Maureen D. Dwyer

Bio: Maureen D. Dwyer is an academic researcher from Vertex Pharmaceuticals. The author has contributed to research in topics: DNA & Binding site. The author has an hindex of 5, co-authored 5 publications receiving 1859 citations.

Active-Site Residues of Escherichia coli DNA Gyrase Required in Coupling ATP Hydrolysis to DNA Supercoiling and Amino Acid Substitutions Leading to Novobiocin Resistance

Abstract: DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP. PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A(2)B(2) gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli.

Crystal structure of calcium-free human sorcin: A member of the penta-EF-hand protein family

Abstract: Sorcin is a 22 kD calcium-binding protein that is found in a wide variety of cell types, such as heart, muscle, brain and adrenal medulla. It belongs to the penta-EF-hand (PEF) protein family, which contains five EF-hand motifs that associate with membranes in a calcium-dependent manner. Prototypic members of this family are the calcium-binding domains of calpain, such as calpain dVI. Full-length human sorcin has been crystallized in the absence of calcium and the structure determined at 2.2 A resolution. Apart from an extended N-terminal portion, the sorcin molecule has a globular shape. The C-terminal domain is predominantly alpha-helical, containing eight alpha-helices and connecting loops incorporating five EF hands. Sorcin forms dimers through the association of the unpaired EF5, confirming this as the mode of association in the dimerization of PEF proteins. Comparison with calpain dVI reveals that the general folds of the individual EF-hand motifs are conserved, especially that of EF1, the novel EF-hand motif characteristic of the family. Detailed structural comparisons of sorcin with other members of PEF indicate that the EF-hand pair EF1-EF2 is likely to correspond to the two physiologically relevant calcium-binding sites and that the calcium-induced conformational change may be modest and localized within this pair of EF-hands. Overall, the results derived from the structural observations support the view that, in sorcin, calcium signaling takes place through the first pair of EF-hands.

The Calpain System

Abstract: The calpain system originally comprised three molecules: two Ca2+-dependent proteases, mu-calpain and m-calpain, and a third polypeptide, calpastatin, whose only known function is to inhibit the two calpains. Both mu- and m-calpain are heterodimers containing an identical 28-kDa subunit and an 80-kDa subunit that shares 55-65% sequence homology between the two proteases. The crystallographic structure of m-calpain reveals six "domains" in the 80-kDa subunit: 1). a 19-amino acid NH2-terminal sequence; 2). and 3). two domains that constitute the active site, IIa and IIb; 4). domain III; 5). an 18-amino acid extended sequence linking domain III to domain IV; and 6). domain IV, which resembles the penta EF-hand family of polypeptides. The single calpastatin gene can produce eight or more calpastatin polypeptides ranging from 17 to 85 kDa by use of different promoters and alternative splicing events. The physiological significance of these different calpastatins is unclear, although all bind to three different places on the calpain molecule; binding to at least two of the sites is Ca2+ dependent. Since 1989, cDNA cloning has identified 12 additional mRNAs in mammals that encode polypeptides homologous to domains IIa and IIb of the 80-kDa subunit of mu- and m-calpain, and calpain-like mRNAs have been identified in other organisms. The molecules encoded by these mRNAs have not been isolated, so little is known about their properties. How calpain activity is regulated in cells is still unclear, but the calpains ostensibly participate in a variety of cellular processes including remodeling of cytoskeletal/membrane attachments, different signal transduction pathways, and apoptosis. Deregulated calpain activity following loss of Ca2+ homeostasis results in tissue damage in response to events such as myocardial infarcts, stroke, and brain trauma.

Flaviviridae :T he Viruses and Their Replication

Abstract: FLAVIVIRUSES 1103 Background and Classification 1103 Structure and Physical Properties of the Virion 1104 Binding and Entry 1105 Genome Structure 1106 Translation and Proteolytic Processing 1107 Features of the Structural Proteins 1108 Features of the Nonstructural Proteins 1109 RNA Replication 1112 Membrane Reorganization and the Compartmentalization of Flavivirus Replication 1112 Assembly and Release of Particles from Flavivirus-infected Cells 1112 Host Resistance to Flavivirus Infection 1113

Treatment with Indinavir, Zidovudine, and Lamivudine in Adults with Human Immunodeficiency Virus Infection and Prior Antiretroviral Therapy

Abstract: Background The new protease inhibitors are potent inhibitors of the human immunodeficiency virus (HIV), and in combination with other antiretroviral drugs they may be able to cause profound and sustained suppression of HIV replication. Methods In this double-blind study, 97 HIV-infected patients who had received zidovudine treatment for at least 6 months and had 50 to 400 CD4 cells per cubic millimeter and at least 20,000 copies of HIV RNA per milliliter were randomly assigned to one of three treatments for up to 52 weeks: 800 mg of indinavir every eight hours; 200 mg of zidovudine every eight hours combined with 150 mg of lamivudine twice daily; or all three drugs. The patients were followed to monitor the occurrence of adverse events and changes in viral load and CD4 cell counts. Results The decrease in HIV RNA over the first 24 weeks was greater in the three-drug group than in the other groups (P<0.001 for each comparison). RNA levels decreased to less than 500 copies per milliliter at week 24 in 28 of...

The Properties of Known Drugs. 1. Molecular Frameworks

Abstract: In order to better understand the common features present in drug molecules, we use shape description methods to analyze a database of commercially available drugs and prepare a list of common drug shapes. A useful way of organizing this structural data is to group the atoms of each drug molecule into ring, linker, framework, and side chain atoms. On the basis of the two-dimensional molecular structures (without regard to atom type, hybridization, and bond order), there are 1179 different frameworks among the 5120 compounds analyzed. However, the shapes of half of the drugs in the database are described by the 32 most frequently occurring frameworks. This suggests that the diversity of shapes in the set of known drugs is extremely low. In our second method of analysis, in which atom type, hybridization, and bond order are considered, more diversity is seen; there are 2506 different frameworks among the 5120 compounds in the database, and the most frequently occurring 42 frameworks account for only one-fou...