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

Lead Research Organisation: University of Warwick
Department Name: Chemistry

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Title Transmembrane domain bacterial expression 
Description We have implemented use of an expression system for transmembrane peptides and proteins as part of this project. Specifically, we have utilized the TrpLE expression vector and protocol for leader peptide cleavage and purification, into our experimenal plans and have obtained excellent expression yields thus far. 
Type Of Material Improvements to research infrastructure 
Provided To Others? Yes  
Impact We can now access a large number of uniformly labelled transmembrane domains in a relatively short timescale and have more flexibility in the types of peptides we investigate. 
 
Description Collaboration with the Drake Laboratory at the Albany Medical Center, New York, USA 
Organisation Albany Medical College
Country United States 
Sector Academic/University 
PI Contribution This collaboration involves design and production of molecules in my group that will be tested in vivo in the Drake laboratory for their ability to modulate BCR function.
Collaborator Contribution The Drake laboratory will provide in vivo testing of molecules designed and prepared in my laboratory for their ability to impact function of the intact BCR in a cell. This work will validate models of the TMD contribution to BCR folding and function.
Impact Drake is listed as a collaborator on this grant, thus this grant is attributable to the collaboration with the Drake lab.
Start Year 2015
 
Description Chemistry workshop with 10 year old school pupils 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 50 10 year olds visited Warwick Chemistry department and spent time in the teaching laboratory, were supervised in participating in several experiments including making an iodine clock and combining various ingredients to try to generate a "slime" of desired consistency. Students liked the idea of "chemicals" being in their homes and "chemical reactions" being a real-world phenomenon and not limited to science classrooms. Encouraged students to think of science as a way of describing the world they live in, not something abstract and boring.
Year(s) Of Engagement Activity 2018
 
Description Chemistry workshop with 10-15 year old school pupils 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 50 10-15 year old students attended for a visit to Warwick Chemistry department, spent time in the teaching laboratories and were assisted in acetylation of salicylic acid, purification steps, and TLC. Led to discussions on chemical modifications, impact on biological uptake and metabolism, and broader discussions of chemistry in the real world (example, why is hydrogen bonding important for life - brought on by observations of aqueous solution in capillary tube). Encouraged students to consider university and science as options in their future.
Year(s) Of Engagement Activity 2018