Structures of the Multidrug Transporter P-glycoprotein Reveal Asymmetric ATP Binding and the Mechanism of Polyspecificity

Lothar Esser,Fei Zhou,Kristen M Pluchino,Joseph Shiloach,Jichun Ma,Wai-Kwan Tang,Camilo Gutierrez,Alex Zhang,Suneet Shukla,James P Madigan,Tongqing Zhou,Peter D Kwong,Suresh V Ambudkar,Michael M Gottesman,Di Xia

doi:10.1074/jbc.m116.755884

Abstract

P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancer; it plays important roles in determining the pharmacokinetics of many drugs. 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. Here we constructed a mutant murine P-gp with a shortened linker to facilitate structural determination. Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker-shortened mutant P-gp possesses basal ATPase activity and binds ATP only in its N-terminal nucleotide-binding domain. Nine independently determined structures of wild type, the linker mutant, and a methylated P-gp at up to 3.3 Å resolution display significant movements of individual transmembrane domain helices, which correlated with the opening and closing 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.

Highlights

P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancer; it plays important roles in determining the pharmacokinetics of many drugs
Functional asymmetry is better demonstrated by non-equivalent nucleotide-binding domain (NBD) in the amino acid sequences of a number of eukaryotic ABC transporters, including the human cystic fibrosis transmembrane regulator [22], multidrug resistance-associated protein-1 [23], transporters associated with antigen processing
Restricting Conformational Flexibility Leads to Dysfunction of P-gp—The function of P-gp requires the flexibility of its two halves, which are joined by a mobile linker that is disordered in all mouse P-gp (mP-gp) structures reported so far

Summary

Edited by Norma Allewell

P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancer; it plays important roles in determining the pharmacokinetics of many drugs. By shortening the linker between the two halves of the mP-gp, we effectively reduced the flexibility of the molecule and obtained a 3.3 Å resolution structure of a linker-shortened mP-gp (⌬lnkmP-gp) This structure helped to accurately determine structures of the wild type and methylated full-length mP-gp (FLmP-gp and FLmP-gpMe) and led to a structure with bound nucleotide to the NBD1, demonstrating for the first time asymmetric nucleotide binding for ABC transporters with two consensus NBDs. This structure helped to accurately determine structures of the wild type and methylated full-length mP-gp (FLmP-gp and FLmP-gpMe) and led to a structure with bound nucleotide to the NBD1, demonstrating for the first time asymmetric nucleotide binding for ABC transporters with two consensus NBDs Analysis of these structures correlates the open-and-close movement of two halves of P-gp with rotation and translation of individual helices in the TM domains of P-gp, suggesting a mechanism of polyspecific substrate recognition by P-gp

Results

Data set

Discussion

Experimental Procedures