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Scientific Report 2008


Molecular Biology




Structural Biology of Integral Membrane Proteins


G. Chang, S. Aller, X. He, A. Karyakin, S. Lieu, P. Szewczk, T. Tuan, A. Ward, J. Yu

Study of the structure of membrane proteins is important for understanding their function. We are interested in 4 areas: (1) the molecular structural basis for the transport of lipids and drugs across the cell membrane by multidrug resistance (MDR) transporters, (2) the crystallography of mammalian MDR transporters and the structural basis of their inhibition, (3) the discovery and rational design of potent MDR reversal agents, and (4) the development and application of a cell-free system capable of producing large quantities of integral membrane proteins. We use several experimental methods, and we collaborate with scientists in other laboratories to achieve our goals.

MDR in the treatment of cancer and infectious disease is often caused by an upregulation of drug efflux pumps imbedded in the cell membrane. The molecular basis of multispecificity and drug efflux by these transporters is not well understood. Through our structural studies, we are elucidating the mechanisms for the transport of amphipathic substrate across the cell membrane in several families of transporters: ATP-binding cassette, small multidrug resistance, major facilitator superfamily, and multiple antimicrobial extrusion. In collaboration with M.G. Finn, Department of Chemistry, and Q. Zhang, Department of Molecular Biology, we are discovering and designing potent inhibitors to be used synergistically with established antibiotics and cancer chemotherapeutics. In collaboration with R.A. Milligan, Department of Cell Biology, we are using electron cryomicroscopy to visualize transporter structures.

We have determined 4 x-ray structures of the ATP-binding cassette transporter MsbA trapped in different conformations: 2 with nucleotide bound and 2 with no nucleotide. Comparisons of the nucleotide-free conformations of MsbA revealed a flexible hinge formed by extracellular loops 2 and 3. The hinge allows the nucleotide-binding domains to disassociate while the ATP-binding half sites remain facing each other. The binding of nucleotide causes a packing rearrangement of the transmembrane helices and changes the accessibility of the transporter from cytoplasmic (inward) facing to extracellular (outward) facing. The inward and outward openings are mediated by 2 different sets of transmembrane helix interactions. Altogether, the conformational changes between these structures suggest that large ranges of motion may be required for substrate transport.

EmrE, an MDR transporter from the small multidrug resistance family, functions as a homodimer of a small 4-transmembrane protein. The membrane insertion topology of the 2 monomers is controversial. EmrE was reported to have a unique orientation in the membrane. Models based on electron microscopy and on several biochemical studies posit an antiparallel dimer. The structures of EmrE in complex with a transport substrate are highly similar to the electron microscopy structure. The first 3 transmembrane helices from each monomer surround the substrate-binding chamber, whereas the fourth helix participates only in dimer formation. Selenomethionine markers clearly indicate an antiparallel orientation for the monomers, supporting
a "dual topology” model.

EmrD, an MDR transporter from the major facilitator superfamily, expels hydrophobic compounds across the inner membrane. The x-ray structure reveals an interior composed of hydrophobic residues, a finding consistent with the role of EmrD in transporting amphipathic molecules that uncouple the proton gradient across the cell membrane. Two long loops extend into the inner leaflet side of the cell membrane and may recognize and bind substrate directly from the lipid bilayer. On the basis of the structure, we propose that multisubstrate specificity, binding, and transport are facilitated by these loop regions and the internal cavity.

 


Geoffrey Chang, Ph.D.
Associate Professor



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