Hybrid Methods for Dynamic Structure Analysis of Proteins from Pathogenic Microorganisms

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PROJECT SUMMARY This research program will investigate the general hypothesis that understanding the conformational diversity of proteins will provide new insights into their biology, and enable medical research. It is directed to two classes of systems: Integral Membrane Proteins (IMPs) and viral-host interactions. IMPs play critical roles as gate keepers, receptors, transporters, homeostasis regulators, and drug targets. These functions are mediated by the conformational plasticity of the IMP in the membrane environment. IMPs are challenging to prepare, and even more challenging to reconstitute in appropriate membrane mimicking environments. Cost-effective technologies for isotope-enrichment in condensed volumes, hybrid approaches combining NMR with evolutionary co-variation (ECs), novel methods of contact prediction, and innovative modeling methods from the protein structure prediction community, will be applied to structure-function studies of IMPs. These IMPs, chosen from important human pathogens, including E. coli, K. pneumoniae, and P. aeruginosa, are potential targets for antibiotic discovery. ECs will also be combined with NMR data to determine structures of multiple “native states” of proteins. The second component of our program is directed to viral – host biomolecular complexes, and antiviral drug discovery. We will utilize innovative paramagnetic NMR methods, together with small angle X-ray scattering (SAXS), electron-electron double resonance spectroscopy (DEER), and Förster resonance energy transfer (FRET), to rigorously define dynamic interdomain structural distributions conferred by the partially-ordered linkers of the murine Moloney Leukemia Virus (MLV) integrase (IN). These data will be interpreted in the context of maximum occupancy probabilities (MaxOcc), and used to probe the role(s) of this flexibility in the gene integration mechanisms of g-retroviruses. Interdomain linkers also function to provide flexibility needed for binding partner promiscuity. We will also determine how the interdomain linker sequences of influenza Non-Structural Protein 1 (NS1) confer appropriate plasticity to define its specificity and affinity for host proteins and RNAs. This structural and functional promiscuity underlies NS1’s mechanisms for suppressing the cellular innate immune response to influenza infection, and rigorous characterization of its dynamic structural basis will provide fundamental information for live-attenuated virus vaccine development. We will also apply our platform to investigate drugs that inhibit SARS-CoV2 virus by binding its main protease (Mpro). We have identified three drugs, already approved for use in humans, originally designed to inhibit the NSP3/4A protease of hepatitis C virus, that also inhibit SARS-CoV2 in viral replication assays at low micromolar concentrations. Our computational docking studies have also identified several other FDA- approved drugs that may inhibit Mpro. Enzyme kinetic, biophysical chemistry, and X-ray crystallography studies will be used to characterize complexes formed between these protease inhibitor drugs and Mpro, and to develop their potential as COVID-19 therapeutics, or as lead compounds for new therapeutic development.