Advancing our knowledge of viral membrane fusion and of IDP-membrane interactions by ESR - For many years Freed has pioneered the development of electron-spin resonance (ESR) methods for the study of proteins and their dynamical effects on membranes. The goal of this proposal is to use those ESR techniques to advance our knowledge of 1) viral membrane fusion and 2) Intrinsic Disordered Protein (IDP)-membrane interactions in conjunction with other biophysical methods. The ongoing COVID19 pandemic has unveiled how limited are our knowledge and methods to combat emerging infectious diseases caused by viral pathogens. A key step in SARS-CoV-2 (“SARS-2”) viral infection is membrane fusion initiated by its fusion peptide (FP) domain in its Spike protein. In fact, membrane fusion is key for all enveloped viruses such as SARS-CoV-1 (“SARS-1”), MERS-CoV, EBOV, influenza and HIV. However, the mechanism of viral membrane fusion is still unclear. We have extensively demonstrated that FP-induced membrane ordering is a prerequisite for viral membrane fusion in all these viruses. We have shown that membrane ordering for some viruses including SARS-1, MERS and EBOV is strongly Ca2+-dependent. Most recently we have shown that SARS-2 FP interacts with membranes more strongly than SARS-1, and it depends very specifically on Ca2+. However, the exact role of Ca2+, as well as the mechanism of membrane ordering are still unclear. The FP only initiates membrane fusion. We have proposed that the transmembrane domain (TMD) of influenza hemagglutinin is important for finalizing membrane fusion. However, this remains to be tested to see if it is applicable to SARS-2. Thus, we plan to continue studies of the mechanism of membrane fusion of SARS-2 as well as other Ca2+-dependent viruses. IDPs lack a stable tertiary structure in solution. After membrane binding, they can either undergo a disorder- to-order transition or still remain in the absence of a stable structure. The viral FPs are such examples. This IDP- membrane interaction localizes IDPs to their target membranes, facilitating interactions with other membrane proteins, and helping to remodel membrane properties. Understanding the structure/function relationships underlying IDP-membrane interactions is a significant challenge because of their highly variable and dynamic nature. We have been using complexin, a key exocytosis regulator related to neurodegeneration, as a model to delineate the IDP-membrane binding mode. We have found that complexins from different organisms have very different modes, which is likely to reflect their different biological functions. We plan to continue to study the membrane-binding C-terminal domain of complexins of several species to determine the mechanisms that govern complexin-membrane binding modes. These findings will be useful for human therapies. ESR is a powerful methodology to study the dynamic and structural properties of SARS FPs and other IDPs, as we have shown. We will employ our well-developed ESR methods to achieve these goals. This will be supplemented by other biophysical methods, including Isothermal Titration Calorimetry and Circular Dichroism.
