The Q loops of the human multidrug resistance transporter ABCB1 are necessary to couple drug binding to the ATP catalytic cycle (original) (raw)

The Q-loop Disengages from the First Intracellular Loop during the Catalytic Cycle of the Multidrug ABC Transporter BmrA

Journal of Biological Chemistry, 2005

The ATP-binding cassette is the most abundant family of transporters including many medically relevant members and gathers both importers and exporters involved in the transport of a wide variety of substrates. Although three high resolution three-dimensional structures have been obtained for a prototypic exporter, MsbA, two have been subjected to much criticism. Here, conformational changes of BmrA, a multidrug bacterial transporter structurally related to MsbA, have been studied. A three-dimensional model of BmrA, based on the "open" conformation of Escherichia coli MsbA, was probed by simultaneously introducing two cysteine residues, one in the first intracellular loop of the transmembrane domain and the other in the Q-loop of the nucleotide-binding domain (NBD). Intramolecular disulfide bonds could be created in the absence of any effectors, which prevented both drug transport and ATPase activity. Interestingly, addition of ATP/Mg plus vanadate strongly prevented this bond formation in a cysteine double mutant, whereas ATP/Mg alone was sufficient when the ATPaseinactive E504Q mutation was also introduced, in agreement with additional BmrA models where the ATP-binding sites are positioned at the NBD/NBD interface. Furthermore, cross-linking between the two cysteine residues could still be achieved in the presence of ATP/Mg plus vanadate when homobifunctional cross-linkers separated by more than 13 Å were added. Altogether, these results give support to the existence, in the resting state, of a monomeric conformation of BmrA similar to that found within the open MsbA dimer and show that a large motion is required between intracellular loop 1 and the nucleotide-binding domain for the proper functioning of a multidrug ATP-binding cassette transporter. ATP-binding cassette (ABC) 3 transporters form one of the largest protein families in all species and are involved in the cellular or subcel-* This work was supported by CNRS Grant PGP 2002 (to A. D. P.), a CNRS Young Investigator ATIP Program grant (to J.-M. J.), and ACI IMPBio Grant IMPB027 from the Ministè re de la Recherche (to A. D. P. and J.-M. J.)

New light on multidrug binding by an ATP-binding-cassette transporter

Trends in Pharmacological Sciences, 2006

ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic microorganisms and human tumour cells by mediating the extrusion of structurally unrelated chemotherapeutic drugs from the cell. The molecular basis by which ABC multidrug transporters bind and transport drugs is far from clear. Genetic analyses during the past 14 years reveal that the replacement of many individual amino acids in mammalian multidrug resistance P-glycoproteins can affect cellular resistance to drugs, but these studies have failed to identify specific regions in the primary amino acid sequence that are part of a defined drug-binding pocket. The recent publication of an X-ray crystallographic structure of the bacterial P-glycoprotein homologue MsbA and an MsbA-based homology model of human P-glycoprotein creates an opportunity to compare the original mutagenesis data with the three-dimensional structures of transporters. Our comparisons reveal that mutations that alter specificity are present in three-dimensional ‘hotspot’ regions in the membrane domains of P-glycoprotein.

Conformational Change Induced by ATP Binding in the Multidrug ATP-Binding Cassette Transporter BmrA †

Biochemistry, 2008

ATP-binding cassette (ABC) transporters are involved in the transport of a wide variety of substrates, and ATP-driven dimerization of their nucleotide binding domains (NBDs) has been suggested to be one of the most energetic steps of their catalytic cycle. Taking advantage of the propensity of BmrA, a bacterial multidrug resistance ABC transporter, to form stable, highly ordered ring-shaped structures ) J. Mol. Biol. 315, 1075-1085, we show here that addition of ATP in the presence of Mg 2+ prevented ring formation or destroyed the previously formed rings. To pinpoint the catalytic step responsible for such an effect, two classes of hydrolysis-deficient mutants were further studied. In contrast to hydrolytically inactive glutamate mutants that behaved essentially as the wild-type, lysine Walker A mutants formed ring-shaped structures even in the presence of ATP-Mg. Although the latter mutants still bound ATP-Mg, and even slowly hydrolyzed it for the K380R mutant, they were most likely unable to undergo a proper NBD dimerization upon ATP-Mg addition. The ATP-driven dimerization step, which was still permitted in glutamate mutants and led to a stable conformation suitable to monitor the growth of 2D crystals, appeared therefore responsible for destabilization of the BmrA ring structures. Our results provide direct visual evidence that the ATP-induced NBD dimerization triggers a conformational change large enough in BmrA to destabilize the rings, which is consistent with the assumption that this step might constitute the "power stroke" for ABC transporters. † This work was supported by CNRS Grant PGP 2002 (to A.D.P.), a CNRS Young Investigator ATIP Program to J.-M.J., and a grant (ANR-06-Blan-0420) from the Agence Nationale de la Recherche to

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

Journal of Biological Chemistry, 2016

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. 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-andclose motion alters the surface topology of P-gp within the drugbinding pocket, providing a mechanistic explanation for the polyspecificity of P-gp in substrate interactions.

The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism

The EMBO …, 2000

The bacterial LmrA protein and the mammalian multidrug resistance P-glycoprotein are closely related ATP-binding cassette (ABC) transporters that confer multidrug resistance on cells by mediating the extrusion of drugs at the expense of ATP hydrolysis. The mechanisms by which transport is mediated, and by which ATP hydrolysis is coupled to drug transport, are not known. Based on equilibrium binding experiments, photoaf®nity labeling and drug transport assays, we conclude that homodimeric LmrA mediates drug transport by an alternating two-site transport (two-cylinder engine) mechanism. The transporter possesses two drug-binding sites: a transport-competent site on the inner membrane surface and a drug-release site on the outer membrane surface. The interconversion of these two sites, driven by the hydrolysis of ATP, occurs via a catalytic transition state intermediate in which the drug transport site is occluded. The mechanism proposed for LmrA may also be relevant for P-glycoprotein and other ABC transporters.

Identification and characterisation of putative drug binding sites in human ATP-binding cassette B5 (ABCB5) transporter

Computational and Structural Biotechnology Journal

The human ATP-binding cassette B5 (ABCB5) transporter, a member of the ABC transporter superfamily, is linked to chemoresistance in tumour cells by drug effluxion. However, little is known about its structure and drug-binding sites. In this study, we generated an atomistic model of the full-length human ABCB5 transporter with the highest quality using the X-ray crystal structure of mouse ABCB1 (Pgp1), a close homologue of ABCB5 and a well-studied member of the ABC family. Molecular dynamics simulations were used to validate the atomistic model of ABCB5 and characterise its structural properties in model cell membranes. Molecular docking simulations of known ABCB5 substrates such as taxanes, anthracyclines, camptothecin and etoposide were then used to identify at least three putative binding sites for chemotherapeutic drugs transported by ABCB5. The location of these three binding sites is predicted to overlap with the corresponding binding sites in Pgp1. These findings will serve as the basis for future in vitro studies to validate the nature of the identified substrate-binding sites in the full-length ABCB5 transporter.

A multidrug ABC transporter with a taste for GTP

Scientific Reports, 2018

During the evolution of cellular bioenergetics, many protein families have been fashioned to match the availability and replenishment in energy supply. Molecular motors and primary transporters essentially need ATP to function while proteins involved in cell signaling or translation consume GTP. ATP-Binding Cassette (ABC) transporters are one of the largest families of membrane proteins gathering several medically relevant members that are typically powered by ATP hydrolysis. Here, a Streptococcus pneumoniae ABC transporter responsible for fluoroquinolones resistance in clinical settings, PatA/ PatB, is shown to challenge this concept. It clearly favors GTP as the energy supply to expel drugs. This preference is correlated to its ability to hydrolyze GTP more efficiently than ATP, as found with PatA/ PatB reconstituted in proteoliposomes or nanodiscs. Importantly, the ATP and GTP concentrations are similar in S. pneumoniae supporting the physiological relevance of GTP as the energy source of this bacterial transporter. Chemical energy is required to sustain life since any cellular task such as DNA maintenance or replication, translation, cell signaling or transport is an energy-driven process. Many proteins have thus been shaped to harness the chemical energy provided by hydrolysis of the β-γ phosphate bond of a nucleotide, mainly ATP or GTP, to mediate their dedicated function 1. They predominantly belong to one of the most ancient protein super-families, the P-loop NTPases 2,3 , and their overwhelming presence in all species, between 10 to 18% of each proteome 4 , reflects their versatile and pivotal functions in many cellular pathways 5. These proteins contain two specific motifs in their sequences: the Walker A motif 6 , G/AX 4 GKT/S, involved in the proper positioning of the polyphosphate moiety of ATP/GTP, and the Walker B motif. This latter is less evident to notice from the sequence because it contains only a conserved aspartate, located at the end of a hydrophobic β-strand, involved in the coordination of a magnesium ion required for catalysis 7. Some of the P-loop NTPases hydrolyze exclusively GTP because they bear an additional motif, the N/TKXD sequence, which mainly dictates a strong specificity for the guanine, as exemplified by the Ras protein 7. In contrast, P-loop ATPases are generally more promiscuous as they can often hydrolyze other nucleotides such as GTP or ITP (e.g. the F1-ATPase 8), albeit at a lower rate than ATP. Yet, this relative lack of specificity may mainly operate in vitro, in particular due to the abundance of ATP as compared to other nucleotides in different organisms 9-11. This seeming lack of specificity of many ATPases is due to limited contact with the base moiety of the nucleotide, often restricted to a stacking interaction between the adenine ring and an aromatic residue 1. The ABC (ATP-Binding Cassette) transporters family is part of the P-loop ATPases super-family and is involved in the vectorial transport, import or export, of a huge variety of compounds including ions, sugars, lipids, peptides and large hydrophobic molecules. The dysfunction of prominent ABC transporters is responsible for severe pathologies such as cystic fibrosis, tangier disease and adrenoleukodystrophy, while the subversion of multidrug ABC transporters confers resistances to therapeutic treatments in malignant cells or pathogenic microorganisms 12,13. These transporters share a common topology with two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). Besides Walker A and B motifs, the NBDs contains several additional motifs involved in ATP binding, including the signature of this family (a stretch of ~12 residues usually starting

Mutations Define Cross-talk between the N-terminal Nucleotide-binding Domain and Transmembrane Helix-2 of the Yeast Multidrug Transporter Pdr5: POSSIBLE CONSERVATION OF A SIGNALING INTERFACE FOR COUPLING ATP HYDROLYSIS TO DRUG TRANSPORT

Journal of Biological Chemistry, 2008

The yeast Pdr5 multidrug transporter is an important member of the ATP-binding cassette superfamily of proteins. We describe a novel mutation (S558Y) in transmembrane helix 2 of Pdr5 identified in a screen for suppressors that eliminated Pdr5-mediated cycloheximide hyper-resistance. Nucleotides as well as transport substrates bind to the mutant Pdr5 with an affinity comparable with that for wild-type Pdr5. Wild-type and mutant Pdr5s show ATPase activity with comparable K m(ATP) values. Nonetheless, drug sensitivity is equivalent in the mutant pdr5 and the pdr5 deletion. Finally, the transport substrate clotrimazole, which is a noncompetitive inhibitor of Pdr5 ATPase activity, has a minimal effect on ATP hydrolysis by the S558Y mutant. These results suggest that the drug sensitivity of the mutant Pdr5 is attributable to the uncoupling of NTPase activity and transport. We screened for amino acid alterations in the nucleotide-binding domains that would reverse the phenotypic effect of the S558Y mutation. A second-site mutation, N242K, located between the Walker A and signature motifs of the N-terminal nucleotide-binding domain, restores significant function. This region of the nucleotide-binding domain interacts with the transmembrane domains via the intracellular loop-1 (which connects transmembrane helices 2 and 3) in the crystal structure of Sav1866, a bacterial ATP-binding cassette drug transporter. These structural studies are supported by biochemical and genetic evidence presented here that interactions between transmembrane helix 2 and the nucleotide-binding domain, via the intracellular loop-1, may define at least part of the translocation pathway for coupling ATP hydrolysis to drug transport.

Structure of a human multidrug transporter in an inward-facing conformation

Journal of Structural Biology, 2010

Multidrug resistance protein 1 (ABCC1) is a member of the 'C' class of ATP-binding cassette transporters, which can give rise to resistance to chemotherapy via drug export from cells. It also acts as a leukotriene C4 transporter, and hence has a role in adaptive immune response. Most Cclass members have an additional NH 2-terminal transmembrane domain versus other ATP-binding cassette transporters, but little is known about the structure and role of this domain. Using electron cryomicroscopy of 2D crystals, data at 1/6 per Å −1 resolution was generated for the full-length ABCC1 protein in the absence of ATP. Analysis using homologous structures from bacteria and mammals allowed the core transmembrane domains to be localised in the map. These display an inward-facing conformation and there is a noteworthy separation of the cytoplasmic nucleotidebinding domains. Examination of non-core features in the map suggests that the additional NH 2terminal domain has extensive contacts on one side of both core domains, and mirrors their inward-facing configuration in the absence of nucleotide.

Molecular Basis of Multidrug Transport by ATP-Binding Cassette Transporters: A Proposed Two-Cylinder Engine Model

2001

ATP-binding cassette multidrug transporters are probably present in all living cells, and are able to export a variety of structurally unrelated compounds at the expense of ATP hydrolysis. The elevated expression of these proteins in multidrug resistant cells interferes with the drug-based control of cancers and infectious pathogenic microorganisms. Multidrug transporters interact directly with the drug substrates. Insights into the structural elements in drug molecules and transport proteins that are required for this interaction are now beginning to emerge. However, much remains to be learned about the nature and number of drug binding sites in the transporters, and the mechanism(s) by which ATP hydrolysis is coupled to changes in affinity and/or accessibility of drug binding sites. This review summarizes recent advances in answering these questions for the human multidrug resistance P-glycoprotein and its prokaryotic homolog LmrA. The relevance of these findings for other ATPbinding cassette transporters will be discussed.