Structural Basis of Biological Membrane Protein Functions and Drug Resistance

Division Of Basic Sciences - Nci

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Abstract

We are working on two mechanisms of cellular drug resistance: target site mutations and reduction of intracellular drug concentration. Target site mutation is one of the most common forms of drug resistance. We use the cytochrome bc1 complex from bovine (Bos taurus) mitochondria (Btbc1, also known as Complex III of the cellular respiratory chain) and the photosynthetic bacterium R. sphaeroides (Rsbc1) as model systems to elucidate the mechanism of drug resistance. Complex III is a validated target for antibiotics targeting pathogenic microbes. Recently, our research has been focusing on whether mitochondrial and bacterial Complex III can be used as surrogates for human and pathogenic organisms for selective targeting. By determining structures of Complex III, from both bovine mitochondria and photosynthetic bacteria, bound with various respiratory inhibitors, we unveiled organism-specific structural differences in cyt bc1 that are sufficiently large to offer potential vulnerabilities to pharmacological exploitation. We also characterized the mechanism of selective inhibition of Complex III by atovaquone and identified pyramoxadone as a fungicide with greater selectivity for Rsbc1. The Q-cycle mechanism is a hypothesis that has been used to describe the electron transfer coupled proton translocation function of Complex III. My group has contributed significantly to the structural elucidation of this mechanism, although some details remain to be resolved. Currently, we are working on a couple of mutants made in Rsbc1 trying to experimentally verify our hypothesis that (1) the control of the ISP conformation is the key to the bifurcated electron transfer at the quinol oxidation site and (2) there exists a communication pathway between quinone reduction site and quinol oxidation site. Our structural data appear to confirm the hypothesis. Reduction of intracellular drug concentration represents another important mechanism of drug resistance. We have been using zebrafish Abcb4 as a model system to study the structure and function of human ABC transporter P-glycoprotein. The hallmark of multidrug resistance conferred by human ABC transporter ABCB1 (hP-gp) is the recognition and efflux of diverse range of drugs, though the precise mechanism of polyspecificity remains unresolved. In aquatic animals such as zebrafish, Abcb4, a functional homolog to hP-gp, plays a vital role in surviving environmental toxicants. Recently, we show that Abcb4 exhibits comparable basal and drug-stimulated ATPase activity to hP-gp. Using cryo-EM, we captured five inward-facing Abcb4 conformations with varying separations between its two lobes, illustrating its open-and-close motion. The range of separation exceeds that seen in published P-gp structures that appear to be conformationally restricted. This global open-and-close motion is coupled with individual helix movement, resulting in a highly fluid substrate-binding pocket. These dynamic changes, likely underlying the polyspecificity of substrate recognition, predict unconventional protein-ligand interactions that are supported by structures of Abcb4 bound to the P-gp inhibitors tariquidar and elacridar, and the substrate vincristine.