The human organic anion transporter (hOAT) family of transmembrane carrier proteins mediate the c... more The human organic anion transporter (hOAT) family of transmembrane carrier proteins mediate the cellular flux of anionic substances, including certain hormones and anti-cancer drugs. hOAT4 is highly expressed at the apical membrane of the renal tubular cell and facilitates drug re-absorption in the kidney. In the present study, the impact of 10 nonsynonymous single nucleotide polymorphisms (SNPs) of hOAT4 on transport function in COS-7 cells was characterized. Experimental approach: Transport uptake assay was used to assess the function of the variant transporters. Cell surface biotinylation and western blot analysis were used to investigate the expression characteristics of the transporter proteins. Comparative modelling was used to interpret the influence of nonsynonymous changes in terms of hOAT4 structure. Key results: Four naturally occurring hOAT4 variants (L29P, R48Y, V155G and T392I) exhibited a significant loss of function. Substitution of leucine-29, which is a conserved residue in OATs, with a proline residue, impaired the synthesis or the apparent stability of the transporter and membrane insertion was disrupted in the R48Y variant. In the case of the V155G and T392I variants, impaired function was due to decreased affinity of the transporter for oestrone sulphate and impaired transportersubstrate turnover respectively. The T392I variant was inhibited more extensively than the wild-type transporter by the cationic substrate tetraethyl ammonium. Conclusions and implications: Several naturally occurring SNPs encode variant hOAT4s that may impair the renal tubular re-absorption of important drug substrates. F Zhou et al British Journal of Pharmacology (2010) 159 419-427 hOAT4 polymorphisms and transport function 424 F Zhou et al British Journal of Pharmacology (2010) 159 419-427
The solute carrier organic anion transporting polypeptide 1A2 (OATP1A2, SLCO1A2) is implicated in... more The solute carrier organic anion transporting polypeptide 1A2 (OATP1A2, SLCO1A2) is implicated in the cellular influx of a number of drugs. We identified five novel single nucleotide polymorphisms (SNPs) in coding exons of the SLCO1A2 gene in a cohort of subjects: G550A, G553A, G673A, A775C, and G862A, that encoded the OATP1A2 variants E184K, D185N, V255I, T259P, and D288N, respectively. The function and expression of these variant transporters were assessed in HEK-293 cells. We found that the novel variants, E184K, D185N, T259P, and D288N, were associated with impaired estrone-3-sulfate, imatinib, and methotrexate transport (∼20-50% of wild-type control); function was retained by OATP1A2-V255I. From biotinylation assays, the decreased function of these variants was due, at least in part, to impaired plasma membrane expression. The four loss-of-function variants were studied further using mutagenesis to produce variants that encode residues with different charges or steric properties. From immunoblotting, the replacement of negatively charged residues at amino acid positions 184 and 185 impaired membrane expression, while either a positive or negative charge at residue 288 supported the correct membrane targeting of OATP1A2. Replacement of T259 with bulky residues disrupted transporter stability. From molecular models, E184, D185, and D288 were located near several charged residues such that intramolecular ionic interactions may stabilize the transporter structure. Individuals who carry these novel SNPs in the SLCO1A2 gene may be at risk from impaired efficacy or enhanced toxicity during treatment with drugs that are substrates for OATP1A2.
The crystal structure of Hg(I1)-plastocyanin h v been determined and refined at a resolution of 1... more The crystal structure of Hg(I1)-plastocyanin h v been determined and refined at a resolution of 1.9 A. The crystals were prepared by soaking crystals of Cu(I1)plastocyanin from poplar leaves (Populus nigra var. italica) in a solution of a mercuric salt. Replacement of the Cu(I1) atom in plastocyanin by Hg(I1) causes only minor changes in the geometry of the metal site, and there are few significant changes elsewhere in the molecule. It is concluded that, as in the case of the native protein, the geometry of the metal site is determined by the polypeptide. The weak metal-S(methionine) bond found in Cu(I1)-plastocyanin remains weak in Hg(I1)-plastocyanin. The "flip" of a proline side chain close to the metal site from a Cy-exo conformation in Cu(I1)-plastocyanin to a C7-endo conformation in Hg(I1)-plastocyanin suggests that this region of the molecule is particularly flexible.
Schizophrenia is a complex neuropsychiatric disorder with limited treatment options and highly de... more Schizophrenia is a complex neuropsychiatric disorder with limited treatment options and highly debilitating symptoms, leading to poor personal, social, and occupational outcomes for an afflicted individual. Our current understanding of schizophrenia suggests that dopaminergic and glutamatergic systems have a significant role in the pathogenesis of the disease. Kynurenic acid, an endogenous glutamate antagonist, is found in elevated concentrations in the prefrontal cortex and cerebrospinal fluid of patients with schizophrenia, and this affects neurotransmitter release in a similar manner to previously observed psychotomimetic agents, such as phencyclidine, underlining the molecular basis to its link in schizophrenia pathophysiology. Kynurenic acid is a breakdown product of tryptophan degradation, through a transamination process mediated by kynurenine aminotransferase (KAT) enzymes. There are four KAT homologues reported, all of which are pyridoxal-5'-phosphate-dependent enzymes....
ABSTRACT In Some Cyanobacteria, Poor Nutrient Conditions Induce A Reduction Of The Phycobilisome ... more ABSTRACT In Some Cyanobacteria, Poor Nutrient Conditions Induce A Reduction Of The Phycobilisome Light-Harvesting System And An Up-Regulation Of A Chlorophyll Antenna Protein, Isia, Which Can Form An 18-Mer Ring Around Photosystem I (Psi) Trimers. We Have Used The Recent High-Resolution Structure Of Photosystem Ii (Psii) To Obtain A New Model Of The Molecular Structure Of Isia, Which Evolved From Cp43. This Model Was Further Refined By Analysis Of Sequence Variation Among Cp43, Isia, And The Nterminal Part Of Psaa, To Produce A Model In Which There Are 16 Chlorophylls (Chls) Per Isia Subunit, Two More Than Previously Predicted. The Structure Of The Isia-Ps I Supercomplex Was Then Examined By Comparing The Potential Of Excitation Transfer, Which Is Done By Optimizing The Chl-Chl Distances.
Hydrogen peroxide (H 2 O 2 ) can act as an intracellular messenger by oxidizing sulfhydryl groups... more Hydrogen peroxide (H 2 O 2 ) can act as an intracellular messenger by oxidizing sulfhydryl groups in cysteines that can be oxidized at neutral pH. The oxidizing agents H 2 O 2 and pyrroloquinoline quinone and the large thiol reagents N-ethylmaleimide and 4-(hydroxymercuri) benzoate each inhibited dipeptidyl peptidase (DP) activity in the intracellular DPIV-related proteins DP8 and DP9 at pH 7.5. In contrast, these treatments did not alter activity in DPIV and fibroblast activation protein. Peptidase inhibition was completely reversed by 2-mercaptoethanol or reduced glutathione. Alkylation of DP8 by the small thiol reagent iodoacetamide prevented inhibition by H 2 O 2, N-ethylmaleimide or pyrroloquinoline quinone. Two cysteines were reactive per peptidase monomer. We exploited these properties to highly purify DP8 by thiol affinity chromatography. Homology modelling of DP8 and DP9 was consistent with the proposal that the mechanism involves decreased protein flexibility caused by intramolecular disulfide bonding. These novel data show that DP8 and DP9 are reversibly inactivated by oxidants at neutral pH and suggest that DP8 and DP9 are H 2 O 2 sensing proteins.
It is estimated that 20% of genes in the human genome encode for integral membrane proteins (IMPs... more It is estimated that 20% of genes in the human genome encode for integral membrane proteins (IMPs) and some estimates are much higher. IMPs control a broad range of events essential to the proper functioning of cells, tissues and organisms. IMPs include the most common targets of clinically useful drugs, such as the G protein coupled receptors (GPCR), the target for more than 50% of prescription drugs . However there is a dearth of high-resolution 3D structural information on the IMPs. The number of the IMPs depositions in the major structural holding, the Protein Data Bank is less than 0.4% of the collection [2]. Therefore good prediction methods of IMPs structures are to be highly valued. In this paper we apply Conditional Random Fields (CRFs) to build a probabilistic model to segment and label sequence data to solve the membrane protein helix prediction problem. The advantage of CRFs is that it allows seamless and principled integration of biological domain knowledge into the model. Our results show that the CRF model outperforms other well known helix prediction approaches on several important measures.
High-molecular-weight arginine-and lysine-specific (Kgp) gingipains are essential virulence facto... more High-molecular-weight arginine-and lysine-specific (Kgp) gingipains are essential virulence factors expressed by the oral pathogen Porphyromonas gingivalis. Haemagglutinin/adhesin (HA) regions of these proteases have been implicated in targeting catalytic domains to biological substrates and in other adhesive functions. We now report the crystal structure of the K3 adhesin domain/module of Kgp, which folds into the distinct b-jelly roll sandwich topology previously observed for K2. A conserved structural feature of K3, previously observed in the Kgp K2 module, is the half-way point anchoring of the surface exposed loops via an arginine residue found in otherwise highly variable sequences. Small-angle X-ray scattering data for the recombinant construct K1K2K3 confirmed a structure comprising a tandem repeat of three homologous modules, K1, K2 and K3 while also indicating an unusual 'y'-shape arrangement of the modules connected by variable linker sequences. Only the K2 and K3 modules and a K1K2 construct were observed to be potently haemolytic. K2, K3 and the K1K2 construct showed preferential recognition of haem-albumin over albumin whereas only low affinity binding was detected for K1 and the K1K2K3 construct. The data indicate replication of some biological functions over the three adhesin domains of Kgp while other functions are restricted.
The International Journal of Biochemistry & Cell Biology, 2009
Rhodopsin was the first G protein-coupled receptor (GPCR) for which a high-resolution crystal str... more Rhodopsin was the first G protein-coupled receptor (GPCR) for which a high-resolution crystal structure was obtained. Several crystal structures have now been solved representing different activation states of the receptor. These structures, together with those from lower resolution techniques (e.g. electron microscopy), shed light on the stepwise process by which energy from an extracellular photon is transduced across the membrane to the intracellular compartment thereby activating signalling mechanisms responsible for very low-level light detection. Controversy remains in several areas including: (i) transmembrane helix movements responsible for the transduction process, (ii) the stoichiometry of coupling to G proteins and their mode of activation, (iii) the role, if any, of receptor oligomerisation and (iv) the suitability of using structures of this GPCR as templates for modelling the structures of other GPCRs, and their mechanisms of activation.
Of the 600+ known proteases identified in mammals, a significant percentage are involved or impli... more Of the 600+ known proteases identified in mammals, a significant percentage are involved or implicated in pathogenic and cancer processes. The Dipeptidyl Peptidase IV (DPIV) gene family, comprising four enzyme members DPIV, Fibroblast Activation Protein (FAP), DP8 and DP9 and two non-enzyme members DP6 (DPL1) and DP10 (DPL2), are interesting in this regard due to their multiple diverse functions, varying patterns of distribution/localisation and subtle but significant differences in structure/substrate recognition. In addition, their engagement in cell biological processes involves both enzymatic and non-enzymatic capabilities. This article examines in detail our current understanding of the biological involvement of this unique enzyme family and their overall potential as therapeutic targets.
Dipeptidyl peptidase IV (DPPIV) is an atypical serine protease that modifies the biological activ... more Dipeptidyl peptidase IV (DPPIV) is an atypical serine protease that modifies the biological activities of certain chemokines and neuropeptides. In addition, human DPPIV, also known as the T-cell activation antigen CD26, binds adenosine deaminase (ADA) to the T-cell surface, thus protecting the T-cell from adenosine-mediated inhibition of proliferation. Mutations were engineered into DPPIV (five point, 16 single point and six deletion mutations) to examine the binding of ADA and 19 monoclonal antibodies. Deletions of C-terminal residues from the 738-residue extracellular portion of DPPIV showed that the 214 residues C-terminal to Ser552 were not required for ADA binding and that peptidase activity could be ablated by deletion of 20 residues from the C-terminus. Point mutations at either of two locations, Leu294 and Val341, ablated ADA binding. Binding by six anti-DPPIV antibodies that inhibited ADA binding was found to require Leu340 to Arg343 and Thr440/Lys441 but not the 214 residues C-terminal to Ser552. The 13 other antibodies studied bound to a truncated DPPIV consisting of amino acids 1-356. Therefore, the binding sites on DPPIV of ADA and antibodies that inhibit ADA binding are discontinuous and overlapping. Moreover, the 47 and 97 residue spacing of amino acids in these binding sites concords with their location on a beta propeller fold consisting of repeated beta sheets of about 50 amino acids.
The community-wide GPCR Dock assessment is conducted to evaluate the status of molecular modeling... more The community-wide GPCR Dock assessment is conducted to evaluate the status of molecular modeling and ligand docking for human G proteincoupled receptors. The present round of the assessment was based on the recent structures of dopamine D3 and CXCR4 chemokine receptors bound to small molecule antagonists and CXCR4 with a synthetic cyclopeptide. Thirty-five groups submitted their receptor-ligand complex structure predictions prior to the release of the crystallographic coordinates. With closely related homology modeling templates, as for dopamine D3 receptor, and with incorporation of biochemical and QSAR data, modern computational techniques predicted complex details with accuracy approaching experimental. In contrast, CXCR4 complexes that had less-characterized interactions and only distant homology to the known GPCR structures still remained very challenging. The assessment results provide guidance for modeling and crystallographic communities in method development and target selection for further expansion of the structural coverage of the GPCR universe.
Proteins: Structure, Function, and Bioinformatics, 2014
BAMLET (Bovine Alpha-lactalbumin Made LEthal to Tumors) is a member of the family of the HAMLET-l... more BAMLET (Bovine Alpha-lactalbumin Made LEthal to Tumors) is a member of the family of the HAMLET-like complexes, a novel class of protein-based anti-cancer complexes that incorporate oleic acid and deliver it to cancer cells. Small angle X-ray scattering (SAXS) was performed on the complex at pH 12, examining the high pH structure as a function of oleic acid added. The SAXS data for BAMLET species prepared with a range of oleic acid concentrations indicate extended, irregular, partially unfolded protein conformations that vary with the oleic acid concentration. Increases in oleic acid concentration correlate with increasing radius of gyration without an increase in maximum particle dimension, indicating decreasing protein density. The models for the highest oleic acid content BAMLET indicate an unusual coiled elongated structure that contrasts with apo-α-lactalbumin at pH 12, which is an elongated globular molecule, suggesting that oleic acid inhibits the folding or collapse of the protein component of BAMLET to the globular form. Circular dichroism of BAMLET and apo-α-lactalbumin was performed and the results suggest that α-lactalbumin and BAMLET unfold in a continuum of increasing degree of unfolded states. Taken together, these results support a model in which BAMLET retains oleic acid by non-specific association in the core of partially unfolded protein, and represent a new type of lipoprotein structure.
ABSTRACT Homology modeling methods have been used to construct models of two proteins--the histid... more ABSTRACT Homology modeling methods have been used to construct models of two proteins--the histidine-containing phosphocarrier protein (HPr) from Mycoplasma capricolum and human eosinophil-derived neurotoxin (EDN). Comparison of the models with the subsequently determined X-ray crystal structures indicates that the core regions of both proteins are reasonably well reproduced, although the template structures are closer to the X-ray structures in these regions--possible enhancements are discussed. The conformations of most of the side chains in the core of HPr are well reproduced in the modeled structure. As expected, the conformations of surface side chains in this protein differ significantly from the X-ray structure. The loop regions of EDN were incorrectly modeled--reasons for this and possible enhancements are discussed.
Juvenile Hormone Esterase (JHE) plays an essential role in the development of insects since it is... more Juvenile Hormone Esterase (JHE) plays an essential role in the development of insects since it is partially responsible for clearing juvenile hormone (JH), one of the hormones that is responsible for insect metamorphosis. JHE is a 60 kDa enzyme that selectively hydrolyzes the ␣/ unsaturated ester of JH. Because of its pivotal role in insect development, we have targeted JHE for use as a biopesticide. In this study, we have constructed a homology-based molecular model of JHE from the agricultural crop pest, Heliothis virescens. JHE is a member of the ␣/ hydrolase fold family of enzymes and was built according to two structures in the same family: acetylcholinesterase from Torpedo californica and lipase from Geotrichum candidum. Analysis of the active site region reveals extensive conservation between JHE and its templates. A surprise was the presence of a conserved Ser near the catalytic triad. Docking of JH III into the active site has provided insight into protein-substrate interactions that are corroborated by experimental observation. The model is being used as a predictive basis to design biopesticides. In this regard, we have identified a site on the protein surface that is suggestive of a recognition site for the putative JHE receptor. Proteins 1999; 34: 184-196. 1999 Wiley-Liss, Inc.
Bacterial elongation factor G (EF-G) physically associates with translocation-competent ribosomes... more Bacterial elongation factor G (EF-G) physically associates with translocation-competent ribosomes and facilitates transition to the subsequent codon through the coordinate binding and hydrolysis of GTP. In order to investigate the amino acid positions necessary for EF-G functions, a series of mutations were constructed in the EF-G structural gene (fusA) of Escherichia coli, specifically at positions flanking the effector domain. A mutated allele was isolated in which the wild-type sequence from codons 29 to 47 ("EFG2947") was replaced with a sequence encoding 28 amino acids from ribosomal protein S7. This mutated gene was unable to complement a fusAts strain when supplied in trans at the nonpermissive temperature. In vitro biochemical analysis demonstrated that nucleotide crosslinking was unaffected in EFG2947, while ribosome binding appeared to be completely abolished. A series of point mutations created within this region, encoding L30A, Y32A, H37A, and K38A were shown to give rise to fully functional proteins, suggesting that side chains of these individual residues are not essential for EF-G function. A sixth mutant, E41A, was found to inefficiently rescue growth in a fusAts background, and was also unable to bind ribosomes normally in vitro. In contrast E41Q could restore growth at the nonpermissive temperature. These results can be explained within the context of a three-dimensional model for the effector region of EF-G. This model indicates that the effector domain contains a negative potential field that may be important for ribosome binding.
The human organic anion transporter (hOAT) family of transmembrane carrier proteins mediate the c... more The human organic anion transporter (hOAT) family of transmembrane carrier proteins mediate the cellular flux of anionic substances, including certain hormones and anti-cancer drugs. hOAT4 is highly expressed at the apical membrane of the renal tubular cell and facilitates drug re-absorption in the kidney. In the present study, the impact of 10 nonsynonymous single nucleotide polymorphisms (SNPs) of hOAT4 on transport function in COS-7 cells was characterized. Experimental approach: Transport uptake assay was used to assess the function of the variant transporters. Cell surface biotinylation and western blot analysis were used to investigate the expression characteristics of the transporter proteins. Comparative modelling was used to interpret the influence of nonsynonymous changes in terms of hOAT4 structure. Key results: Four naturally occurring hOAT4 variants (L29P, R48Y, V155G and T392I) exhibited a significant loss of function. Substitution of leucine-29, which is a conserved residue in OATs, with a proline residue, impaired the synthesis or the apparent stability of the transporter and membrane insertion was disrupted in the R48Y variant. In the case of the V155G and T392I variants, impaired function was due to decreased affinity of the transporter for oestrone sulphate and impaired transportersubstrate turnover respectively. The T392I variant was inhibited more extensively than the wild-type transporter by the cationic substrate tetraethyl ammonium. Conclusions and implications: Several naturally occurring SNPs encode variant hOAT4s that may impair the renal tubular re-absorption of important drug substrates. F Zhou et al British Journal of Pharmacology (2010) 159 419-427 hOAT4 polymorphisms and transport function 424 F Zhou et al British Journal of Pharmacology (2010) 159 419-427
The solute carrier organic anion transporting polypeptide 1A2 (OATP1A2, SLCO1A2) is implicated in... more The solute carrier organic anion transporting polypeptide 1A2 (OATP1A2, SLCO1A2) is implicated in the cellular influx of a number of drugs. We identified five novel single nucleotide polymorphisms (SNPs) in coding exons of the SLCO1A2 gene in a cohort of subjects: G550A, G553A, G673A, A775C, and G862A, that encoded the OATP1A2 variants E184K, D185N, V255I, T259P, and D288N, respectively. The function and expression of these variant transporters were assessed in HEK-293 cells. We found that the novel variants, E184K, D185N, T259P, and D288N, were associated with impaired estrone-3-sulfate, imatinib, and methotrexate transport (∼20-50% of wild-type control); function was retained by OATP1A2-V255I. From biotinylation assays, the decreased function of these variants was due, at least in part, to impaired plasma membrane expression. The four loss-of-function variants were studied further using mutagenesis to produce variants that encode residues with different charges or steric properties. From immunoblotting, the replacement of negatively charged residues at amino acid positions 184 and 185 impaired membrane expression, while either a positive or negative charge at residue 288 supported the correct membrane targeting of OATP1A2. Replacement of T259 with bulky residues disrupted transporter stability. From molecular models, E184, D185, and D288 were located near several charged residues such that intramolecular ionic interactions may stabilize the transporter structure. Individuals who carry these novel SNPs in the SLCO1A2 gene may be at risk from impaired efficacy or enhanced toxicity during treatment with drugs that are substrates for OATP1A2.
The crystal structure of Hg(I1)-plastocyanin h v been determined and refined at a resolution of 1... more The crystal structure of Hg(I1)-plastocyanin h v been determined and refined at a resolution of 1.9 A. The crystals were prepared by soaking crystals of Cu(I1)plastocyanin from poplar leaves (Populus nigra var. italica) in a solution of a mercuric salt. Replacement of the Cu(I1) atom in plastocyanin by Hg(I1) causes only minor changes in the geometry of the metal site, and there are few significant changes elsewhere in the molecule. It is concluded that, as in the case of the native protein, the geometry of the metal site is determined by the polypeptide. The weak metal-S(methionine) bond found in Cu(I1)-plastocyanin remains weak in Hg(I1)-plastocyanin. The "flip" of a proline side chain close to the metal site from a Cy-exo conformation in Cu(I1)-plastocyanin to a C7-endo conformation in Hg(I1)-plastocyanin suggests that this region of the molecule is particularly flexible.
Schizophrenia is a complex neuropsychiatric disorder with limited treatment options and highly de... more Schizophrenia is a complex neuropsychiatric disorder with limited treatment options and highly debilitating symptoms, leading to poor personal, social, and occupational outcomes for an afflicted individual. Our current understanding of schizophrenia suggests that dopaminergic and glutamatergic systems have a significant role in the pathogenesis of the disease. Kynurenic acid, an endogenous glutamate antagonist, is found in elevated concentrations in the prefrontal cortex and cerebrospinal fluid of patients with schizophrenia, and this affects neurotransmitter release in a similar manner to previously observed psychotomimetic agents, such as phencyclidine, underlining the molecular basis to its link in schizophrenia pathophysiology. Kynurenic acid is a breakdown product of tryptophan degradation, through a transamination process mediated by kynurenine aminotransferase (KAT) enzymes. There are four KAT homologues reported, all of which are pyridoxal-5'-phosphate-dependent enzymes....
ABSTRACT In Some Cyanobacteria, Poor Nutrient Conditions Induce A Reduction Of The Phycobilisome ... more ABSTRACT In Some Cyanobacteria, Poor Nutrient Conditions Induce A Reduction Of The Phycobilisome Light-Harvesting System And An Up-Regulation Of A Chlorophyll Antenna Protein, Isia, Which Can Form An 18-Mer Ring Around Photosystem I (Psi) Trimers. We Have Used The Recent High-Resolution Structure Of Photosystem Ii (Psii) To Obtain A New Model Of The Molecular Structure Of Isia, Which Evolved From Cp43. This Model Was Further Refined By Analysis Of Sequence Variation Among Cp43, Isia, And The Nterminal Part Of Psaa, To Produce A Model In Which There Are 16 Chlorophylls (Chls) Per Isia Subunit, Two More Than Previously Predicted. The Structure Of The Isia-Ps I Supercomplex Was Then Examined By Comparing The Potential Of Excitation Transfer, Which Is Done By Optimizing The Chl-Chl Distances.
Hydrogen peroxide (H 2 O 2 ) can act as an intracellular messenger by oxidizing sulfhydryl groups... more Hydrogen peroxide (H 2 O 2 ) can act as an intracellular messenger by oxidizing sulfhydryl groups in cysteines that can be oxidized at neutral pH. The oxidizing agents H 2 O 2 and pyrroloquinoline quinone and the large thiol reagents N-ethylmaleimide and 4-(hydroxymercuri) benzoate each inhibited dipeptidyl peptidase (DP) activity in the intracellular DPIV-related proteins DP8 and DP9 at pH 7.5. In contrast, these treatments did not alter activity in DPIV and fibroblast activation protein. Peptidase inhibition was completely reversed by 2-mercaptoethanol or reduced glutathione. Alkylation of DP8 by the small thiol reagent iodoacetamide prevented inhibition by H 2 O 2, N-ethylmaleimide or pyrroloquinoline quinone. Two cysteines were reactive per peptidase monomer. We exploited these properties to highly purify DP8 by thiol affinity chromatography. Homology modelling of DP8 and DP9 was consistent with the proposal that the mechanism involves decreased protein flexibility caused by intramolecular disulfide bonding. These novel data show that DP8 and DP9 are reversibly inactivated by oxidants at neutral pH and suggest that DP8 and DP9 are H 2 O 2 sensing proteins.
It is estimated that 20% of genes in the human genome encode for integral membrane proteins (IMPs... more It is estimated that 20% of genes in the human genome encode for integral membrane proteins (IMPs) and some estimates are much higher. IMPs control a broad range of events essential to the proper functioning of cells, tissues and organisms. IMPs include the most common targets of clinically useful drugs, such as the G protein coupled receptors (GPCR), the target for more than 50% of prescription drugs . However there is a dearth of high-resolution 3D structural information on the IMPs. The number of the IMPs depositions in the major structural holding, the Protein Data Bank is less than 0.4% of the collection [2]. Therefore good prediction methods of IMPs structures are to be highly valued. In this paper we apply Conditional Random Fields (CRFs) to build a probabilistic model to segment and label sequence data to solve the membrane protein helix prediction problem. The advantage of CRFs is that it allows seamless and principled integration of biological domain knowledge into the model. Our results show that the CRF model outperforms other well known helix prediction approaches on several important measures.
High-molecular-weight arginine-and lysine-specific (Kgp) gingipains are essential virulence facto... more High-molecular-weight arginine-and lysine-specific (Kgp) gingipains are essential virulence factors expressed by the oral pathogen Porphyromonas gingivalis. Haemagglutinin/adhesin (HA) regions of these proteases have been implicated in targeting catalytic domains to biological substrates and in other adhesive functions. We now report the crystal structure of the K3 adhesin domain/module of Kgp, which folds into the distinct b-jelly roll sandwich topology previously observed for K2. A conserved structural feature of K3, previously observed in the Kgp K2 module, is the half-way point anchoring of the surface exposed loops via an arginine residue found in otherwise highly variable sequences. Small-angle X-ray scattering data for the recombinant construct K1K2K3 confirmed a structure comprising a tandem repeat of three homologous modules, K1, K2 and K3 while also indicating an unusual 'y'-shape arrangement of the modules connected by variable linker sequences. Only the K2 and K3 modules and a K1K2 construct were observed to be potently haemolytic. K2, K3 and the K1K2 construct showed preferential recognition of haem-albumin over albumin whereas only low affinity binding was detected for K1 and the K1K2K3 construct. The data indicate replication of some biological functions over the three adhesin domains of Kgp while other functions are restricted.
The International Journal of Biochemistry & Cell Biology, 2009
Rhodopsin was the first G protein-coupled receptor (GPCR) for which a high-resolution crystal str... more Rhodopsin was the first G protein-coupled receptor (GPCR) for which a high-resolution crystal structure was obtained. Several crystal structures have now been solved representing different activation states of the receptor. These structures, together with those from lower resolution techniques (e.g. electron microscopy), shed light on the stepwise process by which energy from an extracellular photon is transduced across the membrane to the intracellular compartment thereby activating signalling mechanisms responsible for very low-level light detection. Controversy remains in several areas including: (i) transmembrane helix movements responsible for the transduction process, (ii) the stoichiometry of coupling to G proteins and their mode of activation, (iii) the role, if any, of receptor oligomerisation and (iv) the suitability of using structures of this GPCR as templates for modelling the structures of other GPCRs, and their mechanisms of activation.
Of the 600+ known proteases identified in mammals, a significant percentage are involved or impli... more Of the 600+ known proteases identified in mammals, a significant percentage are involved or implicated in pathogenic and cancer processes. The Dipeptidyl Peptidase IV (DPIV) gene family, comprising four enzyme members DPIV, Fibroblast Activation Protein (FAP), DP8 and DP9 and two non-enzyme members DP6 (DPL1) and DP10 (DPL2), are interesting in this regard due to their multiple diverse functions, varying patterns of distribution/localisation and subtle but significant differences in structure/substrate recognition. In addition, their engagement in cell biological processes involves both enzymatic and non-enzymatic capabilities. This article examines in detail our current understanding of the biological involvement of this unique enzyme family and their overall potential as therapeutic targets.
Dipeptidyl peptidase IV (DPPIV) is an atypical serine protease that modifies the biological activ... more Dipeptidyl peptidase IV (DPPIV) is an atypical serine protease that modifies the biological activities of certain chemokines and neuropeptides. In addition, human DPPIV, also known as the T-cell activation antigen CD26, binds adenosine deaminase (ADA) to the T-cell surface, thus protecting the T-cell from adenosine-mediated inhibition of proliferation. Mutations were engineered into DPPIV (five point, 16 single point and six deletion mutations) to examine the binding of ADA and 19 monoclonal antibodies. Deletions of C-terminal residues from the 738-residue extracellular portion of DPPIV showed that the 214 residues C-terminal to Ser552 were not required for ADA binding and that peptidase activity could be ablated by deletion of 20 residues from the C-terminus. Point mutations at either of two locations, Leu294 and Val341, ablated ADA binding. Binding by six anti-DPPIV antibodies that inhibited ADA binding was found to require Leu340 to Arg343 and Thr440/Lys441 but not the 214 residues C-terminal to Ser552. The 13 other antibodies studied bound to a truncated DPPIV consisting of amino acids 1-356. Therefore, the binding sites on DPPIV of ADA and antibodies that inhibit ADA binding are discontinuous and overlapping. Moreover, the 47 and 97 residue spacing of amino acids in these binding sites concords with their location on a beta propeller fold consisting of repeated beta sheets of about 50 amino acids.
The community-wide GPCR Dock assessment is conducted to evaluate the status of molecular modeling... more The community-wide GPCR Dock assessment is conducted to evaluate the status of molecular modeling and ligand docking for human G proteincoupled receptors. The present round of the assessment was based on the recent structures of dopamine D3 and CXCR4 chemokine receptors bound to small molecule antagonists and CXCR4 with a synthetic cyclopeptide. Thirty-five groups submitted their receptor-ligand complex structure predictions prior to the release of the crystallographic coordinates. With closely related homology modeling templates, as for dopamine D3 receptor, and with incorporation of biochemical and QSAR data, modern computational techniques predicted complex details with accuracy approaching experimental. In contrast, CXCR4 complexes that had less-characterized interactions and only distant homology to the known GPCR structures still remained very challenging. The assessment results provide guidance for modeling and crystallographic communities in method development and target selection for further expansion of the structural coverage of the GPCR universe.
Proteins: Structure, Function, and Bioinformatics, 2014
BAMLET (Bovine Alpha-lactalbumin Made LEthal to Tumors) is a member of the family of the HAMLET-l... more BAMLET (Bovine Alpha-lactalbumin Made LEthal to Tumors) is a member of the family of the HAMLET-like complexes, a novel class of protein-based anti-cancer complexes that incorporate oleic acid and deliver it to cancer cells. Small angle X-ray scattering (SAXS) was performed on the complex at pH 12, examining the high pH structure as a function of oleic acid added. The SAXS data for BAMLET species prepared with a range of oleic acid concentrations indicate extended, irregular, partially unfolded protein conformations that vary with the oleic acid concentration. Increases in oleic acid concentration correlate with increasing radius of gyration without an increase in maximum particle dimension, indicating decreasing protein density. The models for the highest oleic acid content BAMLET indicate an unusual coiled elongated structure that contrasts with apo-α-lactalbumin at pH 12, which is an elongated globular molecule, suggesting that oleic acid inhibits the folding or collapse of the protein component of BAMLET to the globular form. Circular dichroism of BAMLET and apo-α-lactalbumin was performed and the results suggest that α-lactalbumin and BAMLET unfold in a continuum of increasing degree of unfolded states. Taken together, these results support a model in which BAMLET retains oleic acid by non-specific association in the core of partially unfolded protein, and represent a new type of lipoprotein structure.
ABSTRACT Homology modeling methods have been used to construct models of two proteins--the histid... more ABSTRACT Homology modeling methods have been used to construct models of two proteins--the histidine-containing phosphocarrier protein (HPr) from Mycoplasma capricolum and human eosinophil-derived neurotoxin (EDN). Comparison of the models with the subsequently determined X-ray crystal structures indicates that the core regions of both proteins are reasonably well reproduced, although the template structures are closer to the X-ray structures in these regions--possible enhancements are discussed. The conformations of most of the side chains in the core of HPr are well reproduced in the modeled structure. As expected, the conformations of surface side chains in this protein differ significantly from the X-ray structure. The loop regions of EDN were incorrectly modeled--reasons for this and possible enhancements are discussed.
Juvenile Hormone Esterase (JHE) plays an essential role in the development of insects since it is... more Juvenile Hormone Esterase (JHE) plays an essential role in the development of insects since it is partially responsible for clearing juvenile hormone (JH), one of the hormones that is responsible for insect metamorphosis. JHE is a 60 kDa enzyme that selectively hydrolyzes the ␣/ unsaturated ester of JH. Because of its pivotal role in insect development, we have targeted JHE for use as a biopesticide. In this study, we have constructed a homology-based molecular model of JHE from the agricultural crop pest, Heliothis virescens. JHE is a member of the ␣/ hydrolase fold family of enzymes and was built according to two structures in the same family: acetylcholinesterase from Torpedo californica and lipase from Geotrichum candidum. Analysis of the active site region reveals extensive conservation between JHE and its templates. A surprise was the presence of a conserved Ser near the catalytic triad. Docking of JH III into the active site has provided insight into protein-substrate interactions that are corroborated by experimental observation. The model is being used as a predictive basis to design biopesticides. In this regard, we have identified a site on the protein surface that is suggestive of a recognition site for the putative JHE receptor. Proteins 1999; 34: 184-196. 1999 Wiley-Liss, Inc.
Bacterial elongation factor G (EF-G) physically associates with translocation-competent ribosomes... more Bacterial elongation factor G (EF-G) physically associates with translocation-competent ribosomes and facilitates transition to the subsequent codon through the coordinate binding and hydrolysis of GTP. In order to investigate the amino acid positions necessary for EF-G functions, a series of mutations were constructed in the EF-G structural gene (fusA) of Escherichia coli, specifically at positions flanking the effector domain. A mutated allele was isolated in which the wild-type sequence from codons 29 to 47 ("EFG2947") was replaced with a sequence encoding 28 amino acids from ribosomal protein S7. This mutated gene was unable to complement a fusAts strain when supplied in trans at the nonpermissive temperature. In vitro biochemical analysis demonstrated that nucleotide crosslinking was unaffected in EFG2947, while ribosome binding appeared to be completely abolished. A series of point mutations created within this region, encoding L30A, Y32A, H37A, and K38A were shown to give rise to fully functional proteins, suggesting that side chains of these individual residues are not essential for EF-G function. A sixth mutant, E41A, was found to inefficiently rescue growth in a fusAts background, and was also unable to bind ribosomes normally in vitro. In contrast E41Q could restore growth at the nonpermissive temperature. These results can be explained within the context of a three-dimensional model for the effector region of EF-G. This model indicates that the effector domain contains a negative potential field that may be important for ribosome binding.
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