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Generation of Chimeric P-glycoprotein for Functional and Structural Investigations

Thursday, September 17, 2015 — Poster Session III

3:30 p.m. – 5:00 p.m.
FAES Terrace
NCI
DIR-9

Authors

  • KM Pluchino
  • JK Moen
  • MD Hall
  • L Esser
  • F Zhou
  • EE Chufan
  • SV Amubdkar
  • Di Xia
  • MM Gottesman

Abstract

A major challenge in cancer treatment is acquired or intrinsic multidrug resistance (MDR) to chemotherapeutics. A notorious mediator of MDR is P-glycoprotein (P-gp, ABCB1), which actively effluxes cytotoxic drugs from cancer cells, resulting in sub-therapeutic intracellular concentrations. Understanding how P-gp interacts with drugs has been severely limited by the lack of high-resolution structures of P-gp. Although numerous efforts to obtain a crystal structure have been attempted, human P-gp has never been crystallized. However, mouse P-gp (87% homologous to human P-gp) has been crystallized, and several structures have been recently reported. It is currently unknown why mouse P-gp can be crystallized while human P-gp cannot. We describe the creation of novel chimeras of mouse and human P-gp as an approach to investigate whether specific protein domains are responsible for differences in the ability to form crystals between mouse and human P-gp. A range of chimeras were expressed in mammalian cells and all were found to retain transport function demonstrating that P-gp can tolerate major structural changes. High-level expression of all chimeras was achieved by baculovirus-mediated expression in insect cells for biochemical and structural studies. Initial crystallization screening obtained crystalline materials for multiple of the chimeras, which are currently under optimization. X-ray diffraction experiments using synchrotron radiation showed low-resolution diffraction for crystals of one chimera, indicative of protein crystal. In summary, the approach adopted is a successful strategy, and an advance along the path towards a high-resolution structure of human P-gp.

Category: Institute, Center, and Scientific Directors