Scientific Report 2005

Molecular Biology

Structural Biology of Integral Membrane Proteins

G. Chang, A. Chen, Y. Chen, X. He, O. Pornillos, C.R. Reyes, P. Szewczk, A. Ward, S. Wada, Y. Yin

X-ray crystallography of integral membrane proteins is an exciting and rapidly growing frontier in molecular structural biology. We are interested in 5 areas: (1) the molecular structural basis for lipid and drug transport across the cell membrane by multidrug-resistance (MDR) transporters, (2) the high-resolution structure of yeast and mammalian MDR transporters, (3) signal transduction by receptors, (4) discovery and the structurally based design of potent MDR reversal agents, and (5) the development of an in vitro cell-free system capable of overproducing integral membrane proteins suitable for biophysical study. We use several experimental methods, including detergent/lipid protein biochemistry, 3-dimensional crystallization of integral membrane proteins, and x-ray crystallography. We are developing and using an efficient cell-free membrane protein expression system in collaboration with T. Kudlicki, Invitrogen Corporation, Carlsbad, California, for the overexpression integral membrane proteins for both x-ray crystallography and nuclear magnetic resonance studies.

We are addressing the molecular basis of MDR, a significant challenge in the treatment of infectious disease and cancer. A major cause of MDR in both of these situations is a battery of drug efflux pumps imbedded in the cell membrane. Through our structural studies on MDR transporters, we hope to gain insights into the mechanics of translocating amphipathic substrates across the cell membrane and also the rational design of potent MDR reversal agents.

We are combining chemistry and biology with structure for the discovery and design of potent MDR reversal agents for cancer chemotherapy in collaboration with M.G. Finn, Department of Chemistry; I. Urbatsch, Texas Tech University Health Sciences Center, Lubbock Texas; and S. Reutz, Novartis International AG, Basel, Switzerland. In collaboration with M. Saier, University of California, San Diego, and Q. Zhang, Department of Molecular Biology, we are determining the x-ray structures and mapping the detailed functional components of 3 families of bacterial MDR transporters that are dominant in gram-positive pathogens. In another collaboration, with R.A. Milligan, Department of Cell Biology, we are using electron cryomicroscopy to visualize the low-resolution structures of our transporters. Through these united efforts, we will gain a broader understanding of the structure and function of drug transporters that cause MDR in cancer and bacterial infection.

Recently, we determined a new structure of the MDR ATP-binding cassette transporter homolog MsbA in complex with magnesium, adenosine diphosphate, inorganic vanadate, and rough-chemotype lipopolysaccharide. This structure supports a model involving a rigid-body torque of the 2 transmembrane domains during ATP hydrolysis and suggests a mechanism by which the nucleotide-binding domain communicates with the transmembrane domain. We propose a lipid “flip-flop” mechanism in which the sugar groups are sequestered in the chamber while the hydrophobic tails are dragged through the lipid bilayer (Fig. 1). This posthydrolysis structure of MsbA also gives insight into the possible drug-binding sites for a number of cancer compounds. We are continuing our x-ray structural studies of the small MDR transporter EmrE and of other families of bacterial MDR transporters to better understand the molecular basis of the drug-proton antiport. The x-ray structures of MsbA and EmrE are excellent models for drug efflux systems that confer MDR to cancer cells and infectious microorganisms.

Publications

Ma, C., Chang, G. Crystallography of the integral membrane protein EmrE from Escherichia coli. Acta Crystallogr. D Biol. Crystallogr. 60:2399, 2004.

Reyes, C.L., Chang, G. Structure of the ABC transporter MsbA in complex with ADP•vanadate and lipopolysaccharide. Science 308:1028, 2005.