STRUCTURAL STUDIES OF PROTEINS ASSOCIATED WITH HUMAN DISEASES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Specimen 1: Carbonic anhydrase Carbonic anhydrases (CA)s are zinc-metalloenzyme that catalyzes the reversible inter-conversion of CO2 and HCO3-. Recently, a convincing body of evidence has been accumulated, that suggests the over-expression of CA isozyme IX (CA IX) contributes to the acidication of the extracellular matrix, which is thought to promote growth of certain tumors. In light of these observations, and based on structural alignment homology, using the crystal structure CA II and the sequence of CA IX, we have constructed a double mutant of CA II with Ala 65 replaced by Ser and Asn 67 replace by Gln to resemble the active site of CA IX. This CAIX mimic will be studied using X-ray crystallography, alone and in complex with several clinically used CAs inhibitors and compared to wild-type CA II. Further this structural information will be evaluated in relationship to inhibition and in vitro cytotoxicity assays and a correlated structure-activity relationship will be developed. These studies will provide a useful model to design more isozyme specific CA IX inhibitors that may lead to development of new therapeutic treatments of some cancers. We will also be trying to use high pressure freezing with CO2 (in collaboration with Sol and Chaeun (CHESS) to capture the bound substrate in wild-type and/or apo HCA II. Specimen 2: human Mn SuperOxide Dismutase The function in structure, stability, and catalysis of the interfaces between subunits in manganese superoxide dismutase (MnSOD) will be examined. MnSOD catalyzes the disproportionation of superoxide to produce O2 and H2O2. Human MnSOD is a homotetramer of 22 kDa subunits, a dimer of dimers, with two structurally unique interfaces, a dimeric and a tetrameric interface. The function in catalysis, stability, and structure of these interfaces is uncertain and various mutants MnSOD structures will be studied and correlated to kinetic and stability properties. As mutant forms of MnSOD are prepared as possible antiproliferative agents, such as H30N MnSOD that exhibits less product inhibition compared with wild-type, it should be expected that alteration of other residues at the dimeric interface are likely to diminish catalysis and enhance product inhibition. Specimen 3: HIV-1 subype C protease Human Immunodeficiency Virus type 1 (HIV-1) protease (PR) is an aspartic hydrolase that functions as an obligatory homodimer with 99 amino acids in each subunit (labeled 1-99 and 1¿¿"-99¿¿"). Its role is to cleave the gag and gag/pol polyproteins into structural and enzymatic proteins and to induce the formation of mature, infectious virions. The inhibition of this enzyme yields immature HIV virions, incapable of spreading the infection. Because of its essential role in gaining viral infectivity, HIV-1 PR has been considered an attractive target for discovering new and potent anti-HIV drugs. The structure of the unbound/bound C PR and various unbound/bound multi-drug resistant variant of C PR will be studied to identify structural changes due to the naturally occurring polymorphisms and delineate their implications in antiretroviral drug resistance/susceptibility.