Human ABCB1 with an ABCB11-like degenerate nucleotide binding site maintains transport activity by avoiding nucleotide occlusion (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Biomembranes, 2011
ABC transporters play important roles in all types of organisms by participating in physiological and pathological processes. In order to modulate the function of ABC transporters, detailed knowledge regarding their structure and dynamics is necessary. Available structures of ABC proteins indicate three major conformations, a nucleotide-bound "bottom-closed" state with the two nucleotide binding domains (NBDs) tightly closed, and two nucleotide-free conformations, the "bottom-closed" and the "bottom-open", which differ in the extent of separation of the NBDs. However, it remains a question how the widely open conformation should be interpreted, and whether hydrolysis at one of the sites can drive conformational transitions while the NBDs remain in contact. To extend our knowledge, we have investigated the dynamic properties of the Sav1866 transporter using molecular dynamics (MD) simulations. We demonstrate that the replacement of one ATP by ADP alters the correlated motion patterns of the NBDs and the transmembrane domains (TMD). The results suggest that the hydrolysis of a single nucleotide could lead to extracellular closure, driving the transport cycle. Essential dynamics analysis of simulations suggests that single nucleotide hydrolysis can drive the system toward a "bottom-closed" apo conformation similar to that observed in the structure of the MsbA transporter. We also found significant structural instability of the "bottom-open" form of the transporters in simulations. Our results suggest that ATP hydrolysis at one of the sites promotes transport related conformational changes leading to the "bottom-closed" apo conformation, which could thus be physiologically more relevant for describing the structure of the apo state. j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a m e m
Protein Science, 2011
ABC transporters are a large and important family of membrane proteins involved in substrate transport across the membrane. The transported substrates are quite diverse, ranging from monatomic ions to large biomolecules. Consequently, some ABC transporters are involved in biomedically relevant situations, from genetic diseases to multidrug resistance. The most conserved domains in ABC transporters are the nucleotide binding domains (NBDs), which form a dimer responsible for the binding and hydrolysis of ATP, concomitantly with substrate translocation. To elucidate how ATP hydrolysis structurally affects the NBD dimer, and consequently the transporter, we performed a molecular dynamics study on the NBD dimer of the HlyB ABC exporter. We have observed a change in the contact surface between the monomers after hydrolysis, even though we have not seen dimer opening in any of the five 100 ns simulations. We have also identified specific regions that respond to ATP hydrolysis, in particular the X-loop motif of ABC exporters, which has been shown to be in contact with the coupling helices of the transmembrane domains (TMDs). We propose that this motif is an important part of the NBD-TMD communication in ABC exporters. Through nonequilibrium analysis, we have also identified gradual conformational changes within a short time scale after ATP hydrolysis.
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
Structural and functional diversity calls for a new classification of ABC transporters
FEBS Letters, 2020
Members of the ATP‐binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP‐binding cassette in the nucleotide‐binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs.
ATP-dependent substrate transport by the ABC transporter MsbA is proton-coupled
ATP-binding cassette transporters mediate the transbilayer movement of a vast number of substrates in or out of cells in organisms ranging from bacteria to humans. Current alternating access models for ABC exporters including the multidrug and Lipid A transporter MsbA from Escherichia coli suggest a role for nucleotide as the fundamental source of free energy. These models involve cycling between conformations with inward-and outward-facing substrate-binding sites in response to engagement and hydrolysis of ATP at the nucleotide-binding domains. Here we report that MsbA also utilizes another major energy currency in the cell by coupling substrate transport to a transmembrane electrochemical proton gradient. The dependence of ATP-dependent transport on proton coupling, and the stimulation of MsbA-ATPase by the chemical proton gradient highlight the functional integration of both forms of metabolic energy. These findings introduce ion coupling as a new parameter in the mechanism of this homodimeric ABC transporter.
Insight in eukaryotic ABC transporter function by mutation analysis
Febs Letters, 2006
With regard to structure-function relations of ATPbinding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.
Binding site of ABC transporter homology models confirmed by ABCB1 crystal structure
Theoretical Biology and Medical Modelling, 2009
The human ATP-binding cassette (ABC) transporters ABCB1, ABCC4 and ABCC5 are involved in resistance to chemotherapeutic agents. Here we present molecular models of ABCB1, ABCC4 and ABCC5 by homology based on a wide open inward-facing conformation of Escherichia coli MsbA, which were constructed in order to elucidate differences in the electrostatic and molecular features of their drug recognition conformations. As a quality assurance of the methodology, the ABCB1 model was compared to an ABCB1 X-ray crystal structure, and with published crosslinking and site directed mutagenesis data of ABCB1. Amino acids Ile306 (TMH5), Ile340 (TMH6), Phe343 (TMH6), Phe728 (TMH7), and Val982 (TMH12), form a putative substrate recognition site in the ABCB1 model, which is confirmed by both the ABCB1 X-ray crystal structure and the sitedirected mutagenesis studies. The ABCB1, ABCC4 and ABCC5 models display distinct differences in the electrostatic properties of their drug recognition sites.
Functional Rescue of a Misfolded Eukaryotic ATP-binding Cassette Transporter by Domain Replacement
Journal of Biological Chemistry, 2010
ATP-binding cassette (ABC) transporters are integral membrane proteins that couple ATP binding/hydrolysis with the transport of hydrophilic substrates across lipid barriers. Deletion of Phe-670 in the first nucleotide-binding domain (NBD1) of the yeast ABC transporter, Yor1p, perturbs interdomain associations, reduces functionality, and hinders proper transport to the plasma membrane. Functionality of Yor1p-⌬F was restored upon co-expression of a peptide containing wild-type NBD1. To gain insight into the biogenesis of this important class of proteins, we defined the requirements for this rescue. We show that a misfolding lesion in NBD1 of the full-length protein is a prerequisite for functional rescue by exogenous NBD1, which is mediated by physical replacement of the dysfunctional domain by the soluble NBD1. This association does not restore trafficking of Yor1p-⌬F but instead confers catalytic activity to the small population of Yor1p-⌬F that escapes to the plasma membrane. An important coupling between the exogenous NBD1 and ICL4 within full-length aberrant Yor1p-⌬F is required for functional rescue but not for the physical interaction between the two polypeptides. Together, our genetic and biochemical data reveal that it is possible to modulate activity of ABC transporters by physically replacing dysfunctional domains.
Dimer Opening of the Nucleotide Binding Domains of ABC Transporters after ATP Hydrolysis
Biophysical Journal, 2008
ABC transporters constitute one of the most abundant membrane transporter families. The most common feature shared in the family is the highly conserved nucleotide binding domains (NBDs) that drive the transport process through binding and hydrolysis of ATP. Molecular dynamics simulations are used to investigate the effect of ATP hydrolysis in the NBDs. Starting with the ATP-bound, closed dimer of MalK, four simulation systems with all possible combinations of ATP or ADP-P i bound to the two nucleotide binding sites are constructed and simulated with equilibrium molecular dynamics for ;70 ns each. The results suggest that the closed form of the NBD dimer can only be maintained with two bound ATP molecules; in other words, hydrolysis of one ATP can lead to the opening of the dimer interface of the NBD dimer. Furthermore, we observed that the opening is an immediate effect of hydrolysis of ATP into ADP and P i rather than the dissociation of hydrolysis products. In addition, the opening is mechanistically triggered by the dissociation of the LSGGQ motif from the bound nucleotide. A metastable ADP-P i bound conformational state is consistently observed before the dimer opening in all the simulation systems.
Journal of Biological Chemistry, 2001
ABCR is a member of the ABCA subclass of ATP binding cassette transporters that is responsible for Stargardt macular disease and implicated in retinal transport across photoreceptor disc membranes. It consists of a single polypeptide chain arranged in two tandem halves, each having a multi-spanning membrane domain followed by a nucleotide binding domain. To delineate between several proposed membrane topological models, we have identified the exocytoplasmic (extracellular/lumen) N-linked glycosylation sites on ABCR. Using trypsin digestion, site-directed mutagenesis, concanavalin A binding, and endoglycosidase digestion, we show that ABCR contains eight glycosylation sites. Four sites reside in a 600-amino acid exocytoplasmic domain of the N-terminal half between the first transmembrane segment H1 and the first multi-spanning membrane domain, and four sites are in a 275-amino acid domain of the C half between transmembrane segment H7 and the second multi-spanning membrane domain. This leads to a model in which each half has a transmembrane segment followed by a large exocytoplasmic domain, a multi-spanning membrane domain, and a nucleotide binding domain. Other ABCA transporters, including ABC1 linked to Tangier disease, are proposed to have a similar membrane topology based on sequence similarity to ABCR. Studies also suggest that the N and C halves of ABCR are linked through disulfide bonds.