ORCID Profile
0000-0002-7544-8454
Current Organisation
University of Nottingham
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Publisher: Springer Science and Business Media LLC
Date: 26-06-2003
DOI: 10.1007/S00249-003-0327-6
Abstract: Heterologous expression of domains of eukaryotic proteins is frequently associated with formation of inclusion bodies, consisting of aggregated mis-folded protein. This phenomenon has proved a significant barrier to the characterization of domains of eukaryotic ATP binding cassette (ABC) transporters. We hypothesized that the solubility of heterologously expressed nucleotide binding domains (NBDs) of ABC transporters is dependent on the definition of the domain boundaries. In this paper we have defined a core NBD, and tested the effect of extensions to and deletions of this core domain on protein expression. Of 10 NBDs constructed, only one was expressed as a soluble protein in Escherichia coli, with expression of the remaining NBDs being associated with inclusion body formation. The soluble NBD protein we have obtained corresponds to residues 386-632 of P-glycoprotein and represents an optimally defined domain. The NBD has been isolated and purified to 95% homogeneity by a two-step purification protocol, involving affinity chromatography and gel filtration. Although showing no detectable ATP hydrolysis, the protein retains specific ATP binding and has a secondary structure compatible with X-ray crystallographic data on bacterial NBDs. We have interpreted our results in terms of homology models, which suggest that the N-terminal NBD of P-glycoprotein can be produced as a stable, correctly folded, isolate domain with judicious design of the expression construct.
Publisher: Wiley
Date: 18-01-2010
DOI: 10.1111/J.1742-4658.2009.07486.X
Abstract: One of the Holy Grails of ATP-binding cassette transporter research is a structural understanding of drug binding and transport in a eukaryotic multidrug resistance pump. These transporters are front-line mediators of drug resistance in cancers and represent an important therapeutic target in future chemotherapy. Although there has been intensive biochemical research into the human multidrug pumps, their 3D structure at atomic resolution remains unknown. The recent determination of the structure of a mouse P-glycoprotein at subatomic resolution is complemented by structures for a number of prokaryotic homologues. These structures have provided advances into our knowledge of the ATP-binding cassette exporter structure and mechanism, and have provided the template data for a number of homology modelling studies designed to reconcile biochemical data on these clinically important proteins.
Publisher: Elsevier BV
Date: 08-2004
Publisher: Wiley
Date: 20-08-2010
DOI: 10.1111/J.1742-4658.2010.07789.X
Abstract: Describing the molecular details of the multidrug efflux process of ABCB1, in particular the interdomain communication associated with bioenergetic coupling, continues to prove difficult. A number of investigations to date have implicated transmembrane helix 12 (TM12) in mediating communication between the transmembrane domains and nucleotide-binding domains (NBDs) of ABCB1. The present investigation further addressed the role of TM12 in ABCB1 by characterizing its topography during the multidrug efflux process with the use of cysteine-directed mutagenesis. Cysteines were introduced at various positions along TM12 and assessed for their ability to covalently bind thiol-reactive fluorescent probes with differing physiochemical properties. By analysing each isoform in the basal, ATP-bound and posthydrolytic states, it was possible to determine how the local environment of TM12 alters during the catalytic cycle. Labelling with hydrophobic CM and zwitterionic BM was extensive throughout the helix in the basal, prehydrolytic and posthydrolytic states, suggesting that TM12 is in a predominantly hydrophobic environment. Overall, the carboxy region (intracellular half) of TM12 appeared to be more responsive to changes in the catalytic state of the protein than the amino region (extracellular half). Thus, the carboxy region of TM12 is suggested to be responsive to nucleotide binding and hydrolysis at the NBDs and therefore directly involved in interdomain communication. This data can be reconciled with an atomic-scale model of human ABCB1. Taken together, these results indicate that TM12 plays a key role in the progression of the ATP hydrolytic cycle in ABCB1 and, in particular, in coordinating conformational changes between the NBDs and transmembrane domains.
Publisher: Wiley
Date: 04-2003
DOI: 10.1046/J.1432-1033.2003.03514.X
Abstract: The two nucleotide-binding domains (NBDs) of a number of ATP-binding cassette (ABC) transporters have been shown to be functionally dissimilar, playing different roles in the transport process. A high degree of co-operativity has been determined for the NBDs of the human multidrug transporter, P-glycoprotein. However, the issue of functional symmetry in P-glycoprotein remains contentious. To address this, the NBDs of P-glycoprotein were expressed and purified to 95% homogeneity, as fusions to maltose-binding protein. The NBDs were engineered to contain a single cysteine residue in the Walker-A homology motif. Reactivity of this cysteine residue was demonstrated by specific, time-dependent, covalent labelling with N-ethylmaleimide. No differences in the rates of labelling of the two NBDs were observed. The relative affinity of binding to each NBD was determined for a number of nucleotides by measuring their ability to effect a reduction in N-ethylmaleimide labelling. In general, nucleotides bound identically to the two NBDs, suggesting that there is little asymmetry in the initial step of the transport cycle, namely the recognition and binding of nucleotide. Any observed functional asymmetry in the intact transporter presumably reflects different rates of hydrolysis at the two NBDs or interdomain communications.
Publisher: American Chemical Society (ACS)
Date: 06-06-2003
DOI: 10.1021/BI0341049
Abstract: Our aim is to provide molecular understanding of the mechanisms underlying the (i) interaction between the two nucleotide binding domains (NBDs) and (ii) coupling between NBDs and transmembrane domains within P-glycoprotein (Pgp) during a transport cycle. To facilitate this, we have introduced a number of unique cysteine residues at surface exposed positions (E393C, S452C, I500C, N508C, and K578C) in the N-terminal NBD of Pgp, which had previously been engineered to remove endogenous cysteines. Positions of the mutations were designed using a model based on crystallographic features of prokaryotic NBDs. The single cysteine mutants were expressed in insect cells using recombinant baculovirus and the proteins purified by metal affinity chromatography by virtue of a polyhistidine tag. None of the introduced cysteine residues perturbed the function of Pgp as judged by the characteristics of drug stimulated ATP hydrolysis. The role of residues at each of the introduced sites in the catalytic cycle of Pgp was investigated by the effect of covalent conjugation with N-ethyl-maleimide (NEM). All but one mutation (K578C) was accessible to labeling with [(3)H]-NEM. However, perturbation of ATPase activity was only observed for the derivitized N508C isoform. The principle functional manifestation was a marked inhibition of the "basal" rate of ATP hydrolysis. Neither the extent nor potency to which a range of drugs could affect the ATPase activity were altered in the NEM conjugated N508C isoform. The results imply that the accessibility of residue 508, located in the alpha-helical subdomain of NBD1 in Pgp, is altered by the conformational changes that occur during ATP hydrolysis.
Publisher: Elsevier BV
Date: 2009
DOI: 10.1016/J.ADDR.2008.07.004
Abstract: ABCG2 is best known as a multidrug transporter capable of conferring resistance to cancer cells. However, the protein is also inherently expressed in numerous barrier tissues and intriguingly within hematopoietic stem cells. Unlike its partners ABCB1 and ABCC1, there is considerably less information available on the molecular mechanism of ABCG2. The transporter has a distinct topology and is presumed to function as a homodimer. However, a number of biochemical studies have presented data to suggest that the protein adopts higher order oligomers. This review focuses on this controversial issue with particular reference to findings from low resolution structural data. In addition, a number of molecular models of ABCG2 based on high resolution structures of bacterial ABC transporters have recently become available and are critically assessed. ABCG2 is a structurally distinct member of the triumvirate of human multidrug transporters and continues to evade description of a unifying molecular mechanism.
Publisher: Elsevier
Date: 2012
Publisher: Wiley
Date: 04-2014
DOI: 10.1111/FEBS.12773
Publisher: Wiley
Date: 21-10-2017
DOI: 10.1111/BPH.13882
Publisher: Elsevier BV
Date: 2014
DOI: 10.1016/J.BBAMEM.2013.09.001
Abstract: ATP Binding Cassette (ABC) transporters play prominent roles in numerous cellular processes and many have been implicated in human diseases. Unfortunately, detailed mechanistic information on the majority of ABC transporters has not yet been elucidated. The slow rate of progress of molecular and high resolution structural studies may be attributed to the difficulty in the investigation of integral membrane proteins. These difficulties include the expression of functional, non-aggregated protein in heterologous systems. Furthermore, the extraction of membrane proteins from source material remains a major bottle-neck in the process since there are relatively few guidelines for selection of an appropriate detergent to achieve optimal extraction. Whilst affinity tag strategies have simplified the purification of membrane proteins many challenges remain. For ex le, the chromatographic process and associated steps can rapidly lead to functional inactivation, random aggregation, or even precipitation of the target protein. Furthermore, optimisation of high yield and purity, does not guarantee successful structure determination. Based on this series of potential issues, any investigation into structure-function of membrane proteins requires a systematic evaluation of preparation quality. In particular, the evaluation should focus on function, homogeneity and mono-dispersity. The present investigation provides a detailed assessment of the quality of purified ATP Binding Cassette (ABC) transporters namely ABCB1 (P-gp) and ABCA4 (ABCR). A number of suggestions are provided to facilitate the production of functional, homogeneous and mono-disperse preparations using the insect cell expression system. Finally, the ABCA4 s les have been used to provide structural insights into this essential photo-receptor cell protein.
Publisher: Wiley
Date: 03-2008
Abstract: Expression of the ABC transporter P-glycoprotein (P-gp or ABCB1) is associated with resistance to chemotherapy in cancer. However, early investigations into the regulation of ABCB1 expression revealed that the process is not a classical induction as observed for certain metabolizing enzymes. The process involves the cellular stress response pathway initiated by either inflicted (e.g., chemotherapy damage) or endogenous (e.g., hypoxia) factors. However, ABCB1 is also expressed in a number of noncancerous tissues. In particular, the protein is found at tissues providing a barrier or secretory function. The localization of ABCB1 in normal tissues will impact significantly on drug pharmacokinetics, in particular the absorption and elimination processes. This review also describes the mechanism underlying ABCB1 expression in noncancerous tissue, a process that does not involve the stress response.
Publisher: Elsevier BV
Date: 02-2008
DOI: 10.1016/J.BBAMEM.2007.10.021
Abstract: P-glycoprotein (P-gp) is the most intensively studied eukaryotic ATP binding cassette (ABC) transporter, due to its involvement in the multidrug resistance phenotype of a number of cancers. In common with most ABC transporters, P-gp is comprised of two transmembrane domains (TMDs) and two nucleotide binding domains (NBD), the latter coupling ATP hydrolysis with substrate transport (efflux in the case of P-gp). Biochemical investigations over the past twenty years have attempted to unlock mechanistic aspects of P-glycoprotein through scanning and site-directed mutagenesis of both the TMDs and the NBDs. Contemporaneously, crystallographers have elucidated the atomic structure of numerous ABC transporter NBDs, as well as the intact structure (i.e. NBDs and TMDs) of a distantly related ABC-exporter Sav1866. Significantly, the structure of P-gp remains unknown, and only low resolution electron microscopy data exists. Within the current manuscript we employ crystallographic data for homologous proteins, and a molecular model for P-gp, to perform a structural interpretation of the existing "mutagenesis database" for P-gp NBDs. Consequently, this will enable testable predictions to be made that will result in further in-roads into our understanding of this clinically important drug pump.
Publisher: Wiley
Date: 19-12-2005
DOI: 10.1016/J.FEBSLET.2005.11.083
Abstract: Multidrug transporters are involved in mediating the failure of chemotherapy in treating several serious diseases. The archetypal multidrug transporter P-glycoprotein (P-gp) confers resistance to a large number of chemically and functionally unrelated anti-cancer drugs by mediating efflux from cancer cells. The ability to efflux such a large number of drugs remains a biological enigma and the lack of mechanistic understanding of the translocation pathway used by P-gp prevents rational design of compounds to inhibit its function. The translocation pathway is critically dependent on ATP hydrolysis and drug interaction with P-gp is possible at one of a multitude of allosterically linked binding sites. However, aspects such as coupling stoichiometry, molecular properties of binding sites and the nature of conformational changes remain unresolved or the centre of considerable controversy. The present review attempts to utilise the available data to generate a detailed sequence of events in the translocation pathway for this dexterous protein.
Publisher: Oxford University Press (OUP)
Date: 09-07-2008
Publisher: Springer Science and Business Media LLC
Date: 11-07-2008
DOI: 10.1038/NCB1754
Abstract: Lateral roots originate deep within the parental root from a small number of founder cells at the periphery of vascular tissues and must emerge through intervening layers of tissues. We describe how the hormone auxin, which originates from the developing lateral root, acts as a local inductive signal which re-programmes adjacent cells. Auxin induces the expression of a previously uncharacterized auxin influx carrier LAX3 in cortical and epidermal cells directly overlaying new primordia. Increased LAX3 activity reinforces the auxin-dependent induction of a selection of cell-wall-remodelling enzymes, which are likely to promote cell separation in advance of developing lateral root primordia.
Publisher: Wiley
Date: 15-10-2001
Publisher: Wiley
Date: 28-06-2005
DOI: 10.1016/J.FEBSLET.2005.06.030
Abstract: The transmembrane (TM) domains in P-glycoprotein (P-gp) contain the drug binding sites and undergo conformational changes driven by nucleotide catalysis to effect translocation. However, our understanding of exactly which regions are involved in such events remains unclear. A site-directed labelling approach was used to attach thiol-reactive probes to cysteines introduced into transmembrane segment 6 (TM6) in order to perturb function and infer involvement of specific residues in drug binding and/or interdomain communication. Covalent attachment of coumarin-maleimide at residue 339C within TM6 resulted in impaired ATP hydrolysis by P-gp. The nature of the effect was to reduce the characteristic modulation of basal activity caused by transported substrates, modulators and the potent inhibitor XR9576. Photoaffinity labelling of P-gp with [(3)H]-azidopine indicated that residue 339C does not alter drug binding per se. However, covalent modification of this residue appears to prevent conformational changes that lead to drug stimulation of ATP hydrolysis.
Publisher: Springer Science and Business Media LLC
Date: 09-09-2015
DOI: 10.1007/S00280-015-2858-9
Abstract: Multidrug efflux pumps such as ABCG2 confer drug resistance to a number of cancer types, leading to poor prognosis and outcome. To date, the strategy of directly inhibiting multidrug efflux pumps in order to overcome drug resistance in cancer has been unsuccessful. An alternative strategy is to target proteins involved in the regulation of multidrug efflux pump activity or expression. Pim1 kinase has been demonstrated to phosphorylate ABCG2, promote its oligomerisation and contribute to its ability to confer drug resistance. In the present manuscript, imidazo-pyridazine-based inhibitors of Pim1 were examined for their ability to overcome ABCG2-mediated drug resistance. Drug efficacy was measured as a cytotoxic response or an effect on transport by ABCG2. Protein expression patterns were assessed using western immuno-blotting. The two Pim1 inhibitors increased the potency of flavopiridol, mitoxantrone, topotecan and doxorubicin, specifically in ABCG2-expressing cells. This effect was associated with an increase in the cellular accumulation of [(3)H]-mitoxantrone, suggesting direct impairment of the transporter. However, prolonged pre-incubation with the studied inhibitors greatly enhanced the effect on mitoxantrone accumulation. The inhibitors caused a significant time-dependent reduction in the expression of ABCG2 in the resistant cells, an effect that would improve drug efficacy. Consequently, it appears that the Pim1 inhibitors display a dual-mode effect on ABCG2-expressing cancer cells. This may provide a powerful new strategy in overcoming drug resistance by targeting proteins that regulate expression of efflux pumps.
Publisher: Wiley
Date: 11-2006
Publisher: Elsevier BV
Date: 2017
DOI: 10.1016/J.BCP.2016.10.002
Abstract: The multidrug resistance P-glycoprotein (P-gp) is characterised by the ability to bind and/or transport an astonishing array of drugs. This poly-specificity is imparted by at least four pharmacologically distinct binding sites within the transmembrane domain. Whether or not these sites are spatially distinct has remained unclear. Biochemical and structural investigations have implicated a central cavity as the likely location for the binding sites. In the present investigation, a number of contact residues that are involved in drug binding were identified through biochemical assays using purified, reconstituted P-gp. Drugs were selected to represent each of the four pharmacologically distinct sites. Contact residues important in rhodamine123 binding were identified in the central cavity of P-gp. However, contact residues for the binding of vinblastine, paclitaxel and nicardipine were located at the lipid-protein interface rather than the central cavity. A key residue (F978) within the central cavity is believed to be involved in coupling drug binding to nucleotide hydrolysis. Data observed in this investigation suggest the presence of spatially distinct drug binding sites connecting through to a single translocation pore in the central cavity.
Publisher: Elsevier BV
Date: 11-2006
DOI: 10.1016/J.STR.2006.08.014
Abstract: ABCG2 is a multidrug efflux pump associated with resistance of cancer cells to a plethora of unrelated drugs. ABCG2 is a "half-transporter," and previous studies have indicated that it forms homodimers and higher oligomeric species. In this manuscript, electron microscopic structural analysis directly addressed this issue. An N-terminal hexahistidine-tagged ABCG2(R482G) isoform was expressed to high levels in insect cells. An extensive detergent screen was employed to effect extraction of ABCG2(R482G) from membranes and identified only the fos-choline detergents as efficient. Soluble protein was purified to >95% homogeneity by a three-step procedure while retaining the ability to bind substrates. Cryonegative stain electron microscopy of purified ABCG2(R482G) provided 3D structural data at a resolution of approximately 18 A. Single-particle analysis revealed that the complex forms a tetrameric complex ( approximately 180 A in diameter x approximately 140 A high) with an aqueous central region. We interpret the tetrameric structure as comprising four homodimeric ABCG2(R482G) complexes.
Publisher: Wiley
Date: 07-08-2008
DOI: 10.1111/J.1742-4658.2008.06578.X
Abstract: ABCG2 confers resistance to cancer cells by mediating the ATP-dependent outward efflux of chemotherapeutic compounds. Recent studies have indicated that the protein contains a number of interconnected drug binding sites. The present investigation examines the coupling of drug binding to ATP hydrolysis. Initial drug binding to the protein requires a high-affinity interaction with the drug binding site, followed by transition and reorientation to the low-affinity state to enable dissociation at the extracellular face. [3H]Daunomycin binding to the ABCG2 R482G isoform was examined in the nucleotide-bound and post-hydrolytic conformations. Binding of [3H]daunomycin was displaced by ATP analogues, indicating transition to a low-affinity conformation prior to hydrolysis. The low-affinity state was observed to be retained immediately post-hydrolysis. Therefore, the dissociation of phosphate and/or ADP is likely to be responsible for resetting of the transporter. The data indicate that, like ABCB1 and ABCC1, the 'power stroke' for translocation in ABCG2 R482G is the binding of nucleotide.
Publisher: American Chemical Society (ACS)
Date: 28-02-2008
DOI: 10.1021/BI7023089
Abstract: Reduced intracellular drug accumulation due to the activity of the drug efflux pump ABC (B1) is a major mechanism in the resistance of cancer cells to chemotherapy. ABC (B1) is a poly specific transporter, and the molecular mechanism of its complex translocation process remains to be elucidated. To understand the process will require information on the regions involved in drug binding and those that couple this event to nucleotide hydrolysis. The present investigation focuses on the cytosolic region of transmembrane helix 6 (TM6), which has been widely attributed with a central role in the translocation process. A series of ABC (B1) isoforms containing a unique cysteine within TM6 was constructed and the resultant proteins purified and reconstituted. Accessibility of the cysteines to covalent modification by maleimide reagents was measured for the basal, ATP bound and vanadate trapped conformations of each isoform. Residues at the two extremes of the TM6 region examined (amino acids 344 to 360) were considerably more accessible than the central segment, the latter of which also failed to undergo significant conformational changes during the catalytic cycle. Covalent modification of the cytosolic segment of TM6 did, however, attenuate drug stimulation of ATP hydrolysis and demonstrates an important role for this segment in coupling drug binding to ATP hydrolysis during translocation.
Publisher: American Chemical Society (ACS)
Date: 04-06-2009
DOI: 10.1021/BI900373X
Publisher: American Chemical Society (ACS)
Date: 14-08-2007
DOI: 10.1021/BI700447P
Abstract: Multidrug transporters such as P-glycoprotein require considerable inter-domain communication to couple energy utilization with substrate translocation. Elucidation of the regions or residues involved in these communication pathways is a key step in the eventual molecular description of multidrug transport. We used cysteine-scanning mutagenesis to probe the functional involvement of residues along the cytoplasmic half of transmembrane segment 6 (TM6) and its extension toward the nucleotide binding domain. The mutation of one residue (G346C) in this segment adversely affected drug transport in cells. Further investigation using purified protein revealed that the underlying biochemical effect was a reduction in basal ATP hydrolysis. This G346C mutation also affected the stimulation of ATPase activity in a drug dependent manner but had no effect on drug binding, ATP binding, or ADP release. Homology modeling of P-glycoprotein indicated that the G346C mutation caused a steric interaction between TM5 and TM6, thereby precluding a helical movement required to support ATP hydrolysis.
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Ian Kerr.