Abstract

Multidrug resistance of cancer cells and pathogens is a serious clinical problem. A major factor contributing to drug resistance in cancer is the over-expression of P-glycoprotein, a plasma membrane ATP-binding cassette (ABC) drug efflux pump. Three-dimensional structural data with a resolution limit of approximately 8 A have been obtained from two-dimensional crystals of P-glycoprotein trapped in the nucleotide-bound state. Each of the two transmembrane domains of P-glycoprotein consists of six long alpha-helical segments. Five of the alpha-helices from each transmembrane domain are related by a pseudo-2-fold symmetry, whereas the sixth breaks the symmetry. The two alpha-helices positioned closest to the (pseudo-) symmetry axis at the center of the molecule appear to be kinked. A large loop of density at the extracellular surface of the transporter is likely to correspond to the glycosylated first extracellular loop, whereas two globular densities at the cytoplasmic side correspond to the hydrophilic, nucleotide-binding domains. This is the first three-dimensional structure for an intact eukaryotic ABC transporter. Comparison with the structures of two prokaryotic ABC transporters suggests significant differences in the packing of the transmembrane alpha-helices within this protein family.

Highlights

  • Multidrug resistance of cancer cells and pathogens is a serious clinical problem

  • The minimal functional unit of an ATP-binding cassette (ABC) transporter typically consists of four domains, two hydrophilic nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs), each consisting of several putative membrane-spanning ␣-helices

  • In this study we present the three-dimensional structure for P-gp at ϳ8Å resolution obtained by cryo-electron crystallography of two-dimensional crystals

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Summary

Introduction

Multidrug resistance of cancer cells and pathogens is a serious clinical problem. A major factor contributing to drug resistance in cancer is the over-expression of P-glycoprotein, a plasma membrane ATP-binding cassette (ABC) drug efflux pump. A large loop of density at the extracellular surface of the transporter is likely to correspond to the glycosylated first extracellular loop, whereas two globular densities at the cytoplasmic side correspond to the hydrophilic, nucleotide-binding domains This is the first three-dimensional structure for an intact eukaryotic ABC transporter. In this study we present the three-dimensional structure for P-gp at ϳ8Å resolution obtained by cryo-electron crystallography of two-dimensional crystals These structural data are the highest resolution for any eukaryotic ABC transporter and the first to show the location and packing of the transmembrane ␣-helices. This structure was obtained in the presence of bound nucleotide, enabling a new conformational state of an ABC transporter to be assessed

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