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 sedimentation equilibrium analytical centrifugation, to characterize critical biological assemblies, determine their shape, stoichiometry, and measure their interaction affinity. In collaboration with Jinwei Zhang (LMB-NIDDK), we have studied the interaction of the matrix (MA) domain of HIV-1 Gag with tRNA. HIV-1 Gag is the major structural protein of HIV-1 that recruits the viral genome, viral proteins, and host cell proteins to complete particle envelopment. Gag plays many roles in the viral life cycle, including viral assembly, maturation, and release. Significantly, Gag determines the location of particle assembly through its N-terminal MA domain composed of a basic N-terminus and a C-terminal stalk. The N-terminus of HIV-1 Gag is myristoylated, thus targeting the MA domain to the plasma membrane. Like the nucleocapsid domain that stabilizes the viral RNA within the mature virion, MA binds to RNA, though with a lower affinity. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments in vivo have indicated that MA binds selectively to a subset of tRNAs. Using biophysical methods, including sedimentation velocity analytical ultracentrifugation, isothermal titration calorimetry, and light scattering, we demonstrate that MA forms high affinity 1:1 complexes with select tRNAs, such as tRNA Lys3, tRNA Leu, tRNA Ser, and tRNA Thr. In vivo CLIP experiments using mutated MA constructs and X-ray crystallography identify a series of basic residues on the N-terminus of MA critical for the interaction with the tRNA elbow. The crystal structure also reveals a second tRNA binding site on MA. However, there is no indication that the site is occupied in vitro or in vivo at physiological concentrations. The importance of the MA tRNA interaction is highlighted by in vivo experiments demonstrating that the tRNA binding site on MA is responsible for the subcellular localization of HIV-1 Gag to the plasma membrane. In this manner, tRNA binding competes with Gag membrane interactions. While Gag mutants defective for tRNA binding did not impair virus assembly, loss of this interaction led to decreased viral replication. Overall, host tRNAs negatively regulate HIV-1 Gag membrane interactions and help optimize viral replication.