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Associate Professor Anthony George

Biography

Assoc Prof George has been at the University of Technology since its inception as a university in 1989. He has published 38 papers at UTS and another 12 in his early career. He graduated with Science (Hons), MSc, and PhD degrees from the University of Sydney in 1981 and completed postdoctoral fellowships at Tufts University Medical School (Boston) and the John Curtin School of Medical Research (Canberra).

His tenured academic position at UTS began in 1986. His early research was focused on multidrug resistance in pathogenic bacteria and this work continued at UTS and is still an extant section of his work, which more recently has moved into the study of pathogenic infections in serious medical syndromes such as cystic fibrosis. Although there are several mechanisms and pathways of multidrug resistance (MDR), A/P George’s work is concerned mostly with drug efflux membrane resistance, with experience in a number of techniques, including drug transport assays in cells and everted vesicles, site-directed mutagenesis and cysteine cross-linking to identify crucial residues in drug biding sites.

A/P George’s research has taken two new directions, one beginning in 1995 and the second in 2007. The work in Boston in 1981-83 generated the first report of a chromosome-based multidrug efflux system in bacteria that was under the control of a transcriptional activator/repressor system and encompassed a global network of over 30 genes. In 1996, A/P George published the first and still only successful cloning, expression, and drug efflux for human P-glycoprotein in Eschericia coli. This MDR protein belongs to one of the largest gene/protein families in all species, namely ABC transporters. Most of his work since then has involved structure-function studies of ABC transporters, using traditional laboratory methods and the emerging and powerful computational molecular dynamic simulations that take X-ray crystal structures of proteins and “animate” them in computers to explore function. We have been very successful in this field, having discovered the dimeric structural configuration of ABC transporters and elaborated two extant new models for the function of these proteins. A/P George’s work has been rewarded by prestigious invitations to give keynote talks at international conferences, to Chair a Gordon Research Meeting on multidrug resistance systems and to be invited on to the Scientific Advisory Board of the FEBS ABC Efflux Meetings, held every two years in Austria.

In 2007, A/P George began a new project intended to alleviate the onset and progression of incurable asbestos-related diseases. Research began by studying the effects of asbestos fibres and counteracting agents in human lung cells; and has moved these past two years to an animal model. The work has been rewarded with a Trailblazer Award (2008) and with substantial funding ($712,000) from philanthropic sources. Patent and commercial prospects are possible in the near future.

During his career, A/P George has published trailblazing studies and many scholarly reviews in high impact journals such as Proc Natl Acad Sci USA, Crit Rev Mol Cell Biol, Trend Biochem Sci. He has supervised many Honours, Masters, and PhDs, and has a Research Associate of long standing for his mainstream ABC transporter research. Total research funding and fellowships for all projects exceeds $5m.

Professional

I have held memberships of a number of professional societies including, the Federation of European Microbiologists Society, American Chemical Society, Biophysical Society, and American Society for Biochemistry and Molecular Biology.

Image of Anthony George
Associate Professor, School of Medical and Molecular Biosciences
Associate Member, Centre for Health Technologies
Core Member, ithree institute
B.Sc(Hons), MSc (Syd), PhD (Syd)
 
Phone
+61 2 9514 4158
Room
CB04.06.64

Research Interests

1. ABC transporters and cancer
ABC transporters are involved in diverse cellular processes including resistance to cytotoxic drugs. Inhibition of these proteins will improve the efficacy of primary drug treatment and render these proteins as targets for new drugs. We are using recent atomic structures as the basis for molecular dynamics calculations and cross-linking experiments designed to identify crucial mobile regions, enabling us to identify and test small molecules that interfere with the normal movement of critical regions in these proteins. [Collaboration with Dr Richard Callaghan, Oxford University; Dr Ian Kerr, Nottingham University; and Dr Megan O’Mara, QLD University].

2. Cystic Fibrosis infections
Cystic fibrosis is the most common lethal inherited disorder in Caucasians, affecting 1 in 2,500 births. Whilst median survival has increased from 1-2 years (1960) to a current 36-38 years, chronic lung disease still causes the majority of deaths associated with CF. Most patients are infected chronically with P. aeruginosa. Resistance to chemotherapeutic antibiotics is commonplace. We are testing the efficacy of the combination of tobramycin and amiloride against P. aeruginosa in clinical and laboratory studies.
[in collaboration with Assoc Prof Cythia Whitworth and Dr Lynne Turnbull, IBID/UTS; and Assoc Profs Peter Middleton and Jon Iredell, Sydney University].

3. Ameliorating Asbestosis
When asbestos is mined or pulverized, it becomes suspended in the air as "parachutes", leading to inhalation into the lungs where it can induce fibrosis (asbestosis) or malignancy (mesothelioma). Asbestos fibres generate reactive oxygen radicals that cause cellular and DNA damage. We have made a serendipitous discovery for ameliorating the adverse effects of asbestos fibres that we now want to test in lung cell cultures, and then in animal trials.

I have been involved in a number of teaching subjects at UTS over past years. Currently, I am the subject coordinator for Molecular Biology 1 and DNA Profiling. Most of my teaching is in Molecular Biology 1, a subject that was originally created by me, and whose content has been written, upgraded and revised at regular intervals. It has become one of the largest second year subjects with over 300 students. DNA Profiling in the Forensics Biology Degree is a subject that I inherited from an ex-staff member. The lectures and tutorials are given by external forensic scientists, which allows for an up to date interface with the developments in the subject area. The practical course is my major focus and it has been revised substantially in recent years. I make small contributions in terms of specialist lectures in the subjects Molecular Biology 2 and Biotechnology.

Book Chapters

Callaghan, R., George, A.M. & Kerr, I. 2011, 'Molecular aspects of the translocation process by ABC proteins' in S Ferguson (ed), Comprehensive Biophysics, American Chemical Society, Oxford, pp. 145-173.
George, A.M. 2005, 'Multiple Antimicrobial Resistance' in White, DG; Alekshun, MN; McDermott, PF (eds), Frontiers in Antimicrobial Resistance: a Tribute to Stuart B. Levy, ASM Press, Washington DC, USA, pp. 151-164.
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Journal Articles

Jones, P.M. & George, A.M. 2013, 'Mechanism of the ABC transporter ATPase domains: catalytic models and the biochemical and biophysical record', Critical Reviews in Biochemistry and Molecular Biology, vol. 48, no. 1, pp. 39-50.
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ABC transporters comprise a large, diverse, and ubiquitous superfamily of membrane active transporters. Their core architecture is a dimer of dimers, comprising two transmembrane domains that bind substrate and form the channel, and two ATP-binding cassettes, which bind and hydrolyze ATP to energize the translocase function. The prevailing paradigm for the ABC transport mechanism is the Switch Model, in which the nucleotide binding domains are proposed to dimerise upon binding of two ATP molecules, and thence dissociate upon sequential hydrolysis of the ATP. This idea appears consistent with crystal structures of both isolated subunits and whole transporters, as well as with a significant body of biochemical data. Nonetheless, an alternative Constant Contact Model has been proposed, in which the nucleotide binding domains do not fully dissociate, and ATP hydrolysis occurs alternately at each of the two active sites. Here, we review the biochemical and biophysical data relating to the ABC catalytic mechanism, to show how they may be construed as consistent with a Constant Contact Model, and to assess to what extent they support the Switch Model.
George, A.M. & Jones, P.M. 2013, 'An asymmetric post-hydrolysis state of the ABC transporter ATPase dimer', PLoS One, vol. 8, no. 4, pp. e59854-1-9.
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ABC transporters are a superfamily of enzyme pumps that hydrolyse ATP in exchange for translocation of substrates across cellular membranes. Architecturally, ABC transporters are a dimer of transmembrane domains coupled to a dimer of nucleotide binding domains (NBDs): the NBD dimer contains two ATP-binding sites at the intersubunit interface. A current controversy is whether the protomers of the NBD dimer separate during ATP hydrolysis cycling, or remain in constant contact. In order to investigate the ABC ATPase catalytic mechanism, MD simulations using the recent structure of the ADP+Pi-bound MJ0796 isolated NBD dimer were performed. In three independent simulations of the ADP+Pi/apo state, comprising a total of .0.5 ms, significant opening of the apo (empty) active site was observed; occurring by way of intrasubunit rotations between the core and helical subdomains within both NBD monomers. In contrast, in three equivalent simulations of the ATP/apo state, the NBD dimer remained close to the crystal structure, and no opening of either active site occurred. The results thus showed allosteric coupling between the active sites, mediated by intrasubunit conformational changes. Opening of the apo site is exquisitely tuned to the nature of the ligand, and thus to the stage of the reaction cycle, in the opposite site. In addition to this, in also showing how one active site can open, sufficient to bind nucleotide, while the opposite site remains occluded and bound to the hydrolysis products ADP+Pi, the results are consistent with a Constant Contact Model. Conversely, they show how there may be no requirement for the NBD protomers to separate to complete the catalytic cycle.
Valenzuela, S., Curmi, P.M., George, A.M. & Jones, P.M. 2013, 'Computational Analysis Of The Soluble Form Of The Intracellular Chloride Ion Channel Protein Clic1', BioMed Research International, vol. 2013, pp. 170586-1-170586-14.
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The chloride intracellular channel (CLIC) family of proteins has the remarkable property of maintaining both a soluble form and an integral membrane form acting as an ion channel. The soluble form is structurally related to the glutathione-S-transferase family, and CLIC can covalently bind glutathione via an active site cysteine. We report approximately 0.6 s of molecular dynamics simulations, encompassing the three possible ligand-bound states of CLIC1, using the structure of GSH-bound human CLIC1. Noncovalently bound GSH was rapidly released from the protein, whereas the covalently ligand-bound protein remained close to the starting structure over 0.25 s of simulation. In the unliganded state, conformational changes in the vicinity of the glutathione-binding site resulted in reduced reactivity of the active site thiol. Elastic network analysis indicated that the changes in the unliganded state are intrinsic to the protein architecture and likely represent functional transitions. Overall, our results are consistent with a model of CLIC function in which covalent binding of glutathione does not occur spontaneously but requires interaction with another protein to stabilise the GSH binding site and/or transfer of the ligand. The results do not indicate how CLIC1 undergoes a radical conformational change to form a transmembrane chloride channel but further elucidate the mechanism by which CLICs are redox controlled.
Gong, J., Luk, C.F., Jaiswal, R., George, A.M., Grau, G. & Bebawy, M. 2013, 'Microparticle drug sequestration provides a parallel pathway in the acquisition of cancer drug resistance', European Journal of Pharmacology, vol. 721, no. 1-3, pp. 116-125.
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Expanding on our previous findings demonstrating that microparticles (MPs) spread cancer multidrug resistance, we now show that MPs sequester drugs, reducing the free drug concentration available to cells. MPs were isolated from drug-sensitive and drug-resistant sub-clones of a human breast adenocarcinoma cell line and from human acute lymphoblastic leukemia cells. MPs were assessed for size, mitochondria, RNA and phospholipid content, P-glycoprotein (P-gp) expression and orientation and ATPase activity relative to drug sequestration capacity. Of the drug classes examined, MPs sequestered the anthracycline class to a significant degree. The degree of sequestration was likely due to the size of MPs and thus the amount of cargo they contain, to which the anthracyclines bind. Moreover, a proportion of the P-gp present on MPs was inside-out in orientation, enabling it to influx drugs rather than its typical efflux function. This was confirmed by surface immunofluorescence and by assessment of drug-stimulated ATPase activity following MP permeabilization. Thus we determined that breast cancer MPs carried a proportion of their P-gp oriented inside-out, providing active sequestration within the microvesicular compartment. These results demonstrate a capacity for MPs to sequester chemotherapeutic drugs, which has a predominantly active sequestration component for MPs derived from drug-resistant cells and a predominantly passive component for MPs derived from drug-sensitive cells. This reduction in available drug concentration has potential to contribute to a parallel pathway and complements that of the intercellular transfer of P-gp. These findings lend further support to the role of MPs in limiting the successful management of cancer.
Gong, J., Luk, F., Jaiswal, R., George, A.M., Grau, G. & Bebawy, M. 2013, 'Microparticle drug sequestration provides a parallel pathway in the acquisition of cancer drug resistance', European Journal of Pharmacology, vol. 721, no. 1-3, pp. 116-125.
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Jones, P.M. & George, A.M. 2012, 'Role of the D-loops in allosteric control of ATP hydrolysis in an ABC transporter', Journal of Physical Chemistry A, vol. 116, no. 11, pp. 3004-3013.
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ABC transporters couple ATP hydrolysis to movement of substrates across cell membranes. They comprise two transmembrane domains and two cytosolic nucleotide-binding domains forming two active sites that hydrolyze ATP cooperatively. The mechanism of ATP hydrolysis is controversial and the structural dynamic basis of its allosteric control unknown. Here we report molecular dynamics simulations of the ATP/apo and ATP/ADP states of the bacterial ABC exporter Sav1866, in which the cytoplasmic region of the protein was simulated in explicit water for 150 ns. In the simulation of the ATP/apo state, we observed, for the first time, conformers of the active site with the canonical geometry for an in-line nucleophilic attack on the ATP gamma-phosphate. The conserved glutamate immediately downstream of the Walker B motif is the catalytic base, forming a dyad with the H-loop histidine, whereas the Q-loop glutamine has an organizing role. Each D-loop provides a coordinating residue of the attacking water, and comparison with the simulation of the ATP/ADP state suggests that via their flexibility, the D-loops modulate formation of the hydrolysis-competent state. A global switch involving a coupling helix delineates the signal transmission route by which allosteric control of ATP hydrolysis in ABC transporters is mediated.
George, A.M. & Jones, P.M. 2012, 'Perspectives on the structureefunction of ABC transporters: The Switch and Constant Contact Models', Progress in Biophysics and Molecular Biology, vol. 109, pp. 95-107.
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ABC transporters constitute one of the largest protein families across the kingdoms of archaea, eubacteria and eukarya. They couple ATP hydrolysis to vectorial translocation of diverse substrates across membranes. The ABC transporter architecture comprises two transmembrane domains and two cytosolic ATP-binding cassettes. During 2002-2012, nine prokaryotic ABC transporter structures and two eukaryotic structures have been solved to medium resolution. Despite a wealth of biochemical, biophysical, and structural data, fundamental questions remain regarding the coupling of ATP hydrolysis to unidirectional substrate translocation, and the mechanistic suite of steps involved. The mechanics of the ATP cassette dimer is defined most popularly by the 'Switch Model', which proposes that hydrolysis in each protomer is sequential, and that as the sites are freed of nucleotide, the protomers lose contact across a large solvent-filled gap of 20-30 angstrom: as captured in several X-ray solved structures. Our 'Constant Contact' model for the operational mechanics of ATP binding and hydrolysis in the ATP-binding cassettes is derived from the 'alternating sites' model, proposed in 1995, and which requires an intrinsic asymmetry in the ATP sites, but does not require the partner protomers to lose contact. Thus one of the most debated issues regarding the function of ABC transporters is whether the cooperative mechanics of ATP hydrolysis requires the ATP cassettes to separate or remain in constant contact and this dilemma is discussed at length in this review.
Nolan, L.M., Croft, L., Jones, P.M., George, A.M., Turnbull, L. & Whitchurch, C.B. 2012, 'Extragenic suppressor mutations that restore twitching motility to fimL mutants of Pseudomonas aeruginosa are associated with elevated intracellular cyclic AMP levels', Microbiology Open, vol. 1, no. 4, pp. 490-501.
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Cyclic AMP (cAMP) is a signaling molecule that is involved in the regulation of multiple virulence systems of the opportunistic pathogen Pseudomonas aeruginosa. The intracellular concentration of cAMP in P. aeruginosa cells is tightly controlled at the levels of cAMP synthesis and degradation through regulation of the activity and/or expression of the adenylate cyclases CyaA and CyaB or the cAMP phosphodiesterase CpdA. Interestingly, mutants of fimL, which usually demonstrate defective twitching motility, frequently revert to a wild-type twitching-motility phenotype presumably via the acquisition of an extragenic suppressor mutation(s). In this study, we have characterized five independent fimL twitching-motility revertants and have determined that all have increased intracellular cAMP levels compared with the parent fimL mutant. Whole-genome sequencing revealed that only one of these fimL revertants has acquired a loss-of-function mutation in cpdA that accounts for the elevated levels of intracellular cAMP. As mutation of cpdA did not account for the restoration of twitching motility observed in the other four fimL revertants, these observations suggest that there is at least another, as yet unidentified, site of extragenic suppressor mutation that can cause phenotypic reversion in fimL mutants and modulation of intracellular cAMP levels of P. aeruginosa.
Robinson, M.W., Corvo, I., Jones, P.M., George, A.M., Padula, M.P., To, J., Cancela, M., Rinaldi, G., Tort, J.F., Roche, L. & Dalton, J.P. 2011, 'Collagenolytic Activities of the Major Secreted Cathepsin L Peptidases Involved in the Virulence of the Helminth Pathogen, Fasciola hepatica', Plos Neglected Tropical Diseases, vol. 5, no. 4, pp. 1-13.
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Background: The temporal expression and secretion of distinct members of a family of virulence-associated cathepsin L cysteine peptidases (FhCL) correlates with the entry and migration of the helminth pathogen Fasciola hepatica in the host. Thus, infective larvae traversing the gut wall secrete cathepsin L3 (FhCL3), liver migrating juvenile parasites secrete both FhCL1 and FhCL2 while the mature bile duct parasites, which are obligate blood feeders, secrete predominantly FhCL1 but also FhCL2. Methodology/Principal Findings: Here we show that FhCL1, FhCL2 and FhCL3 exhibit differences in their kinetic parameters towards a range of peptide substrates. Uniquely, FhCL2 and FhCL3 readily cleave substrates with Pro in the P2 position and peptide substrates mimicking the repeating Gly-Pro-Xaa motifs that occur within the primary sequence of collagen. FhCL1, FhCL2 and FhCL3 hydrolysed native type I and II collagen at neutral pH but while FhCL1 cleaved only non-collagenous (NC, non-Gly-X-Y) domains FhCL2 and FhCL3 exhibited collagenase activity by cleaving at multiple sites within the alpha 1 and alpha 2 triple helix regions (Col domains). Molecular simulations created for FhCL1, FhCL2 and FhCL3 complexed to various seven-residue peptides supports the idea that Trp67 and Tyr67 in the S2 subsite of the active sites of FhCL3 and FhCL2, respectively, are critical to conferring the unique collagenase-like activity to these enzymes by accommodating either Gly or Pro residues at P2 in the substrate. The data also suggests that FhCL3 accommodates hydroxyproline (Hyp)-Gly at P3-P2 better than FhCL2 explaining the observed greater ability of FhCL3 to digest type I and II collagens compared to FhCL2 and why these enzymes cleave at different positions within the Col domains. Conclusions/Significance: These studies further our understanding of how this helminth parasite regulates peptidase expression to ensure infection, migration and establishment in host tissues.
Jones, P.M. & George, A.M. 2011, 'Molecular-dynamics simulations of the ATP/apo state of a multidrug ATP-binding cassette transporter provide a structural and mechanistic basis for the asymmetric occluded state', Biophysical Journal, vol. 100, no. 12, pp. 3025-3034.
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ATP-binding cassette transporters use the energy of ATP hydrolysis to transport substrates across cellular membranes. They have two transmembrane domains and two cytosolic nucleotide-binding domains.Biochemical studies have characterized an occluded state of the transporter in which nucleotide is tenaciously bound in one active site, whereas the opposite active site is empty or binds nucleotide loosely. Here, we report molecular-dynamics simulations of the bacterial multidrug ATP-binding cassette transporter Sav1866. In two simulations of the ATP/apo state, the empty site opened substantially by way of rotation of the nucleotide-binding domain (NBD) core subdomain, whereas the ATP-bound site remained occluded and intact. We correlate our findings with elastic network and molecular-dynamics simulation analyses of the 5av1866 NBD monomer, and with existing experimental data, to argue that the observed transition is physiological, and that the final structure observed in the ATP/apo simulations corresponds to the tight/loose state of the NBD dimer characterized experimentally..
Jones, P.M., Robinson, M.W., Dalton, J.P. & George, A.M. 2011, 'The Plasmodium falciparum malaria M1 alanyl aminopeptidase (PfA-M1): Insights of catalytic mechanism and function from MD simulations', PLoS ONE, vol. 6, no. 12, p. e28589.
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Malaria caused by several species of Plasmodium is major parasitic disease of humans, causing 1-3 million deaths worldwide annually. The widespread resistance of the human parasite to current drug therapies is of major concern making the identification of new drug targets urgent. While the parasite grows and multiplies inside the host erythrocyte it degrades the host cell hemoglobin and utilizes the released amino acids to synthesize its own proteins. The P. falciparum malarial M1 alanyl-aminopeptidase (PfA-M1) is an enzyme involved in the terminal stages of hemoglobin digestion and the generation of an amino acid pool within the parasite. The enzyme has been validated as a potential drug target since inhibitors of the enzyme block parasite growth in vitro and in vivo. In order to gain further understanding of this enzyme, molecular dynamics simulations using data from a recent crystal structure of PfA-M1 were performed. The results elucidate the pentahedral coordination of the catalytic Zn in these metallo-proteases and provide new insights into the roles of this cation and important active site residues in ligand binding and in the hydrolysis of the peptide bond. Based on the data, we propose a two-step catalytic mechanism, in which the conformation of the active site is altered between the Michaelis complex and the transition state. In addition, the simulations identify global changes in the protein in which conformational transitions in the catalytic domain are transmitted at the opening of the N-terminal 8 angstrom-long channel and at the opening of the 30 angstrom-long C-terminal internal chamber that facilitates entry of peptides to the active site and exit of released amino acids. The possible implications of these global changes with regard to enzyme function are discussed.
George, A.M. & Jones, P.M. 2011, 'Type II ABC permeases: Are they really so different?', Structure, vol. 19, pp. 1540-1542.
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ATP binding-cassette (ABC) transporters couple ATP hydrolysis to vectorial movement of substrates across cell membranes. Of ancient origin and ubiquitous occurrence, they have been adapted to traffic a vast array of compounds, performing roles such as nutrient import and removal of toxins, antigen presentation, hormone release, signal reception, channel gating, and many others (Higgins, 1992; Jones and George. 2004). Their central roles in many physiological process has brought ABC transporters to the forefront in biomedical research in diverse areas including multidrug resistance in cancers and human genetic disorders, such as cystic fibrosis.
Kerr, I., Jones, P.M. & George, A.M. 2010, 'Multidrug efflux pumps: The structures of prokaryotic ATP-binding cassette transporter efflux pumps and implications for our understanding of eukaryotic P-glycoproteins and homologues.', FEBS Journal, vol. 277, no. 3, pp. 550-563.
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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
George, A.M., Jones, P.M. & Middleton, P. 2009, 'Cystic fibrosis infections: treatment strategies and prospects', FEMS Microbiology Letters, vol. 300, no. 2, pp. 153-164.
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Pseudomonas aeruginosa and Burkholderia cepacia are the two major Gram-negative rods that colonize/infect the lungs of patients with cystic fibrosis (CF). These organisms may cause progressive respiratory failure, although occasionally more rapid infections result in the 'Cepacia' syndrome. Many antibiotics have been used against Pseudomonas and Burkholderia, but once chronic colonization has been established, eradication of these organisms is rare. Drug therapy for CF patients is compromised by a number of bacterial factors that render the infectious agents resistant to antibiotics, including efflux pumps that remove antibiotics, lack of penetration of antibiotics into bacterial biofilms, and changes in the cell envelope that reduce the permeability of antibiotics. Any combination of these mechanisms increases the likelihood of bacterial survival. Therefore, combinations of antibiotics or of antibiotic and nonantibiotic compounds are currently being tested against Pseudomonas and Burkholderia. However, progress has been slow, with only occasional combinations showing promise for the eradication of persistent Gram-negative rods in the airways of CF patients. This review will summarize the current knowledge of CF infections and speculate on potential future pathways to treat these chronic infections.
Jones, P.M. & George, A.M. 2009, 'Opening of the ADP-bound active site in the ABC transporter ATPase dimer: Evidence for a constant contact, alternating sites model for the catalytic cycle', Proteins-Structure Function And Bioinformatics, vol. 75, no. 2, pp. 387-396.
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ABC transporters are ubiquitous, ATP-dependent transmembrane pumps. The mechanism by which ATP hydrolysis in the nucleotide-binding domain (NBD) effects conformational changes in the transmembrane domain that lead to allocrite translocation remains largely unknown. A possible aspect of this mechanism was suggested by previous molecular dynamics simulations of the MJ0796 NBD dimer, which revealed a novel, nucleotide-dependent intrasubunit conformational change involving the relative rotation of the helical and catalytic subdomains. Here, we find that in four of five simulations of the ADP/ATP-bound dimer, the relative rotation of the helical and catalytic subdomains in the ADP-bound monomer results in opening of the ADP-bound active site, probably sufficient or close to sufficient to allow nucleotide exchange. We also observe that in all five simulations of the ADP/ATP-bound dimer, the intimate contact of the LSGGQ signature sequence with the ATP gamma-phosphate is weakened by the intrasubunit conformational change within the ADP-bound monomer. We discuss how these results support a constant contact model for the function of the NBD dimer in contrast to switch models, in which the NBDs are proposed to fully disassociate during the catalytic cycle.
Jones, P.M., O'Mara, M. & George, A.M. 2009, 'ABC transporters: A riddle wrapped in a mystery inside an enigma', Trends in Biochemical Sciences, vol. 34, no. 10, pp. 520-531.
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ATP-binding cassette (ABC) transporters form one of the largest and most ancient of protein families. ABC transporters couple hydrolysis of ATP to vectorial translocation of diverse substrates across cellular membranes. Many human ABC transporters are medically important in causing, for example, multidrug resistance to cytotoxic drugs. Seven complete prokaryotic structures and one eukaryotic structure have been solved for transporters from 2002 to date, and a wealth of research is being conducted on and around these structures to resolve the mechanistic conundrum of how these transporters couple ATP hydrolysis in cytosolic domains to substrate translocation through the transmembrane pore. Many questions remained unanswered about this mechanism, despite a plethora of data and a number of interesting and controversial models.
Kerr, I., Jones, P.M. & George, A.M. 2009, 'Multidrug efflux pumps: The structures of prokaryotic ATP-binding cassette transporter efflux pumps and implications for our understanding of eukaryotic P-glycoproteins and homologues', FEBS Journal, vol. 277, pp. 550-563.
View/Download from: OPUS | Publisher's site
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.
Treerat, P., Widmer, F., Middleton, P., Iredell, J. & George, A.M. 2008, 'In vitro interactions of tobramycin with various nonantibiotics against Pseudomonas aeruginosa and Burkholderia cenocepacia', FEMS Microbiology Letters, vol. 285, no. 1, pp. 40-50.
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Pseudomonas aeruginosa and Burkholderia cepacia are the major pathogens that colonize the airway surface and cause progressive respiratory failure and high mortality, especially in cystic fibrosis (CF) patients. Tobramycin is the treatment of choice, but persistent usage enables the infectious organisms to activate defence mechanisms, making eradication rarely successful. Combinations of antibiotic and nonantibiotic compounds have been tested in vitro against P. aeruginosa and B. cepacia, but with mixed results. Sodium ions interfere with the bacterial tobramycin uptake system, but amiloride partially reverses this antagonism. In this pilot study, we extend previous findings of the effectiveness of tobramycin in combination with amiloride and other nonantibiotics against a P. aeruginosa type strain, and against four P. aeruginosa strains and one Burkholderia cenocepacia strain isolated from CF patients. Significantly, the four clinical P. aeruginosa strains were tobramycin resistant. We also find that Na+ and K+, but not Cl-, are the chief antagonists of tobramycin efficacy. These results suggest that chemotherapy for CF patients might not only be compromised by antibiotic-resistant pathogens alone, but by a lack of penetration of antibiotics caused either by bacterial biofilms or the high sodium flux in the CF lung, or by antagonistic effects of some drug combinations, any of which could allow the persistence of drug-susceptible bacteria.
Jones, P.M. & George, A.M. 2007, 'Nucleotide-dependent allostery within the ABC transporter ATP-binding cassette-A computational study of the MJ0796 dimer', Journal of Biological Chemistry, vol. 282, no. 31, pp. 22793-22803.
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ATP-binding cassette transporters perform energy-dependent transmembrane solute trafficking in all organisms. These proteins often mediate cellular resistance to therapeutic drugs and are involved in a range of human genetic diseases. Enzymological studies have implicated a helical subdomain within the ATP-binding cassette nucleotide-binding domain in coupling ATP hydrolysis to solute transport in the transmembrane domains. Consistent with this, structural and computational analyses have indicated that the helical subdomain undergoes nucleotide-dependent movement relative to the core of the nucleotide-binding domain fold. Here we use theoretical methods to examine the allosteric nucleotide dependence of helical subdomain transitions to further elucidate its role in interactions between the transmembrane and nucleotide-binding domains. Unrestrained 30-ns molecular dynamics simulations of the ATP-bound, ADP-bound, and apo states of the MJ0796 monomer support the idea that interaction of a conserved glutamine residue with the catalytic metal mediates the rotation of the helical subdomain in response to nucleotide binding and hydrolysis. Simulations of the nucleotide-binding domain dimer revealed that ATP hydrolysis induces a large transition of one helical subdomain, resulting in an asymmetric conformation of the dimer not observed previously. A coarse-grained elastic network analysis supports this finding, revealing the existence of corresponding dynamic modes intrinsic to the contact topology of the protein. The implications of these findings for the coupling of ATP hydrolysis to conformational changes in the transmembrane domains required for solute transport are discussed in light of recent whole transporter structures.
Jones, P.M., Turner, K.M., Djordjevic, J.T., Sorrell, T.C., Wright, L.C. & George, A.M. 2007, 'Role of conserved active site residues in catalysis by phospholiphase B1 from Cryptococcus neoformans', Biochemistry, vol. 46, no. 35, pp. 10024-10032.
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Phospholiphase B1 (PLB1), secreted by the pathogenic yeast Crysptococcus neoformans, has an established role in virulence. Although the mechanism of it phospholiphase B, lysophospholipase, and lysophospholipase transacylase activities is unknown, it possesses lipase, subtilisin protease aspartate, and phospholipase motifs containing putative catalytic residues S146, D392 and R108 respectively, conserved in fungal PLBs and essential for human cytosolic phopholiphase A2 (cPLA2) catalysis. To determine the role of these residues in PLB1 catalysis, each was substituted with alanine, and the mutant cDNAs were expressed in Sacchromyces cerevisiae. The mutant PLB1s were deficient in all threee enzymatic activities. As the active site structure of PLB1 is unknown, a homilogy model was developed, based o the X-ray structure of the cPLA2 catalytic domain. This shows that the two proteins share a closely related fold, with the three catalytic residues located in identical positions as part of a single active site with S146 and D392 forming a catalytic dyad. The model suggests that PLB1 lacks the "lid" region which ocucludes the cPLA2 active site and provides a mechanism of interfacial activation. In silico substrate docking studies with cPLA2 reveal the binding mode of the lipid headgroup, confirming the catalytic dyad mechanism ofor the cleavage of the sn-2 ester bond within one of two separate binding tracts for the lipid acyl chains. Residues specific for binding arachidonic and palmitic acids, preferred substrated for cPLA2 and PLB1, respectively. are identified. These results provide an explanation for difference in substrate specificity between lipases sharing the cPLA2 catalytic domain and for the differential effect of inhibitors on PLB1 enzymatic activities.
George, A.M. & Jones, P.M. 2006, 'Molecular dynamics simulations and analysis of ABC transporters', Current Computer-Aided Drug Design, vol. 2, no. 3, pp. 203-214.
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The increasingavailability of atomic-level protein derived from X-ray crystallography and NMR spectroscopy, together with advances in computational power, have ushered in a new era of powerful theoretical apporaches to study protein mechanism,s and, by extension, use a computer-aided structural approach to drug design. Calssical molecular dynamics calculations, in which Newton's equations of motion are solved for all atoms in the system, has emerged as an important tool for analysing protein dynamics at physiologically relevant timescales, in ways that are either very difficult or impossible to do experimentally. Indeed, the computer is becoming a kind of virtual microscope that can observe things not observable by any other means. The availability of more sophisticated parallel computer clusters and program suites will lead to simulations thatw ill be capable of examining entire processes such as polypeptide folding pathways and reaction mechanisms. In this review, the incipient applicationof molecular dynamics analysis of ABC (ATP-Binding Cassette) trasporters is surveyed and discussed, with particular relevance to unresolved and controversial issues.
Jones, P.M. & George, A.M. 2005, 'Multidrug resistance in parasites: ABC transporters, P-glycoproteins and molecular modelling', International Journal For Parasitology, vol. 35, no. 5, pp. 555-566.
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Parasitic diseases, caused by protozoa, helminths and arthropods, rank among the most important problems in human and veterinary medicine, and in agriculture, leading to debilitating sicknesses and loss of life. In the absence of vaccines and with the ge
Jones, P.M. & George, A.M. 2004, 'The ABC transporter structure and mechanism: perspectives on recent research', Cellular and Molecular Life Sciences, vol. 61, pp. 682-699.
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ATP-binding cassetee (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 mocribes to drugs. Elucidation of the sturcture and mechanism of ABC transporters is essential to the rational design of agents to control their fuctiom. While a wealth of high-resolution structures of ABC proteins have been produced in recent years, many fundamental questions regarding the protein's mechanism remain unasnwered. I this review, we examine the recent structural data concerning ABC transporters and related proteins in te ligh of other experimental and theoretical data, and discuss these data in relation to current ideas concerning the transporters' molecular mechanism.
George, A.M. & Hall, R.M. 2002, 'Efflux of chloramphenicol by the Cm1A1 protein', FEMS Microbiology Letter, vol. 209, no. N/A, pp. 209-213.
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Jones, P.M. & George, A.M. 2002, 'Mechanism of ABC transporters: a molecular dynamics simulation of a well characterised nucleotide-binding subunit', Proceedings of the National Academy of Sciences, vol. 99, no. 20, pp. 12639-12644.
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ATP-binding cassette (ABC) transporters are membrane-bound molecular pumps that form one of the largest of all protein families. Several of them are central to phenomena of biomedical interest, including cystic fibrosis and resistance to chemotherapeutic drugs. ABC transporters share a common architecture comprising two hydrophilic nucleotide-binding domains (NBDs) and two hydrophobic transmembrane domains (TMDs) that form the substrate pathway across the membrane. The conformational changes in the NBDs induced by ATP hydrolysis and the means by which they are transmitted to the TMDs to effect substrate translocation remain largely unknown. We have performed a molecular dynamics simulation of HisP, the well studied NBD of the bacterial histidine permease, to identify hinges and switches of the NBD conformational transitions and subunit+subunit interfaces.
Jones, P.M. & George, A.M. 2000, 'Symmetry and Sturcture in P-Glycoprotein and ABC Transporters. What goes Around Comes Around', European Journal fo Biochemistry, vol. 267, no. 0, pp. 5298-5305.
Jones, P.M. & George, A.M. 1998, 'A new structural model for P-glycoprotein', Journal Of Membrane Biology, vol. 166, no. 2, pp. 133-147.
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Multidrug resistance to anti-cancer drugs is a major medical problem. Resistance is manifested largely by the product of the human MDR1 gene, P-glycoprotein, an ABC transporter that is an integral membrane protein of 1280 amino acids arranged into two homologous halves, each comprising 6 putative transmembrane ?-helices and an ATP binding domain. Despite the plethora of data from site-directed, scanning and domain replacement mutagenesis, epitope mapping and photoaffinity labeling, a clear structural model for P-glycoprotein remains largely elusive. In this report, we propose a new model for P-glycoprotein that is supported by the vast body of previous data. The model comprises 2 membrane-embedded 16-strand ?-barrels, attached by short loops to two 6-helix bundles beneath each barrel. Each ATP binding domain contributes 2 ?-strands and 1 ?-helix to the structure. This model, together with an analysis of the amino acid sequence alignment of P-glycoprotein isoforms, is used to delineate drug binding and translocation sites. We show that the locations of these sites are consistent with mutational, kinetic and labeling data.
George, A.M., Hall, R.M. & Stokes, H. 1995, 'Multidrug resistance in Klebsiella pneumoniae: a novel gene, ramA, confers a multidrug resistance phenotype in Escherichia coli', Microbiology-uk, vol. 141, pp. 1909-1920.
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Spontaneous multidrug-resistant (Mdr) mutants of Klebsiella pneumoniae strain ECL8 arose at a frequency of 2-2 x 10-8 and showed increased resistance to a range of unrelated antibiotics, including chloramphenicol, tetracycline, nalidixic acid, ampicillin, norfloxacin, trimethoprim and puromycin. A chromosomal fragment from one such mutant was cloned, and found to confer an Mdr phenotype on Escherichia coli K12 cells that was essentially identical to that of the K. pneumoniae mutant. Almost complete loss of the OmpF porin in the E. coli transformant, and of the corresponding porin in the K. pneumoniae mutant, was observed. The presence of the Mdr mutation in K. pneumoniae or the cloned K. pneumoniae ramA (resistance antibiotic multiple) locus in E. coli also resulted in active efflux of tetracycline, and increased active efflux of chloramphenicol. After transformation of a ramA plasmid into E. coli, expression of chloramphenicol resistance occurred later than expression of resistance to tetracycline, puromycin, trimethoprim and nalidixic acid. The ramA gene was localized and sequenced. It encodes a putative positive transcriptional activator that is weakly related to the E. coli MarA and SoxS proteins. A ramA gene was also found to be present in an Enterobacter cloacae fragment that has previously been shown to confer an Mdr phenotype, and it appears that ramA, rather than the romA gene identified in that study, is responsible for multidrug resistance. The ramA gene from the wild-type K. pneumoniae was identical to that of the mutant strain and also conferred an Mdr phenotype on E. coli, indicating that the mutation responsible for Mdr in K. pneumoniae had not been cloned.