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Principal Investigator: Susan Buchanan
Organization: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
Fiscal Year: 2024
Award: $787
Funding agency: National Institute of Diabetes and Digestive and Kidney Diseases
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.
Our collaborative research utilizes 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 partnership with John Louis (LCP-NIDDK), we furthered our studies on the maturation of the SARS-CoV-2 main protease, MPro, which is indispensable for viral replication and propagation.
A crucial step in the assembly and maturation of SARS-CoV-2 involves the autoactivation of the main protease MPro from the precursor polyproteins. The mature form, which functions as a dimer, is released by autoprocessing the polyprotein chain containing the MPro monomer. Dimerization, an essential process in maturation, is facilitated by Domain III and strengthened by the intermolecular interaction of Domain II with the N-terminal residues of its protomer. 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.
The recombinant expression of a protease precursor results in the processing of its N- and C-termini to yield a mature dimer. Using precursors with targeted mutations that affect protease dimerization, we were able to elucidate the sequence of proteolytic events that lead to maturation and demonstrate a link between dimerization and catalytic activity. Thus, a mutant R298A precursor with a weaker dimerization affinity still has a significant proportion of dimer and shows rapid autoprocessing. However, the predominantly monomeric E290A precursor exhibits a slower time-dependent autoprocessing and accumulates a processed N-terminal site that is subsequently cleaved at the C-terminus. The double E290A, R298A mutant does not dimerize and only cleaves at the N-terminus, as does a truncated MPro (1-199) lacking Domain III. In this manner, we show that MPro autoprocessing is usually a two-step process that starts with intramolecular cleavage at the N-terminus. Subsequent cleavage at the C-terminus is intermolecular and requires a mature dimer.
The initial N-terminal autoprocessing of the polyprotein precursor enables the forming of a mature-like stable dimer interface, concomitant with the active site oxyanion loop equilibrium transitioning to the active conformation (E*) and the onset of catalytic activity.
By mutating residues critical for dimerization and evaluating MPro dimerization brought about by the noncovalent inhibitor ensitrelvir (ESV), we show that processed N-terminal residues Ser1 through Glu14 are essential for dimerization. The combined mutations of G11A, E290A, and R298A restrict dimerization, even upon binding of ESV that induces an E* state. Structural studies indicate a transient dimer structure of the MPro precursor held together through the interactions of the processed N-terminal residues A5-G11 with distinct states of the active sites, E and E*, likely representing an intermediate in autoprocessing. These observations may provide a valuable tool for designing and identifying more effective protease inhibitors targeting the early stages of protease maturation, offering hope for future research and drug development.
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