Bacteria have developed several methods to resist the lethal effects of antibiotics. The broadest spectrum resistance results from the action of Multidrug resistant pumps (MDRs), which extrude a range of compounds of quite diverse chemical structure. The Small Multidrug 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. We propose to determine the structures of the conformations making up the functional cycle of an SMR, and identify the binding determinants for multiple drugs and inhibitors using solution NMR by: 1) Determining the structure of a dimeric SMR in lysolipid micelles in its protonated state, 2) Measuring the affinities of multiple drugs and inhibitors above and below the pKa of the glutamate essential for transport, 3) Identifying the binding determinants for inhibitors and transportable drugs, and 4) Determining the conformational changes induced by substrate and inhibitor binding, and by deprotonation of the critical glutamate. ? ? In addition to the MDR pumps, membrane proteins are responsible for transmembrane signaling, energy transduction, and ion and metabolite transport. These proteins are important in infectious disease, genetic disorders, and cancer. Despite their importance, and the need for structure to understand their function, relatively few such structures are available. For transporters and receptors, ligand binding and transport involve multiple conformations and dynamic changes. Solution NMR is an ideal method for examining these processes. Firmly establishing NMR methods to study larger helical membrane proteins - here a dimer with total of eight transmembrane helices, will have as long-term an impact as the specific findings for an SMR. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM072085-04
Application #
7264495
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Chin, Jean
Project Start
2004-08-01
Project End
2010-07-31
Budget Start
2007-08-01
Budget End
2010-07-31
Support Year
4
Fiscal Year
2007
Total Cost
$310,075
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
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