Clusters of Structurally Similar MHC I HLA-A2 ... - ACS Publications

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Clusters of Structurally Similar MHC I HLA-A2 Molecules, Found with a New Method, Suggest Mechanisms of T‑Cell Receptor Avidity Alexander A. Rashin*,‡,∥ and Robert L. Jernigan∥ ‡

BioChemComp Inc, 543 Sagamore Avenue, Teaneck, New Jersey 07666, United States LH Baker Center for Bioinformatics and Department of Biochemistry, Biophysics and Molecular Biology, 112 Office and Lab Building, Iowa State University, Ames, Iowa 50011-3020, United States

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S Supporting Information *

ABSTRACT: Only α1 and α2 domains of the α-chain of the major histocompatibility complex (MHC) directly bind peptide antigens (Ag-s) and the T-cell receptor (TCR). Significant plasticity was found in the TCR but only minor in (α1 + α2). The α3-domain position variation was noted only in connection to its binding the coreceptor CD8. We apply our methods for identifying functional conformational changes in proteins to a systematic study of similarities between 43 X-ray structures of the entire α chains of MHC-I HLA-A2. Out of 903 different αHLA−A2 pairs 203 show similarities within the earlier determined uncertainty threshold and unexpectedly form three similarity clusters (SCs) with all/most structures in a cluster similar within the uncertainty threshold. Pairs from different SCs always differ above the threshold, mainly due to variations in the α3 position/structure. All structures in SC3 cannot bind the CD8 coreceptor. Strong hydrogen bonds between (α1 + α2) and α3 differ between SC1 and SC2 but are nearly invariant within each SC. Small conformational changes in the (α1 + α2), caused by Ag-s differences, act as an α3 “allosteric switch” between SC2 and SC1. Binding of CD8 to SC2-HLA-A2 (Tax-type Ag-s) changes it to SC1-HLA-A2 (HuD-type Ag-s). HuD binding to HLA-A2 is much less stable than Tax binding. CD8-liganded HLA-A2 preference for binding HuD suggests that CD8-HLA-A2 may present a weakly binding peptide for TCR recognition, supporting the hypothesis that CD8 increases TCR avidity to weak Ag-s. Other HLA-A2 functions may involve α3. TCR-A6liganded-Tax-type-HLA-A2s form two small clusters, similar to either A6-liganded-HuD or A6-liganded-native-Tax HLA-A2s. β-strands of the α1 and α2 domains form a platform supporting antiparallel helices from both domains, separated by a cleft. An 8−10 residue peptide antigen can bind in the cleft and interacts extensively with the β-sheet floor and the helices of the α1 and α2 domains. The β2m chain stabilizes the α1 and α2 domains of the α-chain and interacts with a short piece of the highly conserved α3 domain.1,2,11 There are different alleles of class I MHC (A, B, C, etc.). In Figure 2 are shown the sequence and secondary structure of a human MHC-I A2 (also denoted as HLA-A2)2 for the PDB9 structure 3qfd (such representations (9) for other HLA-A2 structures differ only in rather minor, while sometimes significant, details). It has been found that T-cell receptors (TCR) can recognize multiple polymer-antigens (Ag-s) bound to MHC-I in varying orientations and trigger the immune response.1,2,11 This has led to widely discussed suggestions of “plasticity of TCRs” and of roles of the structures of Ag-s in binding to TCR (refs 1, 2, 12, and 13 and references therein). Conformational variations in

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ell−cell interactions of the adaptive immune response are critically important in protection from pathogens.1−3 The primary components of these interactions, widely studied by protein crystallography,4−7 are the T-cell antigen receptor (TCR), the major histocompatibility complex (MHC) molecule, and T-cell coreceptors CD8 or CD4.3−6 The major function of the TCR is to recognize noncovalently bound antigen within the correct context of the MHC and to initiate transmission of an excitatory signal to the interior of the cell. There are many other factors involved in the transmission of this signal.1−3,8 Class I MHC molecules consists of α- and β-chains. The αchain is usually described as having three domains α1, α2, and α3, comprised of about 90 residues each1,2 (SCOP identifies only two domains: the first one includes α1 and α2, and the second coincides with α3).9,10 Structure 2git9 of the MHC-I molecule is schematically represented as an example in Figure 1 below. The β-chain of about 100 residues, β2-microglobulin, has a well conserved structure in all MHC-I, and its interactions with the α-chain stabilize the water-soluble part of MHC-I. X-ray crystallographic studies of MHC-I show that eight antiparallel © 2015 American Chemical Society

Received: May 22, 2015 Revised: November 23, 2015 Published: November 24, 2015 167

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contrast, β2m (chain) interacts extensively with the underside of the peptide-binding α1 + α2 subdomain. Its position relative to the peptide-binding region is correspondingly well conserved.”4 It has been found (ref 14 and references therein) from comparisons of 13 isolated HLA-A2-peptide complexes that “the α3 domain can vary in its position relative to the rest of the molecule in a fan-like continuum of possible positions covering 3.5°.” It has been also found that binding of the coreceptor CD8αα to the α3 domain changes the domain orientation.14,15 A similar conclusion was suggested in later studies of the murine CD8 binding to the corresponding MHCs.16,17 It was reported that binding of CD8 to the TCR-HLA-A2 complex increases T-cells’ sensitivity to antigen-presenting molecules by about 100-fold1 broadening the spectrum of weak antigens but still triggering the immune response and thus contributing to the T-cell cross-reactivity and promiscuity.18,19 CD8 brings into a proper position the tyrosine kinase Lck which starts a chain of phosphorylation events initiating immunological signal transduction.1 CD8 binds to the α2 and α3 domains of HLA-A2. Some substitutions in the α3 domain cause reduced interaction with CD8.20,21 The role of CD8 in the immune response is still debated. 17−19,22,23 Some importance of CD8 binding to α3 domain is underscored by clinical findings, e.g., (1) the inability of α3 to bind CD8 plays a central role in reactivation of β-herpes virus in immunocompromised patients (while β-herpes is kept in check by a healthy immune system in 60% of all population), and (2) HIV disease progression correlates with the generation of dysfunc̈ T-cells with low cell-surface expression of CD8.24,25 tional naive Before 2013 there were no systematic reports on the flexibility of the HLA-A226a class of molecules critically important for the function of the immune system.1,2 The flexibility of HLA-A2 attracted renewed attention when it was found that HLA-A2 complexed with Tel1p peptide undergoes a significant change upon recognition by A6 TCR.12 It had been called12 “unexpected”, and it had been concluded that “The differences between the Tax and Tel1p ternary complexes could not be predicted from the free peptide-MHC structures and are

Figure 1. Main chain structure of HLA-A2 (PDB file 2git). There are no visible (in color or wire thickness) differences between α1 and α2 “domains” except the cleft between their helices (as mentioned in the text, α1 and α2 are parts of a single “structural domain”, localized, e.g., by SCOP10). This and other wire drawings of HLA-A2 structure images were produced by a combination of visualization programs from PDB9 and PyMOL.33

HLA-A2 antigen-binding α1 + α2 fragment with bound different Ag-s were reported to be only minor,1,2,4,11−13 but between some molecules Cα RMS difference for the fragment 144−151 reaches up to 1 Å.11 It was stated in 1995 that “Among the class I MHC molecules, the α3 domain is itself somewhat variable relative to the rest of the structure. This variation is not, however, likely to reflect differences imposed by different peptide ligands; instead it appears to be a result of different crystal packing and a weak coupling of the α3 domain to the remainder of the protein. In

Figure 2. Sequence and secondary structure representation of HLA-A2 3qfd, taken from the PDB,9 (2012). Arrows denote β-strands, waves are helices (important single turn helices are in gray), and small horseshoes denote turns. Note that sequence numbering in our results is decreased by 3 (compared to this figure) because we excluded the first three residues from all comparisons. 168

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The significance of the findings of widespread flexibility of various extends in HLA-A2 structures, of their clustering, questions raised, and directions of expansion of their studies are discussed in the last section of the paper.

inconsistent with a traditional induced-fit mechanism...” It had been suggested that the differences in flexibility contribute an entropic component to the free energy of the binding recognition between the TCR and the peptide-MHC structures.12 A 2013 paper26 systematically “examined how the geometry of the HLA-A2 α1 and α2 helices varied with the peptide in 51 peptide-HLA-A2 X-ray structures.” The variation was described as histograms of the average bending angles of the α1 and α2 helices and of the width of the peptide binding groove. The results indicated small but significant peptide-dependent variations. The focus of the paper26 was on proving by hydrogen−deuterium exchange and fluorescence methods that “tuning of the flexibility of the MHC proteins by different peptides is a general phenomenon not restricted to only certain peptides or to a single region of the protein.” We recently developed a novel approach for locating and quantifying the functionally meaningful main chain coordinate differences between different X-ray structures of the same molecule.27,28 Our approach (see Methods below) is based upon using the distance difference matrix, DDM, calculated from it root mean square distance difference (RMSDD), and a simple algorithm for the pairwise fitting of the same fragment from two different structures of the same molecule (see ref 27 and Methods below). We found that for a coordinate difference between two structures of the same protein to be meaningful (as opposed to being caused by unquantifiable factors often including crystal contacts) the RMSDD should be larger than a threshold of 0.45 Å (see Methods).27,28 In spring of 2012 we applied our methods27,28 to 37 entire heavy chain HLA-A2 X-ray structures (incorporated into this report). We found significant and varied structural differences beyond the uncertainty threshold, usually considered28−30 functionally important. In this report we present results of our study of similarities and dissimilarities comparing all possible pairs of 43 high resolution X-ray structures of human HLA-A2 including α1, α2, and α3 domains. We found that out of 903 pairs only 203 show similarities within the uncertainty threshold. These 203 pairs unexpectedly form clusters in which all or most of X-ray structures in a cluster are similar to each other within the uncertainty threshold. Structures from different clusters always show RMSDD conformational differences of various magnitude above the uncertainty threshold. We found three such “similarity” clusters and a few structures with RMSDD differences above the uncertainty threshold from practically all other structures. The majority of all conformational differences between pairs of structures with RMSDD differences beyond the uncertainty threshold are due to rigid body movements of various sets of fragments with sizes from three residues to full domains. Most molecules in each cluster share certain common characteristics, but some of such characteristics can be found in other clusters as well. We also reanalyzed earlier described12−14 changes in HLA-A2 upon its binding to CD8 and A6 TCR and found two new similarity clusters among nine A6-liganded Tax-type-HLA-A2s. The types of clustered structural differences, found and described here, may play important roles in the immune response mechanisms, and we indicate some possibilities suggested by our results. We limit ourselves to the crystallographic evidence of the conformational differences and, when possible, find factors that lead to such differences in MHC unbound or bound to TCR or CD8.

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MATERIALS AND METHODS Human Structures Studied. The total number of high resolution structures, 43, was dictated by the maximum size of the tables of readable results. An increase in this number would make this first report too long and difficult to follow. An initial 37 structures were arbitrarily chosen from the literature and additional 6 structures were added from two publications12,26 that reported some differences between HLA-A2 structures. Forty-three studied structures have varying bound native or mimic peptides 8−10 residues long and are solved with resolution from 1.3 to 2 Å (except 1ixa12 with resolution 2.1 Å). The structures are from the crystals with three different symmetries and with one or two HLA-A2 molecules in the unit cell. The PDB9 names of all studied structures and some of their characteristics are given in the tables in the Results and Discussion and in the Supporting Information (SI). For structures with two molecules in the asymmetric unit these two molecules (denoted A and D) from the same structure were compared; however, only molecules denoted A in PDB9 were used in all comparisons of different HLA-A2 structures. Five HLA-A2 structures, 1hhg, 1hhh, 1hhi, 1hhj, 1hhk,11 with lower resolution (2.5−3 Å) not included in 43 high resolution structures lists, were used to check the results for high resolution structures. Twelve structures liganded by TCR A6 or CD8 have a lower resolution (shown in the parentheses): 3pwp (2.69 Å), 3h9s (2.7 Å), 1ao7 (2.6 Å), 1qsf (2.8 Å), 1qse (2.8 Å), 4ftv (2.74 Å), 2gj6 (2.65 Å), 3qfj (2.29 Å), 3d39 (2.81 Å), 3d3v (2.80 Å), 1qrn (2.8 Å), liganded by TCR, and one, 1akj (2.65 Å), liganded by CD8, are considered in separate sections of Results and of Supporting Information (SI). Distance Difference Matrices (DDM). For a protein of N residues the distance matrix (DM) is a square N × N matrix, in which element ij represents the distance between residues i and j (or their Cα atoms as used here). Because the distances i to j and j to i are equal, only half of the DM matrix is presented.27 For two different conformations of the same protein a distance difference matrix, DDM, is constructed as a N × N matrix of differences (DDs) between the corresponding elements of the two DMs.27 We follow our previous work27,28 representing DDMs in three shades (black, gray, and white) based on the ranges of the absolute DD valuesthose below 0.5 Å, between 0.5 and 1 Å, and above 1 Å. Each DDM is characterized by the RMS of all its DDs, denoted as RMSDD, and with the percentage of DD values lying outside the range −1 Å to 1 Å, denoted as Δ. If two pairs of compared structures of the same molecule have identical RMSDD but one of the pairs has the largest differences localized in a smaller area of the DDM, then the other pair would have a larger Δ. We found that such additional controlling parameter was useful27,28 for identifying some reported functional structural changes in enzymes (see below). RMSD vs RMSDD. The similarity between two X-ray structures of the same protein is usually characterized by the root-mean-square difference (RMSD) calculated as the square root of the mean of squares of all distances between the corresponding Cα atoms from these two structures which first have to be somehow fit to one another in 3D. Such fitting, 169

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Fitting of “Rigid” Fragments. For the fitting of “rigid” or “nearly rigid” fragments, we use the fast and accurate algorithm described in our previous paper,27 which uses a superposition of three noncollinear points of a rigid body to superimpose all points of this body, together with quaternion rotations.27 Such a superposition can be represented as an initial superposition of the centers of mass of the fragments (represented by Cα atoms) followed by two rotations around the axes passing through the center of mass and “nearly”27 superimposing two Cα atoms from each fragment. A few residues at proteins’ termini or next to crystallographically unresolved fragments are sometimes excluded from the fitting. Fragments of no fewer than three residues are used. Sequence of Fitting Steps and Evaluation of the Result. In the first step the largest rigid fragment of the second molecule is fit to its coordinates in the first (reference) molecule, and the resultant transformation is applied to coordinates of the entire second molecule (this does not change the RMSDD; however, parameters of all subsequent rigid body movements depend on the choice of the first transformation). Coordinates of bound substrate (or cofactor) were not included in these calculations.27,28 (However, in this work we sometimes modified the PDB file to represent HLAA2+peptide as one chain included in the calculations.) In the following fitting steps the structures of all fragments of the entire sequence of the second molecule should be fit to the structure of the corresponding fragments of the first molecule to verify whether the “movement” is a result of a series of rigid body movements of protein fragments. (Note that some fragments of both structures might fit well after the initial first transformation and might not require any additional individual fitting.) The particular order of fitting steps is arbitrary. For details see our previous paper.27 Examples of some transformation sequences and outputs are provided in the Supporting Information (Section 3.5). Identifying Hydrogen Bonds Between: (a) α and β Subunits, (b) α1 + α2 and α3 Domains. Hydrogen bonds are identified with the module NCONT, version 6.1, of the suite CCP4.31 Explanations of inputs, hydrogen bonds cutoffs,32 and examples of calculated contacts outputs are given in SI4. Fragment 1−181, used in hydrogen bonds search, is location of the (α1 + α2) domain given by SCOP10 as quoted in PDB.9 A commonly used cutoff for the length of a hydrogen bond is 3.4 Å between heavy atoms. However, any sharp cutoff for the presence or absence of a hydrogen bond is necessarily arbitrary,32 and we examined hydrogen bond for cutoffs of 3.0, 3.2, and 3.4 Å. Hydrogen bonds were not searched for in the structures of HLA-A2 liganded by TCR or CD8. These structures are solved at lower resolution and often do not show in their PDB9 files a short helix 225−228 important14 for CD8 binding. Considering the Roles of Crystal Symmetry, Cell Dimension, and Contacts in SCs Formation. Possible influences of interactions in a crystal are often invoked when conformational differences between two structures of the same molecule are difficult to explain. It is possible that in some cases there are a few possible packing arrangements in a crystal allowing accommodation of the molecules of interest and thus dictating their conformations. There is, however, an alternative possibility with a few internally preferred conformations of a given set of molecules, which dictate crystal symmetries and cell dimensions.

however, depends on the method used27 and thus might introduce poorly controlled uncertainty. The alternative characteristic of similarity between two structures of the same molecule is the RMSD between all pairs of internal Ciα−Cjα distances in two molecules (RMSDD), which does not require a preliminary 3D fitting of two structures27 and thus imparts a more objective character to the results of the comparison. To calculate RMSDD, one first builds the distance difference matrix, DDM, as described in the section above, and then takes a square root of the mean of squares of all elements of the DDM. Estimation of Positional Uncertainties. We previously27 determined the range of coordinate uncertainties by calculating and analyzing the DDMs of 1014 pairs of structures (at about room temperature) of bovine ribonuclease A, and whale myoglobin for which the authors did not report any significant conformational changes (“movements”). Each pair was characterized by its DDM, RMSDD and Δ (see above). All of these 1,014 pairs of structures had the RMSDD ≤ 0.44 Å and Δ ≤ 4.05%. However, there are cases in the literature of functionally induced coordinate differences with an RMSDD of 0.45 Å and a Δ of 5% or greater. The values of the RMSDDs and Δs listed above led us to suggest the criteria that a DDM indicates no significant “motion” but only “coordinate uncertainty” when the RMSDD is below 0.46 Å and its Δ is less than 5%.27 Further studies might change these criteria somewhat. In the present study RMSDD ≤ 0.45 Å is always accompanied by Δ < 5% and therefore Δ is not invoked. For further details and discussion see our previous paper.27 Locating Subsets of Similar Structures. First, pick up any pair of structures X1Y1 in which HLA-A2 structures X1 and X2 are similar, i.e., have RMSDD below the uncertainty threshold. This can become the first pair in an SC. Then add to it all pairs of similar structures (RMSDD below uncertainty threshold) of the type X1Z1 and Z2Y1, where X1 and Y1 are the same HLA-A2 structures as in X1Y1. Continue adding pairs of the type Z1Q1 and Z2Q2 (order of structures in a pair does not matter) “overlapping” with the pairs in the subset until no more “overlapping” pairs of similar structures can be found in the total set of pairs of HLA-A2 structures. After no more similar pairs of structures can be found to add to this subset, we discuss whether to exclude from this subset structures which have in it three or less similar pairs (see Results and Discussion below). Then start to build a next similarity cluster (SC) from the HLAA2s remaining in the total set of HLA-A2 pairs. Choice of “Reference” DDMs. There is a wide variety of visual differences among the DDMs of HLA-A2 pairs with RMSDD values above the uncertainty threshold. From these we have selected 16 “reference” DDMs shown in the Results and used for the presentation of results. Visual variations in DDMs corresponding to each “reference” DDM are illustrated in the Supporting Information. Location of “Rigid” Fragments and Blocks. All DDs within a continuous fragment of the protein form a right triangle along the DDM’s diagonal corresponding to this fragment. If this fragment’s conformations in two molecules differ only within the coordinate uncertainty limits, then the right triangle corresponding to this fragment should be almost entirely black containing only minimal white or gray areas. Such a fragment usually is found to change its position in a conformational transition as a rigid body. 170

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Empty cells in the upper table’s triangle indicate pairs with the RMSDD above the threshold. RMSDD of 0.45 are below threshold here because Δ-s are below 5%

a

with P21 symmetry; (d) we verified with Pymol33 and Chimera34 whether there are any clashes between the peptide Ag-s of the substituted molecules in the crystal’s central unit cell and molecules in the crystal cells surrounding the modified central unit cell, which could force the crystal symmetry and cell parameters change (see more in Results and Discussion). Fourth, we used lower resolution (2.5−3 Å) structures,11 not included in the high resolution structures lists, to check the results obtained for SCs with high resolution structures. Graphics. All of the protein images were produced by a combination of graphics programs in the PDB,9 PyMOL,33 and Chimera.34

For the case of clusters of similar structures (SCs, see above) we focus on verifications of this alternative possibility. Table 2 (in the Results) provides cell dimensions of representative structures from SCs. First, by comparing DDMs, we checked whether all structures from a SC with two different crystal symmetries differ in the similar way from representative structures from a SC with a single symmetry. Second, we verified whether all similar structures from the SC with two crystal symmetries can be transformed to the representative structures from the SC with a single symmetry by practically the same conformational transformation using our DDM/RMSDD method (see above and refs 27 and 28). Third, for two HLA-A2 from the same SC but forming crystals with different symmetries we checked a possibility of a particular pressure of the crystal lattice with one symmetry on the embedded single HLA-A2 molecule with coordinates extracted from the crystal structure with a different symmetry but from the same SC. This check included four steps: (a) building a crystal lattice from the coordinates of a molecule from SC2 with crystal symmetry P1; (b) fitting the coordinates of another molecule from SC2 but extracted from a crystal with symmetry P21, including the peptide-Ag-s in the corresponding structures, to the molecule in the built crystal with P1 symmetry; (c) we substituted two molecules in the central unit cell of the crystal with P1 symmetry (with attached Ag-s) by the fitted molecules (with Ag-s attached) from the structure

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RESULTS AND DISCUSSION Initially we calculated RMSDD and DDMs of A−D (PDB notation) α-chains for the structures with two molecules in the unit cell and for all 903 different pairs of α-chains A from all 43 HLA-A2 structures. Then from these 903 pairs we selected a subset of 203 pairs such that in each individual pair the structures were similar (with RMSDD below the uncertainty threshold). 3.1. Presentation of Results for “Similar” Pairs of Structures. In Table 1 we have ordered the similar structures so that filled cells (corresponding to pairs with RMSDDs below the uncertainty threshold) form triangles as full as possible. Each such triangle we tentatively consider as a “cluster of similar structures” (SC). Note that the number of empty cells is 171

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Table 2. Human MHC Class I (HLA-A2), Structure Characteristics and RMSDDs for Two Molecdes from Same Cella

PDB names in the first column are in the same order (shown in the second column) as in Table 1; the third column shows the number of residues given in “molecular description” in PDB; the fourth column shows the number of residues in the α-chain of PDB structural file; the fifth column shows Ag’s name and any mutation in it relative to the wild type; the sixth column gives peptide-Ag’s sequence; the seventh column shows the crystal space group; the eighth, polypeptide chains in the unit cell; the ninth, crystal unit cell dimensions; the 10th, crystallographic resolution of the structure; the 11th, residue mutation in the α-chain; the last column shows RMSDD (in Å) between two identical α-chains in the same unit cell. b Unusual residues: βA−β-alanine; 3Az−3-(aminomethyl)-benzoic acid; Tig−N-(2-aminoethyl)-L-tryptophan; Gic−N-(2-aminoethyl-N-(1H-indol-3YLacetyl)glycine; X−(2R)-amino(2-nitrophenyl)-ethanoic acid; Z−(3S)-3-amino-3-(2-nitrophenyl)-propanoic acid; Sm−selenomethionine; Ps− phosphoserine; Cr−citrullin. a

3.5 times larger than the number of filled cells. Also, liganding by TCR A6 can change structures of two HLA-A2 molecules from “similar” (with RMSDD below the uncertainty threshold) to significantly different (see section 3.9 below). Thus, the clustering might reflect some yet unclarified role of HLA-A2 conformational differences in the immune specificity. The leftmost similarity cluster (referred to below as SC1) is a perfect triangle with all cells filled. The next two clusters (SC2 and SC3) to the right of SC1 are less perfect, having empty cells corresponding to “significant” coordinate differences above

the uncertainty threshold. The rightmost three or four (see below) structures do not seem to belong to any cluster. 3.2. Characteristics of Structures in “Similarity Clusters”. These characteristics given in the PDB9 are tabulated below in Table 2 in the same order of structures as in Table 1. All structures, 1−13, in the cluster SC1 have symmetry 21, two molecules in the unit cells, no mutations in the protein, and RMSDDs between two molecules in the unit cell significantly below the uncertainty threshold. The variation in the peptide−antigen lengths from 8 to 10 apparently does not play an important role. 172

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Figure 3. continued

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Figure 3. continued

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Figure 3. (a−p) Representative distance difference matrices (DDMs). Notations: Short thick bars or segments of thin double lines along the tops and sides of the triangular matrices denote positions of α-helices or β-strands (as given in the PDB file). Tick marks on the top and left are placed at intervals of 20 residues; mostly black triangular areas mean that DDs in them are below 0.5 Å and these areas represent likely rigid blocks in the conformational transitions between the two molecules being compared; the gray areas show DDs between 0.5 and 1 Å; white spaces means that the absolute values of the distance differences (DDs) between the corresponding pair of Cα-atoms in the two structures (e.g., PDB entries) are above 1 Å. Large letters (A to BC′) in bold in DDMs are used to identify their types in Table 3.

Twelve structures in the “similarity cluster” SC2 all have two molecules (denoted as A and D) in a unit cell.9 Ten of the A− D SC2 pairs have RMSDDs within the uncertainty threshold. When A and D molecules from the same cell are compared for 1duz and 2x70 (for DDMs see SI1) A−D pairs have RMSDD slightly above the uncertainty threshold (0.48 and 0.46 Å) suggesting the possibility of crystal induced distortions. SC2 contains structures with two symmetries (P21 and P1) as well as deca- and nonameric bound peptides and a double mutation in 2av1, each of which apparently does not influence the similarity. However, SC2 has eight empty cells indicating coordinate differences with RMSDD above the uncertainty threshold: 1duz3myj, 1duz2x70, 1duz2x4q, 2x703hpj, 2x4q3hpj, 1i7u2x70, 1i7u2x4q, 2v2w2x4q. Seven out of eight of these pairs involve either 2x70 or 2x4q. There are special aspects to the origin and crystal preparation for these two structures. The initial crystallization was performed with the protein complexed

with a low affinity ligand, which subsequently is cleaved off the complex and departs when exposed to UV light, allowing a new ligand to soak in.35,36 This might preserve some structural peculiarities of the structures initially crystallized with low affinity ligands, thus introducing coordinate differences from those structures crystallized directly with their final ligand. RMSDD 2x70A-D (comparing two molecules in the same unit cell) is above the uncertainty threshold, suggesting crystal induced deformations. 2x70 and 2x4q also have bound peptide ligands with unusual residues. All this provides a partial explanation for the empty cells in SC2 but leaves open the question: why are many other structures in SC2 cluster still similar to 2x70 and/or 2x4q with RMSDD below the uncertainty threshold? The top line in the SC2 cluster is for the 1duz structure. It is distinguished by the highest RMSDD (0.48 Å) between two identical molecules in the unit cell (for the DDM see Section 175

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Biochemistry Table 3. RMSDD for All Fairwise Comparisons between 43 Different HLA-A2 Structuresa

a Letters in each cell, marked with the structures on the left and top, indicate the representative DDM from Figure 3, followed by the RMSDD (in Å) for the marked pair of structures. RMSDDs are rounded to one digit after the decimal point; zeros before the decimal point are not shown, e.g., D.8 stands for DDM from Figure 3d with rounded RMSDD = 0.8 Å; A1.2 stands for the representative DDM from Figure 3a and RMSDD = 1.2 Å; BC.5 stands for the representative DDM from Figure 3m with rounded RMSDD = 0.5 Å, etc. “x” indicates similarities within the uncertainty threshold (Table 1).

SI1). This might indicate coordinate differences induced by the interactions in the crystal lattice and contribute to three empty cells involving 1duz. However, in two of these empty cells 1duz is compared to 2x70 and 2x4q as already discussed above. The third empty cell in the top line of SC2 corresponds to the pair 1duz3myj. In 3myj R in the first position of the peptide antigen is substituted with Y, “which alters positions of MHC charged side chains near the peptide-antigen’s N-terminus and reduces the peptide/MHC electrostatic surface potential. These alterations indicate that R1Y variant is an imperfect mimic of the native WT1 peptide (bound to HLA-A2 in 3hpj), and suggests caution in its use...”37 However, 3myj is not dissimilar to 2x70 or 2x4q (their RMSDDs are below the uncertainty threshold) while its analogue 3hpj with the native peptide antigen has RMSDD above the uncertainty threshold when compared to 2x70 or 2x4q. There is also the difference in the crystal symmetries between 3myj and 3hpj. However, to further clarify this requires more comparisons with a wider data set (see Section 3.8 below).

The tentative SC3 cluster of 13 structures is the most difficult to analyze because it has 25 out of 91 cells empty and no publications or even abstracts available for 5 of the 9 included structures named in PDB starting with 3mr. All these nine structures are tentatively included in SC3 because they all are from crystals with the same symmetry (P21), one molecule per unit cell, and have the same mutation (A245V). In SC3 (as in SC2) the protein is complexed with peptide-antigens 9 and 10 residues long. Two more structures with exactly the same characteristics, 3gso and 3ft2, fit well into the SC3 cluster, with only 2 and 0 empty cells, respectively. Structure 3d25 also fits into SC3 with two empty cells. However, it has no mutations. Two more structures, 3bgm and 3bh9, satisfactory fit into SC3. These bring to this cluster 7 and 6 cells indicating coordinate similarity below the uncertainty threshold. However, they have symmetry C2, phosphoserine (SEP) in the fourth position of antigen, and no mutations. The last four structures in Table 1 (1i4f, 3bh8, 3h9h, and 3ixa) have from 0 to 3 similarities with any of the clusters. Note, however, that 3mrk which has only three similarities 176

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HLA-A2 structures having RMSDD above the uncertainty threshold. A straightforward counting of A-type DDM symbols in Table 3 leads to the conclusion that 218 out of 700 DDMs with RMSDD above the uncertainty threshold are of A-type. Almost all of them are from comparisons of structures in SC1 cluster with 16 other structures (3ixa and 3h9h yield together only five of A-type symbols; we will further consider these two structures below). The magnitude of RMSDDs for A-type pairs ranges from 0.8 to 1.3 Å. For comparison, functional coordinate changes in uracil-DNA glycosylase (1akz1ssp) and lactate dehydrogenase (6ldh1ldm) have RMSDDs of 1.14 and 1.25 Å respectively.28−30 The functional coordinates change with the minimum RMSDD of 0.45 Å and Δ = 5.3% (all pairs in this work have Δ < 4%) is for endothiapepsin (4ape5er2).28 The next most abundant symbol (147 occurrences) in Table 3 is for D-type DDM which is found almost exclusively in comparisons between SC1 and SC2 clusters (four occurrences are in the columns for 3h9h and 3ixa). Rounded off RMSDD for D-type varies between 0.6 and 0.9 Å which is within the range expected for function-related coordinate changes.28−30 E-type occurs 40 times mainly within SC3 cluster (four times between SC3 cluster and 3bh8) with RMSDD between 0.5 and 0.8 Å. C-type DDM symbol occurs in Table 3 only 34 times between SC2 and SC3 clusters with RMSDDs between 0.5 and 0.6 Å. The other 10 DDM types occupy about 200 remaining cells in Table 3 corresponding to RMSDD above the uncertainty threshold. Among these the highest RMSDD is 1.3 Å for the Itype symbol in the 3ixa column. 3.4. Pairwise RMSDD for α1 + α2 Comparisons. Most of the attention in crystallographic studies of HLA-A2 has been focused on the changes in the antigen-peptide binding fragment, consisting of domains α1 + α2, and its adaptation to different peptide-Ags. The α3 domain, except for some studies of its binding by CD8, was paid relatively little research attention, becoming a kind of neglected “Cinderella domain”41 among α1, α2, and α3. Most earlier reports (e.g., ref 4) claimed “no significant changes” in the peptide-binding fragment (α1 + α2), while later studies12,26 indicated “small but significant peptide dependent variations” in this fragment. We have verified these differing reports with our DDM/RMSDD method for 1081 α1 + α2 pairs of different 43 HLA-A2 high resolution structures and four lower resolution HLA-A2 structures extracted from the PDB9 structures of TCR A6/HLA-A2 complexes. Among all pairs of high resolution HLA-A2 structures not including mutants 3h9h and 3ixa12 only 14 pairs have RMSDD (shown in parentheses in Å) above the uncertainty threshold:27 3o3a3bh8(0.46), 2clr3mrm(0.46), 2clr3mrk(0.46), 2clr3bh8(0.48), 2av13bh8(0.47), 2x703bgm(0.50), 2x703bh8(0.51), 2x4q3bgm(0.50), 2x4q3bh8(0.51), 1i7u3mrm(0.46), 1i7u3mrk(0.48), 1i7u1i4f(0.46), 2v2w3mrk(0.46), 1i4f3bh8(0.46). Forty-one high resolution structures differ from mutants 3h9h and 3ixa with RMSDD well above the uncertainty threshold (indicating rather significant structural differences).27,28 The same 41 high resolution structures differ with a similarly high RMSDD from the lower resolution (2.7 Å) HLA-A2 3h9s extracted from the PDB structure of its complex with TCR A6. Mutant 3ixa12 does differ within a similar high RMSDD range above the uncertainty threshold from all four low resolution (2.6−2.7 Å) A6-liganded HLA-A2 structures. The high resolution mutants 3ixa and 3h9h12 are similar with RMSDD below the uncertainty

below the uncertainty threshold with SC3 structures is included in that cluster. We retain it there only because it shares structural characteristics with the majority of the structures in SC3. As we will see below, 1i4f has some features that suggest it could be included in SC3 along with 3mrk. 3bh8 has SEP in Ag similarly to 3bgm and 3bh9. 3h9h and 3ixa are artificial mutants.12 It is worth noting that the attempts to rationalize found similarities/dissimilarities between HLA-A2 structures might be only first steps in a very difficult task as we found for a much simpler case of myoglobin structures.38 The fact that almost all HLA-A2 X-ray structures were solved at nonphysiological cryogenic temperatures might make their use in interpretations of data at physiological temperatures more complicated27,39 especially at the binding and catalytic sites.39 While measurements at cryogenic temperatures do introduce some changes (with mean RMSD of 0.13 Å) in the main chain coordinates,38,39 which are mainly used in this study, they are not as large as suggested initially (e.g., see ref 27). It has been suggested39 to redeploy the data collection at room temperature for higher structural reliability especially of the sites linked to ligand binding, catalysis, and allosteric regulation where cryogenic structures show significant coordinate changes. New XFEL methods are expected to soon achieve this goal.40 3.3. Pairwise RMSDD and Deformation Types for All 43 HLA-A2 Structures. Pairwise comparisons of α-chains from 43 structures yielded 700 visually varied DDMs with RMSDDs above the uncertainty threshold. All these 700 DDMs are represented in Figure 3 by visually selected 16 DDMs (see in Section SI2 examples of the variations of the actual DDMs assigned to their representative DDM in Figure 3). The visual selection involves some arbitrariness, and some of the representative DDMs shown in Figure 3 may look similar (e.g., F and C’), while DDMs represented by them are often more clearly different visually (Methods and Figure 2 in SI2). Some of the representative DDMs in Figure 3 require extra comments. White (DDs above 1 Å) represent in some cases distortions of only short segments of the protein chain and occupy only small parts of the DDMs area (e.g., B and C of Figure 3b,c); their contribution to the RMSDD is significant and usually requires one or two movements of rigid fragments. However, some of the DDMs appear as if they were superpositions of two or more simpler representative DDMs. For example, the single white L-shaped strip in Figure 3c indicates a particular important conformational difference at the corner of L between the DDMs from SC3 and other clusters; the white L-shaped strip in Figure 3b indicates a significant conformational distortion at its corner; Figure 3m shows white L-strips similar to those in both Figure 3c and Figure 3b, indicating the presence of both conformational differences between two α-chains. Also, Figure 3d corresponds to a simple rotation of two domains in one structure relative to another; Figure 3n shows a superposition of Figure 3b and Figure 3d, indicating that the two structures differ by both−distortion of the second long helix and a domain rotation. Figure 3l contains in addition to the L-strip from Figure 3c a number of additional relatively large white spots. Therefore, DDMs including combinations of some version of B or C or D require separate representative DDMs (e.g., BC, B′, C′, BD, B′C, BC′), which are shown in Figure 3. The referral to these 16 representative DDMs permits us to present in Table 3 RMSDDs and approximate types of structural differences for all 700 pairwise comparisons of 177

DOI: 10.1021/acs.biochem.5b01077 Biochemistry 2016, 55, 167−185

Article

Biochemistry

Figure 4. Comparison of the change in the SC2 to SC1 α3 position to the change of the toggle position in an electrical switch between three different circuits (one of them is “OFF” see the text). Two superimposed HLA-A2 structures are SC2 Tax HLA-A2 in gray (PDB code 2git) and SC1 HuD HLA-A2 in black (PDB code 3pwl). The change from SC2 to SC1 occurs upon CD8 binding.15 We do not show superimposed any of the structures from SC3 which are CD8 nonbinders.

predominance of the numbers of α3-β2m H-bonds increases with an increase in the H-bond cutoff. Thus, α3 is at least as strongly hydrogen bonded to the β2m chain as twice larger α1 + α2. 3.6. Rigid Fragments Movements. In our earlier study of the functional movements in 17 pairs of globular proteins,28 we found a variety of rigid and nonrigid conformational changes of different scale and magnitude. The procedure can be timeconsuming in many cases, and it is impractical to perform a full study28 of the fragment motions for 700 pairs with RMSDDs above the uncertainty threshold. Thus, we performed such a study for only 6 pairs of the HLA-A2 structures (see SI5). Here we limit ourselves to a brief summary to indicate what conformational changes of a functional magnitude in HLA-A2 fall within the patterns found earlier.28 The simplest rigid body transformation is seen for the Reference DDM C (Figure 3c): the maximal initial DD is 4 Å and after translating a single tripeptide (corresponding to the corner of the white L-shape strip) by 1.9 Å and rotating it by 32° the RMSDD drops to 0.45 Å. The major characteristic of the reference DDM C is an L-shaped white strip with its corner from residue 225 to 228 (the bottom part of the DDM C can be easily seen in DDMs A, F, G, I, etc.) Reference DDM B (Figure 3b, with the white L-angle around the engineered mutation A150P) has a maximum initial DD of 6 Å and after translations and rigid rotations of a tetra- and then tripeptide in the corner of the L-shaped white strip its RMSDD drops to 0.39 Å. DDM D (Figure 3d) has a maximum initial DD of 5 Å

threshold. In agreement with a 2013 study,26 the high resolution mutant 3h9h and low resolution HLA-A2 structure from 3h9s complex with TCR A6 are similar with RMSDD below the uncertainty threshold. The full Table 1 (SI) is available in SI3. Thus, almost all 700 pairs of HLA-A2 structures (Table 3) have differences above the uncertainty threshold mainly due to movements of their α3 domain caused by differences in the bound peptide. 3.5. Hydrogen Bonds between the α-chain Domains and the β2-Chain. It has been stated1,2 that interactions with the β2m subunit stabilize the antigen-binding α1 + α2 domain formed by residues 1−181.9,10 It has also been suggested that variations in the α3 domain position relative to the α1 + α2 domain is a result of the weak coupling of the α3 domain to the remainder of the protein.4 To verify these two statements, we calculate the numbers of hydrogen bonds of α1 + α2 with β2m and α3 with β2m for all 43 HLA-A2 structures for three increasing hydrogen-bond (H-bond) length cutoffs by using CCP431 (see Methods). The numbers of H-bonds found are presented in Table 2 (in SI4), which shows that for all H-bond cutoffs the total number of H-bonds between the α3 and β2m almost always is at least as large but usually larger than the number of H-bonds between the antigen-binding α1 + α2 and β2m. The only exceptions are 2x4q (with selenomethionine at β99 which CCP4 could not process) and 2gtw (one more H-bond for α1+α2 vs α3). The 178

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Article

Biochemistry Table 4. Invariant Short Hydrogen Bonds between α and β Subunits of HLA-A2 in SC1 and SC2a cluster SC1 SC2

α(1 + 2):α3 D30OD2-A211N; T31OG1-Y209OH; R181NH1-G239O; D30OD2-A211N; T31OG1-Y209OH

α(1 + 2):β within 3.0 Å

α3:β within 3.0 Å

Q96OE1-H31NE2; Q32NE2-D53OD2; R48NE-D53OD2; Q96NE2-W60O; D122OD1-W60NE1; Q96OE1-H31NE2; Q32NE2-D53OD2; Q96NE2-W60O; D122OD1-W60NE1; R35NH1-D53OD1

comment

R234NH1-Q8OE1; E232O-Q8NE2; P235O-Y10OH; T190OG1-M99O; R202NH1-M99O; R234NH1-Q8OE1; P235O-Y10OH; E232OE1-S28OG; R234NH1-M99O/OXT

few minor variations; see SI4 2x4q and 2x70 1−2 a:b missed; see SI4

Residue numberings are the same as in PDB files. α(1 + 2):α3 and α(1 + 2):β entries show H-bonds of the peptide-binding domains of the heavy chain [res (1−181)] with its α3 domain and with the β chain; α3:β entries show H-bonds between the α3 domain and the β chain. a

relocating α3 to positions corresponding to SC1 or SC2, which looks like an allosteric effect. We consider this question by analyzing short strong hydrogen bonds (3 Å cutoff) formed by parts of α and β2m chains in SC1 and SC2 that are listed in Table 4. Details can be found in Tables 3−4 (SI) in SI4. We observed “stabilizing” practically invariant strong hydrogen bonds that systematically differ between the structures from SC1 and SC2 (for minor variations in these bonds see footnotes to Tables 3−4 (SI) in SI4). We find that two strong hydrogen bonds, D30OD2-A211N and T31OG1-Y209OH (2.75−2.9 Å long), are practically invariant for all high resolution HLA-A2 structures from SC1, SC2, and SC3. These two hydrogen bonds are the major noncovalent connection between α3 and α1 + α2 for HLA-A2 structures from SC2. Almost all SC1 structures have one more strong hydrogen bond between (α1 + α2) and α3 domains than the SC2 structures. It appears that small conformational changes caused by antigen differences in the β-sheet “floor” of the antigenbinding α1 + α2 act as a “switch” for the α3 position between SC1 and SC2 and for a concomitant change in the hydrogen bonding between the α and β2m subunits of HLA-A2 structures. The change in α3-β2m bonding is more drastic than in (α1+α2)−β2m bonding. The same SC2 to SC1 switch is caused by CD8 binding (Figure 4). We note once again that the structural organization and functioning of the immune response are complex and involve various coreceptors, switches, and effectors. Our aim in this work has been to clarify the possible functioning of a small part of this immensely complex and important human biological system. 3.8. Role of the Crystal Symmetry and Unit Cell Dimensions in the Formation of SCs. There is currently no reliable method to predict a protein’s structure or in what lattice a particular protein would crystallize. However, some observations can be useful. In the section 3.7 of the Results above we report our finding that the strong hydrogen bonds between (α1 + α2) and α3 differ between SC1 and SC2 but are nearly invariant within each of these clusters. This suggests intramolecular reasons for the differences between SC1 and SC2. Figure 5 (SI) in SI7 shows that with only minor differences DDMs of structures from these two SCs are practically identical and are of D-type (Figure 3d). All structures in SC1 have the same crystal symmetry, P21, and the same cell dimensions. Therefore, it is difficult to verify whether crystal interactions could determine the choice of the α-chain structure in SC1. The situation is significantly different in SC2. It contains three structures with the same crystal symmetry, P21, as all structures in SC1, but somewhat different cell dimensions (see Table 2). Other nine structures in SC2 have P1 crystal

and after a translation by 1.9 Å and rotation by 7.3° of a single 97 residues long rigid fragment its RMSDD drops to 0.41 Å. Reference DDM E (Figure 3e) with a maximum initial DD of 5 Å requires 5 transformations of rigid fragments to reduce its RMSDD to 0.42 Å; the longest rigid fragment of 79 residues is translated by 2.6 Å and rotated by 9.1°. Reference DDM F (Figure 3f) with a maximum initial DD of 4 Å requires 9 translations and rotations of rigid fragments to reduce its RMSDD to 0.45 Å; the longest rigid fragment of 79 residues is translated by 2.4 Å and rotated by 9°. Reference DDM A (Figure 3a) with a maximum initial DD of 6 Å requires 10 translations and rotations of rigid fragments to reduce its RMSDD to 0.35 Å; the longest rigid fragment of 89 residues is translated by 1.8 Å and rotated by 9.6°. The described above transformation parameters for rigid body fragments repositioning in HLA-A2 fall into the range of rigid body functional movements in globular proteins.28 Because 2/3 to 3/4 of 700 HLA-A2 pairs with RMSDDs above the uncertainty threshold belong to Reference DDM types A-F (see section 3.3 above) which have been found (see also in SI5) to undergo structural changes through rigid body fragment repositioning, we estimate that at least half of all 700 HLA-A2 structure pairs are interconvertible by similar rigid fragments repositioning. However, it should be noted that sometimes relatively small differences in DDMs might require a number of additional rigid body repositionings.27,28 3.7. Possible Connection between Similarity Clusters (SCs) and α3 Movements between Them. The Cinderella analogy with α3 domain was funny and stimulating and posed a question: are there other stimulating analogies to be found? Images in Figure 4 suggest such an analogy. All (except a few) significant differences between the pairs of HLA-A2 structures with RMSDDs above the uncertainty threshold in Table 3 are due to differences in positions of the α3 domain relative to the (α1 + α2) domain (denoted as domain-1 in SCOP10 and in PDB9 and comprised of residues 1−181). Our finding of three SCs suggests that there are only three different orientations of the α3 domain (two are shown at the bottom of the left image in Figure 4). The similarity of these three positions of the α3 domain (at the bottom of twice larger α1 + α2, binding different antigen-peptides) to an electric switch with three possible toggle positions is certainly an oversimplified but useful analogy. The three-ways electric switch usually has one toggle position corresponding to switching “off” the electric power. HLA-A2s with the mutation A245V or the absent turn of the helix 225−228, characteristic of SC3, do not bind CD815,20,21 and thus might not22,23 let the immunological signal through HLA-A2. Structure 1i4f also lacks the 225−228 helix. SC3 can be looked upon as the “OFF” “switch” position. The next question we addressed is how small differences caused by binding of different peptide-antigens can lead to 179

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Biochemistry Table 5. Five Low Resolution HLA-A2 Structures Compared to Representative Structures from SCsa name PDB

symmetry

peptide-Ag

1hhg 1hhh 1hhi 1hhj 1hhk

P21 P212121 P21 P1 P1

TLTSCNTSV FLPSDFFPSV GILGFVFTL ILKEPVHGV LLFGYPVYV

source of Ag HIV-1 gp hepatitus B influenza A HIV-1 RT HTLV-1 Tax

cell dimensions (edges in Å, angles in °)

res. (Å)

RMSDD 1i7u (Å)

RMSDD 3hpj (Å)

RMSDD 3pwl (Å)

RMSDD 3mrr (Å)

α,β.γ = 90 α,β.γ = 90 α,β.γ = 90 α = 81,β = 76,γ = 77 α = 82,β = 76,γ = 77

2.6 3.0 2.5 2.5 2.5

0.41 0.57 0.35 0.33 0.30

0.32 0.47 0.28 0.30 0.44

0.74 0.83 0.74 0.86 1.00

0.55 0.37 0.53 0.50 0.58

62.7,87.0,79.4; 60.6,81.2,94.8; 63.3,87.7,79.2; 50.4,63.6,74.8; 50.6,63.8,75.1;

a

The last four columns show the RMSDD in comparisons of each HLA-A2s from the leftmost column and each representative HLA-A2 on the top of the last 4 columns (e.g., 1hhg1i7u RMSDD = 0.41 Å).

was further refined and is included in Tables 1−3 as 1duz). Thus, of the total 15 structures in SC2 five have symmetry P21 and 3hpj is not a rare aberration. We studied the model of the crystal of 1i7u from SC2 with the central unit cell surrounded by a shell of the neighboring crystal cells (see Methods). The A and D chains of 1i7u in the central unit cell have been substituted by chains A and D with bound peptides C and F from 3hpj, WT1 (see Table 2) which were fitted to the corresponding chains with bound C and F peptides from 1i7u. There were no clashes of the substituted 3hpj peptides C and F with the α-chains of 1i7u in the crystal unit cells surrounding the central one. When peptides C and F from 3hpj were substituted instead of C and F of the fitted 1i7u complex, the substituted peptides C and F clashed with the corresponding α-chains A and D of the central unit cell (Figure 6 (SI)). This strongly suggests that binding of an alternative peptide can lead to a small intramolecular change at the binding site (in some cases with a responsive α3 movement) but not to a symmetry change caused by the pressure of crystal contacts on the bound peptide-Ag. (It is commonly accepted that binding of different peptide-Ag-s is the main factor in conformational differences between different HLA-A2 structures.) One might suggest that conformational changes in HLA-A2 can be caused by the pressure of crystal contacts on parts of the molecule other than the binding site. However, it was found42 that a “greater number of crystal contacts do not necessarily induce conformational changes, but specific interactions from a smaller number of contacts may stabilize one conformation at the expense of others”. In our study of myoglobins38 we confirmed that significant differences in intercell contacts can produce negligible conformational differences. Here we also found specific invariant intramolecular bonds distinguishing between SC1 and SC2 conformations. Thus, it is much more likely that the HLA-A2 attain some preferred conformation depending on the bound peptide and then crystallize with one or a few suitable symmetries and cell dimensions. Note that selected pair 1i7u3hjp is an excellent choice as DDMs of any combination of chains from this pair of HLA-A2s (e.g., 1i7u(A+B)3hjp(A+B), 1i7u(A+B)3hjp(D+E), 1i7u(D +E)3hjp(A+B), etc.) have RMSDDs within the uncertainty threshold. Structure 1hhh (see Table 5) has symmetry P212121, unique among all HLA-A2 structures studied here. However, it is similar within the uncertainty threshold to 3mrr from SC3 (but not to structures from other SCs) and thus also belongs to SC3 despite its unusual symmetry and cell dimensions. In section 3.2 above we promised to revisit similarity of 3mrk with only three similarities in SC3 and 1i4f which also has three similarities but is not included in SC3. It is clear from Table 1 that 3mrk and 1i4f are similar (RMSDD = 0.24 Å). Both

symmetry with different cell dimensions than structures in SC1 or the three structures with P21 symmetry in SC2 (see Table 2). But all 12 structures from SC2 when compared with structures from SC1 have D-type DDMs (Figures 3d and 5 (SI)). We fit the three structures with P21 symmetry and the five structures with P1 symmetry from SC2 to a typical structure, 3pwl, from SC1. Then we check the results by additional fitting of a few of these eight SC2 structures to a couple of other structures from SC1 (3qfd and 1tvb). The initial superposition of any of the eight SC2 structures onto the SC1 structure was obtained by the fitting the rigid fragment 21−50 with reference residues 23 and 49 using our technique (see Methods) and then applying thus obtained fitting transformation to the entire α-chain. In the next step we apply another transformation (practically the same for all 8 SC2 structures) to the α3 domain from SC2. (For the detailed differences between the two choices of the ends of the moved domain and their reference points, see SI7.) Just this single transformation makes the entire α-chain of an SC2 structure similar (within the uncertainty threshold) to a SC1 structure. In this second transformation all eight fragments (174−271 or 178−271) of structures from SC2 were translated by less than 2 Å and rotated in the range of 5.5−7.4° (see Table 5 (SI) in SI7). Fragments from 3hpj, 2x4q, and 2x70 from SC2 with symmetry P21 (see Table 2) rotated within a somewhat smaller range of 5.5−6.9° with the rotational axis close to being antiparallel to the Y-axis of 3pwl from SC1 (Table 6 (SI) in SI7). Fragments from five other SC2 structures rotate by 6.8−7.4°, and their axes deviated more from axes of 3pwl. Most variations are due to small distortions of the structures around the reference Cα atoms used in the fitting, and their different ranges correlate with the different crystal symmetries. However, the total turn variation is only 1.9°. Thus, regardless of the crystal symmetry, practically the same α3-domain movement transforms all eight structures from SC2 into SC1 structures within the uncertainty threshold. The magnitude of the movement is sufficiently large to be considered functionally important according to widely accepted criteria.28 However, three P21 structures in SC2 include 2x4q and 2x70, which contain peptides with unusual residues, have RMSDD above the uncertainty threshold when compared to 3hpj (see Tables 1 and 3), and introduce additional irregularities in similarities pattern of Table 1 (see section 3.2 above). To check whether 3hpj (the only other P21 structure in SC2) could be just a rare aberration, we examined five earlier published lower resolution structures11 1hhg, 1hhh, 1hhi, 1hhj, 1hhk (see Table 5). Of these five structures four, 1hhg, 1hhi, 1hhj, and 1hhk, are similar within the uncertainty threshold to SC2 structures 1i7u and 3hpj and thus also belong to SC2. Two of them, 1hhg and 1hhi, have symmetry P21. Other two have symmetry P1 (1hhk 180

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Biochemistry Table 6. RMSDDs (Å) of HLA-A2 Tel1p, Tax, HuD, with and without Bound Receptorsa

PDB CD8 1akj 1hhj A6 3pwp HuD A6 3h9s Tel1p A6 1ao7 Tax A6 1qsf Tax (Y8A) A6 1qse Tax (V7R) A6-hi-aff 4ftv Tax A6 2gj6 Tax(Y5K′) A6 3qfj Tax(Y5F) A6 3d39 Tax(Y5F′) A6 1qrn Tax(P6A) 3h7b Tel1p 1duz (Tax) 2git Tax(Y5K′) 3pwl HuD

A6 3pwp HuD

A6 3h9s Tel1p

A6 1ao7 Tax

A6 1qsf Tax (Y8A)

A6 1qse Tax (V7R)

4ftv Tax A6-hiaff

A6 2gj6 Tax (Y5K′)

A6 3qfj Tax (Y5F)

A6 Tax 3d39 (Y5F′)

A6 1qrn Tax (P6A)

3h7b Tellp

0.66

0.74

0.78

0.72

1.02

0.74

0.74

0.47 Å

0.57 Å

0.68

0.63

0.49

0.47

0.47

0.33

0.35

0.35

0.30

0.74

0.71

0.68

0.65

0.65

0.55

0.38

0.38 0.39

0.52 0.46

0.47 0.44

0.51

3pwl HuD

3pwj HuD (2L,9 V)

1hhj HIV1 RT

0.93

0.43

0.92

1.02

0.73

0.54

0.64

0.53

0.63

0.78

0.97

0.79

0.64

0.79

0.89

0.33 0.34

0.60 0.62

0.75 0.81

0.63 0.64

0.63 0.58

0.61 0.62

0.64 0.67

0.52

0.37

0.63

0.82

0.64

0.58

0.63

0.71

0.44

0.35

0.38

0.54

0.73

0.55

0.63

0.54

0.61

0.46

0.42

0.37

0.47

0.68

0.49

0.62

0.48

0.57

0.28

0.43

0.73

0.96

0.73

0.43

0.72

0.82

0.42

0.65

0.87

0.66

0.51

0.65

0.74

0.56

0.76

0.58

0.53

0.56

0.61

0.34

0.16 0.33

0.78 1.01 0.78

0.12 0.35 0.13

0.30 0.41 0.32

0.76

0.86

1duz Tax

2git Tax (Y5K′)

0.93

1.17

0.37

0.53

0.53

0.69

0.59 0.50

0.56 0.50

0.39

0.50

0.31

CD8 or A6 in the cells identifying the HLA-A2 structures show that the coordinates of these structures are extracted from PDB files of complexes of these structures with CD8 or A6. K′ stands for modified lysine and F′ for modified phenylalanine. RMSDDs values within the uncertainty threshold are shown in bold. This table contains one CD8αα liganded Tax HLA-A2, one A6 liganded HuD HLA-2, and one A6 liganded Tel1p; it contains eight A6 liganded native Tax HLA-A2 and its modifications. Of crystallographically solved A6 liganded modified Tax HLA-A2 structure 3d3v is not included, because its RMSDD pattern with only minor differences in some numbers closely resemble RMSDDs of A6 3d39 and because there is no more room in the table. a

structures are also similar to 3mrc and 3mrj and have similar DDMs (see Figure 3 and Table 3). They have the same symmetry and both have type-C L-band indicative of a disruption of an important short helix in the α3 domain. However, it is argued that this disruption in the α3 domain is indirectly caused by the mutation A245V which is present in all 3mr* structures. 1i4f causes a similar disruption without having a mutation. 3bgm and 3bh9, containing phosphoserine, have seven and six similarities in SC3 (Table 1) but have C2 symmetry and very different cell dimensions. Thus, SC3 after adding 1hhh has HLA-A2s with three different symmetries (P21, C2, P212121) and cell dimensions. This again suggests that SCs are not determined by crystal packing effects. While we consider mutants 3h9h and 3ixa in the next section, they are irrelevant in the context of an allostery as even their (α1 + α2) domain is strongly distorted (see section Section 3.4 above and Table 1 (SI) in SI3). 3.9. Comparisons of Tax, Tel1p, HIV-1RT, and HuD HLA-A2 with and without Receptors Bound. RMSDDs of the structures focused on here are presented in Table 6. The unliganded by CD8 HLA-A2 1hhj (2.5 Å resolution) has a bound nine-residue long peptide-Ag from HIV-1 reverse transcriptase (HIV-1 RT, see Table 5). It has RMSDDs within the uncertainty threshold (shown in bold in the last column of Table 6) when 1hhj is compared to 3h7b, 1duz, and 2git from SC2. Because the structure of 1hhj is similar to other structures from SC2, 1hhj HLA-A2 also belongs to SC2. A single RMSDD

in bold script in the top line of Table 6 shows that this 1hhj structure, after being liganded by CD8 in structure 1akj, becomes similar to HuD HLA-A2 (which belongs to SC1); i.e., it has switched to SC1 by the D-type (see section 3.3 above) transformation (Figure 3d and Figure 5a below). However, if CD8 would attempt to bind to the structure 1ao7 (2.6 Å) of 1hhk or 1duz (1duz is a refined structure of 1hhk) liganded by TCR A6 (Figure 5b), CD8 might not be able to do so because HLA-A2 in DDM 1ao71duz shows a distortion in short helix 225−228 similar to distortions of SC3 structures not binding CD8. There is no such α3 distortion in 1akj3pwl (Figure 5c). Figure 5b might indicate a doubt in the universality of the statement43 that α3 binding loop, essential for a CD8 binding, is not significantly changed upon TCR binding of peptideMHC I. It would seem simpler and more function-related if CD8 was to bind to TCR-free HLA-A2 transmitting back to the peptidebinding site, through the α3 domain connections to the rest of the HLA-A2, those structural variations that determined SC1 cluster formation. It had been found earlier that TCR A6 can bind both Tax and HuD peptides with the HuD-HLA-A2 complex being only weakly stable.13 This would fit the hypothetical role for CD8 of increasing the sensitivity of Tcells to even weaker antigens presented by HLA-A2.19,44 This hypothetical suggestion could be supported by a crystallographic finding that CD8 directly binds HuD-HLA-A2 (or another SC1 structure) without any significant change in its α3 domain conformation and orientation relative to the rest of 181

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Figure 5. Comparison of DDMs for structures involved in a receptor binding: (a) between the initial and final HLA-A2 in binding of CD8 (A6 is not bound to HLA-A2); (b) between the initial target of the CD8 binding from SC2 and the same potential target in HLA-A2 liganded by TCR A6; (c) between the X-ray structure of the HLA-A2 bound to CD8 and HuDHLA-A2 unliganded by A6; (d) between HLA-A2s from two A6-liganded Taxlike structures; (e, f) for HLA-A2 from A6-liganded HuD and from 2 Tax-like A6-liganded structures. Notations are the same as in Figure 3.

HLA-A2 structure (which is seen in CD8 binding to hhj-HLAA2). Unfortunately crystallization of CD8-MHC I appears to be forbiddingly difficult with only three structures, one human14,15

and two murine,16,17 having been solved so far. This looks somewhat strange as there is data for the affinities (KD) 182

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Biochemistry between at least 10 CD8/peptide-MHC I44 with values comparable to KD of CD8−1hhj pair. Note that if, however, CD8 would bind to the MHC I already presenting a strong antigen to the TCR, the stronger antigen would be already locked in the complex and CD8 binding would not be able to substitute it with a weaker antigen. These are, of cause, just plausible speculations based on the few relatively low resolution crystallographic studies of CD8-liganded complexes. Next we look at changes in the α-chain upon TCR A6 binding to HLA-A2 shown in Table 6. It is obvious from Table 6 that all but one (3qfj) of the HLA-A2 structures extracted from their complexes with TCR A6 are not similar (within the uncertainty threshold) to any HLA-A2s from the SCs included in Table 6. Thus, binding of A6 in most cases distorts the α3 domains beyond the SCs. However, the Tax HLA-A2 mimic (Y5F) extracted from A6-liganded HLA-A2 structure 3qfj has RMSDD of 0.43 Å when compared to the HLA-A2 HuD structure from SC1. Thus, 3qfj is similar within the uncertainty threshold to 3pwl HuD. Furthermore, the RMSDD 3qfj3pwl is exactly equal to the RMSDD 1akj3pwl where HLA-A2 1akj is extracted from CD8αα liganded HLA-A2 (Figure 5c). Some (related to this study) differences between the Tax and Hud HLA-A2 liganded by TCR A6 as well as the similarities of Tax(Y5F) and HuD HLA-A2 liganded by A6 have been reported previously.45 It is certainly of interest that A6-liganded HuD-HLA-A2 is similar (within the uncertainty threshold) to six (4ftv, 2gj6, 3qfj, 3d39, 3d3v, and 1qrn) out of nine A6-liganded Tax-type HLA-A2 structures in Table 6 (3d3v is not in the Table, see legend). Only three A6-liganded Tax-like-HLA-A2 (1qsf, 1qse, and 1qrn) are similar (within the uncertainty threshold) to 1ao7 (A6-liganded native Tax 1hhk). 1qrn in its turn is almost equally similar (within the uncertainty threshold) to both A61ao7-Tax and A6-3pwp-HuD, as well as to seven more A6liganded Tax-type HLA-A2 structures 1qsf, 1qse, 4ftv, 2gj6, 3qfj, 3d39, and 3d3v (see Table 6 and the legend to it). Presumably, CD8 converts SC2 structure 1hhj to the SC1 HuD-type structure CD8-1akj and then might present it to TCR A6 to be converted to a version of A6-3pwp-HUD structure. However, TCR A6 converts the majority of the Taxlike HLA-A2s (in Table 6) to A6-3pwp-HUD type of structures without any assistance from CD8. It would be interesting to see an X-ray structure of A6-SC2-1hhj prepared with no presence of CD8. If, it would follow the majority of A6-Tax-like structures in Table 6 and be similar to A6-3pwp-HUD type structure, then what CD8 is for in this context? It has been concluded from the study of interactions between affinity enhanced TCR (no X-ray structure available) with CD8 that “binding of the TCR to HLA-A2 (pMHCI) does not transmit structural changes to the pMHCI-CD8 binding site that would alter the subsequent pMHCI/CD8 interactions”.43 Note, however, that liganding of Tax-HLA-A2 with modified high affinity TCR A6 in 4ftv leads to a dramatic difference in the similarity pattern from 1ao7 Tax-HLA-A2 liganded by a regular TCR A6. This again (like Figure 5b) suggests that conclusions from studies with modified TCRs43 might not be universally valid for studies with native TCRs. Thus, we found that A6-liganded Tax-type HLA-A2s form two new similarity clusters: six are similar to A6 liganded HuD and three to A6-liganded native Tax (see legend to Table 6 for one additional structure included in this count but not shown in Table 6). All these peculiarities and similarities between A6-

liganded Tax-type and HuD-type HLA-A2s deserve a more detailed study (see also Figure 5 and SI6). Attempts to crystallize a complex including A6-CD8-HLA-A2 have not been successful so far. Two models built by fitting A6HLA-A2 and CD8αα-HLA-A2 have been published.14,5 In both publications the fitting included superposition of Cα atoms of only the (α1 + α2) domain residues of HLA-A2 while ignored any differences in α3 conformation and position. This certainly is a problem in the fitting (Figure 2c in ref 14) because the 1akj1ao7 pair has an RMSDD of 0.78 Å which is far above the uncertainty threshold. The second fitting (Figure 8c in ref 5) is somewhat better with RMSDD for the pair 1akj1qrn of 0.68 Å (still quite far above the uncertainty threshold). However, if we assume that in the complex HuD from CD8 1akj is already converted by A6 into A6−3pwp, then the RMSDD 3pwp1qrn (Figure 5e) is only 0.37 Å (which is within the uncertainty threshold) and thus makes the fitting more legitimate. (One has to assume, however, that the A6-HLA-A2_CD8 complex still holds after liganding by A6 converts HuD-CD8 to 3pwp.) The top line of Table 6 shows that the lowest RMSDD of 0.47 Å (still above the uncertainty threshold) is between CD8 liganded and A6 liganded HLA-A2-s 1akj and 3qfj (see DDM in SI6, Figure 3 SI). This can be the best candidate for fitting (see above) as 3pwp3qfj has an RMSDD of 0.35 Å (Figure 5f) as well as an RMSDD for 3pwl3qfj of 0.43 Å (both within the uncertainty threshold), possibly suggesting a minimal distortion of α3 HuD upon A6 ligation. In the section below we apply our RMSDD/DDM analysis to the work12 which drew attention to the significant changes in the HLA-A2 (α1 + α2) antigen-binding domain upon TCR A6 binding. Structures of HLA-A2 with bound Tax (1duz) or with bound Tel1p (3h7b) antigen-peptides both belong to the similarity cluster SC2 with RMSDD differences below the uncertainty threshold (Tables 1 and 2). It has been found12 that binding Tel1pHLA-A2 to TCR A6 in the 3h9s complex significantly changes the Tel1pHLA-A2 structure involving modification of the HLA-A2 α-helices around residue Ala150. To verify this finding the mutation A150P was introduced into the HLA-A2 sequences of Tel1pHLA-A2 and TaxHLA-A2 and high resolution crystallographic structures of these mutated unliganded (by TCR A6) structures 3h9h and 3ixa were determined.12 It was found that the mutation strengthened the A6 affinity to Tel1pHLA-A2 and weakened the A6 affinity to the mutated TaxHLA-A2. As expected12 the difference (RMSDD) between the Tel1p in the A6 liganded complex 3h9s and the mutated Tel1p 3h9h is smaller than in the comparison of the A6-liganded Tel1p with the original Tel1p 3h7b. This is reflected in the results (see Table 7) of our DDM/RMSDD27,28,38 approach to MHC structure changes. DDMs in Figure 4a,b (SI) in section SI6 show that A6 binding Table 7. RMSDDs between A6-Liganded and Unliganded Native and Mutated Tax and Tel1p HLA-A2a PDB 3h9s Tel1p A6 1ao7 Tax A6 3h7b Tel1p 1duz (Tax) 3h9h Tel1p m a

183

1ao7 Tax A6

3h7b Tellp

1duz Tax

0.74 Å

0.78 Å 0.60 Å

0.97 Å 0.75 Å 0.34 Å

3h9h Tellp m A150P

3ixa Tax m A150P

0.66 0.71 0.47 0.60

0.54 0.81 0.90 1.12 0.72

Å Å Å Å

Å Å Å Å Å

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to Tel1p also introduces significant changes in the α3 domain. DDMs in Figure 4c,d (SI) also supports previous results12 showing an RMSDD increase from 0.74 to 0.81 Å upon substitution of Tax mutant 3ixa instead of the native 1hhk TaxHLA-A2 in comparison with the A6-liganded native Tax HLAA2, 1ao7. Substituting 1ao7 by the A6-liganded Tax mimic 3qfj in comparisons with 3ixa reduces the RMSDD from 0.81 Å (Figure 4d SI) to 0.61 Å (Figure 4e SI). It is interesting that the comparison of Tel1p liganded by A6 to the mutated Tax structure 3ixa has the lowest RMSDD (0.54 Å) in comparisons with the A6-liganded Tel1p to any other structures in Table 7. DDM in Figure 4f (SI) shows that while the changes in α1+α2 around residue 150 increase compared to Figure 4a,b (SI), the changes related to the α3 movement strongly diminish. The earlier work12 focused only on the (α1 + α2) domain and thus missed these effects involving the α3 domain changes that have been discussed here. Thus, we unexpectedly found that 203 (out of 903) pairs of high resolution HLA-A2 structures, which are similar within the recently determined27,28 uncertainty threshold, form three distinct SCs. Molecules from different clusters differ significantly with their RMSDD values above the uncertainty threshold. Cluster SC3 includes structures with α3 domains which do not bind the CD8 “coreceptor” molecule. Binding of CD8 transforms the initial HLA-A2 from SC2 to HLA-A2 from SC1. The α3 domain connects to the rest of MHC I (β2m subunit and the rest of the α-chain) with more strong hydrogen bonds than the antigen binding α1 + α2 domain. There are sets of α3 hydrogen bonds distinctly different between clusters SC1 and SC2 and almost invariant within each of these SCs. Ligation by A6 of HLA-A2s, with different bound antigens, in most cases changes the α3 orientation and/or structure beyond those observed in the similarity clusters. The analyses of pairs of structures suggest (but do not definitively prove) that the α3 domain may transmit and even magnify small variations in the floor of the antigen-binding groove to the sites of binding coreceptors and other molecules, and initiates feedback changes to the antigen-binding groove, modifying its specificity. Finding that mutations in the Tax antigen peptide lead to formation of two small similarity clusters of A6-liganded-Tax-type-HLA-A2s points in the same direction. All these findings and suggestions seem to be well supported by the analyzed X-ray data but require a further proof. These findings became possible through a careful comparison of the entire α-chains of a large number of HLA-A2s with different bound peptides instead of mainly limiting the comparisons to the antigen binding α1+α2 domains. Further verification of findings and suggestions presented here would require the inclusion into the pairwise comparisons of all known human MHC I structures. Preliminary calculations for murine MHC I indicate a currently insufficient number of high resolution structures for carrying out a similar analysis of the murine MHC I X-ray data.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 201-836-7960. E-mail: [email protected]. Funding

This work has been supported by NIH Grants R01GM072014, and NSF Grant MCB-1021785. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Sambit Mishra at Iowa State University for his assistance in producing protein structures images; we also thank Dr. Brian M. Baker of the University of Notre Dame and Dr. Kannan Natarajan of NIH for their help in finding recent relevant publications in the field.

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ABBREVIATIONS TCR, T-cell receptor; TCR A6, TCR specific to a particular group of antigens; MHC I, major histocompatibility complex, Class I; HLA-A2, most common human MHC I allele expressed by half of humans worldwide; Ag, polypeptide antigen; α1, α2, and α3 domains, terms used in immunological publications for three fragments (about 90 residues each) of the MHC I “heavy” α-chain; β2m, β2 microglobulin of about 100 residues - “light” chain of MHC I; CD8, coreceptor of TCR; PDB, Protein Data Bank; DM, matrix of Cα−Cα distances in a protein conformation; DDM, matrix of differences between elements of two DMs; DD, element of a DDM; RMS, rootmean-square; RMSDD, RMS of all elements of a DDM; Tax, peptide with sequence LLFGYPVYV bound to MHC I HLAA2 protein dimer; HuD, peptide with sequence LGYGFVNYI bound to MHC I HLA-A2 protein dimer; variants of Tax and HuD and other MHC bound peptides are presented in the text; SC1, SC2, SC3, are groups of MHC I HLA-A2 with highly similar conformations of the α-chains bound to different peptide Ag-s

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REFERENCES

(1) Male, D., Brostoff, J., Roth, D., and Roitt, I. Immunology, 7th ed., Elsevier, Amsterdam, 2006. (2) Burmester, G.-R. and Pezzutto, A. Color Atlas of Immunology, Thieme, NY, 2003. (3) Garcia, K. C. (2012) Insights into immune structure, recognition, and signaling. Immunol Rev. 250, 5−9. (4) Madden, D. R. (1995) Annu. Rev. Immunol. 13, 587−622. (5) Rudolph, M. C., Stanfield, R. L., and Wilson, I. A. (2006) How TCRs bind MHCs, peptides, and coreceptors. Annu. Rev. Immunol. 24, 419−466. (6) Garcia, K. C., and Adams, E. J. (2005) How the T cell receptor sees antigen − a structural view. Cell 122, 333−336. (7) Gao, G. F., Rao, Z., and Bell, J. I. (2002) Molecular coordination of αβ T-cell receptors and coreceptors CD8 and CD4 in their recognition of peptide-MHC ligands. Trends Immunol. 23, 408−413. (8) van der Merwe, P. A., and Davis, S. J. (2002) The immunological synapse − a multitasking system. Science 295, 1479−1480. (9) Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N., and Bourne, P. E. (2000) The Protein Data Bank. Nucleic Acids Res. 28, 235−242. (10) Murzin, A. G., Brenner, S. E., Hubbard, T., and Chothia, C. (1995) SCOP: a structural classification of proteins database for the investigation sequences and structures. J. Mol. Biol. 247, 536−540 [PDF]. (11) Madden, D. R., Garbotzi, D. N., and Wiley, D. C. (1993) The antigenic identity of peptide-MHC complexes: a comparison of the

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DOI: 10.1021/acs.biochem.5b01077 Biochemistry 2016, 55, 167−185

Article

Biochemistry conformations of five viral peptides presented by HLA-A2. Cell 75, 693−708. (12) Borbulevych, O., Piepenbrink, K., Gloor, B., Scott, D., Sommese, R., Cole, D., Sewell, A., and Baker, B. M. (2009) T cell receptor crossreactivity directed by antigen-dependent tuning of peptide-MHC molecular flexibility. Immunity 31, 885−896. (13) Borbulevych, O., Piepenbrink, K. H., and Baker, B. M. (2011) Conformational melding permits a conserved binding geometry in TCR recognition of foreign and self molecular mimics. J. Immunol. 186, 2950−2958. (14) Gao, G. F., Tormo, J., Gerth, U. C., Wyer, J. R., McMichael, A. J., Stuart, D. J., Bell, J. I., Jones, E. Y., and Jakobsen, B. K. (1997) Crystal structure of the complex between human CD8αα and HLAA2. Nature 387, 630−634. (15) Gao, G. F., and Jakobsen, B. K. (2000) Molecular interactions of coreceptor CD8 and MHC class I: the molecular basis for functional coordination with the T-cell receptor. Immunol. Today 21, 630−636. (16) Kern, P. S., Teng, M-k., Smolyar, A., Liu, J-h., Liu, L., Hussey, R. E., Spoerl, R., Chang, H.-C., Reinherz, E. L., and Wang, J.-H. (1998) Structural basis of CD8 coreceptor function revealed by crystallographic analysis of a murine CD8αα ectodomain fragment in complex with H-2Kb. Immunity 9, 519−530. (17) Wang, R., Natarajan, K., and Margulies, D. H. (2009) Structural basis of the CD8αβ/MHC class I interactions: focused recognition orients CD8β to a T cell proximal position. J. Immunol. 183, 2554− 2564. (18) Wooldridge, L., van den Berg, H. A., Glick, M., Gostick, E., Laugel, B., Hutchinson, S. H., Milicic, A., Brenchley, J. M., Douek, D. C., Price, D. A., and Sewell, K. (2005) Interaction between the CD8 coreceptor and MHC I stabilizes T cell receptor-antigen complexes on the cell surface. J. Biol. Chem. 280, 27491−27501. (19) Wooldridge, L., Laugel, B., Ekeruche, J., Clement, M., van den Berg, H. A., Price, D. A., and Sewell, K. (2010) CD8 controls T-cell reactivity. J. Immunol. 185, 4625−4632. (20) Salter, R. D., Benjamin, R. J., Wesley, P. K., Buxton, S. E., Garrett, T. P. J., Clayberger, C., Krensky, A. M., Norment, A. M., Littman, D. R., and Parham, P. (1990) A binding site for T-cell Coreceptor CD8 on the α3 domain of HLA-A2. Nature 345, 41−46. (21) Sun, J., Leahy, D. J., and Kavathas, P. B. (1995) Interaction between CD8 and MHC Class I mediated by multiple contact surfaces that include the α2 and α3 domains of MHC Class I. J. Exp. Med. 182, 1275−1280. (22) Cole, D. K., and Gao, G. F. (2004) CD8: adhesion molecule, coreceptor and immuno-modulator. Cell. Mol. Immunol. 1, 81−88. (23) Cheroutre, H., and Lambolez, F. (2008) Doubting the TCR coreceptor function of CD8αα. Immunity 28, 149−159. (24) Gras, S., Saulquin, X., Reiser, J.-B., Debeaupuis, E., Echasserieau, K., Kissenpfennig, A., Legoux, F., Chouquet, A., Le Gorrec, M., Machillot, P., Neveu, B., Thielens, N., Malissen, B., Bonneville, M., and Housset, D. (2009) Structural basis for the affinity-driven selection of a Public TCR against a dominant human cytomegalovirus epitope. J. Immunol. 183, 430−437. (25) Favre, D., Stoddart, C. A., Emu, B., Hoh, R., Martin, J. N., Hecht, F. M., Deeks, S. G., and McCune, J. M. (2011) HIV disease ̈ progression correlates with the generation of dysfunctional naive CD8low T cells. Blood 117, 2189−2199. (26) Hawse, W. F., Gloor, B. E., Ayres, C. M., Kho, K., Nuter, E., and Baker, B. M. (2013) Peptide modulation of class I major histocompatibility complex molecular flexibility and the implications for immune recognition. J. Biol. Chem. 288, 24372−24381. (27) Rashin, A. A., Rashin, A. H. L., and Jernigan, R. (2009) Protein flexibility: coordinate uncertainty and interpretation of structural differences. Acta Crystallogr., Sect. D: Biol. Crystallogr. 65, 1140−1161. (28) Rashin, A. A., Rashin, A. H. L., and Jernigan, R. (2010) Diversity of function-related conformational changes in proteins: coordinate uncertainty, fragment rigidity and stability. Biochemistry 49, 5683− 5704.

(29) Janin, J., and Wodak, S. J. (1983) Structural domains in proteins and their role in the dynamics of protein function. Prog. Biophys. Mol. Biol. 42, 21−78. (30) Gerstein, M., Lesk, A., and Chothia, C. (1994) Structural mechanisms for domain-domain movements in proteins. Biochemistry 33, 6739−6749. (31) Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G. W., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S., Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A., and Wilson, K. S. (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr., Sect. D: Biol. Crystallogr. 67, 235−242. (32) Richardson, J. S. and Richardson, D. C. (1989) Principles and patterns of Protein conformation. In Prediction of Protein Structure and the Principles of Protein Conformation, Fasman, G. D., Ed., Plenum, NY.. (33) The PyMOL Molecular Graphics System, Version 1.5.0.3, Schrödinger, LLC. (34) Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., and Ferrin, T. E. (2004) USCF Chimera − a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605−1612. (35) Celie, P. H. N., Toebes, M., Rodenko, B., Ovaa, H., Perrakis, A., and Schumacher, T. N. M. (2009) Crystal structure of MHC class I HLA-A2.1 bound to a photocleavable peptide. J. Am. Chem. Soc. 131, 12298−12304. (36) Rodenko, B., Toebes, M., Celie, H. N., Perrakis, A., Schumacher, T. N. M., and Ovaa, H. (2009) Crystal structure of MHC class I HLAA2.1 bound to a photocleavable peptide. J. Am. Chem. Soc. 131, 12305−12313. (37) Borbulevych, O., Do, P., and Baker, B. M. (2010) Structures of native and affinity-enhanced WT1 epitopes bound to HLA-A*02101: implications for WT1-based cancer theraputics. Mol. Immunol. 47, 2519−2524. (38) Rashin, A. A., Domagalski, M. J., Zimmermann, M. T., Minor, M., Chruszcz, M., and Jernigan, R. L. (2014) Factors correlating with significant differences between X-ray structures of myoglobin. Acta Crystallogr., Sect. D: Biol. Crystallogr. 70, 481−491. (39) Fraser, J. S., van der Bedem, H., Samelson, A. J., Lang, P. T., Holton, J. M., Echols, N., and Alber, T. (2011) Accessing protein conformational ensembles using room-temperature X-ray crystallography. Proc. Natl. Acad. Sci. U. S. A. 108, 16247−16252. (40) Crystallography at 100. (2014) Science 343, 1091−1116. (41) Rose, H. (2014) The owner of a small book store in Teaneck, NJ, suggested to me this analogy between a neglect of α3 vs α1 and α2 domains and the initial neglect of Cinderella vs her two half sisters. (42) Kondrashov, D. A., Zhang, W., Aranda, A. I. V., Stec, B., and Phillips, G. N., Jr. (2008) Sampling of the native conformational ensemble of myoglobin via structures in different crystalline environments. Proteins: Struct., Funct., Genet. 70, 353−362. (43) Cole, D. K., Dunn, S. M., Sami, M., Boulter, J. M., Jakobsen, B. K., and Sewell, A. K. (2008) T cell receptor engagement of peptidemajor histocompatibility complex class I does not modify CD8 binding. Mol. Immunol. 45, 2700−2709. (44) Cole, D. K., Laugel, B., Clement, M., Price, D. A., Wooldridge, L., and Sewell, A. K. (2012) The molecular determinants of CD8 coreceptor function. Immunology 137, 139−148. (45) Scott, D. R., Borbulevych, O. Y., Piepenbrink, K. H., Corcelli, S. A., and Baker, B. M. (2011) Disparate degrees of hypervariable loop flexibility control T-cell receptor cross-reactivity, specificity, and binding mechanism. J. Mol. Biol. 414, 385−400.

185

DOI: 10.1021/acs.biochem.5b01077 Biochemistry 2016, 55, 167−185