Structure of the catalytic domain of human protein kinase C beta II complexed with a bisindolylmaleimide inhibitor.

The conventional protein kinase C isoform, PKCII, is a signaling kinase activated during the hyperglycemic state and has been associated with the development of microvascular abnormalities associated with diabetes. PKCII, therefore, has been identified as a therapeutic target where inhibitors of its kinase activity are being pursued for treatment of microvascular-related diabetic complications. In this report, we describe the crystal structure of the catalytic domain of PKCbetaII complexed with an inhibitor at 2.

The NS3 proteinase domain of hepatitis C virus is a zinc-containing enzyme.

NS3 proteinase of hepatitis C virus (HCV), contained within the N-terminal domain of the NS3 protein, is a chymotrypsin-like serine proteinase responsible for processing of the nonstructural region of the HCV polyprotein. In this study, we examined the sensitivity of the NS3 proteinase to divalent metal ions, which is unusual behavior for this proteinase class. By using a cell-free coupled transcription-translation system, we found that HCV polyprotein processing can be activated by Zn2+ (and, to a lesser degree, by Cd2+, Pb2+, and Co2+) and inhibited by Cu2+ and Hg2+ ions.

Structure-function analysis of the mammalian DNA polymerase beta active site: role of aspartic acid 256, arginine 254, and arginine 258 in nucleotidyl transfer.

The crystal structure of the catalytic domain of rat DNA polymerase beta revealed that Asp256 is located in proximity to the previously identified active site residues Asp190 and Asp192. We have prepared and kinetically characterized the nucleotidyl transfer activity of wild type and several mutant forms of human and rat pol beta. Herein we report steady-state kinetic determinations of KmdTTP, Km(dT)16, and kcat for mutants in residue Asp256 and two neighboring residues, Arg254 and Arg258, all centrally located on strand beta 7 in the pol beta structure.

Heterologous expression and purification of active human phosphoribosylglycinamide formyltransferase as a single domain.

We report here for the first time that the GART domain of the human trifunctional enzyme possessing GARS, AIRS, and GART activities can be expressed independently in Escherichia coli at high levels as a stable protein with enzymatic characteristics comparable to those of native trifunctional protein. Human trifunctional enzyme is involved in de novo purine biosynthesis, and has long been recognized as a target for antineoplastic intervention. The GART domain was expressed in E.

Proteolytic release and crystallization of the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase.

The RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase was released from recombinant DHFR-RNase H fusion protein by the action of HIV-1 protease and crystallized as large trigonal prisms that diffract x-rays to at least 2.4-A resolution. The protease cleavage occurred 18 residues away from the Phe440-Tyr441 site reported to be processed during maturation of the reverse transcriptase heterodimer.