ABC transporters catalyze transport reactions, such as the high-affinity uptake of micronutrients into bacteria and the export of cytotoxic compounds from mammalian cells. Crystal structures of ABC domains and full transporters have provided a framework for formulating reaction mechanisms of ATP-driven substrate transport, but recent studies have suggested remarkable mechanistic diversity within this protein family. This review evaluates the differing mechanistic proposals and outlines future directions for the exploration of ABC-transporter-catalyzed reactions.

/pdf/mechanistic-diversity-in-atp-binding-cassette-abc-57ofgiae1o.pdf

Mechanistic diversity in ATP-binding cassette (ABC) transporters

All living organisms depend on primary and secondary membrane transport for the supply of external nutrients and removal or sequestration of unwanted (toxic) compounds. Due to the chemical diversity of cellular molecules, it comes as no surprise that a significant part of the proteome is dedicated to the active transport of cargo across the plasma membrane or the membranes of subcellular organelles. Transport against a chemical gradient can be driven by, for example, the free energy change associated with ATP hydrolysis (primary transport), or facilitated by the potential energy of the chemical gradient of another molecule (secondary transport). Primary transporters include the rotary motor ATPases (F-, A-, and V-ATPases), P-type ATPases and a large family of integral membrane proteins referred to as “ABC” (ATP binding cassette) transporters. ABC transporters are widespread in all forms of life and are characterized by two nucleotide-binding domains (NBD) and two transmembrane domains (TMDs). ATP hydrolysis on the NBD drives conformational changes in the TMD, resulting in alternating access from inside and outside of the cell for unidirectional transport across the lipid bilayer. Common to all ABC transporters is a signature sequence or motif, LSGGQ, that is involved in nucleotide binding. Both importing and exporting ABC transporters are found in bacteria, whereas the majority of eukaryotic family members function in the direction of export. Recent progress with the X-ray crystal structure determination of a variety of bacterial and eukaryotic ABC transporters has helped to advance our understanding of the ATP hydrolysis-driven transport mechanism but has also illustrated the large structural and functional diversity within the family.

Structure and mechanism of ABC transporters.

Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this Review, we describe recent advances that have increased our understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria. Clinical and laboratory data indicate that efflux pumps function not only in the drug extrusion process but also in virulence and the adaptive responses that contribute to antimicrobial resistance during infection. The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities.

/pdf/multidrug-efflux-pumps-structure-function-and-regulation-3y0335ts48.pdf

Multidrug efflux pumps: structure, function and regulation

The recently determined C. elegans P-glycoprotein (Pgp) structure revealed significant deviations compared to the original mouse Pgp structure, which suggested possible misinterpreta- tions in the latter model. To address this concern, we generated an experimental electron density map from single-wavelength anomalous dispersion phasing of an original mouse Pgp dataset to 3.8 Aresolution. The map exhibited significantly more detail compared to the original MAD map and revealed several regions of the structure that required de novo model building. The improved drug-free structure was refined to 3.8 Aresolution with a 9.4 and 8.1% decrease in Rwork and Rfree, respectively, (Rwork 521.2%, Rfree 526.6%) and a significant improvement in protein geometry. The improved mouse Pgp model contains ~95% of residues in the favorable Ramachandran region compared to only 57% for the original model. The registry of six transmembrane helices was cor- rected, revealing amino acid residues involved in drug binding that were previously unrecognized. Registry shifts (rotations and translations) for three transmembrane (TM)4 and TM5 and the addi- tion of three N-terminal residues were necessary, and were validated with new mercury labeling and anomalous Fourier density. The corrected position of TM4, which forms the frame of a portal for drug entry, had backbone atoms shifted > 6Afrom their original positions. The drug transloca- tion pathway of mouse Pgp is 96% identical to human Pgp and is enriched in aromatic residues that likely play a collective role in allowing a high degree of polyspecific substrate recognition.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/pro.2387

Refined structures of mouse P‐glycoprotein

One of the major causes of failure in cancer chemotherapy is multidrug resistance (MDR), where cancer cells simultaneously become resistant to different anticancer drugs. Over-expression of membrane efflux pumps like P-glycoprotein (P-gp) that recognizes different chemotherapeutic agents and transports them out of the cell, plays a major role in MDR. The shortcoming of P-gp inhibitors in clinic has been attributed to their non-specific action on P-gp and/or non-selective distribution to non-target organs that leads to intolerable side effects by the P-gp inhibitor at doses required for P-gp inhibition upon systemic administration. Another major issue is the reduced elimination of P-gp substrates (e.g. anticancer drugs) and intolerable toxicities by anticancer drugs when co-administered with P-gp inhibitors. To overcome these shortcomings, new generation of P-gp inhibitors with improved specificity for P-gp have been developed. More recently, attention has been paid to the use of drug delivery systems primarily to restrict P-gp inhibition to tumor and reduce the non-selective inhibition of P-gp in non-target organs. This review will provide an overview and update on the status of P-gp inhibition approaches and the role of drug delivery systems in overcoming P-gp mediated MDR.

P-glycoprotein inhibition as a therapeutic approach for overcoming multidrug resistance in cancer: current status and future perspectives.

Biochemical and structural analysis of the drug transporter P-glycoprotein in Caenorhabditis elegans at a resolution of 3.4 angstroms is used to generate a homology model of the human protein and supports a picture in which P-glycoprotein uses the energy from ATP hydrolysis to expel lipophilic molecules from the inner leaflet of the cell membrane. The ABC (ATP-binding cassette) transporter P-glycoprotein confers multidrug resistance in cancer cells. In this manuscript, the authors biochemically and structurally characterize P-glycoprotein from Caenorhabditis elegans and use that information to generate a homology model for human P-glycoprotein. Their data suggest how P-glycoprotein uses the energy from ATP hydrolysis to expel lipophilic molecules from the inner leaflet of the membrane. P-glycoprotein (P-gp) is an ATP-binding cassette transporter that confers multidrug resistance in cancer cells1,2. It also affects the absorption, distribution and clearance of cancer-unrelated drugs and xenobiotics. For these reasons, the structure and function of P-gp have been studied extensively for decades3. Here we present biochemical characterization of P-gp from Caenorhabditis elegans and its crystal structure at a resolution of 3.4 angstroms. We find that the apparent affinities of P-gp for anticancer drugs actinomycin D and paclitaxel are approximately 4,000 and 100 times higher, respectively, in the membrane bilayer than in detergent. This affinity enhancement highlights the importance of membrane partitioning when a drug accesses the transporter in the membrane4. Furthermore, the transporter in the crystal structure opens its drug pathway at the level of the membrane’s inner leaflet. In the helices flanking the opening to the membrane, we observe extended loops that may mediate drug binding, function as hinges to gate the pathway or both. We also find that the interface between the transmembrane and nucleotide-binding domains, which couples ATP hydrolysis to transport, contains a ball-and-socket joint and salt bridges similar to the ATP-binding cassette importers5, suggesting that ATP-binding cassette exporters and importers may use similar mechanisms to achieve alternating access for transport. Finally, a model of human P-gp derived from the structure of C. elegans P-gp not only is compatible with decades of biochemical analysis6,7,8,9,10,11,12, but also helps to explain perplexing functional data regarding the Phe335Ala mutant13,14. These results increase our understanding of the structure and function of this important molecule.

https://courses.washington.edu/medch582/PDFs/pdfarchive/2013Feb25_Li.pdf

Mi Sun Jin

Papers

Crystal structure of the multidrug transporter P-glycoprotein from Caenorhabditis elegans