Publications by year
In Press
Conners R, León-Quezada RI, McLaren M, Bennett NJ, Daum B, Rakonjac J, Gold VAM (In Press). Cryo-electron microscopy of the f1 filamentous phage reveals a new paradigm in viral infection and assembly.
Abstract:
Cryo-electron microscopy of the f1 filamentous phage reveals a new paradigm in viral infection and assembly
AbstractPhages are viruses that infect bacteria and dominate every ecosystem on our planet. As well as impacting microbial ecology, physiology and evolution, phages are exploited as tools in molecular biology and biotechnology. This is particularly true for the Ff (f1, fd or M13) phages, which represent a widely distributed group of filamentous viruses. Over nearly five decades, Ff has seen an extraordinary range of applications, including in phage display and nanotechnology. However, the complete structure of the phage capsid and consequently the mechanisms of infection and assembly remain largely mysterious. Using cryo-electron microscopy and a highly efficient system for production of short Ff-derived nanorods, we have determined the first structure of a filamentous virus, including the filament tips. Structure combined with mutagenesis was employed to identify domains of the phage that are important in bacterial attack and for release of new phage progeny. These data allow new models to be proposed for the phage lifecycle and will undoubtedly enable the development of novel biotechnological applications.
Abstract.
Conners R, McLaren M, Łapińska U, Sanders K, Stone MRL, Blaskovich MAT, Pagliara S, Daum B, Rakonjac J, Gold VAM, et al (In Press). CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage.
Abstract:
CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage
AbstractThe Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.
Abstract.
Gambelli L, Isupov MN, Conners R, McLaren M, Bellack A, Gold V, Rachel R, Daum B (In Press). New insights into the architecture and dynamics of archaella.
Abstract:
New insights into the architecture and dynamics of archaella
AbstractArchaea swim by means of a unique molecular machine called the archaellum. The archaellum consists of an ATP-powered intracellular motor that drives the rotation of an extracellular filament, allowing the cell to rapidly propel itself through liquid media.The archaellum filament comprises multiple copies of helically organised subunits named archaellins. While in many species several archaellin homologs are encoded in the same operon, structural studies conducted to date have suggested that archaella consist of only one protein species. Thus, the role of the remaining archaellin genes remains elusive.Here we present the structure of the Methanocaldococcus villosus archaellum filament at 3.08 Å resolution. We find that the filament is composed of two alternating archaellins - ArlB1 and ArlB2, suggesting that the architecture and assembly of archaella is more complex than previously thought. Moreover, we identify two major structural elements that enable the archaellum filament to move.Our findings provide new insights into archaeal motility and challenge the current view on the archaellum architecture and assembly.
Abstract.
Gambelli L, McLaren M, Conners R, Sanders K, Gaines MC, Clark L, Gold V, Kattnig D, Sikora M, Hanus C, et al (In Press). Structure of the two-component S-layer of the archaeon <i>Sulfolobus acidocaldarius</i>.
Abstract:
Structure of the two-component S-layer of the archaeon Sulfolobus acidocaldarius
AbstractSurface layers (S-layers) are resilient two-dimensional protein lattices that encapsulate many bacteria and most archaea. In archaea, S-layers usually form the only structural component of the cell wall and thus act as the final frontier between the cell and its environment. Therefore, S-layers are crucial for supporting microbial life. Notwithstanding their importance, little is known about archaeal S-layers at the atomic level. Here, we combined single particle cryo electron microscopy (cryoEM), cryo electron tomography (cryoET) and Alphafold2 predictions to generate an atomic model of the two-component S-layer of Sulfolobus acidocaldarius. The outer component of this S-layer (SlaA) is a flexible, highly glycosylated, and stable protein. Together with the inner and membrane-bound component (SlaB), they assemble into a porous and interwoven lattice. We hypothesize that jackknife-like conformational changes, as well as pH-induced alterations in the surface charge of SlaA, play important roles in S-layer assembly.
Abstract.
2023
Conners R, León-Quezada RI, McLaren M, Bennett NJ, Daum B, Rakonjac J, Gold VAM (2023). Cryo-electron microscopy of the f1 filamentous phage reveals insights into viral infection and assembly.
Nature Communications,
14(1).
Abstract:
Cryo-electron microscopy of the f1 filamentous phage reveals insights into viral infection and assembly
AbstractPhages are viruses that infect bacteria and dominate every ecosystem on our planet. As well as impacting microbial ecology, physiology and evolution, phages are exploited as tools in molecular biology and biotechnology. This is particularly true for the Ff (f1, fd or M13) phages, which represent a widely distributed group of filamentous viruses. Over nearly five decades, Ffs have seen an extraordinary range of applications, yet the complete structure of the phage capsid and consequently the mechanisms of infection and assembly remain largely mysterious. In this work, we use cryo-electron microscopy and a highly efficient system for production of short Ff-derived nanorods to determine a structure of a filamentous virus including the tips. We show that structure combined with mutagenesis can identify phage domains that are important in bacterial attack and for release of new progeny, allowing new models to be proposed for the phage lifecycle.
Abstract.
2022
McLaren M, Gil-Diez P, Isupov M, Conners R, Gambelli L, Gold V, Walter A, Connell S, Williams B, Daum B, et al (2022). <i>In situ</i> structure of a dimeric hibernating ribosome from a eukaryotic intracellular pathogen.
Abstract:
In situ structure of a dimeric hibernating ribosome from a eukaryotic intracellular pathogen
Translational control is an essential process for the cell to adapt to varying physiological or environmental conditions. To survive adverse conditions such as low nutrient levels, translation can be shut down almost entirely by inhibiting ribosomal function. Here we investigated eukaryotic hibernating ribosomes from the microsporidian parasite Spraguea lophii in situ by a combination of cryo-electron tomography (cryoET) and single particle cryoEM. We show that microsporidian spores contain ribosomes primed for host cell invasion and thus shed new light on the infection mechanism of this important pathogen. Prior to host infection, virtually all ribosomes are locked in the 100 S dimeric state, which appears to be formed by a unique dimerization mechanism that is distinct from its bacterial counterparts. Within the dimer, the hibernation factor MDF1 is bound within the E site, locking the L1 stalk in a closed conformation, and thus preventing the translation of mRNAs to polypeptides.
Abstract.
Gambelli L, Isupov MN, Conners R, McLaren M, Bellack A, Gold V, Rachel R, Daum B (2022). An archaellum filament composed of two alternating subunits.
Nature Communications,
13(1).
Abstract:
An archaellum filament composed of two alternating subunits
AbstractArchaea use a molecular machine, called the archaellum, to swim. The archaellum consists of an ATP-powered intracellular motor that drives the rotation of an extracellular filament composed of multiple copies of proteins named archaellins. In many species, several archaellin homologs are encoded in the same operon; however, previous structural studies indicated that archaellum filaments mainly consist of only one protein species. Here, we use electron cryo-microscopy to elucidate the structure of the archaellum from Methanocaldococcus villosus at 3.08 Å resolution. The filament is composed of two alternating archaellins, suggesting that the architecture and assembly of archaella is more complex than previously thought. Moreover, we identify structural elements that may contribute to the filament’s flexibility.
Abstract.
2021
Conners R, McLaren M, Łapińska U, Sanders K, Stone MRL, Blaskovich MAT, Pagliara S, Daum B, Rakonjac J, Gold VAM, et al (2021). CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage.
Nature Communications,
12(1).
Abstract:
CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage
AbstractThe Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.
Abstract.
2020
Hosszu LLP, Conners R, Sangar D, Batchelor M, Sawyer EB, Fisher S, Cliff MJ, Hounslow AM, McAuley K, Leo Brady R, et al (2020). Structural effects of the highly protective V127 polymorphism on human prion protein.
Communications Biology,
3(1).
Abstract:
Structural effects of the highly protective V127 polymorphism on human prion protein
Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). A single point mutation (G127V) in human PrP prevents prion disease, however the structural basis for its protective effect remains unknown. Here we show that the mutation alters and constrains the PrP backbone conformation preceding the PrP β-sheet, stabilising PrP dimer interactions by increasing intermolecular hydrogen bonding. It also markedly changes the solution dynamics of the β2-α2 loop, a region of PrP structure implicated in prion transmission and cross-species susceptibility. Both of these structural changes may affect access to protein conformers susceptible to prion formation and explain its profound effect on prion disease.
Abstract.
2016
Hosszu LLP, Conners R, Sangar D, Batchelor M, McAuley KE, Fisher SJ, Brady RL, Clarke AR, Collinge J (2016). Structural and folding studies of the protective V127 variant of human prion protein.
Author URL.
2011
Agnew C, Borodina E, Zaccai NR, Conners R, Burton NM, Vicary JA, Cole DK, Antognozzi M, Virji M, Brady RL, et al (2011). Correlation of in situ mechanosensitive responses of the Moraxella catarrhalis adhesin UspA1 with fibronectin and receptor CEACAM1 binding.
Proc Natl Acad Sci U S A,
108(37), 15174-15178.
Abstract:
Correlation of in situ mechanosensitive responses of the Moraxella catarrhalis adhesin UspA1 with fibronectin and receptor CEACAM1 binding.
Bacterial cell surfaces are commonly decorated with a layer formed from multiple copies of adhesin proteins whose binding interactions initiate colonization and infection processes. In this study, we investigate the physical deformability of the UspA1 adhesin protein from Moraxella catarrhalis, a causative agent of middle-ear infections in humans. UspA1 binds a range of extracellular proteins including fibronectin, and the epithelial cellular receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Electron microscopy indicates that unliganded UspA1 is densely packed at, and extends about 800 Å from, the Moraxella surface. Using a modified atomic force microscope, we show that the adhesive properties and thickness of the UspA1 layer at the cell surface varies on addition of either fibronectin or CEACAM1. This in situ analysis is then correlated with the molecular structure of UspA1. To provide an overall model for UspA1, we have determined crystal structures for two N-terminal fragments which are then combined with a previous structure of the CEACAM1-binding site. We show that the UspA1-fibronectin complex is formed between UspA1 head region and the 13th type-III domain of fibronectin and, using X-ray scattering, that the complex involves an angular association between these two proteins. In combination with a previous study, which showed that the CEACAM1-UspA1 complex is distinctively bent in solution, we correlate these observations on isolated fragments of UspA1 with its in situ response on the cell surface. This study therefore provides a rare direct demonstration of protein conformational change at the cell surface.
Abstract.
Author URL.
Zaccai NR, Agnew C, Borodina E, Conners R, Burton N, Cole DK, Antognozzi M, Vriji M, Brady RL (2011). Multi-Functional Receptor Binding Activity of the Moraxella Catarrhalis Adhesin UspA1.
Author URL.
2010
Spencer J, Murphy LM, Conners R, Sessions RB, Gamblin SJ (2010). Crystal structure of the LasA virulence factor from Pseudomonas aeruginosa: substrate specificity and mechanism of M23 metallopeptidases.
J Mol Biol,
396(4), 908-923.
Abstract:
Crystal structure of the LasA virulence factor from Pseudomonas aeruginosa: substrate specificity and mechanism of M23 metallopeptidases.
Pseudomonas aeruginosa is an opportunist Gram-negative bacterial pathogen responsible for a wide range of infections in immunocompromized individuals and is a leading cause of mortality in cystic fibrosis patients. A number of secreted virulence factors, including various proteolytic enzymes, contribute to the establishment and maintenance of Pseudomonas infection. One such is LasA, an M23 metallopeptidase related to autolytic glycylglycine endopeptidases such as Staphylococcus aureus lysostaphin and LytM, and to DD-endopeptidases involved in entry of bacteriophage to host bacteria. LasA is implicated in a range of processes related to Pseudomonas virulence, including stimulating ectodomain shedding of the cell surface heparan sulphate proteoglycan syndecan-1 and elastin degradation in connective tissue. Here we present crystal structures of active LasA as a complex with tartrate and in the uncomplexed form. While the overall fold resembles that of the other M23 family members, the LasA active site is less constricted and utilizes a different set of metal ligands. The active site of uncomplexed LasA contains a five-coordinate zinc ion with trigonal bipyramidal geometry and two metal-bound water molecules. Using these structures as a starting point, we propose a model for substrate binding by LasA that explains its activity against a wider range of substrates than those used by related lytic enzymes, and offer a catalytic mechanism for M23 metallopeptidases consistent with available structural and mutagenesis data. Our results highlight how LasA is a structurally distinct member of this endopeptidase family, consistent with its activity against a wider range of substrates and with its multiple roles in Pseudomonas virulence.
Abstract.
Author URL.
2008
Conners R, Hill DJ, Borodina E, Agnew C, Daniell SJ, Burton NM, Sessions RB, Clarke AR, Catto LE, Lammie D, et al (2008). The Moraxella adhesin UspA1 binds to its human CEACAM1 receptor by a deformable trimeric coiled-coil.
EMBO J,
27(12), 1779-1789.
Abstract:
The Moraxella adhesin UspA1 binds to its human CEACAM1 receptor by a deformable trimeric coiled-coil.
Moraxella catarrhalis is a ubiquitous human-specific bacterium commonly associated with upper and lower respiratory tract infections, including otitis media, sinusitis and chronic obstructive pulmonary disease. The bacterium uses an autotransporter protein UspA1 to target an important human cellular receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Using X-ray crystallography, we show that the CEACAM1 receptor-binding region of UspA1 unusually consists of an extended, rod-like left-handed trimeric coiled-coil. Mutagenesis and binding studies of UspA1 and the N-domain of CEACAM1 have been used to delineate the interacting surfaces between ligand and receptor and guide assembly of the complex. However, solution scattering, molecular modelling and electron microscopy analyses all indicate that significant bending of the UspA1 coiled-coil stalk also occurs. This explains how UspA1 can engage CEACAM1 at a site far distant from its head group, permitting closer proximity of the respective cell surfaces during infection.
Abstract.
Author URL.
2007
Conners R, Konarev AV, Forsyth J, Lovegrove A, Marsh J, Joseph-Horne T, Shewry P, Brady RL (2007). An unusual helix-turn-helix protease inhibitory motif in a novel trypsin inhibitor from seeds of Veronica (Veronica hederifolia L.).
J Biol Chem,
282(38), 27760-27768.
Abstract:
An unusual helix-turn-helix protease inhibitory motif in a novel trypsin inhibitor from seeds of Veronica (Veronica hederifolia L.).
The storage tissues of many plants contain protease inhibitors that are believed to play an important role in defending the plant from invasion by pests and pathogens. These proteinaceous inhibitor molecules belong to a number of structurally distinct families. We describe here the isolation, purification, initial inhibitory properties, and three-dimensional structure of a novel trypsin inhibitor from seeds of Veronica hederifolia (VhTI). The VhTI peptide inhibits trypsin with a submicromolar apparent K(i) and is expected to be specific for trypsin-like serine proteases. VhTI differs dramatically in structure from all previously described families of trypsin inhibitors, consisting of a helix-turn-helix motif, with the two alpha helices tightly associated by two disulfide bonds. Unusually, the crystallized complex is in the form of a stabilized acyl-enzyme intermediate with the scissile bond of the VhTI inhibitor cleaved and the resulting N-terminal portion of the inhibitor remaining attached to the trypsin catalytic serine 195 by an ester bond. A synthetic, truncated version of the VhTI peptide has also been produced and co-crystallized with trypsin but, surprisingly, is seen to be uncleaved and consequently forms a noncovalent complex with trypsin. The VhTI peptide shows that effective enzyme inhibitors can be constructed from simple helical motifs and provides a new scaffold on which to base the design of novel serine protease inhibitors.
Abstract.
Author URL.
Crisp J, Conners R, Garrity JD, Carenbauer AL, Crowder MW, Spencer J (2007). Structural basis for the role of Asp-120 in metallo-beta-lactamases.
Biochemistry,
46(37), 10664-10674.
Abstract:
Structural basis for the role of Asp-120 in metallo-beta-lactamases.
Metallo-beta-lactamases (mbetals) are zinc-dependent enzymes that hydrolyze a wide range of beta-lactam antibiotics. The mbetal active site features an invariant Asp-120 that ligates one of the two metal ions (Zn2) and a metal-bridging water/hydroxide (Wat1). Previous studies show that substitutions at Asp-120 dramatically affect mbetal activity, but no consensus exists as to its role in beta-lactam turnover. Here we present crystal structures of the Asn and Cys mutants of Asp-120 of the L1 mbetal from Stenotrophomonas maltophilia. Both mutants retain a dinuclear zinc center with Wat1 present. In the essentially inactive Cys enzyme Zn2 is displaced to a more buried position relative to that in the wild-type enzyme. In the catalytically impaired Asn enzyme the coordination of Zn2 is altered, neither it nor Wat1 is coordinated by Asn-120, and the N-terminal 19 amino acids, important to cooperative interactions between subunits in the wild-type enzyme, are disordered. Comparison with the structure of L1 complexed with the hydrolyzed oxacephem moxalactam suggests that in the Cys mutant Zn2 can no longer make stabilizing interactions with anionic nitrogen species formed in the hydrolytic reaction. The diminished activity of the Asn mutant arises from a combination of loss of intersubunit interactions and impaired proton transfer to, and reduced interaction of Zn2 with, the substrate amide nitrogen. We conclude that, while interactions of Asp-120 with active site water molecules are important to proton transfer and possibly nucleophilic attack by Wat1, its primary role is to optimally position Zn2 for catalytically important interactions with the charged amide nitrogen of substrate.
Abstract.
Author URL.
2006
Conners R, Hooley E, Clarke AR, Thomas S, Brady RL (2006). Recognition of oxidatively modified bases within the biotin-binding site of avidin.
J Mol Biol,
357(1), 263-274.
Abstract:
Recognition of oxidatively modified bases within the biotin-binding site of avidin.
Oxidative damage of DNA results in the formation of many products, including 8-oxodeoxyguanosine, which has been used as a marker to quantify DNA damage. Earlier studies have demonstrated that avidin, a protein prevalent in egg-white and which has high affinity for the vitamin biotin, binds to 8-oxodeoxyguanosine and related bases. In this study, we have determined crystal structures of avidin in complex with 8-oxodeoxyguanosine and 8-oxodeoxyadenosine. In each case, the base is observed to bind within the biotin-binding site of avidin. However, the mode of association between the bases and the protein varies and, unlike in the avidin:biotin complex, complete ordering of the protein in this region does not accompany binding. Fluorescence studies indicate that in solution the individual bases, and a range of oligonucleotides, bind to avidin with micromolar affinity. Only one of the modes of binding observed is consistent with recognition of oxidised purines when incorporated within a DNA oligomer, and from this structure a model is proposed for the selective binding of avidin to DNA containing oxidatively damaged deoxyguanosine. These studies illustrate the molecular basis by which avidin might act as a marker of DNA damage, although the low levels of binding observed are inconsistent with the recognition of oxidised purines forming a major physiological role for avidin.
Abstract.
Author URL.
Booth MPS, Conners R, Rumsby G, Brady RL (2006). Structural basis of substrate specificity in human glyoxylate reductase/hydroxypyruvate reductase.
J Mol Biol,
360(1), 178-189.
Abstract:
Structural basis of substrate specificity in human glyoxylate reductase/hydroxypyruvate reductase.
Human glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is a D-2-hydroxy-acid dehydrogenase that plays a critical role in the removal of the metabolic by-product glyoxylate from within the liver. Deficiency of this enzyme is the underlying cause of primary hyperoxaluria type 2 (PH2) and leads to increased urinary oxalate levels, formation of kidney stones and renal failure. Here we describe the crystal structure of human GRHPR at 2.2 a resolution. There are four copies of GRHPR in the crystallographic asymmetric unit: in each homodimer, one subunit forms a ternary (enzyme+NADPH+reduced substrate) complex, and the other a binary (enzyme+NADPH) form. The spatial arrangement of the two enzyme domains is the same in binary and ternary forms. This first crystal structure of a true ternary complex of an enzyme from this family demonstrates the relationship of substrate and catalytic residues within the active site, confirming earlier proposals of the mode of substrate binding, stereospecificity and likely catalytic mechanism for these enzymes. GRHPR has an unusual substrate specificity, preferring glyoxylate and hydroxypyruvate, but not pyruvate. A tryptophan residue (Trp141) from the neighbouring subunit of the dimer is projected into the active site region and appears to contribute to the selectivity for hydroxypyruvate. This first crystal structure of a human GRHPR enzyme also explains the deleterious effects of naturally occurring missense mutations of this enzyme that lead to PH2.
Abstract.
Author URL.
2005
Hooley E, Conners R, Brady L (2005). Detection of 8-hydroxy-2 ' deoxyadenosine and 8-hydroxy-2 ' deoxyguanosine by Avidin.
Author URL.
Conners R, Schambach F, Read J, Cameron A, Sessions RB, Vivas L, Easton A, Croft SL, Brady RL (2005). Mapping the binding site for gossypol-like inhibitors of Plasmodium falciparum lactate dehydrogenase.
Mol Biochem Parasitol,
142(2), 137-148.
Abstract:
Mapping the binding site for gossypol-like inhibitors of Plasmodium falciparum lactate dehydrogenase.
Gossypol is a di-sesquiterpene natural-product in the form of a functionalised binaphthyl and is isolated from cotton plants. The compound has long been known to exhibit anti-malarial and other biological activities. Previous studies have indicated that compounds of this type target Plasmodium falciparum lactate dehydrogenase (pfLDH), an essential enzyme for energy generation within the parasite. In this study, we report that simple naphthalene-based compounds, the core of the gossypol structure, exhibit weak inhibition of the parasite lactate dehydrogenase. Crystal structures of the complexes formed by binding of these naphthalene-based compounds to their target enzyme have been used to delineate the molecular features likely to form the gossypol binding site. Two modes of binding are observed: one overlapping the pyruvate but not the co-factor site, the other bridging the binding sites for the co-factor nicontinamide group and pyruvate substrate. This latter site encompasses molecular features unique to Plasmodium forms of LDH and is likely to represent the mode of binding for gossypol derivatives that show selectivity for the parasite enzymes. We also report a substrate analogue that unexpectedly binds within the adenine pocket of the co-factor groove. Although these core pharmacophore-like molecules only exhibit low levels of inhibitory activity, these molecular snapshots provide a rational basis for renewed structure-based development of naphthalene-based compounds as anti-malarial agents.
Abstract.
Author URL.
Chaikuad A, Fairweather V, Conners R, Joseph-Horne T, Turgut-Balik D, Brady RL (2005). Structure of lactate dehydrogenase from Plasmodium vivax: complexes with NADH and APADH.
Biochemistry,
44(49), 16221-16228.
Abstract:
Structure of lactate dehydrogenase from Plasmodium vivax: complexes with NADH and APADH.
Malaria caused by Plasmodium vivax is a major cause of global morbidity and, in rare cases, mortality. Lactate dehydrogenase is an essential Plasmodium protein and, therefore, a potential antimalarial drug target. Ideally, drugs directed against this target would be effective against both major species of Plasmodium, P. falciparum and P. vivax. In this study, the crystal structure of the lactate dehydrogenase protein from P. vivax has been solved and is compared to the equivalent structure from the P. falciparum enzyme. The active sites and cofactor binding pockets of both enzymes are found to be highly similar and differentiate these enzymes from their human counterparts. These structures suggest effective inhibition of both enzymes should be readily achievable with a common inhibitor. The crystal structures of both enzymes have also been solved in complex with the synthetic cofactor APADH. The unusual cofactor binding site in these Plasmodium enzymes is found to readily accommodate both NADH and APADH, explaining why the Plasmodium enzymes retain enzymatic activity in the presence of this synthetic cofactor.
Abstract.
Author URL.
2004
Hadfield A, Limpkin C, Read J, Shammas C, Shoemark D, Tranter R, Brady L (2004). Catalytic Cycle of Plasmodium Falciparum Lactate Dehydrogenase.
Author URL.
Cameron A, Read J, Tranter R, Winter VJ, Sessions RB, Brady RL, Vivas L, Easton A, Kendrick H, Croft SL, et al (2004). Identification and activity of a series of azole-based compounds with lactate dehydrogenase-directed anti-malarial activity.
J Biol Chem,
279(30), 31429-31439.
Abstract:
Identification and activity of a series of azole-based compounds with lactate dehydrogenase-directed anti-malarial activity.
Plasmodium falciparum, the causative agent of malaria, relies extensively on glycolysis coupled with homolactic fermentation during its blood-borne stages for energy production. Selective inhibitors of the parasite lactate dehydrogenase (LDH), central to NAD(+) regeneration, therefore potentially provide a route to new antimalarial drugs directed against a novel molecular target. A series of heterocyclic, azole-based compounds are described that preferentially inhibit P. falciparum LDH at sub-micromolar concentrations, typically at concentrations about 100-fold lower than required for human lactate dehydrogenase inhibition. Crystal structures show these competitive inhibitors form a network of interactions with amino acids within the active site of the enzyme, stacking alongside the nicotinamide ring of the NAD(+) cofactor. These compounds display modest activity against parasitized erythrocytes, including parasite strains with known resistance to existing anti-malarials and against Plasmodium berghei in BALB/c mice. Initial toxicity data suggest the azole derivatives have generally low cytotoxicity, and preliminary pharmoco-kinetic data show favorable bioavailability and circulation times. These encouraging results suggest that further enhancement of these structures may yield candidates suitable for consideration as new therapeutics for the treatment of malaria. In combination these studies also provide strong support for the validity of targeting the Plasmodium glycolytic pathway and, in particular, LDH in the search for novel anti-malarials.
Abstract.
Author URL.
2003
Dell G, Wilkinson KW, Tranter R, Parish J, Leo Brady R, Gaston K (2003). Comparison of the structure and DNA-binding properties of the E2 proteins from an oncogenic and a non-oncogenic human papillomavirus.
J Mol Biol,
334(5), 979-991.
Abstract:
Comparison of the structure and DNA-binding properties of the E2 proteins from an oncogenic and a non-oncogenic human papillomavirus.
Human papillomaviruses (HPVS) that infect the genital tract can be divided into two groups: high-risk HPV types, such as HPV 16 and HPV 18, are associated with cancer, low-risk HPV types, such as HPV 6, are associated with benign warts. In both high-risk and low-risk HPV types, the papillomavirus E2 protein binds to four sites within the viral long control region (LCR) and regulates viral gene expression. Here, we present the crystal structure of the minimal DNA-binding domain (DBD) from the HPV 6 E2 protein. We show that the HPV 6 E2 DBD is structurally more similar to the HPV 18 and bovine papillomavirus type 1 (BPV1) E2 proteins than it is to the HPV 16 E2 protein. Using gel retardation assays, we show that the hierarchy of E2 sites within the HPV 16 and HPV 6 LCRs are different. However, despite these differences in structure and site preference, both the HPV 16 and 6 E2 DBDs recognise an extended version of the consensus E2 binding site derived from studies of the BPV1 E2 protein. In both cases, the preferred binding site is 5'AACCGN(4)CGGTT3', where the additional flanking base-pairs are in bold and N(4) represents a four base-pair central spacer. Both of these HPV proteins bind preferentially to E2 sites that contain an A:T-rich central spacer. We show that the preference for an A:T-rich central spacer is due, at least in part, to the need to adopt a DNA conformation that facilitates protein contacts with the flanking base-pairs.
Abstract.
Author URL.
Winter VJ, Cameron A, Tranter R, Sessions RB, Brady RL (2003). Crystal structure of Plasmodium berghei lactate dehydrogenase indicates the unique structural differences of these enzymes are shared across the Plasmodium genus.
Mol Biochem Parasitol,
131(1), 1-10.
Abstract:
Crystal structure of Plasmodium berghei lactate dehydrogenase indicates the unique structural differences of these enzymes are shared across the Plasmodium genus.
As Plasmodium rely extensively on homolactic fermentation for energy production, Plasmodium falciparum lactate dehydrogenase (PfLDH)--the key enzyme in this process--has previously been suggested as a novel target for antimalarials. This enzyme has distinctive kinetic and structural properties that distinguish it from its human homologues. In this study, we now describe the expression, kinetic characterisation and crystal structure determination of the LDH from Plasmodium berghei. This enzyme is seen to have a similar kinetic profile to its P. falciparum counterpart, exhibiting the characteristic lack of substrate inhibition that distinguishes plasmodial from human LDHs. The crystal structure of P. berghei lactate dehydrogenase (PbLDH) shows a very similar active site arrangement to the P. falciparum enzyme. In particular, an insertion of five amino acid residues in the active site loop creates an enlarged volume in the substrate binding site, and characteristic changes in the residues lining the NADH cofactor binding pocket result in displacement of the cofactor relative to its observed position in mammalian and all other LDH structures. These results imply the special features previously described for PfLDH may be shared across the Plasmodium genus, supporting the universal application of therapeutics targeting this enzyme.
Abstract.
Author URL.
2000
Tranter R, Read JA, Jones R, Brady RL (2000). Effector sites in the three-dimensional structure of mammalian sperm beta-acrosin.
Structure,
8(11), 1179-1188.
Abstract:
Effector sites in the three-dimensional structure of mammalian sperm beta-acrosin.
BACKGROUND: Proacrosin is a serine protease found specifically within the acrosomal vesicle of all mammalian spermatozoa. During fertilization proacrosin autoactivates to form beta-acrosin, in which there is a "light" chain cross-linked to a "heavy" chain by two disulphide bonds. beta-acrosin is thought to be multifunctional with roles in acrosomal exocytosis, as a receptor for zona pellucida proteins, and as a protease to facilitate penetration of spermatozoa into the egg. RESULTS: the crystal structures of both ram and boar beta-acrosins have been solved in complex with p-aminobenzamidine to 2.1 a and 2.9 a resolution, respectively. Both enzymes comprise a heavy chain with structural homology to trypsin, and a light chain covalently associated in a similar manner to blood coagulation enzymes. In crystals of boar beta-acrosin, the carboxyl terminus of the heavy chain is inserted into the active site of the neighboring molecule. In both enzyme structures, there are distinctive positively charged surface "patches" close to the active site, which associate with carbohydrate from adjacent molecules and also bind sulfate ions. CONCLUSIONS: from the three-dimensional structure of beta-acrosin, two separate effector sites are evident. First, proteolytic activity, believed to be important at various stages during fertilization, arises from the trypsin-like active site. Activity of this site may be autoregulated through intermolecular associations. Second, positively charged regions on the surface adjacent to the active site may act as receptors for binding zona pellucida glycoproteins. The spatial proximity of these two effector sites suggests there may be synergy between them.
Abstract.
Author URL.
1999
Read JA, Wilkinson KW, Tranter R, Sessions RB, Brady RL (1999). CHLOROQUINE BINDS TO L-LACTATE DEHYDROGENASE IN THE MALARIAL PARASITE (PLASMODIUM FALCIPARUM.
Author URL.
Read JA, Wilkinson KW, Tranter R, Sessions RB, Brady RL (1999). Chloroquine binds in the cofactor binding site of Plasmodium falciparum lactate dehydrogenase.
J Biol Chem,
274(15), 10213-10218.
Abstract:
Chloroquine binds in the cofactor binding site of Plasmodium falciparum lactate dehydrogenase.
Although the molecular mechanism by which chloroquine exerts its effects on the malarial parasite Plasmodium falciparum remains unclear, the drug has previously been found to interact specifically with the glycolytic enzyme lactate dehydrogenase from the parasite. In this study we have determined the crystal structure of the complex between chloroquine and P. falciparum lactate dehydrogenase. The bound chloroquine is clearly seen within the NADH binding pocket of the enzyme, occupying a position similar to that of the adenyl ring of the cofactor. Chloroquine hence competes with NADH for binding to the enzyme, acting as a competitive inhibitor for this critical glycolytic enzyme. Specific interactions between the drug and amino acids unique to the malarial form of the enzyme suggest this binding is selective. Inhibition studies confirm that chloroquine acts as a weak inhibitor of lactate dehydrogenase, with mild selectivity for the parasite enzyme. As chloroquine has been shown to accumulate to millimolar concentrations within the food vacuole in the gut of the parasite, even low levels of inhibition may contribute to the biological efficacy of the drug. The structure of this enzyme-inhibitor complex provides a template from which the quinoline moiety might be modified to develop more efficient inhibitors of the enzyme.
Abstract.
Author URL.
Kampranis SC, Gormley NA, Tranter R, Orphanides G, Maxwell A (1999). Probing the Binding of Coumarins and Cyclothialidines to DNA Gyrase. Biochemistry, 38(7), 1967-1976.
Tranter R, Jones R, Brady L (1999). STRUCTURAL STUDIES ON ACROSIN, a SERINE PROTEASE FOUND IN MAMMALIAN SPERMATOZOA.
Author URL.