basic P3 residues.

The main outcome of our study is that the effect of poly-morphism outside the B pocket~D74Y! on the peptide specific-ity of HLA-B27 is mediated through the conserved Lys70 residue.Depending on the nature of residue 74, Lys70 may adopt different

A

B

Fig. 2. Frequency of H-bonds involving the Lys70 side chain, calculatedfrom 200 ps MD trajectories~1,000 conformations! of B27 peptide com-plexes.A: B*2701-GQAPGYSY complex.B: B*2701-RRYQKSTEL com-plex. Hydrogen bonds were defined using topological criteria: donor~D! toacceptor~A! distance less than 3.25 Å and D-H. . .A angle between 120–1808.

Peptide binding mode of B*2701 1395

A

B

C

Fig. 3. Orientation of a peptide~ball and stick model! in the binding groove of HLA-B27~green ribbons!. A: B*2701, P25 Gln.B: B*2701, P25 Arg. C: B*2705, P25 Arg. Peptide positions are labeled at the Ca atoms from 1~P1! to 9 ~P9!. A peptide model~AQAAAAAAA or ARAAAAAAA ! is indicated as a representative of each peptide subset. Residues differing between B*2701 andB*2705 are underlined. The figure has been obtained with the MOLSCRIPT~Kraulis, 1991! and Raster3D~Merritt & Murphy, 1994!programs. The following color coding was used: carbon, white~protein! or yellow ~peptide!; nitrogen, dark blue; oxygen, red. A boundwater molecule in the B pocket is shown as a light blue ball.

1396 S. Krebs et al.

rotameric states and select different peptide subsets. In most HLA-B27 subtypes, but not in B*2701, residue 74 is Asp~López deCastro, 1998!, which favors a direct H-bond to Lys70. As a con-sequence of this the Lys70 side chain is kept away from the Bpocket and this subsite favors the selection of Arg2-containingpeptides in a binding mode involving a bound water molecule~Madden et al., 1992!. In a previously reported model of Gln2-containing peptides bound to B*2705~Rognan et al., 1997!, Lys70was bound to Asp74 and P2 Gln interacted with Glu45 and Tyr99in the B pocket. However, this interaction was weaker than forArg2, in agreement with experimental binding data~Parker et al.,1994; Villadangos et al., 1995; Galocha et al., 1996!. In B*2701,the bulky Tyr74 is predicted to promote different conformations ofLys70 depending on the bound peptide. If the ligand bears a Gln atP2, Lys70 flips toward pocket B, expels the water molecule, andinteracts with the Gln2 side chain. Thus, the additional positivelycharged Lys70 residue ensures a better electrostatic complemen-tarity and a stronger interaction, through ionized H-bonds, withGln2. When the peptide ligand bears Arg at P2, Lys70 stacks in anintermediate position, H-bonded to Asn97. Since the Ca atom ofresidue 74 is located 16 Å away from the Ca atom of the peptidicP2 residue, this constitutes one of very few examples where along-range effect relayed by different rotameric states of a singleprotein residue controls ligand binding. Strikingly, a conservedresidue~Lys70! is used to mediate the effect of a polymorphic one~Asp0Tyr74! on peptide selection.

Fig. 4. Flow cytometry analysis of surface expression of the B*2701 mu-tant K70A on Hmy.C1R cells~A!. Untransfected Hmy.C1R cells~B! andB*2705 transfectants~C! were used as negative and positive controls,respectively. Cells were stained with the mAb ME1 and a fluorescein-labeled goat antimouse antibody as previously described~Villadangoset al., 1995!.

Fig. 5. Pool sequence analysis of peptides eluted from the HLA-B*2701 mutant K70A. Numbers indicate the picomole amounts ofpth-amino acids recovered in each cycle. Assignment of residues was as previously described~Falk et al., 1991!. In brief, a residuewas assigned when its picomolar yield was increased at least 50% over either of the two previous cycles, but not if it decreased relativeto the previous one. The corresponding residues are underlined. Assigned residues were arbitrarily considered strong when their yieldincreased 300%~P2–P4! or 50%~P5–P9!, respectively, over either of the two previous cycles and if their yield was.5% of the totalamount of pth-amino acid recovered in this cycle. Pool sequencing was performed three times from two independent preparations withsimilar results. Data are shown from one experiment only.

Peptide binding mode of B*2701 1397

Materials and methods

Homology modeling of the B*2701 protein

The antigen-binding domain~a1–a2, residues 1–182! of B*2701was modeled by homology to the X-ray structure of the closelyrelated B*2705 allotype~Madden et al., 1992!. The three occurringmutations~D74Y, D77N, and L81A! were modeled using the BIO-POLYMER module of the SYBYL 6.4 package~Tripos, Assoc.Inc., St. Louis, Missouri! and rotameric states of the newly intro-duced side chains were assigned according to a inhouse three-dimensional database of class I MHC X-ray structures~Rognan,1998!. Hence, Tyr74 and Asn77 side chains were modeled accord-ing to the structure of B*5301~Smith et al., 1996a! and Ala81 sidechain was obtained by simply truncating the original Leu sidechain after its beta carbon.

Peptide-restricted conformational analysisof Lys70 side chain

To sample the whole conformational space accessible to the Lys70side chain in physiological conditions, 28 X-ray structures of classI MHC-bound peptides~Protein Data Bank~PDB! codes: 1a1m,1a1n, 1a1o, 1a9e, 1agb, 1agc, 1agd, 1age, 1agf, 1bz9, 1hhg, 1hhh,1hhi, 1hhj, 1hhk, 1hoc, 1hsa, 1ldp, 1ld9, 1mhc, 1osz, 1tmc, 1vac,1vad, 2vab, 2clr, 2mha, and 2vaa! were first merged in the B*2701structure to reproduce as closely as possible the heterogeneousbulging part~P4 to PC21! of naturally MHC-bound peptides. MHCanchor positions~P1, P2, P3 ,andPC! were then removed from thisset of bound peptides. A peptide model~ARAAAAAAA ! used tomap the electron density map of B*2705-bound peptides was fittedto theP4-PC21 X-ray structures to define a virtual peptide pool ofArg2-containing peptides. After mutating Arg2 into Gln, a newGln2 side chain was modeled and a separate Gln2 peptide poolcreated. Few modifications of the Gln2 side chain~x1 5 257.9,x2 5 165,x3 5 2155! were necessary to orient the terminal amidenitrogen toward MHC H-bond acceptors~Glu45, Glu63!.

For both models~B*2701 in complex with the Arg2 and the Gln2peptide pools!, the five dihedral angles of Lys70 were systemati-cally scanned using 158 increments starting from a random confor-mation. For each rotamer, the internal energy was checked using theTRIPOS force field~Clark et al., 1989! and an energy cutoff of50 kcal0mol above the computed energy minimum. Conformationsproducing intra- or intermolecular~with the virtual peptide pool! ste-ric bumps were discarded from the selection procedure. A total of220 and 198 conformations were finally extracted for Lys70 whenbound to the Arg2 and Gln2 peptide pools, respectively.

Molecular dynamics (MD) simulation of B*2701in complex with two natural ligands

To check the stability of B*2701 peptide interactions depending onpeptide position 2, B*2701 was simulated in complex with twonatural B*2701 ligands: GQAPGYSY and RRYQKSTEL~Garcíaet al., 1997a!. The GQAPGYSY peptide was modeled according tothe B*2705-bound ARAAAAAAA peptide model~PDB code: 1hsa!~Madden et al., 1992! for MHC-anchoring amino acids~P1–P3,Pc! and according to the B*3501-bound HIV-1 nef 75–82 peptide~VPLRPMTY! ~PDB code: 1a1n! ~Smith et al., 1996b! for theP4–P7 bulging part. The RRYQKSTEL peptide was built as pre-viously described~Rognan et al., 1997! from the B*2705-boundARAAAAAAA peptide model by substituting the corresponding

residues for alanine at P1, P3–P9 positions and relaxing each newside chain to its closest energy minimum. Both peptides were thenrefined and annealed as recently described~Krebs et al., 1998!.Briefly, while maintaining protein atoms fixed, the peptide wassimulated at 1,000 K for 50 ps and linearly cooled down to 50 Kover 50 ps to trap low energy conformations. After adding andrefining a shell of water molecules to the last simulated annealingconformer, a 200 ps restrained MD simulation of the solvatedB*2701 peptide complexes was performed using an already re-ported procedure~García et al., 1998!. To avoid large drifts fromthe B27 crystal structure, only protein atoms located in the bindinggroove were allowed to freely move. Other atoms were restrainedto their X-ray coordinates by a weak harmonic constraint of 0.05kcal mol21 Å21.

Site-directed mutagenesis, DNA-mediated gene transfer,and flow cytometry

The construct containing the B*2701 gene was obtained by site-directed mutagenesis of B*2705 genomic DNA and was previouslydescribed~Benjamin et al., 1991!. Site-directed mutagenesis ofB*2701 was performed using the uracil selection method~Kunkel,1985!. Therefore the gene fragment of interest was cut with Bcl Iand ligated into the BamH I site of bacteriophage M13mp18 dsDNA.The resulting phage was grown in thedut2, ung2 Escherichia colistrain CJ236~Biorad, Richmond, California! to produce uracil-containing ssDNA. The complementary strand was synthesizedwith the 59-AGTCTGTGCCGCGGCCTTGCA-39 primer bearingthe mutant codon. Following ligation, the mutant DNA was prop-agated in thedut1 ung1 E. coli strain MV 1190 to suppress uracilcontaining wild-type DNA. After confirming the mutation by di-deoxy sequencing, the mutant fragment was excised with Eco52 Iand BsiW I and religated to the equally digested and dephosphor-ylated B*2701 plasmid.

HMy2.C1R cells were transfected with the mutant DNA as pre-viously described~Calvo et al., 1990!. Surface expression of HLA-B27 molecules was measured by flow cytometry analysis with theME1 mAb, specific for HLA-B27, B7, B22, and B42~Ellis et al.,1982! as previously described~Villadangos et al., 1995!.

Peptide isolation and sequencing

Peptides bound to the mutant HLA-B27 molecules were purified from0.8 to 1.53 1010 cells as previously described~Villadangos et al.,1995! with the exception of using the mAb W6032 ~specific for amonomorphic HLAclass I determinant! ~Barnstable et al., 1978! forpurification of HLA-B27 by affinity chromatography and that thepeptides were eluted with 0.1% trifluoroacetic acid~Sigma, St. Louis,Missouri!. Sequencing of the pooled peptide fractions was carriedout by Edman degradation in an ABI 473A automated sequencer~Applied Biosystems, Foster City, California!. Residue assign-ments were as previously described~Falk et al., 1991!.

Acknowledgments

This work was supported by grants SAF9700182 from the Plan Nacional deI1D and PM95-002 from the Spanish Ministry of Education to JALC, bygrant 31-45504.95 of the Schweizerischer Nationalfond zur Förderung derWissenschaften~SNF! to DR, and by an institutional grant of the Fun-dación Ramón Areces~Madrid! to the CBMSO. SK is a fellow of the SNF.The authors thank Prof. G. Folkers~Department of Pharmacy, ETH Zürich!,for crucial support, Josefa González-Nicolás~Protein Chemistry Depart-ment CBMSO, Madrid! for technical assistance, and the calculation center

1398 S. Krebs et al.

of the ETH Zürich, for allocation of computer time on the Cray J90 andPARAGON supercomputers.

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