Peptide-binding Cleft Research Articles

Mutational Analysis of Conserved Residues in the GCN5 Family of Histone Acetyltransferases

GCN5 is a critical transcriptional co-activator and is the defining member of a large superfamily of N-acetyltransferases. GCN5 catalyzes the transfer of an acetyl group from acetyl-CoA to the epsilon-amino of lysine 14 within the core H3 histone protein. Previous biochemical analyses have indicated a fully ordered kinetic mechanism. Recent structural studies have implicated several conserved residues in catalysis and substrate binding. Here the roles of Glu-173, His-145, and Asp-214 in yeast GCN5 have been evaluated using site-directed mutagenesis, steady state and pre-steady state kinetics, pH analysis, isotope partitioning, and equilibrium binding studies. The results with wild type and E173Q, H145A, and D214A mutants are consistent with chemical catalysis being rate-determining in turnover. All mutants exhibited K(d) values (3.5-8.5 microm) for AcCoA that were similar to wild type enzyme, indicating no functional role for these residues in AcCoA binding. The E173Q mutant demonstrated a approximately 500-600-fold decreases in k(cat) and k(cat)/K(m),(H3), consistent with Glu-173 acting as the general base catalyst as proposed previously. No significant effect was observed on substrate binding steps. His-145 was identified as a residue in the peptide binding cleft that must be unprotonated (pK(a) = 5.8) for peptide binding and likely hydrogen-bonds to the Ser-10 hydroxyl of histone H3. His-145 also contributes to lowering the pK(a) value (by 0.8 units) of general base Glu-173 through a water-mediated hydrogen bond to the carboxylate side chain. Analysis of D214A revealed an obligate protein isomerization step that occurs after AcCoA binding and permits efficient peptide binding. Asp-214 is part of a conformationally flexible loop that mediates the isomerization by stabilizing distinct conformers of the protein.

Regulation of encephalitogenic T cells with recombinant TCR ligands.

We have previously described recombinant MHC class II beta1 and alpha1 domains loaded with free antigenic peptides with potent inhibitory activity on encephalitogenic T cells. We have now produced single-chain constructs in which the peptide Ag is genetically encoded within the same exon as the linked beta1 and alpha1 domains, overcoming the problem of displacement of peptide Ag from the peptide binding cleft. We here describe clinical effects of recombinant TCR ligands (RTLs) comprised of the rat RT1.B beta1alpha1 domains covalently linked to the 72-89 peptide of guinea pig myelin basic protein (RTL-201), to the corresponding 72-89 peptide from rat myelin basic protein (RTL-200), or to cardiac myosin peptide CM-2 (RTL-203). Only RTL-201 possessed the ability to prevent and treat active or passive experimental autoimmune encephalomyelitis. Amelioration of experimental autoimmune encephalomyelitis was associated with a selective inhibition of proliferation response and cytokine production by Ag-stimulated lymph node T cells and a drastic reduction in the number of encephalitogenic and recruited inflammatory cells infiltrating the CNS. The exquisitely selective inhibition could be observed between molecules that differ by a single methyl group (the single amino acid residue difference between RTL-200 (threonine) and RTL-201 (serine) at position 80 of the myelin basic protein peptide). These novel RTLs provide a platform for developing potent and selective human diagnostic and therapeutic agents for treatment of autoimmune disease.

HLA-DQ locus of the human leukocyte antigen complex and type 1 diabetes mellitus: a HuGE review.

The human leukocyte antigen (HLA) complex is located on the short arm of chromosome 6 at p21.3 (1-4). It encompasses approximately 3,500 kilobases of DNA and contains at least 150 genes. It is the primary region of susceptibility for type 1 diabetes mellitus, as well as other autoimmune disorders. Recent genome screens have designated the class II subregion (i.e., the HLA-DR, HLA-DQ, and HLA-DP loci) IDDM1. The DQ locus, which is the focus of this review, consists of two tightly linked genes (DQA1 and DQB1) that encode a and fj glycoproteins, respectively. These molecules combine noncovalently to form functional a-(J heterodimers. The DQA1 and DQB1 genes are highly polymorphic. Allelic variation is observed primarily in the second exon, which corresponds to the peptide-binding cleft. HLA-DQ and other class II molecules present extracellular antigens to helper T cells and stimulate the body's immune response. They have a restricted tissue distribution and are located mainly on macrophages, B cells, and activated T cells. Transcription of DQA1 and DQB1 is complex and involves cisand frans-acting factors. Critical upstream regulatory sequences have been reported for DQA1 and DQB1. Variation in promoter sequences affects gene expression and may also be involved in the pathogenesis of autoimmune disorders. In addition, posttranscription activities appear to influence disease risk. For example, functional DQa$ heterodimers can be formed from the noncovalent association of products of DQA1 and DQB1 genes in cis (5). Alternatively, the combined a and (3 glycoproteins can represent molecules encoded by genes in trans. Hybrid DQ molecules with DR or DP glycoproteins have also been observed.

Determinants of the Peptide-induced Conformational Change in the Human Class II Major Histocompatibility Complex Protein HLA-DR1

The human class II major histocompatibility complex protein HLA-DR1 has been shown previously to undergo a distinct conformational change from an open to a compact form upon binding peptide. To investigate the role of peptide in triggering the conformational change, the minimal requirements for inducing the compact conformation were determined. Peptides as short as two and four residues, which occupy only a small fraction of the peptide-binding cleft, were able to induce the conformational change. A mutant HLA-DR1 protein with a substitution in the beta subunit designed to fill the P1 pocket from within the protein (Gly(86) to Tyr) adopted to a large extent the compact, peptide-bound conformation. Interactions important in stabilizing the compact conformation are shown to be distinct from those responsible for high affinity binding or for stabilization of the complex against thermal denaturation. The results suggest that occupancy of the P1 pocket is responsible for partial conversion to the compact form but that both side chain and main chain interactions contribute to the full conformational change. The implications of the conformational change to intracellular antigen loading and presentation are discussed.

Poor Binding of a HER-2/neu Epitope (GP2) to HLA-A2.1 Is due to a Lack of Interactions with the Center of the Peptide

Class I major histocompatibility complex (MHC) molecules bind short peptides derived from proteins synthesized within the cell. These complexes of peptide and class I MHC (pMHC) are transported from the endoplasmic reticulum to the cell surface. If a clonotypic T cell receptor expressed on a circulating T cell binds to the pMHC complex, the cell presenting the pMHC is killed. In this manner, some tumor cells expressing aberrant proteins are recognized and removed by the immune system. However, not all tumors are recognized efficiently. One reason hypothesized for poor T cell recognition of tumor-associated peptides is poor binding of those peptides to class I MHC molecules. Many peptides, derived from the proto-oncogene HER-2/neu have been shown to be recognized by cytotoxic T cells derived from HLA-A2(+) patients with breast cancer and other adenocarcinomas. Seven of these peptides were found to bind with intermediate to poor affinity. In particular, GP2 (HER-2/neu residues 654-662) binds very poorly even though it is predicted to bind well based upon the presence of the correct HLA-A2.1 peptide-binding motif. Altering the anchor residues to those most favored by HLA-A2.1 did not significantly improve binding affinity. The crystallographic structure shows that unlike other class I-peptide structures, the center of the peptide does not assume one specific conformation and does not make stabilizing contacts with the peptide-binding cleft.

Cis-element dependence and occupancy of the human invariant chain promoter in CIITA-dependent and -independent transcription

The major histocompatibility complex (MHC)-associated invariant chain (Ii) associates with the class II α/β heterodimer during its biosynthesis, inhibiting association of endogenous peptides with the peptide-binding cleft. It is therefore not surprising that there are significant similarities in regulatory mechanisms controlling the expression of the structural class II MHC and Ii genes. One important similarity is that both classes of genes can be expressed via CIITA-dependent or -independent mechanisms. In this report, we have dissected CIITA-dependent and -independent transcription of the Ii gene using an isogenic B-LCL cell pair (Jijoye and clone-13) which do or do not express the class II MHC transactivator (CIITA), respectively. Experiments using mutant or deletion constructs of the Ii gene promoter indicate that while both the X-box and Ii-κB1 elements are critical for CIITA-dependent transcription in B lymphocytes, the Ii-κB1 element is of greater importance for CIITA-independent Ii gene transcription, with the X-box playing a secondary role. Despite these clear differences in cis-element dependence of CIITA-dependent and -independent Ii transcription, there are only subtle differences in the occupancy of these elements in vivo as assessed by genomic footprinting. These differences are restricted to occupancy of the X-box and Y-box, with which the RF-X and NF-Y complexes interact in Ii-positive cells. This difference in the occupancy of the X-box and Y-box in this cell pair indicates that while protein/protein interactions between CIITA and promoter-bound factors stabilize promoter occupancy, these interactions are not absolutely required for occupancy and transcription of the invariant chain gene.

Extensive Alanine Substitutions Increase Binding Affinity of an Influenza Nucleoprotein Peptide to HLA-Aw68 and Do Not Abrogate Peptide-Specific CTL Recognition

Class I MHC molecules bind peptides in the endoplasmic reticulum and present them at the cell surface to circulating CD8+ T cells for analysis. We have examined binding of peptides and stabilization of HLA-Aw68 class I molecules using synthetic peptide variants of an influenza virus nucleoprotein peptide, NP91-99 (KTGGPIYKR). We have demonstrated that insertion of increasing numbers of alanines in the center of the peptide (between P and I), to increase a natural bulging out of the peptide-binding cleft, results in a large decrease in thermal stability. Although there is a great decrease in the t 1/2 of the MHC/peptide complex for NP-1A compared with NP91-99, a T cell line, stimulated by NP91-99, recognizes NP-1A efficiently. Peptide variants with three or more alanines do not show saturable binding to HLA-Aw68 and also are not recognized by the T cell line. Thermal studies show that polyalanine peptides with minimal anchors and nearly all TCR contact residues exchanged stabilized HLA-Aw68 to high temperatures. Additionally, some of these polyalanine peptides are recognized by T cell lines generated against NP91-99. Analysis of the peptide sequences show that the stabilization effects are not due to the hydrophobicity of the peptide. These data suggest that the strength of binding of peptides to HLA-Aw68 is not only dictated by the presence of anchor residues but also by the lack of unfavorable contacts between the peptide ligand and class I MHC-binding cleft.

A Region of Conformational Variability Outside the Peptide-Binding Site of a Class I MHC Molecule

Peptide binding is known to influence the conformation of the surface of class I molecules as detected with mAbs and TCR. A new conformationally sensitive epitope on the mouse class I molecule Kb is defined by mAb AF6-88.5. The recognized structure is affected by amino acid substitutions in any of the three external domains of the class I heavy chain and, in addition, is influenced by the substitution of human for mouse beta2-microglobulin. Interestingly, the epitope for this Ab is not affected by mutations within the peptide-binding cleft or by the nature of the peptide bound. These findings indicate that the effect of a change in one domain of class I can radiate to other parts of the molecule. Furthermore, the existence of conformationally sensitive structures outside of the peptide-binding site suggests the possibility that class I molecules may change their structure in response to binding by receptors and ligands such as the TCR and the coligand CD8. Such structural changes may represent signals that can influence cellular activation events.

Structural Aspects of the Interaction Between Heterogeneic Human Papillomavirus Type 1 E4-Specific T Cell Receptors and the Same Peptide/HLA-DQ8 Complex

TCR usage has been studied in a panel of Th cell clones specific for the same peptide epitope (P N S Q D R G R P R R S D), derived from the human papillomavirus type 1 (HPV1) E4 protein, and restricted through HLA-DQ8. After identifying the V, D, and J genes used by the TCRs and sequencing across the V(D)J junctions, five different alpha-chain sequences and five different beta-chain sequences, comprising six independent clones, were identified. A structural model of our E4 peptide/HLA-DQ8 complex predicted that the guanidinyl side chain on the arginine residue at position 6 of the peptide could exist in different orientations. An intramolecular interaction between this arginine and the glutamine residue at position four appeared to control this orientation. Interacting HPV1 E4-specific TCRs would therefore have to recognize the complex in different conformations, and molecular modeling of the TCRs suggested that this could be achieved by changing the dimensions of the central pocket formed where the CDR3 loops of the TCR alpha- and beta-chains converge. It is known that interactions between bound peptide and amino acid residues lining the peptide-binding cleft of HLA molecules are important for determining the conformation and orientation of the peptide/MHC complex. The suggestion here that intramolecular interactions between amino acids of close proximity on the bound peptide are also important adds a further level of complexity to the mechanism by which TCRs interact with Ag.

T cells discriminate between differentially phosphorylated forms of alphaB-crystallin, a major central nervous system myelin antigen.

Factors such as developmental stage or physiological and infectious stress may change patterns of post-translational protein modification. In order to determine whether such regulated types of modification may influence T cell responsiveness to self proteins we examined the T cell response of SJL (H-2s) mice to alphaB-crystallin, a small heat shock protein that can exist in differentially phosphorylated forms. Epitope mapping revealed the presence of two T cell epitopes that are presented by I-As. One major epitope including residues 41-56 contains an amino acid residue (Ser45) that can be phosphorylated as the result of aging or stress. Accordingly, T cells from SJL mice discriminate between preparations of alphaB-crystallin that differ in their extent of phosphorylation at the level of whole protein as well as at the level of determinant-specific responses. Phosphorylation at Ser45 does not prevent binding of the peptide 41-56 to I-As and computer-assisted modelling of the peptide-MHC complex suggests that the phosphate group of the bound peptide extends outwards from the peptide-binding cleft and may thus be available for direct contact with TCR. Together, our data provide evidence that stress-inducible phosphorylation of alphaB-crystallin creates neo-determinants for T cells and, therefore, may contribute to the breakdown of peripheral tolerance to this self protein.

Evidence that the autoimmune antigen myelin basic protein (MBP) Ac1-9 binds towards one end of the major histocompatibility complex (MHC) cleft.

The NH2-terminal peptide of myelin basic protein (MBP) bound to the class II major histocompatibility complex (MHC) protein I-Au is an immunodominant epitope in experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis. However, the MBP-I-Au complex is very unstable. To investigate this, we performed site-directed mutagenesis of the I-Au MHC protein and the MBP peptide. Biochemical, T cell activation, and molecular modeling studies of mutant complexes demonstrate that the MBP peptide's key residue for MHC binding, lysine 4, is buried in the P6 pocket of I-Au, which is predominantly hydrophobic. This implies that the MBP-I-Au complex differs from more stable complexes in two respects: (a) the peptide leaves the NH2-terminal region of the MHC peptide-binding cleft unoccupied; (b) the peptide is not anchored by typical favorable interactions between peptide side chains and MHC pockets. To test these hypotheses, a modified MBP peptide was designed based on molecular modeling, with the aim of producing strong I-Au binding. Extension of the NH2 terminus of MBP with six amino acids from the ova peptide, and replacement of the lysine side chain in the P6 pocket with an aromatic anchor, results in >1,000-fold increased binding stability. These results provide an explanation for the unusual peptide-MHC-binding kinetics of MBP, and should facilitate an understanding of why mice are not tolerant to this self-peptide- MHC complex.

HLA transgenic mice as humanized mouse models of disease and immunity.

The application of molecular genetics to human disease in the past decade has been helpful in elucidating the genetic influences involved in the induction and pathogenesis of various autoimmune diseases. Among the numerous genes studied for their role in disease development, polymorphisms within HLA class I and class II loci play a significant role in predisposition to disease. HLA molecules are encoded by genes on the short arm of chromosome 6. Crystal structures of MHC molecules show a peptide binding cleft which contains the variable region of MHC molecules. Genetic polymorphism at the MHC determines the specificity and affinity of peptide binding and T cell recognition. HLA molecules play a pivotal role in T cell repertoire selection in the thymus and antigen presentation in the periphery. Analysis of T cell responses in humans has involved the use of T cell lines or clones in vitro from naturally primed individuals. On the other hand, MHC restriction, mapping of epitope recognition, and T cell function in murine systems have been determined by in vivo studies. Various experimental animal models of autoimmune diseases have been studied, contributing greatly to our basic understanding of the disease. For example, type II collagen‐induced arthritis (CIA) 1 in mice and rats has been used as an experimental model for RA. Even though the model differs in the manner polyarthritis is induced, disease expression is broadly similar to RA, with the occurrence of symmetrical peripheral polyarthritis and systemic inflammation. Although growing knowledge of the functions of T and B cells in the immune response has shed light on their role in disease induction, the pathogenic mechanisms of most autoimmune diseases remain unresolved. There are many unanswered questions as to how tolerance to self is usually maintained, because autoreactive T cells can be found in normal as well as diseased individuals. How is tolerance broken in autoimmunity? Do specific autoantigens trigger the immune system to mount tissue-destructive responses? These questions need to be solved for most of the autoimmune diseases. Although the strongest MHC association with an autoimmune disease is between HLA class I B27 and spondyloarthropathies, class II alleles are implicated in most other cases. For example, RA is strongly associated with alleles of the DRB1 locus, whereas diabetes shows a stronger association with DQB1 alleles. HLA-DR and -DQ alleles are inherited en bloc and are known to occur in linkage disequilibrium. To better understand the role of HLA molecules in autoimmune diseases, transgenic animals expressing human HLA genes associated with disease have been developed. The generation of transgenic mice expressing functional HLA molecules has been an important step toward the creation of an in vivo model for enhancing our understanding of the function of human molecules in disease induction and predisposition. The potential value of HLA transgenic animals as a “humanized” disease model was first illustrated in the HLA-B27

Engineering cyclophilin into a proline-specific endopeptidase.

Designing an enzyme requires, among a number of parameters, the appropriate positioning of catalytic machinery within a substrate-binding cleft. Using the structures of cyclophilin-peptide complexes, we have engineered a new catalytic activity into an Escherichia coli cyclophilin by mutating three amino acids, close to the peptide binding cleft, to form a catalytic triad similar to that found in serine proteases. In conjunction with cyclophilin's specificity for proline-bearing peptides, this creates a unique endopeptidase, cyproase 1, which cleaves peptides on the amino-side of proline residues. When acting on an Ala-Pro dipeptide, cyproase 1 has an efficiency (kcat/Km) of 0.7 x 10(4) M(-1) s(-1) and enhances the rate of reaction (kcat/kuncat) 8 x 10(8)-fold. This activity depends upon a deprotonated histidine and is inhibited by nucleophile-specific reagents, as occurs in natural serine proteases. Cyproase 1 can hydrolyse a protein substrate with a proline-specific endoprotease activity.

Rigidification of the alpha2 helix of an MHC class I molecule by a valine to proline mutation in position 165 does not prevent peptide-specific antigen presentation.

Although classical MHC class I glycoproteins bind peptide Ags for display at the cell surface, some MHC class I-related molecules such as the neonatal Fc receptor (FcRn) execute their function without binding peptide ligands. The three-dimensional structure of the FcRn suggested that a substitution of the conserved valine at position 165 of the alpha2 helix by proline contributed to a kink in the position of this helix relative to the alpha1 helix, and resulted in closing of the potential peptide-binding cleft. To test the contribution of proline 165 to the occlusion of the cleft and the binding of potential antigenic peptides, we introduced this mutation into the classical murine MHC class I molecule, H-2Dd, and characterized the ability of such a mutant to present peptide Ags to either a peptide-specific, H-2Dd-restricted T cell hybridoma (B4.2.3), or an allospecific, peptide-dependent, T cell hybridoma (3DT52.5.8). We show that the V165P mutation, expressed at the cell surface either in H-2Dd or in a single chain membrane version of H-2Dd, fails to eliminate recognition of the peptide/MHC complexes by two different T cells. Evaluation of a panel of synthetic substituted peptides suggests that subtle differences in the fine specificity of presentation can be discerned. Thus, the proline substitution at position 165 of FcRn and some other class I-like molecules is not the sole cause of the lack of peptide presentation.

Critical role of Val/Gly86 HLA-DRB dimorphism in the neonatal resistance or susceptibility to maternal hepatitis C virus infection.

There is evidence that hepatitis C virus (HCV) may be vertically transmitted from infected mothers to their children, although it is uncertain to what extent maternal transmission occurs.1 Major maternal risk factors include the HIV-HCV coinfection and a high HCV-RNA titer at delivery; moreover viral subtypes 1b and 3a seem to be most frequently transmitted.2, 3 To evaluate the influence of fetal genetic factors on the acquisition of infection, we examined the polymorphisms of the major histocompatibility complex in 24 mother-infant pairs. We studied 24 children born to HCV-infected mothers, 7 of whom had vertically HCV-infected (PCR for HCV positive and active antibodies to the virus after 12 months of follow-up at least) infants and 17 reverted from seropositivity to seronegativity between 9 and 12 months of age. HLA-DRB1* low resolution genomic typing was performed by PCR-reverse dot-blot whereas PCR-sequence-specific primers was applied for DRB1*, DRB3*, DRB4*, DRB5* high resolution and for DQA1* and DQB1* definition. The most striking difference in HLA gene frequencies of HCV-positive children compared with those of seroreverters was in the HLA-DRB1* 1101 and 1104 genes. The HLA-DRB1*1101 allele was significantly associated with longstanding HCV positivity [21.42% in HCV-positive children vs. 2.9% in HCV-negative children; chi square, 4.44; P = 0.035; odds ratio (OR), 9.00]; conversely the HLA DRB1* 1104 seemed to be related with resistance to infection (0% in HCV-positive vs. 20.5% in HCV-negative; chi square, not significant; P not significant; OR, not determined). No difference was found in the HLA-DQA1 and DQB1 allele distribution. Because the only difference between these two DRB1* 11 subtypes (1101, 1104) is in the glycine/valine dimorphism at position 86 of the antigen-binding site which plays a pivotal role in allorecognition,4 we studied the DRβ86V and DRβ86G frequencies in the HCV-positive children and in the seroreverters. The DRB1*Gly86 frequency was significantly higher in the persistently infected subjects (85.7% vs. 52.9%; chi square, 4.54; P = 0.033; OR = 5.33), whereas valine appeared more frequently in the seroreverters (47.05% vs. 14.3%; chi square, 4.54; P = 0.033; OR = 0.19). We also noticed that the DRβ86VV homozygous genotype was present only in the seroreverters (23.5% vs. 0% in the HCV-positive children); on the contrary the frequency of DRβ86GG appeared increased in the HCV-infected children (71.4% vs. 29.4%) (Table 1).TABLE 1It appears that DRβ86 has a critical role in fetal susceptibility or resistance to maternal HCV infection. We suggest that valine in DRβ86 alters the conformation of the peptidebinding cleft so that it does not permit the arrangement of a steady complex with the viral peptides and results in a low risk of HCV infection. In support of the hypothesis that DRβ86V correlates with infant protection from viral infection, it has been recently observed that in infants born to HIV-infected mothers, DRB1*1501 and DRB1* 1301 alleles (both Val86) were significantly associated with seroreversion status.5 Although our results must be considered preliminary, they could form a useful starting point for more detailed investigations regarding the genetic susceptibility to virus C infection in infants born to infected mothers. Miryam Martinetti, B.S. Ilaria Pacati, M.D. Cristina Daielli, B.S. Laura Salvaneschi, M.D. Anna Maccabruni, M.D. HLA Laboratory; Department of Transfusion Medicine (MM, CD, LS); Institute of Infectious Diseases (IP, AM); University of Pavia; IRCCS Policlinico S. Matteo; Pavia, Italy

Three newly identified A*24 alleles: A*2406, A*2413 and A*2414

Three previously unknown A24-related alleles were identified by PCR-SSO typing and confirmed by DNA sequencing in Australian Aboriginal populations (A*2406, 2413) and in individuals of South American descent (A*2414). A*2406 and A*2413 both have two adjacent (but different) nucleotide substitutions in codon 156 in exon 3 compared to A*2402, resulting in a single amino acid replacement in each allele. The South American A*2414 is apparently a hybrid between A2 and A24 with a segment of the A*24 sequence between codons 95 and 107 in exon 3 replaced with the A*02 sequence. Interallelic sequence exchange is the most likely mechanism in the generation of all three novel alleles. Compared to A*2402, the four amino acid substitutions in the A*2414 molecule would be expected to significantly change the shape of the peptide binding cleft, leading to selection of different peptide ligands. The single amino acid replacements in position 156 of the two Australian Aboriginal A*24 alleles may also have significant functional effects. In particular, Trp replacing Gln in position 156 (A*2406) is predicted to markedly reduce the volume of the peptide binding cleft, influence the interaction of HLA pockets with peptide side chains, and therefore, cause major changes in peptide presentation. These newly defined alleles may reflect the adaptive process of HLA genes to local environments.

A molecular clamp in the crystal structure of the N-terminal domain of the yeast Hsp90 chaperone.

Hsp90 is a highly specific chaperone for many signal transduction proteins, including steroid hormone receptors and a broad range of protein kinases. The crystal structure of the N-terminal domain of the yeast Hsp90 reveals a dimeric structure based on a highly twisted sixteen stranded beta-sheet, whose topology suggests a possible 30-domain-swapped structure for the intact Hsp90 dimer. The opposing faces of the beta-sheets in the dimer define a potential peptide-binding cleft, suggesting that the N-domain may serve as a molecular 'clamp' in the binding of ligand proteins to Hsp90.

HLA-DQB1 codon 57 is critical for peptide binding and recognition.

The association of specific HLA-DQ alleles with autoimmunity is correlated with discrete polymorphisms in the HLA-DQ sequence that are localized within sites suitable for peptide recognition. The polymorphism at residue 57 of the DQB1 polypeptide is of particular interest since it may play a major structural role in the formation of a salt bridge structure at one end of the peptide-binding cleft of the DQ molecules. This polymorphism at residue 57 is a recurrent feature of HLA-DQ evolution, occurring in multiple distinct allelic families, which implies a functional selection for maintaining variation at this position in the class II molecule. We directly tested the amino acid polymorphism at this site as a determinant for peptide binding and for antigen-specific T cell stimulation. We found that a single Ala-->Asp amino acid 57 substitution in an HLA-DQ3.2 molecule regulated binding of an HSV-2 VP-16-derived peptide. A complementary single-residue substitution in the peptide abolished its binding to DQ3.2 and converted it to a peptide that can bind to DQ3.1 and DQ3.3 Asp-57-positive MHC molecules. These binding studies were paralleled by specific T cell recognition of the class II-peptide complex, in which the substituted peptide abolished T cell reactivity, which was directed to the DQ3.2-peptide complex, whereas the same T cell clone recognized the substituted peptide presented by DQ3.3, a class II restriction element differing from DQ3.2 only at residue 57. This structural and functional complementarity for residue 57 and a specific peptide residue identifies this interaction as a key controlling determinant of restricted recognition in HLA-DQ-specific immune response.

Influence on antibody recognition of amino acid substitutions in the cleft of HLA-DQ2 molecules suggestive evidence of peptide-dependent epitopes

The purpose of this study was to identify polymorphic residues of DQ2 β chains which are involved in the epitopes recognized by DQ2-reactive mAbs. Binding studies were performed on a panel of DQ2 mutant cells made by transfection of a DQ (α 1 ∗050l,β 1 ∗0301)-positive B-LCL with DQB1 genes encoding either wildtype or mutated DQβ 1 ∗0202. Several aa substitutions were found to influence the binding of DQ2 mAbs. All but one of the mAbs were clearly sensitive to the introduced aa substitutions, but individual mAbs were differentially influenced, suggesting that they recognized different epitopes on the DQ2 molecule. Substitutions in positions 30 and 37 of the peptide-binding cleft were also found to influence the binding of some mAbs. Second, two mAbs, NFLD.M71 and NFLD.M102, which bound to DQ(α1 ∗0501,β1 ∗0202) expressed in human cells and were sensitive to the introduction of aa substitutions in the cleft, bound weakly to the DQ(α1 ∗0501,β1 ∗0202) heterodimer when expressed in a transfected murine NIH 3T3 cell line. Together this indicates that some DQ2-reactive mAbs may recognize peptide-dependent epitopes.

Sequence and Structural Homologies Between Mycobacterium tuberculosis Chaperonin 10 and the MHC Class I/II Peptide Binding Cleft

The peptide corresponding to the C-terminal half of M.tubeuculosis hsp10 was synthesised based on the prediction that it might represent an independent structural region of the protein. This hypothesis was confirmed by aggregation and CD studies using this peptide and longer sequences of the protein. The peptide shares about 40-50% sequence homology with α2 and β1 chains of MHC class I and II antigens. This and the CD results which indicated that the peptide at acidic pHs folds into an anti-parallel β-sheet were used to generate a 3D model which has the same "W" fold contained in the MHC peptide binding groove. These data suggest that the hypothesis of molecular mimicry proposed to be one of the mechanisms which triggers autoimmune diseases may be extended to hsp10 proteins. Furthermore the suggested evolutionary relationship between hsp′s and MHC antigens may find support from these data.