Office: PHSC 130C Phone: (405) 325-1678 Email: elenaz@ou.edu	Helen I. Zgurskaya Associate Professor B.S. (Dnipropetrovsk State University, Ukraine) 1989 Ph.D. (Russian Academy of Sciences, Moscow) 1992 Postdoctoral Research Fellow (Stanford University Medical School) 1995-1996 Postdoctoral Research Associate (University of California, Berkeley) 1996-1999 OU Junior Faculty Research Program Recipient, 2001
Division: Biochemistry Research Interests Structure and functions of bacterial membrane; mechanisms of multidrug efflux transport systems and periplasmic membrane fusion proteins; designing of experimental approaches to study two-membrane systems.

Opening for Research Assistant Professor

Research Description

Multidrug resistance presents a serious problem in the treatment of bacterial infections. This type of bacterial resistance arises due to the overexpression of  transporters that recognize and efficiently expel from the cells a broad range of structurally unrelated antimicrobial compounds including antibiotics, detergents, dyes and organic solvents. We seek to achieve an understanding of the molecular and biochemical mechanisms of this phenomenon. Specifically, our goal is to elucidate in biochemical terms the mechanism of multidrug transporters of Gram-negative bacteria that cause devastating diseases in animals and human.

The defining feature of Gram-negative bacteria is the presence of two layers of membranes, inner and outer, separated by the periplasmic space. Multidrug transporters of Gram-negative bacteria are constructed in a unique manner: a transporter located in the inner membrane (IM) functions with an outer membrane (OM) channel and a periplasmic “linker” protein (Figure). In this arrangement, efflux complexes traverse both, the inner and the outer, membranes and thus facilitate direct passage of the substrate from the cytoplasm or the inner membrane into the external medium.

Our research is concentrated on two puzzling mechanistic features of these three-component multidrug transporters. First, the IM efflux transporter, which is responsible for multidrug recognition₂, efficiently pumps out from the cells an extraordinary range of lipophilic and amphiphilic compounds. These transporters appear to pump their substrates directly from the phospholipid bilayer of the membrane, rather than across the membrane. Thus, the first line of research in my group focuses on understanding the mechanism of substrate recognition by IM transporters. Second, the drug efflux from IM occurs without drug accumulation in the periplasmic space suggesting a tight coupling of drug efflux with its subsequent export across the outer membrane. This feature poses an apparent mechanistic conundrum, since the outer membrane proteins do not have a direct access to an energy source that is required for directional transport to occur. It was postulated therefore that the periplasmic component of the complex mediates the coupling between IM transporter and the OM channel. Structural studies showed that the periplasmic linkers are highly elongated proteins₁ and might function in the multi-drug efflux across two membranes by creating the adhesion sites between the IM and OM₂. Presently we seek to determine how the three components are assembled into an active efflux complex.

We take two converging approaches. One is based on the purification and reconstitution of proteins in the artificial phospholipid bilayer. In this system we structurally and functionally characterize the individual components of the complex, as well as the overall organization of multidrug transporters by using the hydrodynamic, spectroscopic and enzymological techniques. Our second approach is the in vivo and in vitro biochemical evaluation of the individual components, containing genetically engineered mutations in target residues of interest.

1. Tikhonova, E.B., Devroy, V., Lau, S.Y., Zgurskaya, H.I. Reconstitution of the Escherichia coli macrolide transporter: the periplasmic membrane fusion protein MacA stimulates the ATPase activity of MacB. Mol. Microb. 2007, 63(3): 895-910.

2. Lomovskaya, O., Zgurskaya, H.I., Totrov. M. and Watkins, W.J. Waltzing Transporters and “The Dance Macabre” between Humans and Bacteria. Nat. Rev. Drug Discov., 2007 Jan;6(1):56-65.

3. Dastidar, V., Mao, W., Lomovskaya, O., Zgurskaya H.I. Drug-induced conformational changes in multidrug efflux transporter AcrB from Haemophilus influenzae. J. Bacteriol. 2007, 189(15):5550-8.

4. Zgurskaya, H.I. Molecular Analysis of Efflux Pump Based Antibiotic Resistance. Int. J. Clin. Microbiol., 2002, 292:95-105.

5. Tikhonova, E.B., Wang, Q. and Zgurskaya, H.I. Chimeric analysis of the multicomponent multidrug efflux transporters from gram-negative bacteria. J. Bacteriol., 2002, 184, 6499-6507.
   
   
6. Lomovskaya, O., Zgurskaya, H.I. and Nikaido, H. It takes three to tango. Nature Biotechnol. 2002, 12: 1210-1212.
   
   
7. Zgurskaya, H.I., Krishnamoorthy, G., Tikhonova, E.B., Lau, S.Y. and Stratton, K.L. Mechanism of antibiotic efflux in gram-negative bacteria. Front. Biosci.., 2003, 8:s862-73.
   
   
8. Yu, E.W., McDermott, G., Zgurskaya, H.I, Nikaido, H., Koshland, D.E. Structural basis of multiple drug binding capacity of the AcrB multidrug efflux pump. Science, 2003, 300: 976-80.
   
   
9. Ip, H., Stratton K., Zgurskaya, H. I. and Liu, J. pH-induced conformational changes of AcrA, the membrane fusion protein of Escherichia coli multidrug efflux system. J. Biol. Chem. 2003, 278(50): 50474-82.
   
   
10. Tikhonova E.B. and Zgurskaya, H.I. AcrA, AcrB and TolC of Escherichia coli form a stable intermembrane multidrug efflux complex. J. Biol. Chem., 2004, 279(31):32116-24.
    
    
11. Zakharov S.D., Erukova V.Y., Rokitskaya T.I., Zhalnina M.V., Sharma O., Loll P.J., Zgurskaya H.I., Antonenko Y.N., Cramer W.A. Colicin Occlusion of OmpF and TolC Channels: Outer Membrane Translocons for Colicin Import. Biophys J. 2004, 87(6):3901-11.
    
    
12. Lau, S.Y. and Zgurskaya, H.I. Cell division defects in Escherichia coli deficient of the multidrug transporter AcrEF-TolC. J. Bacteriol. 2005, 187:7815-25.
    
    
13. Mikoloshko, J., Bobyk, K., Zgurskaya, H.I. and Ghosh, P. Conformational flexibility in the multidrug efflux system protein AcrA. Structure, 2006, 21:577-587.