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Research Themes Drug discovery

Membrane Proteome: The ABCs of Transport

SBKB [doi:10.1038/sbkb.2012.106]
Featured Article - November 2012
Short description: Structural analyses of two ABC transporters reveal how diverse transmembrane domain architectures determine substrate specificity and transport mechanisms.

Ribbon diagram of the P-gp protein from C. elegans with the N-terminal and C-terminal halves colored blue and yellow, respectively. Figure courtesy of Jue Chen.

ATP-binding cassette (ABC) transporter proteins are found in all domains of life and use ATP to power the translocation of a wide range of substrates across membranes. ABC transporters are generally composed of two nucleotide-binding domains (NBDs) containing conserved sequence motifs and two variable transmembrane domains (TMDs). ATP hydrolysis by the NBDs transmits conformational changes to the TMDs during transport.

Chen and colleagues investigated the structure and mechanism of P-glycoprotein (P-gp), an ABC transporter that confers drug resistance in cancer cells. The authors focused on P-gp from Caenorhabditis elegans, which has 46% sequence identity with human P-gp. They found that it confers resistance to cytotoxic drugs (actinomycin D and paclitaxel) transported by human P-gp and that these drugs also stimulated the ATPase activity of C. elegans P-gp. The 3.4 Å resolution structure (PDB 4F4C) revealed the architecture of the single-chain P-gp in the absence of nucleotides and substrates. Compared to a murine P-gp structure, worm P-gp shows a similar inward-facing conformation open to the cytoplasm, but with significant differences in conformations or register shifts for half of the TMD helices and, interestingly, only one lateral opening.

The authors speculate that discontinuities in two transmembrane helices lining the lateral opening could create more interaction points for substrates and/or function as flexible gating hinges. The coupling helices at the NBD–TMD interface are augmented by additional conserved salt bridges— probable pivot points for conformational transitions as in other transporters. A homology model of human P-gp is consistent with biochemical data and offers insights into previously unexplained functional data.

Locher and colleagues engineered crosslinks between the NBDs of BtuD to restrict its dynamics, allowing them to solve the 3.5 Å AMP-PNP-bound structure of the vitamin B12 transporter BtuCD in complex with the periplasmic BtuF protein (PDB 4FI3). The NBDs adopt the expected closed sandwich dimer conformation, but unexpected changes in the TMDs of the BtuC subunits allowed the authors to identify a second, potentially conserved, cytoplasmic gate. As the coupling helices move inwards and the first gating helices open in response to nucleotide binding, the loops forming the second gate seal the translocation pathway closed, creating a central cavity between the BtuC subunits. Although the cavity is large enough to bind vitamin B12, no substrate associated with BtuCD-F in detergent solution due to conformational flexibility. Using BtuF, the authors succeeded in delivering and trapping vitamin B12 in the BtuCD cavity in the presence of AMP-PNP, the first experimental observation of bound substrate for this transporter class. The structural and biochemical experiments allowed the authors to propose a detailed model for a peristaltic transport mechanism.

Both studies highlight how ABC transporters use conserved NBD structures combined with divergent and dynamic TMD architectures to affect unique transport mechanisms.

Michael A. Durney

References

  1. M.S. Jin et al. Crystal structure of the multidrug transporter P-glycoprotein from Caenorhabditis elegans.
    Nature. (23 September 2012). doi:10.1038/nature11448

  2. V.M. Korkhov et al. Structure of AMP-PNP-bound vitamin B12 transporter BtuCD-F.
    Nature. (23 September 2012). doi:10.1038/nature11442

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