Structural Basis of Biological Membrane Protein Functions and Drug Resistance

Division Of Basic Sciences - Nci

Investigators

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Abstract

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 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 belew. (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.