Since atomic structure is key to understanding mechanisms and to designing modulators, the long- term objective of this proposal is to establish the basis for understanding four functional classes of membrane proteins at this level of molecular structure and mechanism. In the aquaporin family (AQPs) specific goals include (1) determining the basis of selectivity using mutations in four AQPs whose structures have already determined in our laboratory, (2) expression and structure determination of a human aquaporin AQP4, with impact for drug design against collateral damage in stroke, and the malaria parasite aquaporin PfAQP, a new anti-malarial drug-target. A chemical 'tethering'approach will identify compounds that can be used to prove the principle of the malarial aquaporin as a drug target. Building on our landmark first molecular structure and mechanism of a transmembrane Ammonia transporter, AmtB, research is to establish the atomic basis for function using structural and functional analysis of mutant proteins, designed on the basis of our structure. A second goal in this family is determining the basis for AmtB regulation by GlnK. Structures of related family members that include the Rh subfamily present in eukaryotes will be sought. A third class is that of urea transporters where we seek to determine structures of a urea channel from the human pathogen Helicobacter pylorisince this channel is essential for pathogenicity in stomach ulcer. A second target is the urea transporter from P. aeruginosa. This target is homolgous to human urea transporters, particularly in the transmembrane regions, and thus presents an entry into this new membrane protein fold. Lastly we seek to refine conditions for expression in a membrane fraction, of a functional but simplified version of the neuronal nicotinic acetylcholine receptor obtained by heterologous expression of a unique single subunit, a7, that forms fully functional homopentameric AcChR channels. The techniques used in this research are evolving forefront methods for expression of membrane proteins, extraction and purification, crystallization, and structure determination of membrane proteins. Structural models will be tested and refined by mutational analysis coupled to structural and functional measurements on mutant proteins. The structural and functional analyses of native and mutant proteins will lead to procedures that can directly evaluate and/or establish their therapeutic advantage as drug targets, and provide a key to advancing their successful implementation as targets. While 40% of all targets for today's drugs are membrane proteins there is not yet a single atomic structure for any one such drug target membrane protein. The structures of these proteins will establish their mechanism of action and provide templates for drug discovery aimed at those of particular therapeutic advantage, and the essential elements to understand their selectivity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM024485-33
Application #
7752584
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Chin, Jean
Project Start
1979-04-01
Project End
2011-12-31
Budget Start
2010-01-01
Budget End
2010-12-31
Support Year
33
Fiscal Year
2010
Total Cost
$440,878
Indirect Cost
Name
University of California San Francisco
Department
Biochemistry
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Kapoor, Khyati; Finer-Moore, Janet S; Pedersen, Bjørn P et al. (2016) Mechanism of inhibition of human glucose transporter GLUT1 is conserved between cytochalasin B and phenylalanine amides. Proc Natl Acad Sci U S A 113:4711-6
Thurtle-Schmidt, Bryan H; Stroud, Robert M (2016) Structure of Bor1 supports an elevator transport mechanism for SLC4 anion exchangers. Proc Natl Acad Sci U S A 113:10542-6
Kintzer, Alexander F; Stroud, Robert M (2016) Structure, inhibition and regulation of two-pore channel TPC1 from Arabidopsis thaliana. Nature 531:258-62
Salo-Ahen, Outi M H; Tochowicz, Anna; Pozzi, Cecilia et al. (2015) Hotspots in an obligate homodimeric anticancer target. Structural and functional effects of interfacial mutations in human thymidylate synthase. J Med Chem 58:3572-81
Tochowicz, Anna; Santucci, Matteo; Saxena, Puneet et al. (2015) Alanine mutants of the interface residues of human thymidylate synthase decode key features of the binding mode of allosteric anticancer peptides. J Med Chem 58:1012-8
Johri, Atul K; Oelmüller, Ralf; Dua, Meenakshi et al. (2015) Fungal association and utilization of phosphate by plants: success, limitations, and future prospects. Front Microbiol 6:984
Kim, JungMin; Wu, Shenping; Tomasiak, Thomas M et al. (2015) Subnanometre-resolution electron cryomicroscopy structure of a heterodimeric ABC exporter. Nature 517:396-400
Rosenberg, Oren S; Dovala, Dustin; Li, Xueming et al. (2015) Substrates Control Multimerization and Activation of the Multi-Domain ATPase Motor of Type VII Secretion. Cell 161:501-512
Kumar, Hemant; Finer-Moore, Janet S; Kaback, H Ronald et al. (2015) Structure of LacY with an ?-substituted galactoside: Connecting the binding site to the protonation site. Proc Natl Acad Sci U S A 112:9004-9
Monk, Brian C; Tomasiak, Thomas M; Keniya, Mikhail V et al. (2014) Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer. Proc Natl Acad Sci U S A 111:3865-70

Showing the most recent 10 out of 75 publications