Andrea Ferrante

Cutting Edge: HLA-DM–Mediated Peptide Exchange Functions Normally on MHC Class II–Peptide Complexes That Have Been Weakened by Elimination of a Conserved Hydrogen Bond

The mechanism by which HLA-DM (DM) promotes exchange of peptides bound to HLA-DR (DR) is still unclear. We have shown that peptide interaction with DR1 can be considered a folding process as evidenced by cooperativity. However, in DM-mediated ligand exchange, prebound peptide release is noncooperative, which could be a function of the breaking of a critical interaction. The hydrogen bond (H-bond) between β-chain His81 and the peptide backbone at the −1 position is a candidate for such a target. In this study, we analyze the exchange of peptides bound to a DR1 mutant in which formation of this H-bond is impaired. We observe that DM still functions normally. However, as expected of a cooperative model, this H-bond contributes to the overall energetics of the complex and its disruption impacts the ability of the exchange ligand to fold with the binding groove into a stable complex.

Cooperativity of hydrophobic anchor interactions: evidence for epitope selection by MHC class II as a folding process.

Peptide binding to MHC class II (MHCII) molecules is stabilized by hydrophobic anchoring and hydrogen bond formation. We view peptide binding as a process in which the peptide folds into the binding groove and to some extent the groove folds around the peptide. Our previous observation of cooperativity when analyzing binding properties of peptides modified at side chains with medium to high solvent accessibility is compatible with such a view. However, a large component of peptide binding is mediated by residues with strong hydrophobic interactions that bind to their respective pockets. If these reflect initial nucleation events they may be upstream of the folding process and not show cooperativity. To test whether the folding hypothesis extends to these anchor interactions, we measured dissociation and affinity to HLA-DR1 of an influenza hemagglutinin-derived peptide with multiple substitutions at major anchor residues. Our results show both negative and positive cooperative effects between hydrophobic pocket interactions. Cooperativity was also observed between hydrophobic pockets and positions with intermediate solvent accessibility, indicating that hydrophobic interactions participate in the overall folding process. These findings point out that predicting the binding potential of epitopes cannot assume additive and independent contributions of the interactions between major MHCII pockets and corresponding peptide side chains.

ENTHALPY-ENTROPY COMPENSATION AND COOPERATIVITY AS THERMODYNAMIC EPIPHENOMENA OF STRUCTURAL FLEXIBILITY IN LIGAND-RECEPTOR INTERACTIONS

Ligand binding is a thermodynamically cooperative process in many biochemical systems characterized by the conformational flexibility of the reactants. However, the contribution of conformational entropy to cooperativity of ligation needs to be elucidated. Here, we perform kinetic and thermodynamic analyses on a panel of cycle-mutated peptides, derived from influenza H3 HA(306-319), interacting with wild type and a mutant HLA-DR. We observe that, within a certain range of peptide affinity, this system shows isothermal entropy-enthalpy compensation (iEEC). The incremental increases in conformational entropy measured as disruptive mutations are added in the ligand or receptor are more than sufficient in magnitude to account for the experimentally observed lack of free-energy decrease cooperativity. Beyond this affinity range, compensation is not observed, and therefore, the ability of the residual interactions to form a stable complex decreases in an exponential fashion. Taken together, our results indicate that cooperativity and iEEC constitute the thermodynamic epiphenomena of the structural fluctuation that accompanies ligand-receptor complex formation in flexible systems. Therefore, ligand binding affinity prediction needs to consider how each source of binding energy contributes synergistically to the folding and kinetic stability of the complex in a process based on the trade-off between structural tightening and restraint of conformational mobility.

Peptide-MHC Class II Complex Stability Governs CD4 T Cell Clonal Selection

The clonal composition of the T cell response can affect its ability to mediate infection control or to induce autoimmunity, but the mechanisms regulating the responding TCR repertoire remain poorly defined. In this study, we immunized mice with wild-type or mutated peptides displaying varying binding half-lives with MHC class II molecules to measure the impact of peptide-MHC class II stability on the clonal composition of the CD4 T cell response. We found that, although all peptides elicited similar T cell response size on immunization, the clonotypic diversity of the CD4 T cell response correlated directly with the half-life of the immunizing peptide. Peptides with short half-lives focused CD4 T cell response toward high-affinity clonotypes expressing restricted public TCR, whereas peptides with longer half-lives broadened CD4 T cell response by recruiting lower-affinity clonotypes expressing more diverse TCR. Peptides with longer half-lives did not cause the elimination of high-affinity clonotypes, and at a low dose, they also skewed CD4 T cell response toward higher-affinity clonotypes. Taken collectively, our results suggest the half-life of peptide-MHC class II complexes is the primary parameter that dictates the clonotypic diversity of the responding CD4 T cell compartment.

HLA-DM Mediates Epitope Selection by a “Compare-Exchange” Mechanism when a Potential Peptide Pool Is Available

Background HLA-DM (DM) mediates exchange of peptides bound to MHC class II (MHCII) during the epitope selection process. Although DM has been shown to have two activities, peptide release and MHC class II refolding, a clear characterization of the mechanism by which DM facilitates peptide exchange has remained elusive. Methodology/Principal Findings We have previously demonstrated that peptide binding to and dissociation from MHCII in the absence of DM are cooperative processes, likely related to conformational changes in the peptide-MHCII complex. Here we show that DM promotes peptide release by a non-cooperative process, whereas it enhances cooperative folding of the exchange peptide. Through electron paramagnetic resonance (EPR) and fluorescence polarization (FP) we show that DM releases prebound peptide very poorly in the absence of a candidate peptide for the exchange process. The affinity and concentration of the candidate peptide are also important for the release of the prebound peptide. Increased fluorescence energy transfer between the prebound and exchange peptides in the presence of DM is evidence for a tetramolecular complex which resolves in favor of the peptide that has superior folding properties. Conclusion/Significance This study shows that both the peptide releasing activity on loaded MHCII and the facilitating of MHCII binding by a candidate exchange peptide are integral to DM mediated epitope selection. The exchange process is initiated only in the presence of candidate peptides, avoiding possible release of a prebound peptide and loss of a potential epitope. In a tetramolecular transitional complex, the candidate peptides are checked for their ability to replace the pre-bound peptide with a geometry that allows the rebinding of the original peptide. Thus, DM promotes a “compare-exchange” sorting algorithm on an available peptide pool. Such a “third party”-mediated mechanism may be generally applicable for diverse ligand recognition in other biological systems.

A Peptide/MHCII conformer generated in the presence of exchange peptide is substrate for HLA-DM editing

The mechanism of HLA-DM (DM) activity is still unclear. We have shown that DM-mediated peptide release from HLA-DR (DR) is dependent on the presence of exchange peptide. However, DM also promotes a small amount of peptide release in the absence of exchange peptide. Here we show that SDS-PAGE separates purified peptide/DR1 complexes (pDR1) into two conformers whose ratio is peptide Kd-dependent. In the absence of exchange peptide, DM only releases peptide from the slower migrating conformer. Addition of exchange peptide converts the DM-resistant conformer to the slower migrating conformer, which is DM labile. Thus, exchange peptide generates a conformer of pDR1 which constitutes the intermediate for peptide exchange and the substrate for DM activity. The resolution of the intermediate favors the highest affinity peptide. However, once folded into the DM-resistant conformer, even low affinity peptides can be presented in the absence of free peptide, broadening the repertoire available for presentation.

Naive T Cell Repertoire Skewing in HLA-A2 Individuals by a Specialized Rearrangement Mechanism Results in Public Memory Clonotypes

How the naive T cell repertoire arises and forms the memory repertoire is still poorly understood. This relationship was analyzed by taking advantage of the focused TCR usage in HLA-A2–restricted CD8 memory T cell responses to influenza M158–66. We analyzed rearranged BV19 genes from CD8 single-positive thymocytes, a surrogate for the naive repertoire, from 10 HLA-A2 individuals. CDR3 amino acid sequences associated with response to influenza were observed at higher frequencies than expected by chance, an indicator of preselection. We propose that a rearrangement mechanism involving long P-nucleotide addition from the J2.7 region explains part of this increase. Special rearrangement mechanisms can result in identical T cells in different individuals, referred to as public responses. Indeed, the rearrangements utilizing long P nucleotide additions were commonly observed in the response to the M158–66 epitope in 30 HLA-A2 middle-aged adults. Thus, in addition to negative and positive selection, special rearrangement mechanisms may influence the composition of the naive repertoire, resulting in more robust responses to a pathogen in some individuals.

For many but not for all: how the conformational flexibility of the peptide/MHCII complex shapes epitope selection

The adaptive immune response starts when CD4+ T cells recognize peptide antigens presented by class II molecules of the Major Histocompatibility Complex (MHCII). Two outstanding features of MHCII molecules are their polymorphism and the ability of each allele to bind a large panoply of peptides. The ability of each MHCII molecule to interact with a limited, though broad, range of amino acid sequences, or “permissive specificity” of binding, is the result of structural flexibility. This flexibility has been identified through biochemical and biophysical studies, and molecular dynamic simulations have modeled the conformational rearrangements that the peptide and the MHCII undergo during interaction. Moreover, there is evidence that the structural flexibility of the peptide/MHCII complex correlates with the activity of the “peptide-editing” molecule DM. In light of the impact that these recent findings have on our ability to predict MHCII epitopes, a review of the structural and thermodynamic determinants of peptide binding to MHCII is proposed.

The Thermodynamic Mechanism of Peptide–MHC Class II Complex Formation Is a Determinant of Susceptibility to HLA-DM

Peptides bind MHC class II molecules through a thermodynamically nonadditive process consequent to the flexibility of the reactants. Currently, how the specific outcome of this binding process affects the ensuing epitope selection needs resolution. Calorimetric assessment of binding thermodynamics for hemagglutinin 306–319 peptide variants to the human MHC class II HLA-DR1 (DR1) and a mutant DR1 reveals that peptide/DR1 complexes can be formed with different enthalpic and entropic contributions. Complexes formed with a smaller entropic penalty feature circular dichroism spectra consistent with a non–compact form, and molecular dynamics simulation shows a more flexible structure. The opposite binding mode, compact and less flexible, is associated with greater entropic penalty. These structural variations are associated with rearrangements of residues known to be involved in HLA-DR (DM) binding, affinity of DM for the complex, and complex susceptibility to DM-mediated peptide exchange. Thus, the thermodynamic mechanism of peptide binding to DR1 correlates with the structural rigidity of the complex, and DM mediates peptide exchange by “sensing” flexible complexes in which the aforementioned residues are rearranged at a higher frequency than in more rigid ones.

HLA-DM: arbiter conformationis

The recognition by CD4+ T cells of peptides bound to class II MHC (MHCII) molecules expressed on the surface of antigen‐presenting cells is a key step in the initiation of an adaptive immune response. Presentation of peptides is the outcome of an intracellular selection process occurring in dedicated endosomal compartments involving, among others, an MHCII‐like molecule named HLA‐DM (DM). The impact of DM on the epitope selection machinery has been known for more than 15 years. However, the mechanism by which DM skews the presented repertoire in favour of kinetically stable complexes has remained elusive. Here, a review of the most recent observations in the field is presented, pointing to the possibility that DM decides the survival of a peptide–MHCII complex (pMHCII) on the basis of its conformational flexibility, which is a function of the ‘tightness’ of interaction between the peptide and the MHCII at a specific region of the binding site.