The ABC transporter structure and mechanism: perspectives on recent research

ATP-binding cassette (ABC) transporters are multidomain integral membrane proteins that utilise the energy of ATP hydrolysis to translocate solutes across cellular membranes in all phyla. ABC transporters form one of the largest of all protein families and are central to many important biomedical phenomena, including resistance of cancers and pathogenic microbes to drugs. Elucidation of the structure and mechanism of ABC transporters is essential to the rational design of agents to control their function. While a wealth of high-resolution structures of ABC proteins have been produced in recent years, many fundamental questions regarding the proteins mechanism remain unanswered. In this review, we examine the recent structural data concerning ABC transporters and related proteins in the light of other experimental and theoretical data, and discuss these data in relation to current ideas concerning the transporters molecular mechanism.Received 29 August 2003; received after revision 19 November 2003; accepted 28 November 2003     

Nitrate and nitrite transport in bacteria

The topological arrangements of nitrate and nitrite reductases in bacteria necessitate the synthesis of transporter proteins that carry the nitrogen oxyanions across the cytoplasmic membrane. For assimilation of nitrate (and nitrite) there are two types of uptake system known: ABC transporters that are driven by ATP hydrolysis, and secondary transporters reliant on a proton motive force. Proteins homologous to the latter type of transporter are also involved in nitrate and nitrite transport in dissimilatory processes such as denitrification. These proteins belong to the NarK family, which is a branch of the Major Facilitator Superfamily. The mechanism and substrate specificity of transport via these proteins is unknown, but is discussed in the light of sequence analysis of members of the NarK family. A hypothesis for nitrate and nitrite transport is proposed based on the finding that there are two distinct types of NarK.     

The role of P-glycoprotein/cellular prion protein interaction in multidrug-resistant breast cancer cells treated with paclitaxel

We previously reported that treatment with P-glycoprotein (P-gp) substrates promotes in vitro invasion in multidrug-resistant (MDR) breast cancer cells. This effect is initiated by the P-gp pump function and mediated by interaction of P-gp with some unknown component(s). However, the underlying mechanism(s) remains poorly understood. Here we confirm a novel physical interaction between P-gp and cellular prion protein (PrP^c). Blocking P-gp activity or depletion of PrP^c inhibited paclitaxel (P-gp substrate)- induced invasion. Paclitaxel further facilitated the formation of P-gp/PrP^c clusters residing in caveolar domains and promoted the association of P-gp with caveolin-1. Both caveolin-1 and the integrity of caveolae were required for the drug-induced invasion. In addition, the P-gp/PrP^c complex also played an important role in anti-apoptotic activity of MCF7/Adr cells.These data provide new insights into the mode by which MDR breast cancers evade cytotoxic attacks from P-gp substrates and also suggest a role for P-gp/ PrP^c interaction in this process. Received 4 September 2008; received after revision 16 November 2008; accepted 18 November 2008     

MDR1, cholesterol certification and cell growth: a comparative study in normal and multidrug-resistant KB cell lines

The product of the MDR1 gene (P-gp) has been implicated in the transport of cholesterol from plasma membrane to endoplasmic reticulum for esterification. In previous studies on leukemia cell lines, we suggested that cholesterol esterification may regulate the rate of cell growth and that the MDR1 gene might be involved in this process by modulating intracellular cholesterol esters levels. To further investigate this matter, the rate of cell growth, cholesterol metabolism, expression of the MDR1 gene, and P-gp activity were compared in KB cell lines displaying differences in expression and function of P-gp (drug-sensitive phenotype versus MDR phenotype). The rate of cell growth correlated with cholesterol esterification in all KB cell lines, whereas the over-expression of MDR1 observed in the MDR cell lines was not always associated with an increased capacity of cells to esterify cholesterol. Two known inhibitors of P-gp activity, progesterone and verapamil, strongly inhibited both cholesterol esterification and cell proliferation in all KB cell lines, but they affected intracellular accumulation of labeled vinblastine only in MDR cell lines. These results further support a role for cholesterol esters in the regulation of cell growth and suggest that the P-gp expressed in MDR KB cells is not involved in the general process leading to cholesterol esterification. Received 14 February 2000; received after revision 10 April 2000; accepted 8 May 2000     

An overview of cancer multidrug resistance: a still unsolved problem

Although various mechanisms involved in anticancer multidrug resistance (MDR) can be identified, it remains a major problem in oncology. Beyond that, the introduction of new “targeted” drugs have not solved the problem. On the contrary, it has been demonstrated that the “classical” MDR-associated mechanisms are similar or identical to those causing resistance to these novel agents. These mechanisms include the enhanced activity of drug pumps, i.e. ABC or alternative transporters; modulation of cellular death pathways; alteration and repair of target molecules; and various less common mechanisms. Together they build a complex network of cellular pathways and molecular mechanisms mediating an individual MDR phenotype. Although the application of new high throughput “-omics” technologies have identified multiple new gene-/protein expression signatures or factors associated with drug resistance, so far none of these findings has been useful for creating improved diagnostic assays, for prediction of individual therapy response, or for development of updated chemosensitizers. Received 05 March 2008; received after revision 21 May 2008; accepted 23 May 2008     

sHsps and their role in the chaperone network

Small Hsps (sHsps) encompass a widespread but diverse class of proteins. These low molecular mass proteins (15—42 kDa) form dynamic oligomeric structures ranging from 9 to 50 subunits. sHsps display chaperone function in vitro, and in addition they have been suggested to be involved in the inhibition of apoptosis, organisation of the cytoskeleton and establishing the refractive properties of the eye lens in the case of α-crystallin. How these different functions can be explained by a common mechanism is unclear at present. However, as most of the observed phenomena involve nonnative protein, the repeatedly reported chaperone properties of sHsps seem to be of key importance for understanding their function. In contrast to other chaperone families, sHsps bind several nonnative proteins per oligomeric complex, thus representing the most efficient chaperone family in terms of the quantity of substrate binding. In some cases, the release of substrate proteins from the sHsp complex is achieved in cooperation with Hsp70 in an ATP-dependent reaction, suggesting that the role of sHsps in the network of chaperones is to create a reservoir of nonnative refoldable protein.     

Molecular models of the open and closed states of the whole human CFTR protein

Cystic fibrosis transmembrane conductance regulator (CFTR), involved in cystic fibrosis (CF), is a chloride channel belonging to the ATP-binding cassette (ABC) superfamily. Using the experimental structure of Sav1866 as template, we previously modeled the human CFTR structure, including membrane-spanning domains (MSD) and nucleotide-binding domains (NBD), in an outward-facing conformation (open channel state). Here, we constructed a model of the CFTR inward-facing conformation (closed channel) on the basis of the recent corrected structures of MsbA and compared the structural features of those two states of the channel. Interestingly, the MSD:NBD coupling interfaces including F508 (ΔF508 being the most common CF mutation) are mainly left unchanged. This prediction, completed by the modeling of the regulatory R domain, is supported by experimental data and provides a molecular basis for a better understanding of the functioning of CFTR, especially of the structural features that make CFTR the unique channel among the ABC transporters.     

The ATP-binding cassette family: a structural perspective

The ATP-binding cassette family is one of the largest groupings of membrane proteins, moving allocrites across lipid membranes, using energy from ATP. In bacteria, they reside in the inner membrane and are involved in both uptake and export. In eukaryotes, these transporters reside in the cell’s internal membranes as well as in the plasma membrane and are unidirectional—out of the cytoplasm. The range of substances that these proteins can transport is huge, which makes them interesting for structure–function studies. Moreover, their abundance in nature has made them targets for structural proteomics consortia. There are eight independent structures for ATP-binding cassette transporters, making this one of the best characterised membrane protein families. Our understanding of the mechanism of transport across membranes and membrane protein structure in general has been enhanced by recent developments for this family.     

A dynamic view of peptides and proteins in membranes

Biological membranes are highly dynamic supramolecular arrangements of lipids and proteins, which fulfill key cellular functions. Relatively few high-resolution membrane protein structures are known to date, although during recent years the structural databases have expanded at an accelerated pace. In some instances the structures of reaction intermediates provide a stroboscopic view on the conformational changes involved in protein function. Other biophysical approaches add dynamic aspects and allow one to investigate the interactions with the lipid bilayers. Membrane-active peptides fulfill many important functions in nature as they act as antimicrobials, channels, transporters or hormones, and their studies have much increased our understanding of polypeptide-membrane interactions. Interestingly several proteins have been identified that interact with the membrane as loose arrays of domains. Such conformations easily escape classical high-resolution structural analysis and the lessons learned from peptides may therefore be instructive for our understanding of the functioning of such membrane proteins. Received 11 March 2008; received after revision 2 May 2008; accepted 5 May 2008     

Insights into acylphosphatase structure and catalytic mechanism

Acylphosphatase is one of the smallest enzymes known (about 98 amino acid residues). It is present in organs and tissues of vertebrate species as two isoenzymes sharing over 55% of sequence homology; these appear highly conserved in differing species. The two isoenzymes can be involved in a number of physiological processes, though their effective biological function is not still certain. The solution and crystal structures of different isoenzymes are known, revealing a close packed protein with a fold similar to that shown by other phosphate-bind ing proteins. The structural data, together with an extended site-directed mutagenesis investigation, led to the identification of the residues involved in enzyme catalysis. However, it appears unlikely that these residues are able to perform the full catalytic cycle: a substrate-assisted catalytic mechanism has therefore been proposed, in which the phosphate moiety of the substrate could act as a nucleophile activating the catalytic water molecule. Received 12 November 1996; accepted 27 November 1996     

ABCA2: a candidate regulator of neural transmembrane lipid transport

Studies in the past years have implicated multispan transmembrane transport molecules of the ATP binding cassette (ABC) transporter family in cellular lipid export processes. The prototypic ABC transporter ABCA1 has recently been demonstrated to act as a major facilitator of cellular cholesterol and phospholipid export. Moreover, the transporter ABCA4 (ABCR) plays a pivotal role in retinaldehyde processing, and ABCA3 has recently implicated in lung surfactant processing. These pioneering observations have directed considerable attention to the A subfamily of ABC proteins. ABCA2 is the codefining member of the ABC A-transporter subclass. Although known for some time, it was not until recently that its complete molecular structure was established. Unlike other ABC A-subfamily members, ABCA2 is predominantly expressed in the brain and neural tissues. The unique expression profile together with available structural data suggest roles for this largest known ABC protein in neural transmembrane lipid export. Received 31 January 2002; received after revision 11 March 2002; accepted 11 March 2002     

Function and molecular evolution of multicopper blue proteins

Multicopper blue proteins (MCBPs) are multidomain proteins that utilize the distinctive redox ability of copper ions. There are a variety of MCBPs that have been roughly classified into three different groups, based on their domain organization and functions: (i) nitrite reductase-type with two domains, (ii) laccase-type with three domains, and (iii) ceruloplasmin-type with six domains. Together, the second and third group are often commonly called multicopper oxidases (MCOs). The rapid accumulation of genome sequence information in recent years has revealed several new types of proteins containing MCBP domains, mainly from bacteria. In this review, the recent research on the functions and structures of MCBPs is summarized, mainly focusing on the new types. The latter half of this review focusses on the twodomain MCBPs, which we propose as the evolutionary intermediate of the MCBP family.Received 25 February 2005; received after revision 23 May 2005; accepted 31 May 2005     

Nucleotide-binding domains of human cystic fibrosis transmembrane conductance regulator: detailed sequence analysis and three-dimensional modeling of the heterodimer

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is encoded by the gene that is defective in cystic fibrosis, the most common lethal inherited disease among the Caucasian population. CFTR belongs to the ABC transporter superfamily, whose members form macromolecular architectures composed of two membrane-spanning domains and two nucleotide-binding domains (NBDs). The experimental structures of NBDs from several ABC transporters have recently been solved, opening new avenues for understanding the structure/function relationships and the consequences of some disease-causing mutations of CFTR. Based on a detailed sequence/structure analysis, we propose here a three-dimensional model of the human CFTR NBD heterodimer. This model, which is in agreement with recent experimental data, highlights the specific features of the CFTR asymmetric active sites located at the interface between the two NBDs. Moreover, additional CFTR-specific features can be identified at the subunit interface, which may play critical roles in active site interdependence and are uncommon in other NBD dimers.Received 16 October 2003; received after revision 16 November 2003; accepted 21 November 2003     

The function of apolipoproteins L

The function of the proteins of the apolipoprotein L (apoL) family is largely unknown. These proteins are classically thought to be involved in lipid transport and metabolism, mainly due to the initial discovery that a secreted member of the family, apoL-I, is associated with high-density lipoprotein particles. However, the other members of the family are believed to be intracellular. The recent unravelling of the mechanism by which apoL-I kills African trypanosomes, as well as the increasing evidence for modulation of apoL expression in various pathological processes, provides new insights about the functions of these proteins. ApoLs share structural and functional similarities with proteins of the Bcl-2 family. Based on the activity of apoL-I in trypanosomes and the comparison with Bcl-2 proteins, we propose that apoLs could function as ion channels of intracellular membranes and be involved in mechanisms triggering programmed cell death. Received 28 February 2006; received after revision 18 May 2006; accepted 2 June 2006     

Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and related ABC transporters

Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.     

Progress in biomimetic carbohydrate recognition

The importance of carbohydrate recognition in biology, and the unusual challenges involved, have lead to great interest in mimicking saccharide-binding proteins such as lectins. In this review, we discuss the design of artificial carbohydrate receptors, focusing on those which work under natural (i.e. aqueous) conditions. The problem is intrinsically difficult because of the similarity between substrate (carbohydrate) and solvent (water) and, accordingly, progress has been slow. However, recent developments suggest that solutions can be found. In particular, the “temple” family of carbohydrate receptors show good affinities and excellent selectivities for certain all-equatorial substrates. One example is selective for O-linked β-N-acetylglucosamine (GlcNAc, as in the O-GlcNAc protein modification), while another is specific for β-cellobiosyl and closely related disaccharides. Both show roughly millimolar affinities, matching the strength of some lectin–carbohydrate interactions.     

Investigating metal-binding in proteins by nuclear magnetic resonance

Metal ions play a key role for the function of many proteins. The interaction of the metal ion with the protein and its involvement in the function of the protein vary widely. In some proteins, the metal ion is bound tightly to the ligand residues and may be the key player in the function of the protein, as in the case of blue copper proteins. In other proteins, the metal ion is bound only temporarily and loosely to the protein, as in the case of some metalloenzymes and other proteins where the metal ion acts as a cofactor necessary for the function of the protein. Such proteins are often known as metal ion-activated proteins. The review focuses on recent nuclear magnetic resonance (NMR) studies of a series of metal-dependent proteins and the characterization of the metal-binding sites. In particular, we focus on NMR techniques for studying metal binding to proteins such as chemical shift mapping, paramagnetic NMR and changes in backbone dynamics upon metal binding. Received 12 October 2006; received after revision 30 November 2006; accepted 5 February 2007