This proposal combines my two NIGMS grants. Our work on the M2 proton channel from influenza A virus (GM56423) currently focuses on the mechanism of proton movement through the channel. M2 is also the target of the amantadine class of influenza drugs, and most isolates of influenza A virus are now amantadine- resistant. Crystallographic structures of M2 in various functional states will be solved at very high resolution using the X-ray free electron laser to enable structure determination at room temperature. Parallel, collaborative studies use single-molecule measurement and 2DIR to probe dynamics. Very high-resolution structures of a series of small molecule inhibitors in complex with amantadine-resistant mutants of M2 are being determined, to enable our collaborators to conduct structure-based drug design. De novo protein design (GM54616) provides a means to test and refine our understanding of protein structure and function. We address questions of sequence-specific recognition in membranes. A variety of methods exist for the design or selection of antibodies and other reagents that recognize the water-soluble regions of proteins. However, companion methods for targeting Transmembrane (TM) regions are not generally available. Therefore, we are developing methods for the computational design of peptides that target TM helices in a sequence-specific manner, focusing on EGF receptors (collaboration with Natalia Jura) and integrins (collaboration with A. Orr). To elucidate the mechanisms by which proton-coupled transporters function, we have designed model proteins that use proton gradients to drive transport of transition metal ions up a gradient. We are increasing the efficiency of these minimal models and also expanding our methods to allow design of phosphate transporters and lipid flippases. We propose to continue work on the design of model diiron proteins to determine how a protein tunes the properties of these cofactors to affect diverse O2- dependent processes such as substrate oxidation and radical formation. We are designing water and membrane-soluble versions of the protein; by varying the identity and geometry of ligands and the water- accessibility of the center to determine how these parameters they define reactivity. We are studying the mechanisms by which bacterial histidine kinases transmit conformational information through multi-domain TM proteins. HKs are widely used by bacteria to sense and respond to diverse environmental cues such as nutrients or noxious substances. Crystal structures of various truncated domains of HKs have been solved. However, there are no high-resolution structures for HK membrane- spanning domains or full-length HKs, and their signaling mechanism is a matter of debate. By integrating structural information from diverse experimental techniques and functional measurements of HKs we seek to elucidate the mechanism of signaling in HKs. 59

Public Health Relevance

Our lab uses de novo protein design to test the principles of membrane protein structure and function ? if we understand membrane proteins we should be able to design them from scratch. We also study the structure and inhibition of M2, a transmembrane proton transporter from influenza A virus, which is the target of amantadine. Finally, we study transmembrane histidine kinases, which are used by bacteria to sense their environment. 59

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Unknown (R35)
Project #
3R35GM122603-02S1
Application #
9699960
Study Section
Program Officer
Preusch, Peter
Project Start
2017-05-01
Project End
2022-04-30
Budget Start
2018-05-01
Budget End
2019-04-30
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94118
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Yang, Bing; Wu, Haifan; Schnier, Paul D et al. (2018) Proximity-enhanced SuFEx chemical cross-linker for specific and multitargeting cross-linking mass spectrometry. Proc Natl Acad Sci U S A 115:11162-11167
Chino, Marco; Zhang, Shao-Qing; Pirro, Fabio et al. (2018) Spectroscopic and metal binding properties of a de novo metalloprotein binding a tetrazinc cluster. Biopolymers 109:e23339
Dang, Bobo; Wu, Haifan; Mulligan, Vikram Khipple et al. (2017) De novo design of covalently constrained mesosize protein scaffolds with unique tertiary structures. Proc Natl Acad Sci U S A 114:10852-10857
Thomaston, Jessica L; Woldeyes, Rahel A; Nakane, Takanori et al. (2017) XFEL structures of the influenza M2 proton channel: Room temperature water networks and insights into proton conduction. Proc Natl Acad Sci U S A 114:13357-13362
Polizzi, Nicholas F; Wu, Yibing; Lemmin, Thomas et al. (2017) De novo design of a hyperstable non-natural protein-ligand complex with sub-Å accuracy. Nat Chem 9:1157-1164