Browsing by Subject "Crystal structure"
Now showing 1 - 3 of 3
Results Per Page
Sort Options
Item Approaching the crystal structure of the polymerase γ catalytic complex(2011-08) Meng, Qingchao, master of arts in cell and molecular biology; Yin, Yuhui Whitney; Molineux, IanIn this thesis, a 4.7Å crystal structure of the human mitochondria DNA polymerase γ catalytic complex is reported. Though the DNA substrate-binding site is not identifiable in the structure, two conformational changes in the enzyme architecture are described: 1) rotation of the distal monomer of the accessory subunit towards the catalytic subunit, and 2) shift of the thumb motif of the polymerase domain towards the active site. Both conformational changes suggest a structure of Pol γ in the DNA-bound state and in its active site “closed” conformation.Item Structural Studies and Evaluation of Inhibitors of Mycobacterium tuberculosis H37Rv Shikimate Dehydrogenase (MtSDH)(2014-03-21) Lalgondar, MallikarjunShikimate dehydrogenase (SDH) is a reversible enzyme catalyzing the reduction of 3-dehydroshikimate (3DHS) to shikimate (SKM) utilizing NADPH cofactor in the shikimate pathway, a central route for biosynthesis of aromatic amino acids, folates and ubiquinones in microogransims, plants and parasites, which renders the enzymes of this essential pathway as attractive targets for developing antimicrobials, herbicides and antiparasitic agents. In this study, the crystal structure of Mycobacterium tuberculosis SDH (MtSDH) was determined in the apo-form and in complex with a ligand, SKM. The overall structure of MtSDH contains two structural domains with ?/? architecture. The N-terminal substrate binding domain and C-terminal cofactor binding domain are interconnected by two helices forming an active site groove where catalysis occurs. In MtSDH, a series of helices connecting ?10 and ?11 strands replace a long loop found in other known SDH structures and this region may undergo structural changes upon cofactor binding. NADP^(+) was modeled reliably in the cofactor binding site to gain insight into specific interactions. The analysis reveals that NADP(H) binds in anti conformation and in addition to residues in ?basic patch?, Ser125 within the glycine rich loop may interact with the 2'-phosphate of adenine ribose and form a novel cofactor binding microenvironment in SDH family of enzymes. Biochemically, five inhibitors identified previously from a high-throughput enzyme assay screen were evaluated. The IC_(50) values of these compounds range from 2.8-4.6 ?M. Further investigation indicates that these compounds display non-competitive or mixed inhibition mode with both substrate and cofactor. This study is expected to provide better understanding of MtSDH structural features and a framework for rational design of inhibitors based on initially characterized compounds.Item Structural studies of Gαq signaling and regulation(2008-12) Shankaranarayanan, Aruna; Tesmer, John; Hackert, Marvin L.Gαq signaling is implicated in a number of physiological processes that include platelet activation, cardiovascular development and smooth muscle function. Historically, Gαq is known to function by activating its effector, phospholipase Cβ. Desensitization of Gαq signaling is mediated by G-protein coupled receptor kinases (GRK) such as GRK2 that phosphorylates the activated receptor and also sequesters activated Gαq and Gβγ subunits. Our crystal structure of Gαq-GRK2-Gβγ complex shows that Gαq forms effector-like interactions with the regulator of G-protein signaling (RGS) homology domain of GRK2 involving the classic effector-binding site of Gα subunits, raising the question if GRK2 can itself be a Gáq effector and initiate its own signaling cascade. In the structure, Gα and Gβγ subunits are completely dissociated from one another and the orientation of activated Gαq with respect to the predicted cell membrane is drastically different from its position in the inactive Gαβγ heterotrimer. Recent studies have identified a novel Gαq effector, p63RhoGEF that activates RhoA. Our crystal structure of the Gαq-p63RhoGEF-RhoA complex reveals that Gαq interacts with both the Dbl homology (DH) and pleckstrin homology (PH) domains of p63RhoGEF with its C-terminal helix and its effector-binding site, respectively. The structure predicts that Gαq relieves auto-inhibition of the catalytic DH domain by the PH domain. We show that Gαq activates p63RhoGEF-related family members, Trio and Kalirin, revealing several conduits by which RhoA is activated in response to Gq-coupled receptors. The Gαq effector-site interaction with p63RhoGEF/GRK2 does not overlap with the Gαq-binding site of RGS2/RGS4 that function as GTPase activating proteins (GAPs). This suggests that activated G proteins, effectors, RGS proteins, and activated receptors can form high-order complexes at the cell membrane. We confirmed the formation of RGS-Gαq-effector complexes and our results suggest that signaling pathways initiated by GRK2 and p63RhoGEF are regulated by RGS proteins via both allosteric and GAP mechanisms. Our structural studies of Gαq signaling provide insight into protein-protein interactions that induce profound physiological changes. Understanding such protein interfaces is a key step towards structure-based drug design that can be targeted to treat diseases concerned with impaired Gαq signaling.