Our research focuses on the structural biology of proteins involved in signal transduction (STATs, cytokines, hepatocyte growth factor) and regulation of gene expression (STATs and Nus proteins). We have also collaborated on a novel antiviral entry inhibitory protein, scytovirin. Using three-dimensional (3D) solution structures and dynamics of proteins and complexes, involving protein-protein, protein-nucleic, and protein-carbohydrate interactions, we elucidate the mechanism of action for these systems and investigate ways to modulate the function. In the STAT system of proteins, we have determined the solution structure of the N-terminal domain of STAT4 and found evidence for a new dimerization interface. Corroborating evidence for this interface is being pursued via investigation of the N-terminal domains (NTDs) of all seven STAT family members. A manuscript describing these studies is being revised for publication. The interactions indicated in the NTD studies have also enabled us to examine the heterodimerization of STAT1 and STAT2 NTDs. These studies are key to understanding the variety of modes of combination among the STAT proteins that can explain the involvement of the seven STAT proteins in literally hundreds of signaling/DNA recognition processes. The studies are being supplemented with further structural determinations for other STAT NTDs. We have continued our studies of transcriptional regulation via antitermination in prokaryotes. Our studies have focused on the protein:protein and protein:RNA interactions associated with the NusB and NusE proteins and the boxA RNA. We have determined the solution structure of NusB from Aquifex aeolicus, shown it to be consistent with all NusB homologues, and investigated the biophysics of NusB:NusE interactions. These studies have revealed the binding site of NusE on NusB and provide an explanation for the increased affinity for boxA RNA binding to the binary NusB:NusE complex compared to boxA RNA:NusB binding. This work is under revision for publication in the Journal of Molecular Biology. Additionally, the solution structure of the E. coli NusB:boxA RNA binary complex is nearly completed. This complex reveals new insight into the ternary complex of NusB:NusE:boxA RNA. These studies are enhanced by analyses of the binding interactions for various known mutations of NusB. The overall study will enable a more detailed and global picture of the interactions critical to the process of antitermination than have been available before. In collaboration with the Molecular Targets Development Program, CCR, we have reported the solution structure of the novel anti-viral protein Scytovirin, published in the Journal of Molecular Biology. Our studies included examination of the oligosaccharide binding, which is the key element of the entry-inhibition mechanism proposed for Scytovirin. This protein exhibits remarkable anti-viral activity against HIV and Ebola. Animal studies are underway to guide future development and will be combined with structurally guided engineering studies. We are continuing to study the details of the oligosaccharide binding. Through the use of site engineering, we hope to generate a protein with only one active binding site, that would facilitate both a detailed structural analyses and be valuable in dissecting the interaction with viral gp41 and gp120. Our studies of the ubiquitin-protein ligase E3, called gp78, is being conducted in collaboration with Dr. Allan Weissman, CCR. The gp78 protein has a role in sarcoma metastasis, and it is an excellent target for a combined structural and molecular biological investigation of mechanism and intervention. These studies are underway and involve both investigations of individual domains of gp78 as well as interactions with other critical components, ubiquitin and the mammalian E2 associated with this protein. New results are emerging from these studies and will be reported on in the coming year. We devote a part of our efforts to the development of improved NMR techniques and hardware, as well as protein engineering. Recent work in our laboratory has developed new approaches to tagging macromolecules with paramagnetic centers and obtaining unique structural information about intermolecular interactions and structures of multi-component complexes. This effort is continuing as a subset of the various structural biology projects described above
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