- Email: [email protected]
On the molecular nature of ⪡restrictive⪢ antigenic elements present on major histocompatibility complex (MHC) proteins
(~) ELSEVIER Paris 1989
Res. lmmuno!o 1989, 140, 145-158
HYPOTHESIS
ON THE MOLECULAR NATURE OF ~> ANTIGENIC ELEMENTS PRESENT ON MAJOR HIS]If)COMPATIBILITY COMPLEX (MHC) PROTEINS J. Novotny (1) (o), RoE. Brucco|eri (l) (.) and P. KourHsky (2)
(1) Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114 (USA), and (2) lnstitut Pasteur, 75724 Paris Cedex 15
Summary. By analogy with the way in which antibodies recognize their specific antigens, it appears likely that T-cell receptors recognize peptides presented by MHC molecules as composite epitopes involving the presented peptide and portions of the MHC moiecuies. We extend here the analogy to attempt to define which portions of the MHC molecules are most accessible to the TcR and thereby most likely to participate in the binding. We suggest that the alpha chain segments 56-60, 73-77, 149-153 and 158-162, which are most protruding according to accessibility calculations, are likely candidates for the interaction. Again, by analogy with antibodies, we further propose that TcR recognition may involve: (1) an <> recognition process, focussed on a few residues which provide the major contribution to the binding energy, and (2) a <~passive >>recognition process, in which the relatively large contact areas between TcR and the composite epitope primarily need to be compatible with one another rather than to contribute significantly to binding energy. KEY-WORDS: MHC, T lymphocyte, Antibody antigenicity; T-cell receptors, Large probe accessibility, CONGEN conformational search.
Received February 28, 1989. (*) Present address: The Squibb Institute for Medical Research, Princeton, NJ, 08543-4000 (USA).
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Introduction. Protein antigens foreign to a vertebrate body challenge its immune system by two different routes. In humoral immunity, surface areas (epitopes) of intact antigens are specifically recognized by antibodies, leading to the formation of soluble antigen-antibody complexes. Protein antigens are also internalized by so-called antigen-presenting cells, degraded, and short peptides thereof are presented on their surface in association with the highly polymorphic class I and class II molecules of the major histocompatibility complex (MHC). The MHC-bound epitopes are recognized by T-cell receptors (TcR). It has been found that this recognition is usually limited to antigen-presenting cells and T cells derived from the same organism. This restriction has been traced down to the identity of the presenting MHC molecule. That is, as the T cells of a given organism interact with the peptidic epitope, they also have somehow ~ learned >>to recognize the MHC molecules of that organism. The learning process probably takes place in the thymus and involves a selection for a subset of T cells that constitute a repertoire somehow biased towards self MHC recognition (reviewed in Schwartz, 1985, 1986; Marrack and Kappler, 1988; Kourilsky and Claverie, 1989a, b). The phenomenon of MHC restriction is not fully understood yet. Current molecular hypotheses are based on two recent Findings. First, it has been shown that, for a given antigen, certain peptides are presented by certain alleles of MHC molecules, but not by other alleles. At least for class II MHC molecules, this property correlates to a certain extent with the capacity of the peptides to bind to purified MHC molecules (reviewed in Buus et al., 1987). Secondly, the HLA-A2 allele of the human class I MHC was purified and crystallized, and its three-dimensional structure solved by X-ray diffraction. The s. t. r. u. .c .t .u.r.e. diqnlav~ nl_k., h^l:^_, segments on top of r---~ . a .groove . . in h,~,n,o..., .--,.,.., two a,r,~a-~u~a, an eight-stranded beta-sheet (Bjorkman et al., 1987a,b). Polymorphic amino acid residues map into this presumptive peptide-binding site, or close to it. Accordingly, it is believed that the groove is endowed with some specificity and is selective in two respects: it preferentially binds certain peptides and possibly determines the conformation of the bound peptide. Three-dimensional structures of the TcR, of the peptide bound to MHC antigen or, afortiori, of their tri-molecular complex, are not available at present. It is possible however, based on structural information available, to build and analyse molecular models. For TcR, the leading structural hypothesis, as suggested by us (Novotny et al., 1986a) and others (Chothia et al., 1988; Bjorkman and Davies, 1988; Claverie et al., 1989) and as supported by limited experimental evidence (Hedrick et al., 1988; Marchalonis et al., 1988), maintains that TcR and antibodies have close structural similarity. That is, amino
M H C = major histocompatibility complex. r.m.s. = root mean square.
I
TcR = T-cell receptor.
M O L E C U L A R N A T U R E O F M H C A N T I G E N I C ELE_MENTE:.~ !47
acid residues known to define dimensions and overall size of antibody-binding sites are known to be conserved in TcR variable domains, and the TcR and antibodies are likely to accommodate, in their respective binding sites, antigens of the same size range. Both molecules are also expected to recognize surfaces of their respective protein antiigens in an analogous manner (Novotny et al., 1986a; Chothia ~.' al., 1988). Molecular details of antigenic epitopes recognized by antibodies have been emerging from the crystal structure of Fab fragments complexed with their specific antigens (Amit et al., 1986, Sheriff et al., 1987; Mariuzza et al., 1987; Alzarri et al., 1988; Davies et al., 1988). The data have shown that contacts are made over a rather large surface (of about 8 nm2), as common to many other protein-protein interactions (Miller et al., 1987a,b). Accordingly, it appears likely that TcR recognize both the presented peptide and the neighbouring regions of the presenting MHC molecule (Poljak, 1987; Ajitkumar et al., 1988). The possible molecular nature of such a composite epitope is the subject of the present communication. We first discuss which regions of the presenting MHC molecules are most likely to be involved in interactions with TcR. Then, we propose a distinction, based upon considerations on binding energies, between ~ active ~ and ~ passive >~recognition. These suggestions are anticipated to be useful in the elaboration of critical experiments.
Regions of MI-IC molecules which are most likely to interact with TcR.
Under the assumption that TcR and antibodies have similar structures and modes of recognizing their antigenic partner, it follows that TcR should, in general, ~'~-*o"* ~ t a r , ~.~uu~ ~o;a .... in • addition • ,.,,..,,,,., ,,lll,~ to rc~uu~ --:-' .... of the presented peptide (Poljak, 1987). Whether the contacted MHC residues are polymorphic or not is a separate issue which will be discussed later. If the contact area in the MHC-peptide-TcR complex is on the order of 20 × 30 ~ both alpha helices in the MHC molecule can provide contact zones, as suggested by Ajitkumar et al. (1988). This does not imply that the intact alpha helices are involved in the presumed interaction. In the absence of clearcut experimental data, we used the molecular model of HLA-A2 to identify the regions most likely to interact. We reasoned that the most protruding part of MHC molecules in the vicinity of the bound peptide would be the most likely targets for TcR interaction in the same way as such protruding parts in an antigen are usually recognized by an antibody. We previously showed that protein segments most accessible to a large probe (comparable in size to antibody domains) correlate well with known antigenic epitopes (Novotny et al., 1986b, 1987). Here, we extend the large probe accessibility calculations to MHC molecules. We carried out the large probe accessibility calculations both on the alphacarbon coordinates of the human HLA-A2 molecule (the original crystallographic data of Bjorkman et al., 1987a) and a complete atomic model
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constructed by us as described in the ~nnex. It is clear that the results should be viewed with caution: (1) they relate to an approximate, imperfect model of the HLA-A2 molecule; (2) any ge~aeralization to other MHC molecules lies in the assumption that the structme is conserved in the regions identified as important; (3) the large probe men,hod is itself, a crude approach to the identification of epitopes. With these reservations in mind, we found results of the calculation (given in the Annex) interesting in that they revealed to us peculiar features of the HLA-A2 molecule surface. There me two obvious kinks in the HLA-A2 helices, centered around residues 56-50 in alpha-1 and 158-162 in alpha-2. In addition, the calculation suggests th~ segments 73-77 in alpha-1 and 149-153 in alpha-2 are protuberant as well (see figures 1 and 2 in ~ Annex >>). Indeed, each segment contains one or several ~esidues which point up in the HLA-A2 crystal structure (58, 73, 76, 150, 151,159, 162)(Bjorkman et aL, 1987a, b). Given the overall conservation of amino acid residues in these segments, it is not impossible that these four calc Mated epitopes exist in all class I antigens and, assuming the correctness of the Ia molecule model of Brown et al. (1988), in class II antigens as well. Indeed, due to the periodicity of alpha-helical structures, certain positions in these segments are easily accessible, while others (i.e. approximately every two other residues) are buried at the helix-sheet interface. Maay positions are highly conserved and very few are actually variable, as cot ld be anticipated, since the HLA-A2 crystal structure apparently displays cnly 2-4 polymorphic residues (62, 65, 66, 163) pointing up from the alpha helices in a position to interact with TcR. Certain residues (in or out of tht,se four segments) that point into the groove are nevertheless accessible to a k:rge probe (66, 152, 156), which raises the possibility that certain MHC residues Darticivate both in vevtide bindin~ and TcR contacts. The uncertainties inLerent i n o u r approach are Such tha'[ we do not think it useful to discuss the results of the calculations in more details.
Proposed molecular nature of the composite epitope. Based upon the above considerations, we propose that the epitopes seen by the TcR usually consist of two moieties: (1) side chain(s) from at least some of the four short alpha-helical segments (56-60, 73-77, 149-153, 158-162); and (2)side chain(s) of the foceign MHC-bound peptide. The MHC molecule/foreign peptide antigenicity is thus viewed as a special case of protein antigenicity, retaining the key attributes of noncontiguity and exceptional surface exposure of the epitopes of soluble proteins° As already mentioned, many of the residues belonging to these four helical segments are poorly or not at all polymorphic, emphasizing the possible role of non-polymorphic residues in making contacts with TcR. Whether all the presumptive contact regions are simultaneously recognized by any given TcR is an open question at this stage.
M O L E C U L A R N A T U R E OF A~'HC A N T I G E N I C ELEIffz~N1 ;b" i49 In pursuing the analogy between recognition of antibodies and TcR, we now make use of the finding that, although antibodies bind antigen over a relatively large surface, the energy of binding may well result from a much more limited area. Thus, in the lysozyme-antibody complexes, the contact area is on the order of 8 nm 2. However, energy calculations suggest that the energetically defined epitope is less than 3 nm 2 in surface and cortsists of side chains (and occasionally backbones) of no more than 2 to 4 resit~.ues (Novotny et al., 1989). On the antibody side, the most important re~ion in terms of binding energy, is the part of the ar, tigen-binding site where residues coded by D and J gene segments significantly contribute (Novotny et al.~ 1989). Under the assumption that these f'mdings (thus far valid for the three antigen-antibody complexes of known crystal structures) are general, their transposition to T-cell recognition has the following important implication: in the composite MHCpeptide epitope, we anticipate that the energetically relevant epitope will involve just a few side chains from the peptide and occasionally from the MHC molecule (usually involving MHC residues homologous t~3 those listed in table I). Accordingly, in the recognition of the composite epitope by TcR, we propose to distinguish between ~ active >>and ~ passive >>recognition. ~ Active >> recognition is limited to the relatively few non-covalent bor.ds which contribute significantly towards the energy of binding• ~ Passive >>recognition refers to the compatibility of surface which can establish contacts without major energetic contribution to binding. The bound peptide must participate in active recognition. Whether or not the MHC molecule also participates in active recognition in a systematic or non-systematic fashion is an open question. It is relevant to the problem of positive selection in the thymus (Singer et al., 1QR~ • Mftrrflelr
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TABLE I. - - Amino acid sequence conservation in the four most prominently exposed segments of HLA-A2 alpha-hdix 1 and 2.
Residue number 58 59 60 73 76 77
Alpha-helix- 1 Amino acid
Residue number
GLU, Gly, Arg TYR TRP Variable VAL, Glu Variable
150 15i 152 159 150 t62
Predominating residues are given in boldfa,ze.
Alpha-helix-2 Amino acid ALA GLY, His, Arg Variable TYR LEU GLY, Ala
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Recognition by TcR. As reviewed by Bougueleret and Claverie (1987), Davies and Bjorkman (1988) and Claverie et al. (1989), most TcR polypeptide chain variability is accounted for by junctional variability between the J, D and V gene segments. Most of the antigen-specific diversity of TcR molecules is therefore expected to be located around the bottom part of its binding-site cavity, where amino acids created by the gene junctional events reside. Davis and Bjorkman (1988) further speculated that <>. Similarly, Chothia et al. (1988) suggested that the TcR residues 25-31 (in both the alpha and beta chains) might contact the alpha-helices present in the MHC antigens. In line with the above discussion, we wish to emphasize several points. 1) We do not perceive the surface of the MHC protein-peptide complex as being flat. On the contrary, we associate the conspicuously protruding parts on this surface, identified by large probe accessibility calculations, with the most prominent antigenic epitopes potentially recognized by the receptor. 2) We do not require T-cell specificity to be strictly partitioned into MHC antigen-specific CDR1 and CDR2 regions and foreign peptide-specific CDR3 region. Although we do not exclude such a possibility apriori, we see no reason to envision any preferred orientation of the T-cell receptor binding site on the surface of the MHC molecule. It is . . . . :~.1.. k ......... that V-alpha and V-beta primary structure subgroups developed which are biased towards s. p e. c i .f i c.
i. n t. e r. a e. t i.n n. w i t ~,~,~ h t h p 1 V l t - I ~ Le.~~ v,~a, jn[ ~~, x ,x n~ , ~~ , , ~v l,, .aL~l ~ ;. ~o, , nl L *l l ,oO !;~*o,4 J d I . O I b ~ , ~ ;,~ ~ l / L ,L~ ,gk ~ , llI .J l~ ~
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case, weak correlations of such sequence variants with TcR gene subgroups should be expected (cf., e.g., Matis et al., 1987). 3) In analogy to antibodies, we expect the bottom part of the T-cell receptor binding cavity to largely determine specificity of binding, in the energetic sense. This would agree with the fact that TcR variability is concentrated predominantly in the third hypervariable regions of its polypeptide chains (Bougueleret and Claverie, 1987). Davis and Bjorkman (1988) ascribe a different functional importance to sequence hypervariability found in the V-D-J junctional sections of T-cell receptor genes. They suggest that the increased variability of these segments reflects preferential association with highly variable sequences of foreign peptides. This variability pattern is to be contrasted with that seen in, for example, mouse immunoglobulin sequences, where the variability is more evenly distributed among the six hypervariable (CDR) loops. The genetic mechanisms by which variability is created may, however, have little bearing on geometric details of macromolecular recognition. For example, the chicken genome is known to contain only one immunoglobulin VL gene and is believed to have only a few VH chain genes, i.e. significantly less than the number of mouse VH genes (Reynaud et ai.,
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1985). The chicken VL variability repertoire is generated in ontogenesis by putative cross-over events in which 25 pseudogenes take part (Reynaud et al., 1987). This genetic mechanism and the resulting variability pattern are distinctly different from those of mouse or human VL polypeptide chains, yet there is no reason to believe that the antigenic repertoire encountered by chickens is significantly different from that confronted by mice and men. In summary, we view the MHC peptide/TcR interaction as primarily focussed on a small area involving, on one hand, the bottom cavity of TcR and, on the other, a few peptide residues and, occasionally or systematically, a few residues of the MHC molecule. In addition to this so-called ((active>~ recognition, surface compatibility over a larger area would be necessary in what we have termed ((passive >>recognition. MHC residues in the areas which, by calculation, we usually anticipate ~o be relevant in (
ANNEX
Alpha-carbon coordinates of the human HLA-A2 extracellular domains were obtained from the Brookhaven Protein Data Bank (Bernstein et al., 1977). The complete backbone, i.e. the missing coordinates of the N, C and O atoms, were reconstructed by adapting CONGEN, a conformational search program (Bruccoleri and Karplus, 1987) to find peptide conformations whose alpha~arhnn~ hegt fit the erv~f~]|nor~nhlo d~t~ ~v~il~hl~ ]~nr nr~vlnll~ ~nn|ie~flnn~ J ~ , . , , ~ ,~/. /~ ,/.,./..~n~...~.. ~..~ ~
CONGEN has been used to find conformations for loops where each anchor point (i.e. N- and C-terminus) was known. This was done by first sampling phi and psi torsion angles in the backbones of the first n-3 residues, where n is the number of residues in the loop, and then using the modified G5 and Scheraga chain closure algorithm (G5 and Scheraga, 1979; Bruccoleri and Karplus, 1985) to complete the loop. However, here we used just the backbone sampling to thread the peptide backbone through the points specified by the crystallographic alpha-carbon positions. In order to perform this search efficiently, it was necessary to modify the search algorithm. Without the chain closure criterion, a complete sampling for even a small number of backbone residues would require prohibitive amounts of computer time. However, by using a different method of searching the tree of conformations produced by CONGEN, a so-called directed search, we can generate good confrontations early in the search process and can thus truncate the search and greatly reduce computer time requirements. To understand the modifications in the search algorithm, we must envision the conformational search as a tree (Bruccoleri and Karplus, 1987). The rcat of the tree represents the molecule at the beginning of the search pro-
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tess; each of the leaves of the tree represents the final conformations and the intermediate nodes represent partially constructed conformations. The connection between nodes represents a selection of torsion angles for a particular amino acid residue in ~he backbone. The search process begins with just the root and samples the backbone torsions for the first residue, i.e. expansion of the root node. A number of nodes is generated, each of which can be expanded to yield nodes which represent conformations that have two residues constructed. These expansions contribute u n il the leaves (final conformations) are reached. In the normal CONGEN search, the expansion process proceeds depth first, namely, the first node generated by an expansion is selected for the next expansion, and so on until the leaf node is generated and the next node at the lowest level is expanded, etc. This method is acceptable for a complete search of the tree. However, in a directed search, the node chosen for the next expansion is always the one which has the ~ best >> conformation, where ~ best>> in this case means smallest r.m.s, deviation to the alpha-carbon coordinates. Generally, t~s directed expansion of nodes will result in the lowest r.m.s. deviation conformations being generated first. However, because of errors inherent in the crystallographic data and because of the discrete sampling used, it is possible for the directed search to stall while it explores conformations that do not yield high quality conformations. Thus, we used a protocol whereby we constructed 10 residues at a time, and if no results were generated within two hours of CPU time, we reduced the length of the constructed segments to 5. The specification of the phi and psi backbone angles provides the information necessary to construct the beta carbon positions, since the direction of the C-alpha-C-beta bonds is uniquely determined by the locations of the uu:~l ~-eupnet ~uosutucnLs, i.e. the N and t: atoms. Side chain atoms beyond the beta carbons were constructed by CONGEN as described before (Novotny et al., 1988). We expected side chain orientations to be biased by occasional errors, as observed before ill CONGEN-reconstructed side chains of hemerythrin and immunoglobulin VL domain (Novotny et al., 1988). Charged residues and aromatic rings were most often misplaced in the CONGEN side chain placement protocol that used the CHARMM in vacuo potential energy function (Brooks et al., 1983), as was used in this work. .-~L.~--
P
--1--1.
. . . .
L--J.-'~-
. . . .
--
The final model was energy-minimized with harmonic constraints of 20 kcal (83.6 k J) applied to the positions of alpha-carbons. The r.m.s, shift between t.~e crystallographic C-alpha coordinates atoms and those of the final, energyminimi~.d" model, was 0.9 ,~ for the heavy chain and 1.5 .~ for the beta-2-microglobulin. Large probe accessibility was computed as described before, using the accessibi;;.ty algorithm of Lee and Richards (1971) as modified by R.E. Bruccoleri (Novotny et al., 1986b) and the probe radius of 10 ~ . To minimize the influence of side chain placement errors in the HLA-A2 model upon antigenicity analysis, large probe accessibility was also computed on the naked
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backbone. The rationale for this approach is based on the observation of Thornton et al. (1986) that antigenicity (protrusiveness) of peptide segments in proteins is essentially determined by the shape of the backbone. The contact surface of the HLA-A2 extraceUular domains was calculated using the spherical probe of 1-nm radius (numerical results are available upon request) and smoothed over 7-residue segments as described before (Novotny et al., 1986b). Contact surface profiles of the complete HLA-A2 alphachain model and its virtual C-alplla backbone are shown in figure 1. It can be seen that the essential features of both curves coincide, although some differences are apparent (e.g. the mutual shifts of maxima around positions 75 and 147). We feel that the C-alpha-generated profile is more reliable, and the above discussion makes use of it.
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FIG. 1. - - Large probe accessibility profile o f the alpha chain o f the human class I M H C antigen allele HLA-A2. Heavy line: data obtained with alpha-carbon coordinates only; light line: data obtained with the whole molecular model, constructed as described in the ¢ Annex,. The asterisks denote locations of residues recognized by specific monoclonal antibodies on various human and mouse class I alleles (including the HLA-A2), as determined by Parham's group ana aescnbed by Bjorkman et al. (1987b). Results of Abastado et al. (1987), Hogan et al. (1988), Toubert et al. (1988), Sire et ai. (1988) and Santos-Agaudo et al. (1988) have also been included.
I
FIG. 2B (bottom). - - A stereoscopic alpha-carbon tracing of backbone of alpha-I and alpha-2 domains from the HLA-A2 molecule. Location of prominent calculated epitopes (cf. fig. 1) close to interhelical groove (putative foreign peptide binding site of Bjorkman et aL, 1987b) is emphasized by heavy lines.
FIG. 2A (top). - - A color-coded computer-graphics representation of polypeptMe backbone of HLA-A2 alpha-1 and alpha-2 domains (see ¢~Annex)> f~r details o f backbone construction). The four putative ~ antigenic )) segments 56-60, 73-77, 149-153 and 158-162 (cf. fig. 1) are rendered in green.
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B
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I ~ O L E C U L A R N A T U R E OF M H C A N T I G E N I C E L E M E N T S
155
As expected, positions of the major contact surface maxima correspond to locations of bends and turns of the polypeptide chain between adjacent beta-strands or between strands and helices (peaks around positions 20, 43, 90, 110, 130, 140 and 180). These, however, are relatively distant from the (ttop>> surface of the extracellular domains created by the two long helical runs and the foreign peptide-binding groove. More interestingly, irregular breaks pcesent in the helical runs in the alpha-1 domain (50-84) as well as the alpha-2 domain (138-180) stand up as major protruding parts of the surface. The surface maxima centered on residues 58 and 151 rank among the most prominent features of the whole profile. The other two maxima, centered on residues 76 and 159, are less pronounced. Figure 2 shows the alpha-1 alpha-2 domain dimer with the segments 56-60, 73-77, 149-153 and 158-162 emphasized. A comparison between calculated epitopes and class I MHC residues identified as important in the recognition by a variety of w.onoclonal antibodies can be seen in figure 1. The correlation is reasonably good but far from complete. However, there is a bias in the comparison, because the serological experiments have only characterized polymorphic residues; virtually nothing is known about the participation oi the non-polymorphic ones in epitopes recognized by antibodies. The experimental evidence dealing with the identification of MHC residues possibly involved in making contacts with TcR is relatively complex and its interpretation is difficult (reviewed in Kourilsky and Claverie, 1989a). Table I shows the 12 positions which are accessible in the four segments that are most exposed to the large probe, and indicates the variability at each position.
REFERENCES ABASTADO,J.P., JAULIN,C., SCHUTZE,M.-P., LANGLADE-DEMOYEN,P., PLATA,F., OZATO, K. 8[: KOURILSKY,P. (1987), Fine mapping of epitopes in intradomain Kd/Dd recombinants. J. exp. Med., 166, 327-340. AJITKUMAR, P., GEIER, S.S., KESARI, K.V., BORRIELLO, F., NAKAGAWA,~I., BLUESTONE,J.A., SAPER, M.A., WILEY, D.C. & NATHENSON,S.G. (1988), Evidence that multiple residues on both the alpha-helices of the class I MHC molecule are simultaneously recognized by the T-cell receptor. Ceil, 54, 47-56. ALZARI,P.M., LASCOMBE,M.B. & POLJAK,R.J. (1988), Three dimensional structure of antibodies. Ann. Rev. Immunol., 6, 555-580. AMIT, A., MARIUZZA,R., PHILLIPS,S.E.V. & POLJAK,R. (1986), Three-dimensional structure of an antigen-antibody complex at 2.8 A resolution. Science, 233, 747-752. BARLOW,D.W., EDWARDS,M.S. & THORNTON,J.M. (1986), Continuous and discontinuous protein antigenic determinants. Nature (Lond.), 322, 747-748. BENJAMIN, D.C., BERZOFSKY, J.A., EAST, I.J., GURD, J.G., MILLER, A., PRAGER,E.M., REICHLIN,M., SERCARZ,E.E., SMITH-GILL,S.J., TODD,P.E. WILSON,A.C. (1984), The antigenic structure of proteins. A reappraisal. Ann. Rev. Immunol., 2, 67-101.
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BERNSTEIN, F.C., KOETZLE,T.F., WILLIAMS,G.J.B., MEYER, E.F., BRICE, M.D., RODGERS,J.R., ~ A R D , O., SHIMANOUCHI,T. & TASUMhM.J. (1977), The Protein Data Bank" a computer-base archival file for macromolecular structures. J. tool. Biol., 112, 535-542. BJORKMAN,P.J., SAPER,M.A., SAMRAOUI,B., BENNETT,W.S., STROMINGER,J.L. & WILEY, D.C. (1987a), Structure of the human class I histocompatibility antigen, HLA-A2. Nature (Lond.), 329, 506-512. B~OP.KMAN,P.J., SAPER,M.A., SAMgAOUI,B., BENNETT,W.S., STROMINGER,J.L. & WIL~Y, D.C. (1987b), The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature (Lond.), 329, 512-518. BOUGUERELET,L. & CLAVERIE,J.M. (1987), Variability analysis of the human and mouse T-cell ,,.,...~,,,..*-'-~...,o ..,.~........,,,~. ~h°;~ Immunogenetics, 26, 304-308. BROOKS,B.R., BRUCCOLEm,R.E., OLAFSOS,B.D., STATES,D.J., SWAMINSTHAN,S. & KAgPLUS,M. (1983), CHARMM, A program for macromolecular energy, minimization, and dynamics calculations. J. comput. Chem., 4, 187-217 BROWN, J.H., JARDETZKY,T., SAPER, M.A., SAMRAOOI,B., B~ORKMAN, P.J. & WILEY, D.C. (1988), A hypothetical model of the foreign antigen binding site of class II histocompatibii~ty molecules. Nature (Lond.), 332, 845-850. BgucCOLERI, R.E. & KAgPLUS,M. (1985), Chain-closure with bond angle variations• Macromolecules, 18, 2767-2773. BRUCCOLERI,R.E. & KARPLUS,M. (1987), Prediction of the folding of short polypeptide segments by uniform conformational sampling. Biopolymers, 2T6, 137-168. Buus, S., SETTE,A. & Gl~V, H.M. (1987), The interaction between protein-derived immunogenic peptides and I a. lmmunoi. Rev., 98, 115-141. CHOTHIA, C., BOSWELL,D.R. & LESK, A.M. (1988), The outline structure of the T-cell receptor. EMBO J., 7, 3745-3755. CLAVERIE, J.-M., PROCnNICKA-CnALUFOUR,A. & BOUOUELERET, L. (1989), Immunological implications of a Fab-like structure for the T-cell receptor. Immunol. Today, 10~ 10-14. CLAVaERGER, C., PARnAM, F., ROrnBARD, J., LUDWXG,D.S., SCHOOLNIK,G.K. & KR~NSICY,A.M. (1987), HLA-A2 peptides can regulate cytolysis by human allo~eneic T l v r n n h n c v t e _ ~ ]gTnturo ( I ~ n r i ~ "~'~ "/K~_"/K~ DAVIES,D.R., SHERIFF,S. & PADLAN,E. (1988), Antigen antibody complexes. J. biol. Chem., 263, 10541-19544. DAVIS,M.M. & BJORgMAN,P. (1988), T-cell antigen receptor gene and T-cell recognition. Nature (Lond.), 334, 395-401. GAY, D., Buus, S., PASTERNAK,J., KAPPLER,J. & MARRACK,P. (1988), The T-cell accessory molecule CD4 recognizes a monomorphic determinant on isolated Ia. Proc. nat. Acad. Sci. (Wash.), 8S, 5629-5633. G~, N. & SCI-mRAGA,H.A. (1970), Ring closure and local conformational deformations of chain molecules. Macromolecules, 3, 178-187. HEDmCK, S.M., ENGEL, I., MCELLIOOTT, D.L., FINK, P.J., MEILING, H.S.U., tD~SBURG, D. & MATIS, L.A. (1988), Selection of amino acid and the beta chain of the T cell antigen receptor. Science, 239, 1541-1544. HOGAN, K.T., CLAYBERGER, C., BERNHARD, E.J., WALK, S.F., RIDCE, J.P., P~, P., IC~NSKY,A.M. & ENGLEI~P.D,V.H. (!988), Identification by site-directed mutagenesis of amino acid residues contributing to serologic and CTL-defined epitope differences between HLA-A2.1 and HLA-A2.3. J. Immunol., 141, 2519-2525. KotnuLSiCV, P., CI~OUAT, G., 1L~OUgDIN-COMBE,C. & CLAVERIE, J.-M. (1987), Working principles in the immune system implied by the ~peptidic self)> model. Proc. nat. Acad. Sci. (Wash.), 84, 3400-3404. KotnuLSIC¢,P. & CLAVERm,J.-M. (1989a), MHC-aatigen interaction- what does the T cell receptor se.e? Adv. Immunol., 45, 107-193. d ----.lff--~d'
~k,a--,'wwat~4.,],
.tt,¢~,
• I,¢~--
I I,F~"
•
MOLECULAR NATURE OF MHC ANTIGENIC ELEMENTS
157
KOURILSKY, P. & CLAVERIE, J.-M. (1989b), MHC restriction, alloreactivity and thymic education: a common link ? Cell, 56, 327-329. LEE, B.K. & RICHARDS,F.M. (1971), Interpretation of protein structures: estimation of static accessibility. J. tool Biol., 55, 379-400. MARCHALONIS,J.J., SCHLUTER,S.F., HUBBARD,R.A., MCCABE, C. & ALLEN, R.C. (1988), Immunog!obulin epitopes defined by synthetic peptides corresponding to joining region sequence: conservation of determinants and dependence upon the presence of an arginyl or lysyl residue for cross-reaction between light chains and T-cell receptor chains. Moi. Immunol., 25, 771-784. MARRACK, P. & KAPPLER, J. (1988), The T-cell repertoire for antigen and MHC. Immunol. Today, 9, 308-315. MARYANSKI,J.L., ABASTADO,J.-P. & KOURILSKV,P. (1987), Specificity of peptide presentation by a set of hybrid mouse class I MHC molecules. Nature (Lond.), 330, 660-662. MATIS, L.A., SORGER,S.B., MCELLIGOTT,D.L., FINr, P.J. & HEDRICK,S.M. (1987), The molecular basis of alloreactivity in antigen-specific, major histocompatibility complex-restricted T cell clones. Cell, 51, 59-69. MILLER, S., LESK, A.M., JANIN, J. & CHOTHIA, C. (1987a), The accessible surface area and stability of oligomeric proteins. Nature (Lond.), 328, 834-836. MILLER, S., JANIN, J., LESK,A.M. & CHOTHIA,C. (1987b), Interior and surface of monomeric proteins. J. tool Biol., 196, 641-656. NOVOTNY,J., TONEGAWA,S., SAITO,H., KRANZ,D.M. & EISEN, H.N. (1986a), Secondary, tertiary and quaternary structure of T-cell-specific itnmunoglobulinlike polypeptide chains. Proc. nat. Acad. Sci. (Wash.), 83, 742-746. NOVOTNY,J., HANDSCHUMACHER,M., HABER,E., BRUCCOLERI,R.E., CARLSON,D.W., FANNING,D.W., SMITH,J.A. & ROSE, G.D. (1986b), Antigenic determinants in proteins coincide with surface regions accessible to large probes (antibody domains). Proc. nat. Acad. Sci. (Wash.), 83, 226-230. NOVOTNY, J., HANDSCHUMACHER,M. & BRUCCOLERI,R.E. (1987), Protein antigenicity: a static surface property. Immunol. Today, 8, 26-31. NOVOTNY, J., RASHIN,A.A. & BRUCCOLERI,R.E. (1988), Criteria that discriminate between native proteins and incorrectly folded models. Proteins, 4, 19-30. NOVOTNY,J., BRUCCOLERI,R.E. & SAUL, F.A. (1989), On the attribution of binding energy in antigen-antibody complexes McPC 603, D1.3 and HyHEL-5. Biochemistry, in press. POLJAK,R.J. (1987), Hypothesis: a molecular model for the MHC-restricted recognition of antigens by the T-cell receptor. Ann. Inst. Pasteur/Immunol., 138, 175-180. REYNAUD,C.A., ANQUEZ,V., DAHAN,A. & WEILL,J.C. (1985), A single rearrangement event generates most of the chicken immunoglobulin light chain diversity. Cell, 40, 283-291. REYNAUD,C.A., ANQUEZ,V., GRIMAL,H. & WEILL,J.C. (1987), A hyperconversion mechanism generates the chicken light chain pre~mmune repertoire. Cell, 48, 379-388. SANTOS-AGUADO,J., BARBOSA,J.A., BIRO,A. & STROMINGER,J.L. (1988), Molecular characterization of serologic recognition sites in the human HLA-A2 molecule. J. Immunol., 141, 2811-2818. SCHWARTZ,R.H. (1985), T-lymphocyte recognition of antigen in association with gene products of the major histocompatibility complex. Ann. Hey. immunol., 3, 237-261. SCHWARTZ,R.H. (1986), Immune response (Ir) genes of the murine major histocompatibility complex. Adv. Immunol., 38, 31-201. SINGER,A.., MIZUOCHI,T., MUNITZ,T.I. & GRESS, R.E. (1986), Role of self antigens m the selection of the developing T cell repertoire, in: ¢¢Progress in immunology VI~, VIth International Congress of Immunology, B. Cinader & R.G. Miller, Eds., Academic Press Inc., p. 60.
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SIRE, J., CHIMINI, G., BORETTO, J., TOUBERT, A., KAHN-PERLES, B., LAYET, C., SODOYER, R., LEMONNIER,F. & JORDAN,B. (1988), Hybrid genes between HLA-A2 and HLA-A3 constructed by in vivo recombination allow mapping of HLA-A2 and HLA-A3 polymorphic antigenic determinants. J. Immunol., 140, 2422-2430. TOUBERT, A., RAFFOUX, C., BORETTO, J., SIRE, J., SODOYER, R., THURAU, S.R., AMOR, B., COLOMBANI, J., LEMONNIER, F.A. & JORDAN, B.R. (1988), Epitope mapping of HLA-B27 and HLA-B7 antigens by using intradomain recombinants. J. Immunol., 141, 2503-2509. THORNTON,J., EDWARDS,M.S., TAYLOR,W.R. & BARLOW,D.J. (1986), Location of ~ continuous >>antigenic determinants in the protruding regions or prot~.ins. EMBO J., 5, 409-413.
Res. lmmuno!o 1989, 140, 145-158
HYPOTHESIS
ON THE MOLECULAR NATURE OF ~
(1) Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114 (USA), and (2) lnstitut Pasteur, 75724 Paris Cedex 15
Summary. By analogy with the way in which antibodies recognize their specific antigens, it appears likely that T-cell receptors recognize peptides presented by MHC molecules as composite epitopes involving the presented peptide and portions of the MHC moiecuies. We extend here the analogy to attempt to define which portions of the MHC molecules are most accessible to the TcR and thereby most likely to participate in the binding. We suggest that the alpha chain segments 56-60, 73-77, 149-153 and 158-162, which are most protruding according to accessibility calculations, are likely candidates for the interaction. Again, by analogy with antibodies, we further propose that TcR recognition may involve: (1) an <
Received February 28, 1989. (*) Present address: The Squibb Institute for Medical Research, Princeton, NJ, 08543-4000 (USA).
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Introduction. Protein antigens foreign to a vertebrate body challenge its immune system by two different routes. In humoral immunity, surface areas (epitopes) of intact antigens are specifically recognized by antibodies, leading to the formation of soluble antigen-antibody complexes. Protein antigens are also internalized by so-called antigen-presenting cells, degraded, and short peptides thereof are presented on their surface in association with the highly polymorphic class I and class II molecules of the major histocompatibility complex (MHC). The MHC-bound epitopes are recognized by T-cell receptors (TcR). It has been found that this recognition is usually limited to antigen-presenting cells and T cells derived from the same organism. This restriction has been traced down to the identity of the presenting MHC molecule. That is, as the T cells of a given organism interact with the peptidic epitope, they also have somehow ~ learned >>to recognize the MHC molecules of that organism. The learning process probably takes place in the thymus and involves a selection for a subset of T cells that constitute a repertoire somehow biased towards self MHC recognition (reviewed in Schwartz, 1985, 1986; Marrack and Kappler, 1988; Kourilsky and Claverie, 1989a, b). The phenomenon of MHC restriction is not fully understood yet. Current molecular hypotheses are based on two recent Findings. First, it has been shown that, for a given antigen, certain peptides are presented by certain alleles of MHC molecules, but not by other alleles. At least for class II MHC molecules, this property correlates to a certain extent with the capacity of the peptides to bind to purified MHC molecules (reviewed in Buus et al., 1987). Secondly, the HLA-A2 allele of the human class I MHC was purified and crystallized, and its three-dimensional structure solved by X-ray diffraction. The s. t. r. u. .c .t .u.r.e. diqnlav~ nl_k., h^l:^_, segments on top of r---~ . a .groove . . in h,~,n,o..., .--,.,.., two a,r,~a-~u~a, an eight-stranded beta-sheet (Bjorkman et al., 1987a,b). Polymorphic amino acid residues map into this presumptive peptide-binding site, or close to it. Accordingly, it is believed that the groove is endowed with some specificity and is selective in two respects: it preferentially binds certain peptides and possibly determines the conformation of the bound peptide. Three-dimensional structures of the TcR, of the peptide bound to MHC antigen or, afortiori, of their tri-molecular complex, are not available at present. It is possible however, based on structural information available, to build and analyse molecular models. For TcR, the leading structural hypothesis, as suggested by us (Novotny et al., 1986a) and others (Chothia et al., 1988; Bjorkman and Davies, 1988; Claverie et al., 1989) and as supported by limited experimental evidence (Hedrick et al., 1988; Marchalonis et al., 1988), maintains that TcR and antibodies have close structural similarity. That is, amino
M H C = major histocompatibility complex. r.m.s. = root mean square.
I
TcR = T-cell receptor.
M O L E C U L A R N A T U R E O F M H C A N T I G E N I C ELE_MENTE:.~ !47
acid residues known to define dimensions and overall size of antibody-binding sites are known to be conserved in TcR variable domains, and the TcR and antibodies are likely to accommodate, in their respective binding sites, antigens of the same size range. Both molecules are also expected to recognize surfaces of their respective protein antiigens in an analogous manner (Novotny et al., 1986a; Chothia ~.' al., 1988). Molecular details of antigenic epitopes recognized by antibodies have been emerging from the crystal structure of Fab fragments complexed with their specific antigens (Amit et al., 1986, Sheriff et al., 1987; Mariuzza et al., 1987; Alzarri et al., 1988; Davies et al., 1988). The data have shown that contacts are made over a rather large surface (of about 8 nm2), as common to many other protein-protein interactions (Miller et al., 1987a,b). Accordingly, it appears likely that TcR recognize both the presented peptide and the neighbouring regions of the presenting MHC molecule (Poljak, 1987; Ajitkumar et al., 1988). The possible molecular nature of such a composite epitope is the subject of the present communication. We first discuss which regions of the presenting MHC molecules are most likely to be involved in interactions with TcR. Then, we propose a distinction, based upon considerations on binding energies, between ~ active ~ and ~ passive >~recognition. These suggestions are anticipated to be useful in the elaboration of critical experiments.
Regions of MI-IC molecules which are most likely to interact with TcR.
Under the assumption that TcR and antibodies have similar structures and modes of recognizing their antigenic partner, it follows that TcR should, in general, ~'~-*o"* ~ t a r , ~.~uu~ ~o;a .... in • addition • ,.,,..,,,,., ,,lll,~ to rc~uu~ --:-' .... of the presented peptide (Poljak, 1987). Whether the contacted MHC residues are polymorphic or not is a separate issue which will be discussed later. If the contact area in the MHC-peptide-TcR complex is on the order of 20 × 30 ~ both alpha helices in the MHC molecule can provide contact zones, as suggested by Ajitkumar et al. (1988). This does not imply that the intact alpha helices are involved in the presumed interaction. In the absence of clearcut experimental data, we used the molecular model of HLA-A2 to identify the regions most likely to interact. We reasoned that the most protruding part of MHC molecules in the vicinity of the bound peptide would be the most likely targets for TcR interaction in the same way as such protruding parts in an antigen are usually recognized by an antibody. We previously showed that protein segments most accessible to a large probe (comparable in size to antibody domains) correlate well with known antigenic epitopes (Novotny et al., 1986b, 1987). Here, we extend the large probe accessibility calculations to MHC molecules. We carried out the large probe accessibility calculations both on the alphacarbon coordinates of the human HLA-A2 molecule (the original crystallographic data of Bjorkman et al., 1987a) and a complete atomic model
148
J. N O V O T N Y E T A L .
constructed by us as described in the ~nnex. It is clear that the results should be viewed with caution: (1) they relate to an approximate, imperfect model of the HLA-A2 molecule; (2) any ge~aeralization to other MHC molecules lies in the assumption that the structme is conserved in the regions identified as important; (3) the large probe men,hod is itself, a crude approach to the identification of epitopes. With these reservations in mind, we found results of the calculation (given in the Annex) interesting in that they revealed to us peculiar features of the HLA-A2 molecule surface. There me two obvious kinks in the HLA-A2 helices, centered around residues 56-50 in alpha-1 and 158-162 in alpha-2. In addition, the calculation suggests th~ segments 73-77 in alpha-1 and 149-153 in alpha-2 are protuberant as well (see figures 1 and 2 in ~ Annex >>). Indeed, each segment contains one or several ~esidues which point up in the HLA-A2 crystal structure (58, 73, 76, 150, 151,159, 162)(Bjorkman et aL, 1987a, b). Given the overall conservation of amino acid residues in these segments, it is not impossible that these four calc Mated epitopes exist in all class I antigens and, assuming the correctness of the Ia molecule model of Brown et al. (1988), in class II antigens as well. Indeed, due to the periodicity of alpha-helical structures, certain positions in these segments are easily accessible, while others (i.e. approximately every two other residues) are buried at the helix-sheet interface. Maay positions are highly conserved and very few are actually variable, as cot ld be anticipated, since the HLA-A2 crystal structure apparently displays cnly 2-4 polymorphic residues (62, 65, 66, 163) pointing up from the alpha helices in a position to interact with TcR. Certain residues (in or out of tht,se four segments) that point into the groove are nevertheless accessible to a k:rge probe (66, 152, 156), which raises the possibility that certain MHC residues Darticivate both in vevtide bindin~ and TcR contacts. The uncertainties inLerent i n o u r approach are Such tha'[ we do not think it useful to discuss the results of the calculations in more details.
Proposed molecular nature of the composite epitope. Based upon the above considerations, we propose that the epitopes seen by the TcR usually consist of two moieties: (1) side chain(s) from at least some of the four short alpha-helical segments (56-60, 73-77, 149-153, 158-162); and (2)side chain(s) of the foceign MHC-bound peptide. The MHC molecule/foreign peptide antigenicity is thus viewed as a special case of protein antigenicity, retaining the key attributes of noncontiguity and exceptional surface exposure of the epitopes of soluble proteins° As already mentioned, many of the residues belonging to these four helical segments are poorly or not at all polymorphic, emphasizing the possible role of non-polymorphic residues in making contacts with TcR. Whether all the presumptive contact regions are simultaneously recognized by any given TcR is an open question at this stage.
M O L E C U L A R N A T U R E OF A~'HC A N T I G E N I C ELEIffz~N1 ;b" i49 In pursuing the analogy between recognition of antibodies and TcR, we now make use of the finding that, although antibodies bind antigen over a relatively large surface, the energy of binding may well result from a much more limited area. Thus, in the lysozyme-antibody complexes, the contact area is on the order of 8 nm 2. However, energy calculations suggest that the energetically defined epitope is less than 3 nm 2 in surface and cortsists of side chains (and occasionally backbones) of no more than 2 to 4 resit~.ues (Novotny et al., 1989). On the antibody side, the most important re~ion in terms of binding energy, is the part of the ar, tigen-binding site where residues coded by D and J gene segments significantly contribute (Novotny et al.~ 1989). Under the assumption that these f'mdings (thus far valid for the three antigen-antibody complexes of known crystal structures) are general, their transposition to T-cell recognition has the following important implication: in the composite MHCpeptide epitope, we anticipate that the energetically relevant epitope will involve just a few side chains from the peptide and occasionally from the MHC molecule (usually involving MHC residues homologous t~3 those listed in table I). Accordingly, in the recognition of the composite epitope by TcR, we propose to distinguish between ~ active >>and ~ passive >>recognition. ~ Active >> recognition is limited to the relatively few non-covalent bor.ds which contribute significantly towards the energy of binding• ~ Passive >>recognition refers to the compatibility of surface which can establish contacts without major energetic contribution to binding. The bound peptide must participate in active recognition. Whether or not the MHC molecule also participates in active recognition in a systematic or non-systematic fashion is an open question. It is relevant to the problem of positive selection in the thymus (Singer et al., 1QR~ • Mftrrflelr
~nrl
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will not be discussed here.
TABLE I. - - Amino acid sequence conservation in the four most prominently exposed segments of HLA-A2 alpha-hdix 1 and 2.
Residue number 58 59 60 73 76 77
Alpha-helix- 1 Amino acid
Residue number
GLU, Gly, Arg TYR TRP Variable VAL, Glu Variable
150 15i 152 159 150 t62
Predominating residues are given in boldfa,ze.
Alpha-helix-2 Amino acid ALA GLY, His, Arg Variable TYR LEU GLY, Ala
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J. N O V O T N Y
ET AL.
Recognition by TcR. As reviewed by Bougueleret and Claverie (1987), Davies and Bjorkman (1988) and Claverie et al. (1989), most TcR polypeptide chain variability is accounted for by junctional variability between the J, D and V gene segments. Most of the antigen-specific diversity of TcR molecules is therefore expected to be located around the bottom part of its binding-site cavity, where amino acids created by the gene junctional events reside. Davis and Bjorkman (1988) further speculated that <
i. n t. e r. a e. t i.n n. w i t ~,~,~ h t h p 1 V l t - I ~ Le.~~ v,~a, jn[ ~~, x ,x n~ , ~~ , , ~v l,, .aL~l ~ ;. ~o, , nl L *l l ,oO !;~*o,4 J d I . O I b ~ , ~ ;,~ ~ l / L ,L~ ,gk ~ , llI .J l~ ~
T l i e T.,~ l i l J [ ÷k..+ Lllg:;l[,L
case, weak correlations of such sequence variants with TcR gene subgroups should be expected (cf., e.g., Matis et al., 1987). 3) In analogy to antibodies, we expect the bottom part of the T-cell receptor binding cavity to largely determine specificity of binding, in the energetic sense. This would agree with the fact that TcR variability is concentrated predominantly in the third hypervariable regions of its polypeptide chains (Bougueleret and Claverie, 1987). Davis and Bjorkman (1988) ascribe a different functional importance to sequence hypervariability found in the V-D-J junctional sections of T-cell receptor genes. They suggest that the increased variability of these segments reflects preferential association with highly variable sequences of foreign peptides. This variability pattern is to be contrasted with that seen in, for example, mouse immunoglobulin sequences, where the variability is more evenly distributed among the six hypervariable (CDR) loops. The genetic mechanisms by which variability is created may, however, have little bearing on geometric details of macromolecular recognition. For example, the chicken genome is known to contain only one immunoglobulin VL gene and is believed to have only a few VH chain genes, i.e. significantly less than the number of mouse VH genes (Reynaud et ai.,
M O L E C U L A R N A T U R E OF M H C A N T I G E N I C E L E M E N T S
151
1985). The chicken VL variability repertoire is generated in ontogenesis by putative cross-over events in which 25 pseudogenes take part (Reynaud et al., 1987). This genetic mechanism and the resulting variability pattern are distinctly different from those of mouse or human VL polypeptide chains, yet there is no reason to believe that the antigenic repertoire encountered by chickens is significantly different from that confronted by mice and men. In summary, we view the MHC peptide/TcR interaction as primarily focussed on a small area involving, on one hand, the bottom cavity of TcR and, on the other, a few peptide residues and, occasionally or systematically, a few residues of the MHC molecule. In addition to this so-called ((active>~ recognition, surface compatibility over a larger area would be necessary in what we have termed ((passive >>recognition. MHC residues in the areas which, by calculation, we usually anticipate ~o be relevant in (
ANNEX
Alpha-carbon coordinates of the human HLA-A2 extracellular domains were obtained from the Brookhaven Protein Data Bank (Bernstein et al., 1977). The complete backbone, i.e. the missing coordinates of the N, C and O atoms, were reconstructed by adapting CONGEN, a conformational search program (Bruccoleri and Karplus, 1987) to find peptide conformations whose alpha~arhnn~ hegt fit the erv~f~]|nor~nhlo d~t~ ~v~il~hl~ ]~nr nr~vlnll~ ~nn|ie~flnn~ J ~ , . , , ~ ,~/. /~ ,/.,./..~n~...~.. ~..~ ~
CONGEN has been used to find conformations for loops where each anchor point (i.e. N- and C-terminus) was known. This was done by first sampling phi and psi torsion angles in the backbones of the first n-3 residues, where n is the number of residues in the loop, and then using the modified G5 and Scheraga chain closure algorithm (G5 and Scheraga, 1979; Bruccoleri and Karplus, 1985) to complete the loop. However, here we used just the backbone sampling to thread the peptide backbone through the points specified by the crystallographic alpha-carbon positions. In order to perform this search efficiently, it was necessary to modify the search algorithm. Without the chain closure criterion, a complete sampling for even a small number of backbone residues would require prohibitive amounts of computer time. However, by using a different method of searching the tree of conformations produced by CONGEN, a so-called directed search, we can generate good confrontations early in the search process and can thus truncate the search and greatly reduce computer time requirements. To understand the modifications in the search algorithm, we must envision the conformational search as a tree (Bruccoleri and Karplus, 1987). The rcat of the tree represents the molecule at the beginning of the search pro-
152
J. N O V O T N Y
ET AL.
tess; each of the leaves of the tree represents the final conformations and the intermediate nodes represent partially constructed conformations. The connection between nodes represents a selection of torsion angles for a particular amino acid residue in ~he backbone. The search process begins with just the root and samples the backbone torsions for the first residue, i.e. expansion of the root node. A number of nodes is generated, each of which can be expanded to yield nodes which represent conformations that have two residues constructed. These expansions contribute u n il the leaves (final conformations) are reached. In the normal CONGEN search, the expansion process proceeds depth first, namely, the first node generated by an expansion is selected for the next expansion, and so on until the leaf node is generated and the next node at the lowest level is expanded, etc. This method is acceptable for a complete search of the tree. However, in a directed search, the node chosen for the next expansion is always the one which has the ~ best >> conformation, where ~ best>> in this case means smallest r.m.s, deviation to the alpha-carbon coordinates. Generally, t~s directed expansion of nodes will result in the lowest r.m.s. deviation conformations being generated first. However, because of errors inherent in the crystallographic data and because of the discrete sampling used, it is possible for the directed search to stall while it explores conformations that do not yield high quality conformations. Thus, we used a protocol whereby we constructed 10 residues at a time, and if no results were generated within two hours of CPU time, we reduced the length of the constructed segments to 5. The specification of the phi and psi backbone angles provides the information necessary to construct the beta carbon positions, since the direction of the C-alpha-C-beta bonds is uniquely determined by the locations of the uu:~l ~-eupnet ~uosutucnLs, i.e. the N and t: atoms. Side chain atoms beyond the beta carbons were constructed by CONGEN as described before (Novotny et al., 1988). We expected side chain orientations to be biased by occasional errors, as observed before ill CONGEN-reconstructed side chains of hemerythrin and immunoglobulin VL domain (Novotny et al., 1988). Charged residues and aromatic rings were most often misplaced in the CONGEN side chain placement protocol that used the CHARMM in vacuo potential energy function (Brooks et al., 1983), as was used in this work. .-~L.~--
P
--1--1.
. . . .
L--J.-'~-
. . . .
--
The final model was energy-minimized with harmonic constraints of 20 kcal (83.6 k J) applied to the positions of alpha-carbons. The r.m.s, shift between t.~e crystallographic C-alpha coordinates atoms and those of the final, energyminimi~.d" model, was 0.9 ,~ for the heavy chain and 1.5 .~ for the beta-2-microglobulin. Large probe accessibility was computed as described before, using the accessibi;;.ty algorithm of Lee and Richards (1971) as modified by R.E. Bruccoleri (Novotny et al., 1986b) and the probe radius of 10 ~ . To minimize the influence of side chain placement errors in the HLA-A2 model upon antigenicity analysis, large probe accessibility was also computed on the naked
153
M O L E C U L A R N A T U R E OF M H C A N T I G E N I C E L E M E N T S
backbone. The rationale for this approach is based on the observation of Thornton et al. (1986) that antigenicity (protrusiveness) of peptide segments in proteins is essentially determined by the shape of the backbone. The contact surface of the HLA-A2 extraceUular domains was calculated using the spherical probe of 1-nm radius (numerical results are available upon request) and smoothed over 7-residue segments as described before (Novotny et al., 1986b). Contact surface profiles of the complete HLA-A2 alphachain model and its virtual C-alplla backbone are shown in figure 1. It can be seen that the essential features of both curves coincide, although some differences are apparent (e.g. the mutual shifts of maxima around positions 75 and 147). We feel that the C-alpha-generated profile is more reliable, and the above discussion makes use of it.
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FIG. 1. - - Large probe accessibility profile o f the alpha chain o f the human class I M H C antigen allele HLA-A2. Heavy line: data obtained with alpha-carbon coordinates only; light line: data obtained with the whole molecular model, constructed as described in the ¢ Annex,. The asterisks denote locations of residues recognized by specific monoclonal antibodies on various human and mouse class I alleles (including the HLA-A2), as determined by Parham's group ana aescnbed by Bjorkman et al. (1987b). Results of Abastado et al. (1987), Hogan et al. (1988), Toubert et al. (1988), Sire et ai. (1988) and Santos-Agaudo et al. (1988) have also been included.
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FIG. 2B (bottom). - - A stereoscopic alpha-carbon tracing of backbone of alpha-I and alpha-2 domains from the HLA-A2 molecule. Location of prominent calculated epitopes (cf. fig. 1) close to interhelical groove (putative foreign peptide binding site of Bjorkman et aL, 1987b) is emphasized by heavy lines.
FIG. 2A (top). - - A color-coded computer-graphics representation of polypeptMe backbone of HLA-A2 alpha-1 and alpha-2 domains (see ¢~Annex)> f~r details o f backbone construction). The four putative ~ antigenic )) segments 56-60, 73-77, 149-153 and 158-162 (cf. fig. 1) are rendered in green.
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I ~ O L E C U L A R N A T U R E OF M H C A N T I G E N I C E L E M E N T S
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As expected, positions of the major contact surface maxima correspond to locations of bends and turns of the polypeptide chain between adjacent beta-strands or between strands and helices (peaks around positions 20, 43, 90, 110, 130, 140 and 180). These, however, are relatively distant from the (ttop>> surface of the extracellular domains created by the two long helical runs and the foreign peptide-binding groove. More interestingly, irregular breaks pcesent in the helical runs in the alpha-1 domain (50-84) as well as the alpha-2 domain (138-180) stand up as major protruding parts of the surface. The surface maxima centered on residues 58 and 151 rank among the most prominent features of the whole profile. The other two maxima, centered on residues 76 and 159, are less pronounced. Figure 2 shows the alpha-1 alpha-2 domain dimer with the segments 56-60, 73-77, 149-153 and 158-162 emphasized. A comparison between calculated epitopes and class I MHC residues identified as important in the recognition by a variety of w.onoclonal antibodies can be seen in figure 1. The correlation is reasonably good but far from complete. However, there is a bias in the comparison, because the serological experiments have only characterized polymorphic residues; virtually nothing is known about the participation oi the non-polymorphic ones in epitopes recognized by antibodies. The experimental evidence dealing with the identification of MHC residues possibly involved in making contacts with TcR is relatively complex and its interpretation is difficult (reviewed in Kourilsky and Claverie, 1989a). Table I shows the 12 positions which are accessible in the four segments that are most exposed to the large probe, and indicates the variability at each position.
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MOLECULAR NATURE OF MHC ANTIGENIC ELEMENTS
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