Cell surface clusters of MHC class I molecules: origin, structure, and functions

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MHC (major histocompatibility complex) class I proteins transport peptide fragments of the cellular proteome to the plasma membrane to present them to cytotoxic T cells, which can recognize non-self peptides and kill the presenting cells in case of virus infection or tumorigenic aberration. At the cell surface, MHC class I heavy chains can lose the ligand peptide and the light chain (beta-2 microglobulin) and form non-covalent associations, as we have shown. We have characterized these 'MHC class I free heavy chain clusters' with co-immunoprecipitation, with a novel antibody micropatterning two-hybrid assay, with fluorescence recovery after photobleaching (FRAP), and with single-molecule fluorescence microscopy. Our preliminary data point to the alpha-3 domain of the class I heavy chain as the interaction site and suggests that the clusters are transient. We hypothesize that the class I free heavy chain clusters are mostly dimers of heavy chains, and that they play a role in the highly efficient endocytic sorting of the different conformations of class I at the cell surface, the mechanism of which is so far unknown. Another potential function is the signaling to T cells and NK cells, for which there is evidence in the literature. The first objective of this work is to understand which MHC allotypes form MHC free heavy chain clusters, and how. We will test different murine and human allotypes for cluster formation and mutagenize and truncate them to find the interaction site. To detect the clusters, we will use the micropatterning assay (Springer and Lanzerstorfer groups) and FRAP (Lanzerstorfer group). The second objective is to establish the molecular structure and dynamics of the clusters. We will generate in silico models with the help of our collaborator Martin Zacharias (Technical University of Munich, Computational Biology) and produce the clusters from recombinant proteins for X-ray crystallography. We'll use the advanced spectroscopy techniques of our collaborators Jacob Piehler (Osnabrück University, for quantitative single-molecule microscopy) and Gerhard Schütz (Vienna Technical University, for artificial membrane systems) to investigate the size, dynamics, and affinity of the clusters in live cells and on membranes. The third objective is to characterize the biological function of the clusters. To investigate their role in class I endocytic sorting, we will monitor the rate of endocytosis and cell surface return of the clusters and compare them with peptide-loaded (monomeric) class I molecules. To investigate their role in signaling, we will stimulate reporter T and NK cells with cells expressing clusters, vs. control cells. We will also test whether free heavy chain clusters plays a role in the formation of covalent dimers of HLA-B*27:05, which are involved in the pathogenesis of inflammatory autoimmune diseases.