Multidrug resistance (MDR) is a long-standing clinic challenge in cancer therapies 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. A major cause of MDR is the over expression of efflux ABC transporters such as human P-glycoproteins (hP-gp) on cell surface. The prospect of reversing the function of hP-gp to overcome MDR in cancer therapy has driven 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, it is not known how these inhibitors work to this day. We believe the solution is to obtain the structure 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 elucidation of the structure at atomic resolution of hP-gp for a long time in our attempts to uncover the mechanism of P-gp function from a structural perspective. Understanding the structural basis of P-gp substrate polyspecificity has been hampered by its intrinsic flexibility, which is facilitated by a 75-residue linker that connects the two halves of P-gp. We shortened the linker to facilitate structure determination of a linkage-shortened mP-gp at 3.3 A resolution, which we used to determine the structure and movement of wild-type P-gp. Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker mutant P-gp possessed basal ATPase activity and bound ATP in its N-terminal nucleotide-binding domain. Structures of wild-type, linker mutant, and a methylated P-gp displayed significant movements of individual transmembrane-domain helices, which correlated with the open-and-close motion of the two halves of P-gp. The open-and-close motion alters the surface topology of P-gp within the drug-binding pocket, providing a mechanistic explanation for the polyspecificity of P-gp in substrate interactions. This work afford us the ability to analyze the structural basis of P-gp function, assuming both proteins works by a similar mechanism. 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. My lab also engages in molecular modeling studies of ABC transporters, which has become a tool to gain structural and functional insights into those conformations that are not accessible by experimental means. We have determined the structures of the cytochrome bc1 complex from bovine mitochondria (Mtbc1) and the photosynthetic bacterium R. sphaeroides (Rsbc1) in various forms, proposed an hypothesis for the mechanism of the surface-affinity modulated iron-sulfur protein (ISP) conformation switch to account for the bifurcated electron transfer (ET) at the quinol oxidation (QP) site, provided experimental evidence to support this hypothesis, and identified substrate ubiquinol (QH2) in the QP site for the first time. All these achievements were rooted in our relentless pursuit of better diffracting crystals. The structure solution of Rsbc1 accomplishes one of our goals in establishing a model system to systematically study the bc1 complex by combining structural, genetic, and biochemical techniques. Our structural studies of bovine bc1 led us to propose that the key to the bifurcated ET at the QP site is the control of the ISP-ED movement, which regulates the distance between the 2Fe2S cluster and c1 heme. The distance is too long to permit ET between the two sites when ISP-ED is in the fixed conformation; ET is only possible when ISP-ED is in the mobile conformation. We hypothesized that by modulating the shape of the binding surface, the cyt b subunit effectively controls its affinity for the ISP-ED, the movement of the ISP, and thereby the directions of the two electrons from the substrate ubiquinol. Data from reports in the literature and new experiments from our lab and from others support this hypothesis. Currently we are focusing on demonstrating the control mechanism in experiment in the absence of inhibitors, which is more relevant to physiological conditions. Over a decade of intensive post 3D-structure 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 bc1 by components of the TCA cycle and by molecular oxygen. Molecular oxygen enhances the electron transfer activity of bc1 by 82% depending on the intactness of the complex. The effect of oxygen on the reaction sequence of the cytochrome bc1 complex is at the step of heme bL reduction during the bifurcated oxidation of ubiquinol via the Q-cycle mechanism. Specific interactions between TCA cycle enzymes, malate dehydrogenase (MDH) and aconitase (ACON), have been demonstrated by co-precipitation and their ability to enhance bc1 activity. Crystallograpic studies of these interactions are underway.
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