The matrix protein M2 is a 97-residue homotetrameric protein that performs many important functions for the influenza virus. The primary function of this protein is to conduct protons to acidify the interior of the virus for the release of viral RNA from the capsid for replication and the transmission of infectious particles (11- 14). The transmembrane domain constitutes the functional core of the M2 protein, essential for the tetramerization and proton conductance (15), while the N- and C-terminal domains are necessary for viral budding (13, 14, 16, 17). From a biophysical perspective, M2 is also an interesting system to probe proton diffusion and pH-dependent conformational change as many of these structural and functional studies would yield insight into the proton conduction pathways of more complex proton channels and pumps. Because this protein is crucial for the viral life cycle, a detailed understanding of the M2 protein structure and its mechanisms of proton conduction will not only address the very fundamental processes of proton conduction and stabilization in membrane proteins, but will also aid in the development of targeted M2 channel blockers. The proposed research strategy herein seeks to elucidate the proton conduction mechanism in both AM2 and BM2 as well as probe its interactions with another matrix protein M1 to better understand the roles of these proteins in the replication and transmission of infectious influenza viruses. In this research, we propose to: 1. Link the proton-coupled conformational changes of AM2 to the kinetics of proton conduction and elucidate the hydrogen-bonding network of pore-lining residues and waters that stabilize the proton as it diffuses to the His tetrad to obtain a detailed picture of the conduction pathway through this protein. 2. Confirm the specificity and stoichiometry of binding of full-length AM2 to its interaction partner M1 and obtain a high-resolution structure of the resulting complex. 3. Determine the high-resolution structure of the functional core and of full-length BM2 to establish the conduction pathway through the protein and carry out a comparative analysis of the structures and mechanisms of proton conduction of AM2 and BM2.
This project focuses on elucidating the structures and mechanisms of function of matrix proteins M2 from influenza A and B viruses in order to establish a better understanding of the roles of these proteins in viral replication, assembly and budding, to understand the mechanisms of proton conduction in membrane proteins, and to advance the development of targeted therapeutics. A complete picture of the proton-coupled conformational changes and the hydrogen-bonding and water networks within the pore of the AM2 and BM2 proton channels from a combination of high-resolution x-ray crystallography and spectroscopy will not only yield insight into the proton conduction mechanism that these channels employ to acidify the interior of the virus for the release of viral RNA into the host cell, but will also address fundamental questions of proton diffusion and stabilization in membrane proteins and advance the design of viral channel pore blockers. Similarly, preliminary pull-down studies have suggested that M2 plays another important role in viral budding upon binding to its interaction partner M1, which necessitates studies that probe the direct binding of these two proteins to determine the specificity and stoichiometry of binding to elucidate the role of this complex in the replication, budding, and transmission of infectious viruses.
