Publications by authors named "Alexander D Cameron"

33 Publications

Transfer of stabilising mutations between different secondary active transporter families.

FEBS Open Bio 2021 May 1. Epub 2021 May 1.

Department of Life Sciences, Imperial College London, UK.

Integral membrane transporters play essential roles in the movement of substrates across biological membranes. One approach to produce transporters suitable for structural studies is to introduce mutations that reduce conformational flexibility and increase stability. However, it can be difficult to predict which mutations will result in a more stable protein. Previously, we stabilised the uric acid-xanthine transporter, UapA, a member of the SLC23 family, through introduction of a single-point mutation, G411V, trapping the protein in the inward-facing conformation. Here, we attempted to stabilise the structurally related BOR1 transporter from Arabidopsis thaliana, a member of the SLC4 family, by introducing the equivalent substitution. We identified possible residues, P362 and M363, in AtBOR1, likely to be equivalent to the G411 of UapA, and generated four mutants, P362V or L and M363F or Y. Stability analysis using heated Fluorescent Size Exclusion Chromatography indicated that the M363F/Y mutants were more stable than the WT AtBOR1 and P362V/L mutants. Furthermore, functional complementation analysis revealed that the M363F/Y mutants exhibited reduced transport activity compared to the P362V/L and WT proteins. Purification and crystallisation of the M363F/Y proteins yielded crystals that diffracted better than WT (5.5 vs 7 Å). We hypothesise that the increased bulk of the F and Y substitutions limits the ability of the protein to undergo the conformational rearrangements associated with transport. These proteins represent a basis for future studies on AtBOR1.
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http://dx.doi.org/10.1002/2211-5463.13168DOI Listing
May 2021

Structural basis of trehalose recognition by the mycobacterial LpqY-SugABC transporter.

J Biol Chem 2021 Jan 18:100307. Epub 2021 Jan 18.

School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK. Electronic address:

The Mycobacterium tuberculosis (Mtb) LpqY-SugABC ATP-binding cassette transporter is a recycling system that imports trehalose released during remodelling of the Mtb cell-envelope. As this process is essential for the virulence of the Mtb pathogen it may represent an important target for tuberculosis drug and diagnostic development, but the transporter specificity and molecular determinants of substrate recognition are unknown. To address this, we have determined the structural and biochemical basis of how mycobacteria transport trehalose using a combination of crystallography, STD NMR, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays and the synthesis of trehalose analogues. This analysis pinpoints key residues of the LpqY substrate binding lipoprotein that dictate substrate-specific recognition and has revealed which disaccharide modifications are tolerated. These findings provide critical insights into how the essential Mtb LpqY-SugABC transporter reuses trehalose and modified analogues, and specifies a framework that can be exploited for the design of new anti-tubercular agents and/or diagnostic tools.
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http://dx.doi.org/10.1016/j.jbc.2021.100307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949145PMC
January 2021

Light-Activated Electron Transfer and Catalytic Mechanism of Carnitine Oxidation by Rieske-Type Oxygenase from Human Microbiota.

Angew Chem Int Ed Engl 2021 02 28;60(9):4529-4534. Epub 2020 Dec 28.

School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK.

Oxidation of quaternary ammonium substrate, carnitine by non-heme iron containing Acinetobacter baumannii (Ab) oxygenase CntA/reductase CntB is implicated in the onset of human cardiovascular disease. Herein, we develop a blue-light (365 nm) activation of NADH coupled to electron paramagnetic resonance (EPR) measurements to study electron transfer from the excited state of NADH to the oxidized, Rieske-type, [2Fe-2S] cluster in the AbCntA oxygenase domain with and without the substrate, carnitine. Further electron transfer from one-electron reduced, Rieske-type [2Fe-2S] center in AbCntA-WT to the mono-nuclear, non-heme iron center through the bridging glutamate E205 and subsequent catalysis occurs only in the presence of carnitine. The electron transfer process in the AbCntA-E205A mutant is severely affected, which likely accounts for the significant loss of catalytic activity in the AbCntA-E205A mutant. The NADH photo-activation coupled with EPR is broadly applicable to trap reactive intermediates at low temperature and creates a new method to characterize elusive intermediates in multiple redox-centre containing proteins.
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http://dx.doi.org/10.1002/anie.202012381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986066PMC
February 2021

Structural basis of carnitine monooxygenase CntA substrate specificity, inhibition and inter-subunit electron transfer.

J Biol Chem 2020 Nov 6. Epub 2020 Nov 6.

University of Warwick, United Kingdom.

Microbial metabolism of carnitine to trimethylamine (TMA) in the gut can accelerate atherosclerosis and heart disease and these TMA-producing enzymes are therefore important drug targets. Here, we report the first structures of the carnitine oxygenase CntA, an enzyme of the Rieske oxygenase family. CntA exists in a head-to-tail a3 trimeric structure. The two functional domains (the Rieske and the catalytic mononuclear iron domains) are located > 40 Å apart in the same monomer but adjacent in two neighbouring monomers. Structural determination of CntA and subsequent electron paramagnetic resonance measurements uncover the molecular basis of the so-called bridging glutamate (E205) residue in inter-subunit electron transfer. The structures of the substrate-bound CntA help to define the substrate pocket. Importantly, a tyrosine residue (Y203) is essential for ligand recognition through a π-cation interaction with the quaternary ammonium group. This interaction between an aromatic residue and quaternary amine substrates allows us to delineate a subgroup of Rieske oxygenases (group V) from the prototype ring-hydroxylating Rieske oxygenases involved in bioremediation of aromatic pollutants in the environment. Furthermore, we report the discovery of the first known CntA inhibitors and solve the structure of CntA in complex with the inhibitor, demonstrating the pivotal role of Y203 through a π-π stacking interaction with the inhibitor. Our study provides the structural and molecular basis for future discovery of drugs targeting this TMA-producing enzyme in human gut.
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http://dx.doi.org/10.1074/jbc.RA120.016019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948474PMC
November 2020

Strategies for successful isolation of a eukaryotic transporter.

Protein Expr Purif 2020 02 23;166:105522. Epub 2019 Oct 23.

Department of Life Sciences, Imperial College London, Exhibition Road, London, SW7 2AZ, UK. Electronic address:

The isolation of integral membrane proteins for structural analysis remains challenging and this is particularly the case for eukaryotic membrane proteins. Here we describe our efforts to isolate OsBOR3, a boron transporter from Oryza sativa. OsBOR3 was expressed as both full length and a C-terminally truncated form lacking residues 643-672 (OsBOR3). While both express well as C-terminal GFP fusion proteins in Saccharomyces cerevisiae, the full length protein isolates poorly in the detergent dodecyl-β-d-maltoside (DDM). The OsBOR3 isolated in DDM in large quantities but was contaminated with GFP tagged protein, indicated incomplete protease removal of the tag. Addition of the reducing agent dithiothreitol (DTT) had no effect on isolation. Detergent screening indicated that the neopentyl glycol detergents, LMNG, UDMNG and DMNG conferred greater stability on the OsBOR3 than DDM. Isolation of OsBOR3 in LMNG both in the presence and absence of DTT produced large quantities of protein but contaminated with GFP tagged protein. Isolation of OsBOR3 in DMNG + DTT resulted in protein sample that does not contain any detectable GFP but elutes at a higher retention volume than that seen for protein isolated in either DDM or LMNG. Mass spectrometry confirmed that the LMNG and DMNG purified protein is OsBOR3 indicating that the DMNG isolated protein is monomer compared to the dimer isolated using LMNG. This was further supported by single particle electron microscopic analysis revealing that the DMNG protein particles are roughly half the size of the LMNG protein particles.
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http://dx.doi.org/10.1016/j.pep.2019.105522DOI Listing
February 2020

Cryo-EM of multiple cage architectures reveals a universal mode of clathrin self-assembly.

Nat Struct Mol Biol 2019 10 3;26(10):890-898. Epub 2019 Oct 3.

School of Life Sciences, University of Warwick, Coventry, UK.

Clathrin forms diverse lattice and cage structures that change size and shape rapidly in response to the needs of eukaryotic cells during clathrin-mediated endocytosis and intracellular trafficking. We present the cryo-EM structure and molecular model of assembled porcine clathrin, providing insights into interactions that stabilize key elements of the clathrin lattice, namely, between adjacent heavy chains, at the light chain-heavy chain interface and within the trimerization domain. Furthermore, we report cryo-EM maps for five different clathrin cage architectures. Fitting structural models to three of these maps shows that their assembly requires only a limited range of triskelion leg conformations, yet inherent flexibility is required to maintain contacts. Analysis of the protein-protein interfaces shows remarkable conservation of contact sites despite architectural variation. These data reveal a universal mode of clathrin assembly that allows variable cage architecture and adaptation of coated vesicle size and shape during clathrin-mediated vesicular trafficking or endocytosis.
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http://dx.doi.org/10.1038/s41594-019-0292-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7100586PMC
October 2019

Structural Basis of Glycerophosphodiester Recognition by the Substrate-Binding Protein UgpB.

ACS Chem Biol 2019 09 21;14(9):1879-1887. Epub 2019 Aug 21.

School of Life Sciences , University of Warwick , Coventry , West Midlands CV4 7AL , United Kingdom.

() is the causative agent of tuberculosis (TB) and has evolved an incredible ability to survive latently within the human host for decades. The pathogen encodes for a low number of ATP-binding cassette (ABC) importers for the acquisition of carbohydrates that may reflect the nutrient poor environment within the host macrophages. UgpB (Rv2833c) is the substrate binding domain of the UgpABCE transporter that recognizes glycerophosphocholine (GPC), indicating that this transporter has a role in recycling glycerophospholipid metabolites. By using a combination of saturation transfer difference (STD) NMR and X-ray crystallography, we report the structural analysis of UgpB complexed with GPC and have identified that UgpB not only recognizes GPC but is also promiscuous for a broad range of glycerophosphodiesters. Complementary biochemical analyses and site-directed mutagenesis precisely define the molecular basis and specificity of glycerophosphodiester recognition. Our results provide critical insights into the structural and functional role of the UgpB transporter and reveal that the specificity of this ABC-transporter is not limited to GPC, therefore optimizing the ability of to scavenge scarce nutrients and essential glycerophospholipid metabolites via a single transporter during intracellular infection.
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http://dx.doi.org/10.1021/acschembio.9b00204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6757277PMC
September 2019

The Leishmania PABP1-eIF4E4 interface: a novel 5'-3' interaction architecture for trans-spliced mRNAs.

Nucleic Acids Res 2019 02;47(3):1493-1504

Warwick Integrative Synthetic Biology Centre (WISB) and School of Life Sciences, University of Warwick, Gibbet Hill, Coventry CV4 7AL, UK.

Trans-splicing of trypanosomatid polycistronic transcripts produces polyadenylated monocistronic mRNAs modified to form the 5' cap4 structure (m7Gpppm36,6,2'Apm2'Apm2'Cpm23,2'U). NMR and X-ray crystallography reveal that Leishmania has a unique type of N-terminally-extended cap-binding protein (eIF4E4) that binds via a PAM2 motif to PABP1. This relies on the interactions of a combination of polar and charged amino acid side-chains together with multiple hydrophobic interactions, and underpins a novel architecture in the Leishmania cap4-binding translation factor complex. Measurements using microscale thermophoresis, fluorescence anisotropy and surface plasmon resonance characterize the key interactions driving assembly of the Leishmania translation initiation complex. We demonstrate that this complex can accommodate Leishmania eIF4G3 which, unlike the standard eukaryotic initiation complex paradigm, binds tightly to eIF4E4, but not to PABP1. Thus, in Leishmania, the chain of interactions 5'cap4-eIF4E4-PABP1-poly(A) bridges the mRNA 5' and 3' ends. Exceptionally, therefore, by binding tightly to two protein ligands and to the mRNA 5' cap4 structure, the trypanosomatid N-terminally extended form of eIF4E acts as the core molecular scaffold for the mRNA-cap-binding complex. Finally, the eIF4E4 N-terminal extension is an intrinsically disordered region that transitions to a partly folded form upon binding to PABP1, whereby this interaction is not modulated by poly(A) binding to PABP1.
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http://dx.doi.org/10.1093/nar/gky1187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379680PMC
February 2019

Structural and functional determination of homologs of the -acetylglucosamine-6-phosphate deacetylase (NagA).

J Biol Chem 2018 06 4;293(25):9770-9783. Epub 2018 May 4.

From the School of Life Sciences and

The () pathogen encodes a GlcNAc-6-phosphate deacetylase enzyme, NagA (Rv3332), that belongs to the amidohydrolase superfamily. NagA enzymes catalyze the deacetylation of GlcNAc-6-phosphate (GlcNAc6P) to glucosamine-6-phosphate (GlcN6P). NagA is a potential antitubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments. Here, we report the structural and functional characterization of NagA from (MSNagA) and (MMNagA), close relatives of Using a combination of X-ray crystallography, site-directed mutagenesis, and biochemical and biophysical assays, we show that these mycobacterial NagA enzymes are selective for GlcNAc6P. Site-directed mutagenesis studies revealed crucial roles of conserved residues in the active site that underpin stereoselective recognition, binding, and catalysis of substrates. Moreover, we report the crystal structure of MSNagA in both ligand-free form and in complex with the GlcNAc6P substrate at 2.6 and 2.0 Å resolutions, respectively. The GlcNAc6P complex structure disclosed the precise mode of GlcNAc6P binding and the structural framework of the active site, including two divalent metals located in the α/β binuclear site. Furthermore, we observed a cysteine residue located on a flexible loop region that occludes the active site. This cysteine is unique to mycobacteria and may represent a unique subsite for targeting mycobacterial NagA enzymes. Our results provide critical insights into the structural and mechanistic properties of mycobacterial NagA enzymes having an essential role in amino-sugar and nucleotide metabolism in mycobacteria.
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http://dx.doi.org/10.1074/jbc.RA118.002597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6016474PMC
June 2018

Transporter oligomerization: form and function.

Biochem Soc Trans 2016 12;44(6):1737-1744

Department of Life Sciences, Imperial College London, London SW7 2AZ, U.K.

Transporters are integral membrane proteins with central roles in the efficient movement of molecules across biological membranes. Many transporters exist as oligomers in the membrane. Depending on the individual transport protein, oligomerization can have roles in membrane trafficking, function, regulation and turnover. For example, our recent studies on UapA, a nucleobase ascorbate transporter, from Aspergillus nidulans, have revealed both that dimerization of this protein is essential for correct trafficking to the membrane and the structural basis of how one UapA protomer can affect the function of the closely associated adjacent protomer. Here, we review the roles of oligomerization in many particularly well-studied transporters and transporter families.
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http://dx.doi.org/10.1042/BST20160217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5134999PMC
December 2016

Structure of eukaryotic purine/H(+) symporter UapA suggests a role for homodimerization in transport activity.

Nat Commun 2016 Apr 18;7:11336. Epub 2016 Apr 18.

Department of Life Sciences, Imperial College London, London SW7 2AZ, UK.

The uric acid/xanthine H(+) symporter, UapA, is a high-affinity purine transporter from the filamentous fungus Aspergillus nidulans. Here we present the crystal structure of a genetically stabilized version of UapA (UapA-G411VΔ1-11) in complex with xanthine. UapA is formed from two domains, a core domain and a gate domain, similar to the previously solved uracil transporter UraA, which belongs to the same family. The structure shows UapA in an inward-facing conformation with xanthine bound to residues in the core domain. Unlike UraA, which was observed to be a monomer, UapA forms a dimer in the crystals with dimer interactions formed exclusively through the gate domain. Analysis of dominant negative mutants is consistent with dimerization playing a key role in transport. We postulate that UapA uses an elevator transport mechanism likely to be shared with other structurally homologous transporters including anion exchangers and prestin.
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http://dx.doi.org/10.1038/ncomms11336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4837479PMC
April 2016

Crystal structures reveal the molecular basis of ion translocation in sodium/proton antiporters.

Nat Struct Mol Biol 2016 Mar 1;23(3):248-55. Epub 2016 Feb 1.

Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.

To fully understand the transport mechanism of Na(+)/H(+) exchangers, it is necessary to clearly establish the global rearrangements required to facilitate ion translocation. Currently, two different transport models have been proposed. Some reports have suggested that structural isomerization is achieved through large elevator-like rearrangements similar to those seen in the structurally unrelated sodium-coupled glutamate-transporter homolog GltPh. Others have proposed that only small domain movements are required for ion exchange, and a conventional rocking-bundle model has been proposed instead. Here, to resolve these differences, we report atomic-resolution structures of the same Na(+)/H(+) antiporter (NapA from Thermus thermophilus) in both outward- and inward-facing conformations. These data combined with cross-linking, molecular dynamics simulations and isothermal calorimetry suggest that Na(+)/H(+) antiporters provide alternating access to the ion-binding site by using elevator-like structural transitions.
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http://dx.doi.org/10.1038/nsmb.3164DOI Listing
March 2016

Crystal structure of the anion exchanger domain of human erythrocyte band 3.

Science 2015 Nov;350(6261):680-4

Japan Science and Technology Agency (JST), Exploratory Research for Advanced Technology (ERATO) Human Receptor Crystallography Project, Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan. JST, Research Acceleration Program, Membrane Protein Crystallography Project, Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan. Department of Cell Biology, Kyoto University Faculty of Medicine, Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan. Division of Molecular Biosciences, Membrane Protein Crystallography group, Imperial College London, London SW7 2AZ, UK. Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Chilton, Oxfordshire OX11 0DE, UK. Research Complex at Harwell Rutherford, Appleton Laboratory, Harwell Oxford, Didcot, Oxfordshire OX11 0FA, UK. Platform for Drug Discovery, Informatics, and Structural Life Science, Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.

Anion exchanger 1 (AE1), also known as band 3 or SLC4A1, plays a key role in the removal of carbon dioxide from tissues by facilitating the exchange of chloride and bicarbonate across the plasma membrane of erythrocytes. An isoform of AE1 is also present in the kidney. Specific mutations in human AE1 cause several types of hereditary hemolytic anemias and/or distal renal tubular acidosis. Here we report the crystal structure of the band 3 anion exchanger domain (AE1(CTD)) at 3.5 angstroms. The structure is locked in an outward-facing open conformation by an inhibitor. Comparing this structure with a substrate-bound structure of the uracil transporter UraA in an inward-facing conformation allowed us to identify the anion-binding position in the AE1(CTD), and to propose a possible transport mechanism that could explain why selected mutations lead to disease.
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http://dx.doi.org/10.1126/science.aaa4335DOI Listing
November 2015

GFP-based expression screening of membrane proteins in insect cells using the baculovirus system.

Methods Mol Biol 2015 ;1261:197-209

Institute of Biochemistry, National Chung Hsing University, Taichung, 40227, Taiwan,

A key step in the production of recombinant membrane proteins for structural studies is the optimization of protein yield and quality. One commonly used approach is to fuse the protein to green fluorescent protein (GFP), enabling expression to be tracked without the need to purify the protein. Combining fusion to green fluorescent protein with the baculovirus expression system provides a useful platform for both screening and production of eukaryotic membrane proteins. In this chapter we describe our protocol for the expression screening of membrane proteins in insect cells using fusion to GFP as a reporter. We use both SDS-PAGE with in-gel fluorescence imaging and fluorescence-detection size-exclusion chromatography (FSEC) to screen for expression.
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http://dx.doi.org/10.1007/978-1-4939-2230-7_11DOI Listing
August 2015

Crystal structure of the sodium-proton antiporter NhaA dimer and new mechanistic insights.

J Gen Physiol 2014 Dec;144(6):529-44

Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, England, UK Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE, England, UK Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell, Oxford, Didcot, Oxfordshire OX11 0FA, England, UK School of Life Sciences, University of Warwick, Coventry CV4 7AL, England, UK

Sodium-proton antiporters rapidly exchange protons and sodium ions across the membrane to regulate intracellular pH, cell volume, and sodium concentration. How ion binding and release is coupled to the conformational changes associated with transport is not clear. Here, we report a crystal form of the prototypical sodium-proton antiporter NhaA from Escherichia coli in which the protein is seen as a dimer. In this new structure, we observe a salt bridge between an essential aspartic acid (Asp163) and a conserved lysine (Lys300). An equivalent salt bridge is present in the homologous transporter NapA, but not in the only other known crystal structure of NhaA, which provides the foundation of most existing structural models of electrogenic sodium-proton antiport. Molecular dynamics simulations show that the stability of the salt bridge is weakened by sodium ions binding to Asp164 and the neighboring Asp163. This suggests that the transport mechanism involves Asp163 switching between forming a salt bridge with Lys300 and interacting with the sodium ion. pKa calculations suggest that Asp163 is highly unlikely to be protonated when involved in the salt bridge. As it has been previously suggested that Asp163 is one of the two residues through which proton transport occurs, these results have clear implications to the current mechanistic models of sodium-proton antiport in NhaA.
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http://dx.doi.org/10.1085/jgp.201411219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4242812PMC
December 2014

Molecular mechanism of ligand recognition by membrane transport protein, Mhp1.

EMBO J 2014 Aug 21;33(16):1831-44. Epub 2014 Jun 21.

School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK

The hydantoin transporter Mhp1 is a sodium-coupled secondary active transport protein of the nucleobase-cation-symport family and a member of the widespread 5-helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5-substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5-substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5-(2-naphthylmethyl)-L-hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1.
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http://dx.doi.org/10.15252/embj.201387557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4195764PMC
August 2014

A two-domain elevator mechanism for sodium/proton antiport.

Nature 2013 Sep 1;501(7468):573-7. Epub 2013 Sep 1.

Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK.

Sodium/proton (Na(+)/H(+)) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets. The best understood model system for Na(+)/H(+) antiport is NhaA from Escherichia coli, for which both electron microscopy and crystal structures are available. NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein. Like many Na(+)/H(+) antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur. The only reported NhaA crystal structure so far is of the low pH inactivated form. Here we describe the active-state structure of a Na(+)/H(+) antiporter, NapA from Thermus thermophilus, at 3 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding directly, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 20° against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second, Na(+)/H(+) antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.
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http://dx.doi.org/10.1038/nature12484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3914025PMC
September 2013

The structure of the yeast NADH dehydrogenase (Ndi1) reveals overlapping binding sites for water- and lipid-soluble substrates.

Proc Natl Acad Sci U S A 2012 Sep 4;109(38):15247-52. Epub 2012 Sep 4.

Division of Molecular Biosciences, Imperial College, London, United Kingdom.

Bioenergy is efficiently produced in the mitochondria by the respiratory system consisting of complexes I-V. In various organisms, complex I can be replaced by the alternative NADH-quinone oxidoreductase (NDH-2), which catalyzes the transfer of an electron from NADH via FAD to quinone, without proton pumping. The Ndi1 protein from Saccharomyces cerevisiae is a monotopic membrane protein, directed to the matrix. A number of studies have investigated the potential use of Ndi1 as a therapeutic agent against complex I disorders, and the NDH-2 enzymes have emerged as potential therapeutic targets for treatments against the causative agents of malaria and tuberculosis. Here we present the crystal structures of Ndi1 in its substrate-free, NAD(+)- and ubiquinone- (UQ2) complexed states. The structures reveal that Ndi1 is a peripheral membrane protein forming an intimate dimer, in which packing of the monomeric units within the dimer creates an amphiphilic membrane-anchor domain structure. Crucially, the structures of the Ndi1-NAD(+) and Ndi1-UQ2 complexes show overlapping binding sites for the NAD(+) and quinone substrates.
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http://dx.doi.org/10.1073/pnas.1210059109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458368PMC
September 2012

Stabilizing the heterologously expressed uric acid-xanthine transporter UapA from the lower eukaryote Aspergillus nidulans.

Mol Membr Biol 2013 Feb 14;30(1):32-42. Epub 2012 Jun 14.

Division of Molecular Biosciences, Department of Life Sciences, Imperial College London, UK.

Despite detailed genetic and mutagenic analysis and a recent high-resolution structure of a bacterial member of the nucleobase-ascorbate transporter (NAT) family, understanding of the mechanism of action of eukaryotic NATs is limited. Preliminary studies successfully expressed and purified wild-type UapA to high homogeneity; however, the protein was extremely unstable, degrading almost completely after 48 h at 4°C. In an attempt to increase UapA stability we generated a number of single point mutants (E356D, E356Q, N409A, N409D, Q408E and G411V) previously shown to have reduced or no transport activity, but correct targeting to the membrane. The mutant UapA constructs expressed well as GFP fusions in Saccharomyces cerevisiae and exhibited similar fluorescent size exclusion chromatography (FSEC) profiles to the wild-type protein, following solubilization in 1% DDM, LDAO or OM + 1 mM xanthine. In order to assess the relative stabilities of the mutants, solubilized fractions prepared in 1% DDM + 1 mM xanthine were heated at 45°C for 10 min prior to FSEC. The Q408E and G411V mutants gave markedly better profiles than either wild-type or the other mutants. Further FSEC analysis following solubilization of the mutants in 1% NG ± xanthine confirmed that G411V is more stable than the other mutants, but showed that Q408E is unstable under these conditions. G411V and an N-terminally truncated construct G411VΔ1-11 were submitted to large-scale expression and purification. Long-term stability analysis revealed that G411VΔ1-11 was the most stable construct and the most suited to downstream structural studies.
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http://dx.doi.org/10.3109/09687688.2012.690572DOI Listing
February 2013

Alternating access mechanism in the POT family of oligopeptide transporters.

EMBO J 2012 Aug 1;31(16):3411-21. Epub 2012 Jun 1.

Department of Biochemistry, University of Oxford, Oxford, UK.

Short chain peptides are actively transported across membranes as an efficient route for dietary protein absorption and for maintaining cellular homeostasis. In mammals, peptide transport occurs via PepT1 and PepT2, which belong to the proton-dependent oligopeptide transporter, or POT family. The recent crystal structure of a bacterial POT transporter confirmed that they belong to the major facilitator superfamily of secondary active transporters. Despite the functional characterization of POT family members in bacteria, fungi and mammals, a detailed model for peptide recognition and transport remains unavailable. In this study, we report the 3.3-Å resolution crystal structure and functional characterization of a POT family transporter from the bacterium Streptococcus thermophilus. Crystallized in an inward open conformation the structure identifies a hinge-like movement within the C-terminal half of the transporter that facilitates opening of an intracellular gate controlling access to a central peptide-binding site. Our associated functional data support a model for peptide transport that highlights the importance of salt bridge interactions in orchestrating alternating access within the POT family.
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http://dx.doi.org/10.1038/emboj.2012.157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3419923PMC
August 2012

G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody.

Nature 2012 Jan 29;482(7384):237-40. Epub 2012 Jan 29.

Iwata Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.

G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A(2A) adenosine receptor (A(2A)AR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain. Here we report the raising of a mouse monoclonal antibody against human A(2A)AR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A(2A)AR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A(2A)AR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure and to CDR-3 of the nanobody in the active β(2)-adrenergic receptor structure, but locks A(2A)AR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.
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http://dx.doi.org/10.1038/nature10750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3303121PMC
January 2012

Crystal structure of a bacterial homologue of the bile acid sodium symporter ASBT.

Nature 2011 Oct 5;478(7369):408-11. Epub 2011 Oct 5.

Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK.

High cholesterol levels greatly increase the risk of cardiovascular disease. About 50 per cent of cholesterol is eliminated from the body by its conversion into bile acids. However, bile acids released from the bile duct are constantly recycled, being reabsorbed in the intestine by the apical sodium-dependent bile acid transporter (ASBT, also known as SLC10A2). It has been shown in animal models that plasma cholesterol levels are considerably lowered by specific inhibitors of ASBT, and ASBT is thus a target for hypercholesterolaemia drugs. Here we report the crystal structure of a bacterial homologue of ASBT from Neisseria meningitidis (ASBT(NM)) at 2.2 Å. ASBT(NM) contains two inverted structural repeats of five transmembrane helices. A core domain of six helices harbours two sodium ions, and the remaining four helices pack in a row to form a flat, 'panel'-like domain. Overall, the architecture of the protein is remarkably similar to the sodium/proton antiporter NhaA, despite having no detectable sequence homology. The ASBT(NM) structure was captured with the substrate taurocholate present, bound between the core and panel domains in a large, inward-facing, hydrophobic cavity. Residues near this cavity have been shown to affect the binding of specific inhibitors of human ASBT. The position of the taurocholate molecule, together with the molecular architecture, suggests the rudiments of a possible transport mechanism.
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http://dx.doi.org/10.1038/nature10450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3198845PMC
October 2011

Crystal structure of glucansucrase from the dental caries pathogen Streptococcus mutans.

J Mol Biol 2011 Apr 25;408(2):177-86. Epub 2011 Feb 25.

Department of Food and Nutritional Sciences, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-Ku, Shizuoka 422-8526, Japan.

Glucansucrase (GSase) from Streptococcus mutans is an essential agent in dental caries pathogenesis. Here, we report the crystal structure of S. mutans glycosyltransferase (GTF-SI), which synthesizes soluble and insoluble glucans and is a glycoside hydrolase (GH) family 70 GSase in the free enzyme form and in complex with acarbose and maltose. Resolution of the GTF-SI structure confirmed that the domain order of GTF-SI is circularly permuted as compared to that of GH family 13 α-amylases. As a result, domains A, B and IV of GTF-SI are each composed of two separate polypeptide chains. Structural comparison of GTF-SI and amylosucrase, which is closely related to GH family 13 amylases, indicated that the two enzymes share a similar transglycosylation mechanism via a glycosyl-enzyme intermediate in subsite -1. On the other hand, novel structural features were revealed in subsites +1 and +2 of GTF-SI. Trp517 provided the platform for glycosyl acceptor binding, while Tyr430, Asn481 and Ser589, which are conserved in family 70 enzymes but not in family 13 enzymes, comprised subsite +1. Based on the structure of GTF-SI and amino acid comparison of GTF-SI, GTF-I and GTF-S, Asp593 in GTF-SI appeared to be the most critical point for acceptor sugar orientation, influencing the transglycosylation specificity of GSases, that is, whether they produced insoluble glucan with α(1-3) glycosidic linkages or soluble glucan with α(1-6) linkages. The structural information derived from the current study should be extremely useful in the design of novel inhibitors that prevent the biofilm formation by GTF-SI.
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http://dx.doi.org/10.1016/j.jmb.2011.02.028DOI Listing
April 2011

Structure and mechanism of the chalcogen-detoxifying protein TehB from Escherichia coli.

Biochem J 2011 Apr;435(1):85-91

Membrane Protein Laboratory, Imperial College London, Diamond Light Source, Chilton, Oxfordshire OX11 0DE, UK.

The oxyanion derivatives of the chalcogens tellurium and selenium are toxic to living organisms even at very low levels. Bacteria have developed mechanisms to overcome their toxicity by methylating them. The structure of TehB from Escherichia coli has been determined in the presence of the cofactor analogues SAH (S-adenosylhomocysteine) and sinefungin (a non-hydrolysable form of S-adenosyl-L-methionine) at 1.48 Å (1 Å=0.1 nm) and 1.9 Å respectively. Interestingly, our kinetic data show that TehB does not discriminate between selenium or tellurite oxyanions, making it a very powerful detoxifying protein. Analysis of the active site has identified three conserved residues that are capable of binding and orientating the metals for nucleophilic attack: His176, Arg177 and Arg184. Mutagenesis studies revealed that the H176A and R184A mutants retained most of their activity, whereas the R177A mutant had 65% of its activity abolished. Based on the structure and kinetic data we propose an SN2 nucleophilic attack reaction mechanism. These data provide the first molecular understanding of the detoxification of chalcogens by bacteria.
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http://dx.doi.org/10.1042/BJ20102014DOI Listing
April 2011

Benchmarking membrane protein detergent stability for improving throughput of high-resolution X-ray structures.

Structure 2011 Jan;19(1):17-25

Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London SW7 2AZ, UK.

Obtaining well-ordered crystals is a major hurdle to X-ray structure determination of membrane proteins. To facilitate crystal optimization, we investigated the detergent stability of 24 eukaryotic and prokaryotic membrane proteins, predominantly transporters, using a fluorescent-based unfolding assay. We have benchmarked the stability required for crystallization in small micelle detergents, as they are statistically more likely to lead to high-resolution structures. Using this information, we have been able to obtain well-diffracting crystals for a number of sodium and proton-dependent transporters. By including in the analysis seven membrane proteins for which structures are already known, AmtB, GlpG, Mhp1, GlpT, EmrD, NhaA, and LacY, it was further possible to demonstrate an overall trend between protein stability and structural resolution. We suggest that by monitoring membrane protein stability with reference to the benchmarks described here, greater efforts can be placed on constructs and conditions more likely to yield high-resolution structures.
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http://dx.doi.org/10.1016/j.str.2010.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3111809PMC
January 2011

The alternating access mechanism of transport as observed in the sodium-hydantoin transporter Mhp1.

J Synchrotron Radiat 2011 Jan 5;18(1):20-3. Epub 2010 Nov 5.

Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London SW7 2AZ, UK.

Secondary active transporters move molecules across cell membranes by coupling this process to the energetically favourable downhill movement of ions or protons along an electrochemical gradient. They function by the alternating access model of transport in which, through conformational changes, the substrate binding site alternately faces either side of the membrane. Owing to the difficulties in obtaining the crystal structure of a single transporter in different conformational states, relatively little structural information is known to explain how this process occurs. Here, the structure of the sodium-benzylhydantoin transporter, Mhp1, from Microbacterium liquefaciens, has been determined in three conformational states; from this a mechanism is proposed for switching from the outward-facing open conformation through an occluded structure to the inward-facing open state.
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http://dx.doi.org/10.1107/S0909049510032449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3004247PMC
January 2011

Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2.

EMBO J 2011 Jan 3;30(2):417-26. Epub 2010 Dec 3.

Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, London, UK.

PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di- and tri-peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepT(So), a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 Å resolution, reveals a ligand-bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide-binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepT(So) represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.
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http://dx.doi.org/10.1038/emboj.2010.309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3025455PMC
January 2011

Thieno[3,2-c]pyrazoles: a novel class of Aurora inhibitors with favorable antitumor activity.

Bioorg Med Chem 2010 Oct 25;18(19):7113-20. Epub 2010 Jul 25.

Nerviano Medical Sciences-Oncology, via Pasteur 10, 20014 Nerviano, Milan, Italy.

A novel series of 3-amino-1H-thieno[3,2-c]pyrazole derivatives demonstrating high potency in inhibiting Aurora kinases was developed. Here we describe the synthesis and a preliminary structure-activity relationship, which led to the discovery of a representative compound (38), which showed low nanomolar inhibitory activity in the anti-proliferation assay and was able to block the cell cycle in HCT-116 cell line. This compound demonstrated favorable pharmacokinetic properties and good efficacy in the HL-60 xenograft tumor model.
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http://dx.doi.org/10.1016/j.bmc.2010.07.048DOI Listing
October 2010

Molecular basis of alternating access membrane transport by the sodium-hydantoin transporter Mhp1.

Science 2010 Apr;328(5977):470-3

Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London SW7 2AZ, UK.

The structure of the sodium-benzylhydantoin transport protein Mhp1 from Microbacterium liquefaciens comprises a five-helix inverted repeat, which is widespread among secondary transporters. Here, we report the crystal structure of an inward-facing conformation of Mhp1 at 3.8 angstroms resolution, complementing its previously described structures in outward-facing and occluded states. From analyses of the three structures and molecular dynamics simulations, we propose a mechanism for the transport cycle in Mhp1. Switching from the outward- to the inward-facing state, to effect the inward release of sodium and benzylhydantoin, is primarily achieved by a rigid body movement of transmembrane helices 3, 4, 8, and 9 relative to the rest of the protein. This forms the basis of an alternating access mechanism applicable to many transporters of this emerging superfamily.
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http://dx.doi.org/10.1126/science.1186303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2885435PMC
April 2010

Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter.

Science 2008 Oct 16;322(5902):709-13. Epub 2008 Oct 16.

Membrane Protein Laboratory, Diamond Light Source Limited, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire OX11 0DE, UK.

The nucleobase-cation-symport-1 (NCS1) transporters are essential components of salvage pathways for nucleobases and related metabolites. Here, we report the 2.85-angstrom resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, 10 of which are arranged in two inverted repeats of five helices. The structures of the outward-facing open and substrate-bound occluded conformations were solved, showing how the outward-facing cavity closes upon binding of substrate. Comparisons with the leucine transporter LeuT(Aa) and the galactose transporter vSGLT reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite sides of the membrane. The reciprocal opening and closing of these cavities is synchronized by the inverted repeat helices 3 and 8, providing the structural basis of the alternating access model for membrane transport.
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http://dx.doi.org/10.1126/science.1164440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2885439PMC
October 2008