Journal of Molecular Biology
Volume 346, Issue 5, 11 March 2005, Pages 1367-1379
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Thermodynamic and Structural Equivalence of Two HLA-B27 Subtypes Complexed with a Self-peptide

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The F pocket of major histocompatibility complex (in humans HLA) class I molecules accommodates the C terminus of the bound peptide. Residues forming this pocket exhibit considerable polymorphism, and a single difference (Asp116 in HLA-B*2705 and His116 in HLA-B*2709 heavy chains) confers differential association of these two HLA-B27 subtypes to the autoimmune disease ankylosing spondylitis. As peptide presentation by HLA molecules is of central importance for immune responses, we performed thermodynamic (circular dichroism, differential scanning calorimetry, fluorescence polarization) and X-ray crystallographic analyses of both HLA-B27 subtypes complexed with the epidermal growth factor response factor 1-derived self-peptide TIS (RRLPIFSRL) to understand the impact of the Asp116His exchange on peptide display. This peptide is known to be presented in vivo by both subtypes, and as expected for a self-peptide, TIS-reactive cytotoxic T lymphocytes are absent in the respective individuals. The thermodynamic analyses reveal that both HLA-B27:TIS complexes exhibit comparable, relatively high thermostability (Tm∼60 °C) and undergo multi-step unfolding reactions, with dissociation of the peptide in the first step. As shown by X-ray crystallography, only subtle structural differences between the subtypes were observed regarding the architecture of their F pockets, including the presence of distinct networks of water molecules. However, no consistent structural differences were found between the peptide presentation modes. In contrast to other peptides displayed by the two HLA-subtypes which show either structural or dynamical differences in their peptide presentation modes, the TIS-complexed HLA-B*2705 and HLA-B*2709 subtypes are an example for thermodynamic and structural equivalence, in agreement with functional data.

Introduction

Major histocompatibility complex (MHC) class I molecules are heterotrimeric protein complexes consisting of a heavy chain (HC) that is non-covalently bound to β2-microglobulin (β2m), and a peptide which is accommodated in a binding groove exhibiting six pockets (A to F) that are shaped by polymorphic residues of the HC.1 The ensemble of the two α-helices of the HC and the peptide provides epitopes for T cell receptors on cytotoxic T lymphocytes (CTL), and MHC class I antigens play a crucial role in the immune system by presenting intracellularly derived peptides to these cells.2 The rules for peptide binding by MHC class I molecules have been established through X-ray crystallography1 and analysis of peptide sequences.3 The most important positions for a well-anchored peptide are located near its termini.4 In case of the human MHC class I antigen HLA-B27, the peptide residues in position p2 and pΩ (the C terminus) are involved in anchoring the peptide to the binding groove.5 The remaining residues either protrude from the binding groove or fine-tune peptide binding to the MHC molecule by interacting with HC pockets A to F.6, 7

The human HLA-B27 gene is of special interest as it is strongly associated with a group of autoimmune diseases, in particular ankylosing spondylitis (AS).8, 9 The molecular mechanisms underlying this association are still elusive.9, 10, 11, 12, 13, 14 However, a hint for understanding the disease association may come from the discovery that not all HLA-B27 subtypes are connected to AS.8, 9 In particular, the B*2709 subtype which is not associated to AS differs from the prototypical and strongly disease associated subtype B*2705 only in a single residue located at the floor of the peptide binding groove.15 This amino acid (Asp116 in B*2705, His116 in B*2709) is involved in anchoring pΩ within the F pocket. The exchange of residue 116 influences the repertoire of bound peptides16, 17 as well as the mode of presentation of the same peptide by the two subtypes.18, 19, 20 Since the development of distinct T cell receptor repertoires by individuals bearing these subtypes depends on this polymorphism as well,21 all biophysical and functional differences between these HLA-B27 subtypes are due to the Asp116His replacement, although residue 116 is inaccessible to ligands on effector cells.

A variety of different methods have been employed for the biophysical characterization of equilibrium and kinetic parameters of the interaction between selected MHC molecules and their respective peptide ligands.22, 23, 24, 25, 26, 27, 28, 29, 30, 31 These investigations included also an analysis of the thermal unfolding of HLA-B27 antigens using circular dichroism (CD) spectroscopy,25, 26 demonstrating the importance of peptide residues at p1, p3 and pΩ as well as the B-pocket residue Cys67. In addition, a pairwise comparison of the B*2705 and B*2709 subtypes has recently been carried out using differential scanning calorimetry (DSC)30 and time-resolved fluorescence depolarization combined with molecular dynamics simulations.31 The results of these studies show that a seemingly minor structural difference (Asp116His) in the HLA-B27 HC can exert pronounced effects on the stability and thermodynamic properties of MHC class I molecules, and also provide information on the interdependence of HC, β2m, and peptide during unfolding of these antigens. Studies employing a peptide similarly bound to both subtypes under natural conditions would serve a most useful role as reference for the analysis of peptides that exhibit distinct thermodynamic properties like m9 (GRFAAAIAK)30, 31 or are differentially presented by B*2705 and B*2709, such as the Epstein Barr-virus-derived pLMP2 (RRRWRRLTV).20

Therefore, we have extended our studies to the nonameric self-peptide TIS (RRLPIFSRL). TIS is derived from the epidermal growth factor (EGF)-response factor 1 (ERF-1, TIS11B protein). This peptide was first shown to be naturally displayed by B*2705,32 then by B*2709,33 and finally confirmed as shared ligand of both HLA-B27 subtypes.16 TIS-specific CTL cannot be detected in individuals typing as B*2705 or B*2709,21 indicating that thymic negative T cell selection acts appropriately on TIS-reactive CTL. The studies described here are aimed at an understanding of whether the F pocket polymorphism influences the thermodynamic and structural properties of the two HLA-B27:TIS complexes.

Section snippets

Thermostabilities of HLA-B27:TIS complexes monitored by CD and DSC

The two HLA-B27:TIS complexes were prepared from inclusion bodies of separately expressed HC and β2m, followed by refolding in the presence of excess TIS peptide as described in Materials and Methods. The melting properties of the two HLA-B27:TIS complexes were first determined by CD measurements (see Figure 1(a) for representative experiments), showing similar Tm values of 58.2±1.8 °C and 61.3±2.4 °C for B*2705:TIS and B*2709:TIS, respectively, the difference between the mean values being within

Discussion

An MHC class I molecular complex is a complicated entity in terms of thermodynamic analyses: it is composed of a peptide and two protein chains, β2m and HC, and the latter is built of various structural domains. The calorimetric unfolding enthalpy ΔHcal as obtained from DSC measurements is proportional to the area under the experimental excess heat capacity curve, and deconvolution of this curve can provide further insight into the unfolding process. However, the two “two-state” transitions, as

Protein preparation

The TIS peptides were synthesized by standard solid phase procedures, purified by HPLC, and obtained either from Alta Bioscience, Birmingham, UK (native peptide (RRLPIFSRL)), or from Biosynthan, Berlin, Germany (C6-LY- or C8-LY-modified TIS peptides). Heterotrimeric HLA-B27:TIS complexes (HC/β2m/peptide) were prepared and purified as described.30, 41, 42 They were used for crystallization at concentrations of 20 mg ml−1 (B*2705:TIS) and 18 mg ml−1 (B*2709:TIS), respectively, in 10 mM Tris–HCl buffer

Acknowledgements

We are grateful to M. Rühl and A. Neubert for excellent technical assistance, C. Rückert for help with sample preparation, M. P. Heyn for support, and U. Müller (BESSY-II, Berlin), for help at the synchrotron facility. This work was financially supported by the Deutsche Forschungsgemeinschaft through SFB 449 (TP A5, B6), Sonnenfeld-Stiftung, Berlin, and Fonds der Chemischen Industrie.

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