Chapter Three - Molecular Basis of the Polyspecificity of P-Glycoprotein (ABCB1): Recent Biochemical and Structural Studies

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Abstract

ABCB1 (P-glycoprotein/P-gp) is an ATP-binding cassette transporter well known for its association with multidrug resistance in cancer cells. Powered by the hydrolysis of ATP, it effluxes structurally diverse compounds. In this chapter, we discuss current views on the molecular basis of the substrate polyspecificity of P-gp. One of the features that accounts for this property is the structural flexibility observed in P-gp. Several X-ray crystal structures of mouse P-gp have been published recently in the absence of nucleotide, with and without bound inhibitors. All the structures are in an inward-facing conformation exhibiting different degrees of domain separation, thus revealing a highly flexible protein. Biochemical and biophysical studies also demonstrate this flexibility in mouse as well as human P-gp. Site-directed mutagenesis has revealed the existence of multiple transport-active binding sites in P-gp for a single substrate. Thus, drugs can bind at either primary or secondary sites. Biochemical, molecular modeling, and structure–activity relationship studies suggest a large, common drug-binding pocket with overlapping sites for different substrates. We propose that in addition to the structural flexibility, the molecular or chemical flexibility also contributes to the binding of substrates to multiple sites forming the basis of polyspecificity.

Introduction

ATP-binding cassette (ABC) transporters such as ABCB1 (P-glycoprotein/P-gp), ABCG2, and ABCC1 are well known for their association with multidrug resistance (MDR), effluxing structurally diverse compounds, powered by the hydrolysis of ATP (Ambudkar et al., 1999). P-gp also plays an important role in the pharmacokinetics of many drugs, altering their absorption, distribution, and excretion. P-gp has been extensively studied since 1976, when it was identified as the multidrug efflux pump in Chinese hamster ovary cells that had been selected for resistance to colchicine (Juliano & Ling, 1976). It is a 170 kDa single polypeptide chain consisting of two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). It is believed that this transporter functions through an alternate access mechanism involving two different conformations (Hollenstein et al., 2007, Jardetzk, 1966, Senior et al., 1995, van Wonderen et al., 2014). Drug binding occurs when the protein adopts an inward-facing conformation (inverted V appearance) observed in P-gps such as those found in mouse and Caenorhabditis elegans P-gp X-ray structures obtained in the absence of nucleotide (Aller et al., 2009, Jin et al., 2012). This is followed by a significant structural change to an outward-facing conformation (V-shaped in appearance) such as the one exhibited by the X-ray structure of SAV1866 with bound nucleotide (ADP) (Dawson & Locher, 2006), when drug release takes place. Hydrolysis of ATP is believed to reset the protein to the inward-facing form to begin a new cycle of drug binding and release (Callaghan et al., 2006, Sauna and Ambudkar, 2000). The switch from inward to outward form certainly requires a highly flexible structure.

Substrate “promiscuity” or polyspecificity is a well-known characteristic of P-gp and the subject of much research. Attempts have been made to understand the ability of P-gp to recognize various chemically and structurally diverse substrates through biochemical investigations and structural studies. Despite all these studies, the molecular basis of this unusual property still remains poorly understood and is a matter of intense debate. Other review articles have been published recently on the subject (Gutmann et al., 2010, Sharom, 2014, Wong et al., 2014). In this review, we will focus on recent biochemical and structural studies of P-gp, with discussion primarily on its substrate polyspecificity.

Section snippets

Structural flexibility revealed by X-ray crystallography

In 2009, Aller et al. (2009) were the first to report an X-ray structure of a mammalian ABC transporter. They solved the structure of mouse P-gp at a resolution of 3.8–4.4 Å, in the absence of nucleotide, with and without bound inhibitors. In the absence of nucleotide or drug-substrate, the mouse P-gp structure (3G5U.pdb) was observed to adopt an inward-facing conformation. It has a large internal cavity of about 6000 Å3 open to both the cytoplasm and the membrane inner leaflet, with a wide

Molecular Modeling Studies

Since the publication of the first X-ray crystal structures of mouse P-gp in 2009 (Aller et al., 2009), a number of laboratories have employed molecular modeling studies trying to identify the drug-binding sites of P-gp. The high similarity between mouse (mdr1a) and human P-gp sequences (87% identity, 94% similarity) has encouraged many research groups to build homology models of human P-gp based on the structures of mouse P-gp and many modeling studies have been published. In 2012, the

Conclusions and Perspectives

The ability of P-gp to recognize a wide variety of substrates is remarkable and thus it has been the subject of intense research for almost 40 years. The field has certainly shown advances, as we know today (i) the overall molecular structure of P-gp, (ii) the structure is highly flexible, (iii) the substrates are transported through a central cavity, and (iv) the alternate access mechanism of transport that enjoys a significant consensus in the ABC transporter community. It seems evident that

Acknowledgments

We thank George Leiman for editorial assistance in the preparation of the chapter and the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research, for financial support. H.-M. S. was supported in part by a NUS-OPF fellowship from the National University of Singapore. A critical reading of the chapter by Drs. John Golin and Atish Patel is also gratefully acknowledged.

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