This project seeks to determine the mechanisms, structures, and structure change of integral transmembrane proteins that govern critical transmembrane processes, at the level that can lead to improved therapeutics for human disease. The premise is that alterations in molecular structures are necessary for the function of transmembrane transporters and gated channels, and are coordinated by regulatory functions. The hypothesis is that understanding the linkage between structure change and function provides a roadmap for therapeutic intervention by organic compounds or Fab fragments generated to stabilize conformational states. A major innovation is the technology and ability to determine atomic structures of membrane proteins and eukaryotic, or human membrane proteins at a resolution sufficient to instruct in the development of therapeutic development of compounds. Principal technologies include X-ray diffraction, electron cryomicroscopy, transport assays, electrophysiology.
Three aims focus on different classes of transmembrane proteins.
Aim 1 focuses on elaborating the mechanisms of a recently discovered class of intracellular channels that govern the release of ions and nutrients from the vacuole in plants or fungi, or the endolysosome in animals.
One aim i s to build on our atomic structure determination of a two-pore channel TPC1 from plants, and to determine how regulation of ion transport by voltage, by calcium ions, and by phosphorylation is brought about.
The aim moves toward human TPC1 where an inhibitor seen in our structure can cure mice of Ebola virus that enters the cell through the endolysosome, and to another intracellular channel human TRPML where mutations cause a lysosomal storage disease.
Aim 2 seeks to determine the mechanisms that govern secondary transmembrane transporters and their sister uniporters.
The aim focuses first on a high affinity phosphate transporter where we obtained high resolution structure, made 22 mutations and recorded transport properties Vmax and Km, and effects on growth of yeast deleted of its own phosphate transporters, expressing the mutants in the plasma membrane. We also focus on mutants in the lactose transporter that for the first time converted the structure between states in the biological transport cycle. The goal is to understand how the binding and release of substrates is coupled to the transport of a driving ion, protons, and to see if this surprising mechanism is common throughout secondary transporters.
This aim also addresses a human glucose transporter where we showed how drug leads block the uniporter. This transporter is relevant to many cancers.
Aim 3 aims to leverage our atomic structure of human brain aquaporin 4, to understand the binding by patient antibodies with the autoimmune, sometimes lethal disease neuromyelitis optica. This will open the way to ask how we may alter this interaction to therapeutic benefit.

Public Health Relevance

Biological membranes of the eukaryotic cell, both outside and inside, govern transport of ions, nutrients, and insulate from toxic compounds. The mechanisms of transport or gating require a conformational cycle of opening and closing on alternate sides of the membrane. These conformational cycles therefore offer several avenues for modulation by drugs, or biotherapeutics. We focus on eukaryotic and human membrane proteins because of their roles in diseases, here including viral infection, lysosomal diseases, glucose acquisition and autoimmunity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024485-43
Application #
9842614
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Preusch, Peter
Project Start
1979-04-01
Project End
2020-12-31
Budget Start
2020-01-01
Budget End
2020-12-31
Support Year
43
Fiscal Year
2020
Total Cost
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
94118
Kintzer, Alexander F; Green, Evan M; Dominik, Pawel K et al. (2018) Structural basis for activation of voltage sensor domains in an ion channel TPC1. Proc Natl Acad Sci U S A 115:E9095-E9104
Finer-Moore, Janet S; Lee, Tom T; Stroud, Robert M (2018) A Single Mutation Traps a Half-Sites Reactive Enzyme in Midstream, Explaining Asymmetry in Hydride Transfer. Biochemistry 57:2786-2795
Kumar, Hemant; Finer-Moore, Janet S; Jiang, Xiaoxu et al. (2018) Crystal Structure of a ligand-bound LacY-Nanobody Complex. Proc Natl Acad Sci U S A 115:8769-8774
Kintzer, Alexander F; Stroud, Robert M (2018) On the structure and mechanism of two-pore channels. FEBS J 285:233-243
Boswell-Casteel, Rebba C; Johnson, Jennifer M; Stroud, Robert M et al. (2016) Integral Membrane Protein Expression in Saccharomyces cerevisiae. Methods Mol Biol 1432:163-86
Kintzer, Alexander F; Stroud, Robert M (2016) Structure, inhibition and regulation of two-pore channel TPC1 from Arabidopsis thaliana. Nature 531:258-62
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
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
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

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