Structural Basis of Biological Membrane Protein Functions and Drug Resistance

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Cellular drug resistance is rendered by several mechanisms. From structural perspectives, my group focuses our study on the two most common ones: target site mutations and reduction of intracellular drug concentration. Target site mutation is one of the most common forms of drug resistance. My lab has been studying the structure and function of 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) in various forms. Complex III is a validated target for antibiotics targeting pathogenic microbes. Based on our structural and functional studies, we have proposed a hypothesis to address central mechanistic question of the Q-cycle mechanism for Complex III function. This hypothesis, termed the surface-affinity modulated iron-sulfur protein (ISP) conformation switch, addresses the mechanism for the bifurcated electron transfer (ET) at the quinol oxidation (QP) site of the cytochrome bc1 complex. We have provided further experimental evidence to support our hypothesis by structure determinations of various Rsbc1 structures in complex with different inhibitors, which showed the switching of the conformation of iron-sulfur protein in the presence of different inhibitors. Over a decade of extensive studies have arguably resolved most questions regarding the structure-function relationship of the cytochrome bc1 complex, setting the stage for integrating knowledge of this vital complex into a broader bioenergetics landscape that includes the regulation of cyt bc1 by components of the TCA cycle such as malate dehydrogenase (MDH), aconitase (ACON) and succinate-ubiquinol dehydrogenase (Complex II) and by small molecules such as molecular oxygen. These studies are ongoing. We have also been studying Complex III biogenesis by elucidating the structures of bcs1, a mitochondrial membrane protein that is critical in assisting insertion of the ISP subunit into core assembly of the Complex III. Reduction of intracellular drug concentration represents another important mechanism of drug resistance. Multidrug resistance (MDR) is a long-standing clinic challenge in cancer chemotherapies and in treatment of microbial infections; it is defined by a simultaneous resistance or cross resistance to various unrelated therapeutic agents by cancers or microbial pathogens. One mechanism of MDR is the over expression of efflux ABC transporters such as human P-glycoproteins (hP-gp) on the cell surface. The prospect of reversing the function of hP-gp in order to overcome MDR in cancer therapy has been driving development of P-gp specific inhibitors. However, such efforts have so far been unsuccessful, despite extensive studies designed to elucidate the underlying mechanism of function of these P-gp inhibitors. One issue is clearly related to the lack of detailed structural knowledge of P-gp relating to various steps along its catalytic pathway and the solution is to obtain the structures of hP-gp in complex with these inhibitors such that detailed interactions can be revealed. As a first step, we must obtain the structure(s) of hP-gp in its native form and in various conformations. My lab has been working on the elucidation of the structure at atomic resolution of hP-gp and mP-gp (mouse P-gp) for a long time, and more recently ZfP-gp (Zebra fish P-gp), in our attempts to uncover the mechanism of P-gp function, especially their ability to recognize structurally diverse compounds, from a structural perspective. Some of the questions we would like to address are (1) understanding the structural basis of P-gp substrate polyspecificity, (2) the coupling of ATP hydrolysis to the substrate translocation, and (3) the mechanism of P-gp inhibition. For many years, the structure determination of P-gp by the crystallographic method has been hampered by its intrinsic flexibility that is facilitated by a 75-residue linker connecting the two halves of P-gp. We shortened the linker to facilitate the structure determination of mP-gp, which were subsequently used for successful structure determination of many other mP-gp structures. These structures lead to some very interesting findings outlined below. (1) Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker-shortened mutant P-gp possesses a basal ATPase activity but has lost the drug-stimulated ATPase activity. (2) The linker-shortened mutant is structurally intact and surprisingly still has the same inward-facing conformation as that observed in the full-length P-gp, which suggests that the loss of function of the linker-shortened mutant is due to the loss of flexibility of the protein. (3) In the absence of substrate, P-gp only binds ATP asymmetrically in the NBD1, which is supported by our protective methylation experiment. (4) Analyses of a series of structures of wild-type, linker mutant, and a methylated P-gp showed individual transmembrane-domain helices of P-gp undergoing significant movements, which, importantly, correlates strongly with the opening-and-closing movement of the two lobes of P-gp. Thus, the opening-and-closing motion of the two halves of P-gp alters the surface topology within its drug-binding pocket, providing a mechanistic explanation for the polyspecificity of P-gp in substrate interactions. This work affords us the ability to analyze the structural basis of P-gp function. More importantly, this success offered us an opportunity to investigate the differences in solution behavior between human and mouse P-gp, which, as we hope, may lead to the structure solution of hP-gp.