Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multi-Drug Resistance pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multi-Drug Resistance pumps (SMRs) are 100-110 residue, dimeric proton-drug antiporters that contain the full multidrug transport machinery, stripped to its barest essentials. Hence they are ideal transporters for a comprehensive structural and functional understanding of drug transport and inhibition in a medically important MDR. Unfortunately, the conformational plasticity that enables these proteins to transport such diverse substrates has led to very different structural models and structural interpretations, particularly for the protein in detergent micelles. Thus we propose to determine the lipid bilayer-embedded structures of the conformations making up the functional cycle of the SMR from Staph aureus that provides resistance against common disinfectants, and identify the binding determinants for multiple drugs and inhibitors using solution NMR by: 1) Developing a high performance discoidal peptide-lipid bilayer """"""""nanodiskette"""""""" system for NMR studies of membrane proteins, 2) Establishing the relative topology for the Smr monomers (parallel vs inverted), followed by full structure determination of the apo-form of the protein in nanodiskettes, 3) Identifying the binding determinants for transportable drugs and inhibitors, 4) Defining the structural changes in the substrate-bound, and inhibitor-bound Smr transporter, and 5) Characterizing the fold of the most controversial family member, EmrE, in lipid nanodiskettes as a function of protonation state and TPP binding.
The Small Multidrug Resistance transporter protein protects strains of Staph aureus that carry it from common hospital disinfectants. We will determine the three dimensional structure of the protein by itself, and in complex with the compounds that it transports, in order to understand how it functions, and ultimately guide the design of inhibitors of the transporter.
|Liu, Nina; Tao, Yisong; Brenowitz, Michael D et al. (2015) Structural and Functional Studies on the Marburg Virus GP2 Fusion Loop. J Infect Dis 212 Suppl 2:S146-53|
|Laage, Ségolène; Tao, Yisong; McDermott, Ann E (2015) Cardiolipin interaction with subunit c of ATP synthase: solid-state NMR characterization. Biochim Biophys Acta 1848:260-5|
|Dai, Zhou; Tao, Yisong; Liu, Nina et al. (2015) Conditional trimerization and lytic activity of HIV-1 gp41 variants containing the membrane-associated segments. Biochemistry 54:1589-99|
|Padlan, Camille S; Malashkevich, Vladimir N; Almo, Steve C et al. (2014) An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates. RNA 20:447-61|
|Regula, Lauren K; Harris, Richard; Wang, Fang et al. (2013) Conformational properties of peptides corresponding to the ebolavirus GP2 membrane-proximal external region in the presence of micelle-forming surfactants and lipids. Biochemistry 52:3393-404|
|Poget, Sébastien F; Harris, Richard; Cahill, Sean M et al. (2010) 1H, 13C, 15N backbone NMR assignments of the Staphylococcus aureus small multidrug-resistance pump (Smr) in a functionally active conformation. Biomol NMR Assign 4:139-42|
|Wu, Haiyan; Shekar, S Chandra; Flinn, Rory J et al. (2009) Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110alpha and are disrupted in oncogenic p85 mutants. Proc Natl Acad Sci U S A 106:20258-63|
|Shekar, S Chandra; Backer, Jonathan M; Girvin, Mark E (2008) Effectively doubling the magnetic field in spin-1/2-spin-1, HSQC, HDQC, coupled HSQC, and coupled HDQC in solution NMR. J Chem Phys 128:184501|
|Poget, Sebastien F; Girvin, Mark E (2007) Solution NMR of membrane proteins in bilayer mimics: small is beautiful, but sometimes bigger is better. Biochim Biophys Acta 1768:3098-106|
|Poget, Sebastien F; Cahill, Sean M; Girvin, Mark E (2007) Isotropic bicelles stabilize the functional form of a small multidrug-resistance pump for NMR structural studies. J Am Chem Soc 129:2432-3|