Structural and Functional Roles of Transmembrane Domains in B-cell Receptor Signalling

The B-cell receptor (BCR) complex, comprised of an antigen-binding subunit (membrane immunoglobulin, mIg) and a signalling subunit (the CD79a-b heterodimer), is one of the most important immune receptors in humans and controls B-cell development, activity, selection and death. Yet the mechanism of BCR signalling remains a matter of speculation, partly because of a lack of atomic-level structural data that reveals regions of the complex critical for functional receptor assembly, transport, and signal transmission, namely the transmembrane domains (TMDs). We hypothesise that the TMDs of the three proteins in the BCR complex are mediators of strong and specific (yet uncharacterised) interactions, and outline here work that would yield a molecular-level description of the structures and interactions of the BCR TMDs for the first time. Our hypothesis is based on reports dating back over 25 years which implicate the TM domains as sites of functionally essential protein-protein and protein-lipid interactions, but have thus far not revealed a molecular level understanding of this role. This proposal outlines a molecular-level, biophysical investigation of the structures and interactions of the BCR TMDs, which we will use to design molecules to modulate BCR function. Specifically, we will (i) characterise the strength and sequence dependence of TMD interactions within and between components of the BCR complex in a natural membrane, (ii) utilise a panel of biophysical methods to characterise the structure, stability, stoichiometry and atomic details of TMD interactions in multiple (synthetic) lipid environments, and (iii) use this new information to design and test ability of molecules to disrupt interactions and modify BCR signalling and function in situ. This work will yield the first map of interactions between the BCR TMDs and the first structural data for the BCR TMDs in different lipid environments, thus enhancing our mechanistic understanding of BCR signalling and supporting efforts in basic immunology. The molecules we design would act as a proof of concept that BCR modulation is possible using this approach, and provide a platform for a more comprehensive drug discovery programme in the future towards new theraputic treatments for autoimmune diseases, B-cell leukaemias and lymphomas.

We undertake here to investigate the transmembrane domain (TMD) interactions involved in assembly of the B cell receptor complex, since these regions have been discussed as sites of functionally essential (but poorly understood) protein-protein and protein-lipid interactions for the past 25 years. We hypothesise that the TMDs of the three proteins in the BCR complex are mediators of strong and specific (yet uncharacterised) interactions. The aim of the work described in our proposal is to provide the first comprehensive map of TMD-TMD interactions possible in the BCR complex in natural membranes as well as synthetic membranes with varying lipid composition. The three main objectives of the work are (i) characterisation of the strength (stability) and sequence dependence of TMD interactions within (e.g IgM homodimers, CD79a-b heterodimer) and between (e.g. CD79-IgM) components of the BCR complex in a natural membrane using the TOXCAT and GALLEX assays and scanning mutagenesis, (ii) utilisation of a panel of biophysical methods (including circular dichroism and fluorescence spectroscopy, analytical ultracentrifugation and solution state NMR) to characterise the structure, stability, stoichiometry and atomic details of TMD interactions in multiple (synthetic) lipid environments, and (iii) use of this new information to design and test ability of molecules to disrupt TMD interactions and modify BCR signalling and function in situ in the lab of Prof. James Drake (Albany Medical College, New York). Molecules will be applied to B cell lines of murine splenic B cells to determine their impact on signalling and endocytosis. Commercial viability of downstream applications of our work (e.g. molecules for modulation of BCR function) will be assessed with support from Warwick Ventures, our technology transfer office.

Potential beneficiaries from this work range from academics and researchers in the fields of structural biology, membrane protein biophysics and experimental immunology to medical researchers, the commercial sector (e.g. pharmaceutical companies) and members of the public seeking treatment solutions for a range of autoimmune diseases. Our long-term goal is mechanistically-driven development of novel therapeutic molecules for modulation of B cell receptor (BCR) signalling, to be used in the treatment of e.g. autoimmune diseases and B-cell lymphoproliferative disorders. Autoimmune diseases are a major health issue, with numbers in the US alone eclipsing both cancer and heart disease. Current therapeutic treatments have shown high toxicity and can lead to long-term side effects, thus alternative treatments are actively being sought. If successful, these molecules would provide such an alternative and form the basis of a larger drug discovery programme that would have commercial economic impact. The information about transmembrane domain (TMD) structure and protein / lipid interactions we collect will underpin this development, and would in its own right, inform the fields of membrane protein folding and structural biology. Overall, we propose a new target, i.e. the TMDs of key immune complexes, may provide a promising new area of discovery.