Biophysics of Macromolecular Complexes

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Chromatin structure and architecture. Histone proteins package and condense genomic DNA into chromatin within the cell nucleus. Proteins such as the CCCTC binding factor (CTCF) help direct chromatin higher-order organization through passive and active mechanisms by imposing topological constraints, mediating long-range genomic interactions, and participating in transcriptional events. CTCF is a highly conserved DNA binding protein found exclusively in bilaterians. In humans, the protein consists of an eleven zinc-finger DNA binding domain, flanked by conserved N-terminal and C-terminal tails constituting about 57 percent of the protein. While the zinc-fingers specifically recognize DNA motifs and interact with RNA, the roles of the N- and C-terminal domains remain primarily unknown. These termini interact with cohesin and help stabilize the complexes delineating topologically associated chromatin domains. However, it is unlikely that this is their sole role. We have shown that these N- and C-terminal domains are intrinsically disordered in solution, a property shared by many nuclear and DNA binding proteins. Current work focuses on identifying protein partners that bind to the N- and C-termini of CTCF and a study of the complexes formed to understand how CTCF regulates higher-order genome organization within the eukaryotic nucleus. Human CTCF has an ortholog CTCFL, primarily associated with spermatogenesis and some cancer types. While CTCF and CTCFL have a highly conserved eleven zinc-finger DNA binding domain and recognize identical DNA motifs, they differ significantly in their N- and C-termini suggesting that the diverse roles for these proteins arise from their termini. Similarly, while conserved among bilaterians and across evolution with a core zinc-finger DNA binding domain, CTCF may have divergent termini across phyla. We are interested in characterizing protein partners for CTCF from select species to dissect further the multiple roles that the protein plays in organizing the genome. Macromolecular assemblies of biological interest. We utilize hydrodynamic methods, particularly sedimentation velocity and equilibrium analytical centrifugation, to characterize critical biological assemblies, determine their shape and stoichiometry, and measure their interaction affinity. In collaboration with John Louis (LCP-NIDDK), we are studying the maturation of the dimeric SARS-CoV-2 main protease (MPro) and the effects of transition-state analog inhibitors. MPro functions as a dimer and is indispensable for viral replication and propagation. MPro is formed as a monomer, as part of a polyprotein chain. MPro dimerization is required for its release and the generation of non-structural proteins necessary for viral replication and transcription. Oral COVID-19 therapies recently granted emergency use authorization by the FDA target the main protease MPro, highlighting its importance. Using a carefully designed monomeric form of MPro(M) and comparing its properties to that of the wild-type dimeric MPro(WT), we show that protease activity requires the dimeric form of the enzyme. GC376, a transition-state analog protease inhibitor used in veterinary applications to treat feline coronavirus infections, interacts with MPro(M) and MPRo(WT). Interestingly, MPro(M) binds to GC376 with a higher affinity than MPro(WT). Furthermore, using activity assays and hydrodynamic methods, we show that GC376 promotes MPro(M) dimerization in a concentration-dependent manner. GC376 does not restore the catalytic activity observed for MPro(WT), reflecting structural differences in the monomeric MPro(M) design. However, data demonstrate that the binding of the transition state analog favors the dimer form of the enzyme, enhancing catalytic activity. These observations may provide a valuable tool for designing and identifying more effective protease inhibitors.