Structural studies of DCoH, a bifunctional enzyme and protein-binding transcriptional coactivator

Although protein-DNA interactions have been extensively characterized in numerous structure determinations, the protein-protein interactions that must also play a critical role in the regulation of transcription are less well understood at a detailed atomic level. The work described in this doctoral thesis centers on the crystal structure of a protein known as Dimerization Cofactor of HNF-1 (DCoH), a coactivator of transcription. Coactivators, a diverse class of proteins, are thought in many cases to stimulate transcription via protein-protein interactions that bridge DNA-binding transcription factors and the basal transcriptional apparatus. The DCoH structure, which provides the first atomic level picture of a coactivator, unexpectedly bears a striking resemblance to TATA-binding protein (TBP). Both DCoH and TBP possess a distinctive "saddle" motif formed by a curved, antiparallel {dollar}beta{dollar}-sheet. The structural similarity with TBP and the RNP RNA-binding motif raised the possibility that DCoH could participate in interactions with nucleic acids, and has suggested hypotheses regarding its mechanism of coactivation.; The work detailed in this thesis demonstrates that a complex between a peptide corresponding to the N-terminus of HNF-{dollar}1alpha{dollar} and DCoH does not form under native conditions. However, if DCoH--a 48 kD homotetramer under native conditions--is allowed to associate with the peptide under refolding conditions, a 31 kD species forms. This complex corresponds to a heterotetramer comprised of two monomers each of the peptide and DCoH. Formation of the complex precludes homotetramerization of DCoH, and vice-versa. Combined with an analysis of the DCoH structure, the results support a model for the interaction of DCoH with HNF-{dollar}1alpha.{dollar} The alternate states of DCoH, the homotetramer and the heterotetramer with HNF-1{dollar}alpha,{dollar} may be relevant to the regulation of DCoH activity in vivo.; Another aspect of this work derives from the fact that DCoH also is an enzyme. It catalyses the dehydration of a carbinolamine form of tetrahydrobiopterin, a step in the regeneration of this cofactor, which is required for aromatic amino acid hydroxylases and nitric oxide synthase. To directly visualize the active site for this enzymatic activity and to define the mechanism of catalysis, the crystal structure of the complex of DCoH with a pterin product analog was determined. Comparison of the DCoH-pterin structure with the other available structural information for pterin-binding proteins shows that DCoH employs a novel mode of recognition for this ligand. At least two mechanistic proposals are consistent with the DCoH-pterin structure.; DCoH may provide a paradigm for transcriptional activation by a coactivator. The proximity of the pterin binding site to the saddle motif raises the possibility of a connection between the enzymatic and coactivation functions. The functioning of DCoH homologs in a variety of contexts, from bacteria to Xenopus embryos to mammalian liver, and the similarity with TBP supports the possibility of interactions with other targets, including nucleic acids. The proposed model for HNF-{dollar}1alpha{dollar} binding accords with this idea, in that the TBP-like saddle remains available for another interaction. The significance of the present work also lies in large measure in the ability of structure to guide formulation of testable hypotheses concerning the modes by which a protein exerts its biological effects. (Abstract shortened by UMI.)...