Assembly of MHC class II molecules: A structural investigation of protein-protein and protein-lipid interactions within the lipid bilayer

• Applicant: Professorin Dr. Britta Brügger
• Subject Area: Biochemistry
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 283963326

The function of membrane proteins depends on interactions both inside and outside the lipid bilayers into which they are embedded. While protein-protein interaction modules within the membrane are described, much less is known about specific interactions of protein transmembrane domains (TMDs) with membrane lipids. The huge diversity of membrane lipid species suggests that they fulfil specific functions beyond their role as structural components of the bilayer. Recent data demonstrate a novel role of individual lipid species, which can function as modulators of membrane protein activity. One example is our recent discovery of a direct interaction of a type I membrane protein with a sphingolipid. Employing a photoaffinity approach, we have identified a highly specific interaction of p24, a protein cycling in the secretory pathway, with a single sphingomyelin species, involving both the head group and the C18 fatty acyl chain of the lipid. Cellular, in vitro and in silico characterisation of this protein-sphingolipid interaction allowed us to define a lipid-binding motif within the TMD of p24. Bioinformatics analysis of a mammalian dataset resulted in a number of protein candidates containing this putative sphingolipid-binding motif. One of these proteins is the human MHC class II protein DQ alpha 1 that forms a heterodimer with the DQ beta 1 chain. In its peptide-loaded state this heterodimer is translocated to the plasma membrane of antigen-presenting cells to enable presentation of foreign peptides to CD4-positive T helper cells. Analysis of the transmembrane domain sequences of MHC class II DQ alpha and beta chains revealed the presence of a conserved GXXXG dimerisation module in both the alpha and beta chains. In addition, the DQ alpha chains contain the putative sphingolipid-binding motif, and a sequence reminiscent of this sphingolipid-binding motif is found in the beta chains. Here we aim i) to structurally characterise the TMD heterodimerisation module, ii) to investigate sphingolipid-binding of alpha and beta chains, and iii) to understand at a structural level the molecular interactions underlying lipid binding. To this end we will investigate this interaction using a number of biophysical approaches, including multidimensional solid-state and solution NMR spectroscopy in phospholipid bilayer and bicellar environments, combined with in vitro and in silico approaches to study TMD-lipid interactions of the individual alpha/beta chains and the heterodimeric alpha/beta complex. In addition, we will characterise protein variants compromised in dimerisation and/or lipid binding. Together, these investigations will contribute to our understanding of contributions of TMD-TMD and TMD-lipid interactions to the function of MHC class II molecules.

• DFG Programme: Research Grants
• International Connection: France
• Partner Organisation: Agence Nationale de la Recherche / The French National Research Agency
• Cooperation Partner: Professor Dr. Burkhard Bechinger