Biophysics of Macromolecular Complexes

CTCF与染色质结构。

CTCF and chromatin structure. 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. The mammalian form 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 unknown for the most part. 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-termini 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 highly conserved eleven zinc-finger DNA binding domains 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 sedimentation equilibrium analytical centrifugation, to characterize critical biological assemblies, determine their shape and stoichiometry, and measure their interaction affinities. In collaboration with John Louis (LCP-NIDDK), we are studying the maturation of the SARS-CoV-2 main protease (MPro) and the effects of therapeutic covalent inhibitors. The mature form of MPro functions as a dimer and is indispensable for viral replication and propagation. MPro is formed as a monomer, part of a polyprotein chain, and auto-processing leads to its release and subsequent dimerization. Structural studies of the MPro monomer reveal that it adopts a native-like fold with an unwound oxyanion loop conformation (E), defining the catalytically inactive state. Enzymatic assays indicate that this majority inactive conformer is in equilibrium with an active minor conformer (E*). The covalent inhibitors, GC373 and nirmatrelvir (NMV), approved for treating feline and human coronavirus infections, respectively, bind to the MPro monomer. Sedimentation studies indicate that these inhibitors, particularly NMV, promote the weak dimerization of MPro and that this requires the presence of the N-terminal residues. Structural studies on the complex demonstrate the conversion of the E conformation to an active form (E*) resembling that observed for the mature wild-type dimer. The N-terminus's organized structure and the protein's improved conformational stability predispose the NMV complex to dimerize. The observed auto-processing of a mini-precursor at the N-terminus indicates that binding of the N-terminal cleavage site sequence in the precursor stabilizes the E* conformation, leading to the liberation of the N-terminus and predisposing MPro for maturation as a dimer. These observations may provide a valuable tool for designing and identifying more effective protease inhibitors.