RESUMO
In the context of periodontal disease, cysteine proteinases or gingipains from Porphyromonas gingivalis have been implicated in the hydrolysis of cytokines, including gamma interferon (IFN-γ). This cytokine plays a crucial role in host defenses, in part, by regulating expression of major histocompatibility complex molecules. Our recent analysis has identified three structurally defined modules, K1, K2, and K3, of the hemagglutinin region of the lysine gingipain Kgp. These three structurally homologous domains have a common ß-sandwich topology that is similar to that found in a superfamily of adhesins and carbohydrate binding domains. The three Kgp hemagglutinin modules are distinguished by variation in some of the loops projecting from the ß-sandwich core. Recombinant products corresponding to both single and multidomain regions as well as native Kgp bound IFN-γ with similar affinities. Among the adhesin domain constructs, only the K1K2 polypeptide inhibited the upregulation of HLA-1 expression in a human erythroleukemia (K562) line induced by both recombinant and native IFN-γ. The K1K2 polypeptide also inhibited HLA-DR expression induced by IFN-γ in human umbilical vein endothelial cells. These effects were competitively inhibited by coincubation with sodium or potassium chloride solution. The N-terminal residues of IFN-γ were implicated in mediating the effect of K1K2, while antibody binding to loop 1 of K2 blocked the action of K1K2. The findings indicate the potential significance of structurally defined Kgp adhesin modules in the inactivation of IFN-γ but also the potential of K1K2 in locating the target for the catalytic domain of Kgp.
Assuntos
Adesinas Bacterianas/metabolismo , Cisteína Endopeptidases/metabolismo , Antígenos HLA/metabolismo , Interferon gama/metabolismo , Peptídeos/metabolismo , Porphyromonas gingivalis/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Animais , Anticorpos Antibacterianos/imunologia , Células Cultivadas , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Cisteína Endopeptidases Gingipaínas , Antígenos HLA/genética , Humanos , Interferon gama/genética , Células K562 , Modelos Moleculares , Peptídeos/genética , Peptídeos/imunologia , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/imunologia , Ligação Proteica , Conformação Proteica , Coelhos , Proteínas Recombinantes , Fatores de TempoRESUMO
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 ß-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.
Assuntos
Adesinas Bacterianas/química , Cisteína Endopeptidases/química , Hemaglutininas/química , Porphyromonas gingivalis/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Adesinas Bacterianas/ultraestrutura , Albuminas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Membrana Celular , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/ultraestrutura , Cisteína Endopeptidases Gingipaínas , Hemaglutininas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Porphyromonas gingivalis/metabolismo , Ligação Proteica , Subunidades Proteicas/química , Alinhamento de Sequência , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismoRESUMO
Aspergillus nidulans amine oxidase (ANAO) has the unusual ability among the family of copper and trihydroxyphenylalanine quinone-containing amine oxidases of being able to oxidize the amine side chains of lysine residues in large peptides and proteins. We show here that in common with the related enzyme from the yeast Pichia pastoris, ANAO can promote the cross-linking of tropoelastin and oxidize the lysine residues in α-casein proteins and tropoelastin. The crystal structure of ANAO, the first for a fungal enzyme in this family, has been determined to a resolution of 2.4 Å. The enzyme is a dimer with the archetypal fold of a copper-containing amine oxidase. The active site is the most open of any of those of the structurally characterized enzymes in the family and provides a ready explanation for its lysine oxidase-like activity.
Assuntos
Amina Oxidase (contendo Cobre)/isolamento & purificação , Aspergillus nidulans/enzimologia , Proteínas Fúngicas/isolamento & purificação , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/ultraestrutura , Sequência de Aminoácidos , Aspergillus nidulans/genética , Aspergillus nidulans/ultraestrutura , Domínio Catalítico/genética , Cristalografia por Raios X , Dimerização , Proteínas Fúngicas/genética , Proteínas Fúngicas/ultraestrutura , Glicosilação , Humanos , Oxigenases de Função Mista/química , Dados de Sequência Molecular , Dobramento de Proteína , Multimerização Proteica , Especificidade por Substrato/genética , Tropoelastina/química , Tropoelastina/metabolismo , Tropoelastina/ultraestruturaRESUMO
Porphyromonas gingivalis is an obligately anaerobic bacterium recognized as an aetiological agent of adult periodontitis. P. gingivalis produces cysteine proteinases, the gingipains. The crystal structure of a domain within the haemagglutinin region of the lysine gingipain (Kgp) is reported here. The domain was named K2 as it is the second of three homologous structural modules in Kgp. The K2 domain structure is a 'jelly-roll' fold with two anti-parallel beta-sheets. This fold topology is shared with adhesive domains from functionally diverse receptors such as MAM domains, ephrin receptor ligand binding domains and a number of carbohydrate binding modules. Possible functions of K2 were investigated. K2 induced haemolysis of erythrocytes in a dose-dependent manner that was augmented by the blocking of anion transport. Further, cysteine-activated arginine gingipain RgpB, which degrades glycophorin A, sensitized erythrocytes to the haemolytic effect of K2. Cleaved K2, similar to that found in extracted Kgp, lacks the haemolytic activity indicating that autolysis of Kgp may be a staged process which is artificially enhanced by extraction of the protein. The data indicate a functional role for K2 in the integrated capacity conferred by Kgp to enable the porphyrin auxotroph P. gingivalis to capture essential haem from erythrocytes.
Assuntos
Adesinas Bacterianas/química , Cisteína Endopeptidases/química , Proteínas Hemolisinas/química , Hemólise , Porphyromonas gingivalis/enzimologia , Adesinas Bacterianas/farmacologia , Adesinas Bacterianas/fisiologia , Sequência de Aminoácidos , Células Cultivadas , Cristalografia por Raios X , Cisteína Endopeptidases/farmacologia , Cisteína Endopeptidases/fisiologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/patologia , Cisteína Endopeptidases Gingipaínas , Proteínas Hemolisinas/farmacologia , Proteínas Hemolisinas/fisiologia , Humanos , Lisina/química , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Copper-containing amine oxidases (CAOs) require a protein-derived topaquinone cofactor (TPQ) for activity. TPQ biogenesis is a self-processing reaction requiring the presence of copper and molecular oxygen. Recombinant human diamine oxidase (hDAO) was heterologously expressed in Drosophila S2 cells, and analysis indicates that the purified hDAO contains substoichiometric amounts of copper and TPQ. The crystal structure of a complex of an inhibitor, aminoguanidine, and hDAO at 2.05 Å resolution shows that the aminoguanidine forms a covalent adduct with the TPQ and that the site is â¼75% occupied. Aminoguanidine is a potent inhibitor of hDAO with an IC(50) of 153 ± 9 nM. The structure indicates that the catalytic metal site, normally occupied by copper, is fully occupied. X-ray diffraction data recorded below the copper edge, between the copper and zinc edges, and above the zinc edge have been used to show that the metal site is occupied approximately 75% by copper and 25% by zinc and the formation of the TPQ cofactor is correlated with copper occupancy.
Assuntos
Amina Oxidase (contendo Cobre)/química , Amina Oxidase (contendo Cobre)/metabolismo , Benzoquinonas , Sítios de Ligação , Cobre/química , Di-Hidroxifenilalanina/análogos & derivados , Humanos , Metais , Quinonas/química , Quinonas/metabolismo , Proteínas Recombinantes , Difração de Raios X , ZincoRESUMO
Copper amine oxidases (CAOs) are ubiquitous in nature and catalyse the oxidative deamination of primary amines to the corresponding aldehydes. Humans have three viable CAO genes (AOC1-3). AOC1 encodes human diamine oxidase (hDAO), which is the frontline enzyme for histamine metabolism. hDAO is unique among CAOs in that it has a distinct substrate preference for diamines. The structure of hDAO in space group P2(1)2(1)2(1) with two molecules in the asymmetric unit has recently been reported. Here, the structure of hDAO refined to 2.1 A resolution in space group C222(1) with one molecule in the asymmetric unit is reported.
Assuntos
Amina Oxidase (contendo Cobre)/química , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Domínio Catalítico , Linhagem Celular , Cristalografia por Raios X , Drosophila melanogaster , Humanos , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Homologia Estrutural de ProteínaRESUMO
Human vascular adhesion protein 1 (VAP-1) is involved in lymphocyte-endothelial cell adhesion and has been implicated in many human inflammatory diseases. VAP-1 is a member of the copper amine oxidase family of enzymes with a trihydroxyphenylalanine quinone (TPQ) cofactor. Previously characterized crystals of VAP-1 suffered from anisotropy and contained disordered regions; in addition, one form was consistently twinned. In an effort to grow crystals that diffracted to higher resolution for inhibitor-binding studies, a construct with an N-terminal deletion was made and expressed in the Chinese hamster ovary (CHO) glycosylation mutant cell line Lec8. Screening produced crystals that displayed some anisotropy and contained seven molecules per asymmetric unit. These crystals belonged to space group C2, with unit-cell parameters a=394.5, b=115.8, c=179.3â Å, ß=112.3°. The structure was refined to a resolution of 2.9â Å, with Rcryst and Rfree values of 0.250 and 0.286, respectively.
Assuntos
Amina Oxidase (contendo Cobre)/química , Moléculas de Adesão Celular/química , Animais , Células CHO , Domínio Catalítico , Cricetinae , Cricetulus , Cristalografia por Raios X , Cisteína/química , Dissulfetos/química , Glicosilação , Humanos , Modelos Moleculares , Ligação Proteica , Multimerização ProteicaRESUMO
Humans have three functioning genes that encode copper-containing amine oxidases. The product of the AOC1 gene is a so-called diamine oxidase (hDAO), named for its substrate preference for diamines, particularly histamine. hDAO has been cloned and expressed in insect cells and the structure of the native enzyme determined by X-ray crystallography to a resolution of 1.8 A. The homodimeric structure has the archetypal amine oxidase fold. Two active sites, one in each subunit, are characterized by the presence of a copper ion and a topaquinone residue formed by the post-translational modification of a tyrosine. Although hDAO shares 37.9% sequence identity with another human copper amine oxidase, semicarbazide sensitive amine oxidase or vascular adhesion protein-1, its substrate binding pocket and entry channel are distinctly different in accord with the different substrate specificities. The structures of two inhibitor complexes of hDAO, berenil and pentamidine, have been refined to resolutions of 2.1 and 2.2 A, respectively. They bind noncovalently in the active-site channel. The inhibitor binding suggests that an aspartic acid residue, conserved in all diamine oxidases but absent from other amine oxidases, is responsible for the diamine specificity by interacting with the second amino group of preferred diamine substrates.
Assuntos
Amina Oxidase (contendo Cobre)/química , Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Sítios de Ligação , Cálcio/metabolismo , Cobre/metabolismo , Cristalografia por Raios X , Dimerização , Diminazena/análogos & derivados , Diminazena/metabolismo , Drosophila/enzimologia , Humanos , Cinética , Metalotioneína/genética , Modelos Moleculares , Pentamidina/metabolismo , Regiões Promotoras Genéticas , Conformação Proteica , Especificidade por Substrato , Difração de Raios XRESUMO
Periodontal disease is an inflammatory process affecting supporting tissues surrounding the teeth. The anaerobic gram-negative bacterium Porphyromonas gingivalis is implicated in the disease. This organism requires the uptake of porphyrins most apparently as haem 1 from local haemorrhage and it has a HA2 receptor on the outer membrane for this purpose that provides the opportunity to achieve selective anti-microbial activity. Uniquely, this receptor is based on recognition of porphyrin macrocycle and on a propionic acid side-chain rather than recognition of the coordinated metal ion through chelation, a process used by other organisms with the HasA porphyrin receptor. Porphyrin-antibiotic conjugates 11, 12, 13a and 13b were designed as potential highly selective P. gingivalis inhibitors, a key point being that they are based on the use of free-base porphyrins to render them unpalatable to other organisms. These compounds were synthesised from metronidazole 4 and deuteroporphyrin IX 3. Conjugates 11, 12, 13a and 13b are all recognised by the HA2 receptor of P. gingivalis, bind as strongly as haem 1 to HA2 and are highly effective. For example, the amide-linked mono-metronidazole mono-acid adducts 11 and 12 have the same growth inhibitory activity towards P. gingivalis and both are two-fold more active than metronidazole 4 and ten- to twenty-fold more effective than the metronidazole derivative, amine 5. The methyl esters 9 and 10, in contrast, are not recognised by HA2 and are ineffective in inhibiting P. gingivalis, leading to the conclusion that capture by HA2 may be necessary for activity of the adducts. Preliminary growth inhibition assays involving a range of bacteria have demonstrated the high selectivity of conjugates 13a and 13b towards P. gingivalis.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Metronidazol/química , Metronidazol/farmacologia , Porfirinas/química , Porphyromonas gingivalis/efeitos dos fármacos , Amidas/química , Antibacterianos/síntese química , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Ésteres/química , Metronidazol/síntese química , Metronidazol/metabolismo , Porphyromonas gingivalis/citologiaRESUMO
In this study, we report two high-resolution structures of the pyridoxal 5' phosphate (PLP)-dependent enzyme kynurenine aminotransferase-I (KAT-I). One is the native structure with the cofactor in the PLP form bound to Lys247 with the highest resolution yet available for KAT-I at 1.28 Å resolution, and the other with the general PLP-dependent aminotransferase inhibitor, aminooxyacetate (AOAA) covalently bound to the cofactor at 1.54 Å. Only small conformational differences are observed in the vicinity of the aldimine (oxime) linkage with which the PLP forms the Schiff base with Lys247 in the 1.28 Å resolution native structure, in comparison to other native PLP-bound structures. We also report the inhibition of KAT-1 by AOAA and aminooxy-phenylpropionic acid (AOPP), with IC50s of 13.1 and 5.7 µM, respectively. The crystal structure of the enzyme in complex with the inhibitor AOAA revealed that the cofactor is the PLP form with the external aldimine linkage. The location of this oxime with the PLP, which forms in place of the native internal aldimine linkage of PLP of the native KAT-I, is away from the position of the native internal aldimine, with the free Lys247 substantially retaining the orientation of the native structure. Tyr101, at the active site, was observed in two conformations in both structures.
Assuntos
Ácido Amino-Oxiacético/química , Fosfato de Piridoxal/química , Transaminases/antagonistas & inibidores , Transaminases/química , Cristalografia por Raios X , Humanos , Domínios ProteicosRESUMO
The study of protein-protein interactions can be hampered by the instability of one or more of the protein complex components. In this study, we showed that intein-mediated cyclization can be used to engineer an artificial intramolecular cyclic protein complex between two interacting proteins: the largely unstable LIM-only protein 4 (LMO4) and an unstructured domain of LIM domain binding protein 1 (ldb1). The X-ray structure of the cyclic complex is identical to noncyclized versions of the complex. Chemical and thermal denaturation assays using intrinsic tryptophan fluorescence and dynamic light scattering were used to compare the relative stabilities of the cyclized complex, the intermolecular (or free) complex, and two linear versions of the intramolecular complex (in which the interacting domains of LMO4 and ldb1 were fused, via a flexible linker, in either orientation). In terms of resistance to denaturation, the cyclic complex is the most stable variant and the intermolecular complex is the least stable; however, the two linear intramolecular variants show significant differences in stability. These differences appear to be related to the relative contact order (the average distance in sequence between residues that make contacts within a structure) of key binding residues at the interface of the two proteins. Thus, the restriction of the more stable component of a complex may enhance stability to a greater extent than restraining less stable components.
Assuntos
Ciclização , Inteínas/fisiologia , Complexos Multiproteicos/química , Desnaturação Proteica/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Homeodomínio/química , Proteínas com Domínio LIM , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Fatores de Transcrição/química , Tripsina/metabolismoRESUMO
Directed evolution is a common tool employed to generate enzymes suitable for industrial use. High thermal stability is often advantageous or even a requirement for biocatalysts, as such the evolution of protein stability is of practical as well as academic interest. Even when evolving enzymes for new or improved catalytic functions, stability is an important factor since it can limit the accumulation rate and number of desired active site mutations. Dienelactone hydrolase, a small monomeric protein, has been previously evolved via a three-stage process to possess enhanced activity and specificity toward non-physiological substrates. In addition to seven active site mutations there were three surface mutations that were thought to increase the stability of the enzyme and compensate for the destabilizing active site mutations. Here, the individual influence of the three surface mutations--Q110L, Y137C and N154D--on the thermal and chemical stability of DLH has been assessed. While the Q110L and N154D mutations improved the thermal stability, the influence of the Y137C mutation was more complex. Individually it was destabilizing both thermally and chemically, but when in the presence of the Q110L and N154D mutations its effect was neutralized in relation to thermal but not chemical stability. In the context of a directed evolution experiment, these compensatory surface mutations play important roles. However, our results show that detrimental mutations can arise, thus the simultaneous monitoring of stability changes while evolving enzymes for enhanced catalytic properties can be beneficial.
Assuntos
Substituição de Aminoácidos , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Evolução Molecular Direcionada , Mutação de Sentido Incorreto , Estabilidade Enzimática/genética , Estrutura Secundária de ProteínaRESUMO
The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/ß hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Evolução Molecular Direcionada/métodos , Bioengenharia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Cristalização , Cinética , Modelos Moleculares , Nitrofenóis/metabolismo , Conformação Proteica , Dobramento de Proteína , Bibliotecas de Moléculas Pequenas , Especificidade por SubstratoRESUMO
We demonstrate the importance of properly accounting for London dispersion and basis-set-superposition error (BSSE) in quantum-chemical optimizations of protein structures, factors that are often still neglected in contemporary applications. We optimize a portion of an ensemble of conformationally flexible lysozyme structures obtained from highly accurate X-ray crystallography data that serve as a reliable benchmark. We not only analyze root-mean-square deviations from the experimental Cartesian coordinates, but also, for the first time, demonstrate how London dispersion and BSSE influence crystallographic R factors. Our conclusions parallel recent recommendations for the optimization of small gas-phase peptide structures made by some of the present authors: Hartree-Fock theory extended with Grimme's recent dispersion and BSSE corrections (HF-D3-gCP) is superior to popular density functional theory (DFT) approaches. Not only are statistical errors on average lower with HF-D3-gCP, but also the convergence behavior is much better. In particular, we show that the BP86/6-31G* approach should not be relied upon as a black-box method, despite its widespread use, as its success is based on an unpredictable cancellation of errors. Using HF-D3-gCP is technically straightforward, and we therefore encourage users of quantum-chemical methods to adopt this approach in future applications.
Assuntos
Modelos Moleculares , Proteínas/química , Cristalografia por Raios X , Muramidase/química , Conformação Proteica , Teoria QuânticaRESUMO
Dienelactone hydrolase (DLH) is a monomeric protein with a simple α/ß-hydrolase fold structure. It readily crystallizes in space group P212121 from either a phosphate or ammonium sulfate precipitation buffer. Here, the structure of DLH at 1.85â Å resolution crystallized in space group C2 with two molecules in the asymmetric unit is reported. When crystallized in space group P212121 DLH has either phosphates or sulfates bound to the protein in crucial locations, one of which is located in the active site, preventing substrate/inhibitor binding. Another is located on the surface of the enzyme coordinated by side chains from two different molecules. Crystallization in space group C2 from a sodium citrate buffer results in new crystallographic protein-protein interfaces. The protein backbone is highly similar, but new crystal contacts cause changes in side-chain orientations and in loop positioning. In regions not involved in crystal contacts, there is little change in backbone or side-chain configuration. The flexibility of surface loops and the adaptability of side chains are important factors enabling DLH to adapt and form different crystal lattices.
Assuntos
Proteínas de Bactérias/química , Hidrolases de Éster Carboxílico/química , Pseudomonas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Citratos/química , Cristalografia por Raios X , Modelos Moleculares , Fosfatos/química , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Pseudomonas/enzimologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Citrato de Sódio , Sulfatos/químicaRESUMO
The crystal structure of GcnA, an N-acetyl-beta-D-glucosaminidase from Streptococcus gordonii, was solved by multiple wavelength anomalous dispersion phasing using crystals of selenomethionine-substituted protein. GcnA is a homodimer with subunits each comprised of three domains. The structure of the C-terminal alpha-helical domain has not been observed previously and forms a large dimerisation interface. The fold of the N-terminal domain is observed in all structurally related glycosidases although its function is unknown. The central domain has a canonical (beta/alpha)(8) TIM-barrel fold which harbours the active site. The primary sequence and structure of this central domain identifies the enzyme as a family 20 glycosidase. Key residues implicated in catalysis have different conformations in two different crystal forms, which probably represent active and inactive conformations of the enzyme. The catalytic mechanism for this class of glycoside hydrolase, where the substrate rather than the enzyme provides the cleavage-inducing nucleophile, has been confirmed by the structure of GcnA complexed with a putative reaction intermediate analogue, N-acetyl-beta-D-glucosamine-thiazoline. The catalytic mechanism is discussed in light of these and other family 20 structures.
Assuntos
Acetilglucosamina/análogos & derivados , Acetilglucosaminidase/antagonistas & inibidores , Acetilglucosaminidase/química , Endocardite Bacteriana/enzimologia , Endocardite Bacteriana/microbiologia , Streptococcus gordonii/enzimologia , Tiazóis/química , Tiazóis/farmacologia , Acetilglucosamina/química , Acetilglucosamina/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Ligantes , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
(R)-N(delta)-(N'-Sulfodiaminophosphinyl)-L-ornithine (PSorn) is the active component of a phytotoxin, called phaseolotoxin, produced by Pseudomonas savastanoi pv. phaseolicola. PSorn acts as a potent transition state (TS) inhibitor of ornithine transcarbamoylase (OTCase, E.C. 2.1.3.3) that binds to the OTCase from Escherichia coli (ARGI) with a dissociation constant of 1.6 pM. While inhibition of OTCase can lead to arginine auxotrophy, P. savastanoi pv. phaseolicola is able to synthesize toxin while growing on minimal medium. This is achieved by the expression during toxin production of a second gene encoding OTCase activity that is not inhibited by PSorn (ROTCase). ROTCase is orthologous to other OTCases, but it has substitutions to key conserved amino acids, particularly to those around the carbamoyl phosphate (CP) binding site and in the ornithine binding "SMG" loop. This suggests that the topology of the CP binding site and the closure of the SMG loop may be different in ROTCase. Steady-state kinetics indicate that ROTCase has an ordered mechanism, and the (13)C kinetic isotope effect (IE) in CP indicates that it is the first substrate to bind. However, unlike other OTCases, there is a random element to the mechanism since the second substrate ornithine (Orn) was unable to completely suppress the IE to unity. The most striking difference with ROTCase is the reduction of k(cat) to between 1% and 2% of other OTCases. This is consistent with the large IE that ROTCase exhibits (3.4%) at near-zero Orn. These results suggest that the chemistry of the reaction is rate limiting for ROTCase. ROTCase has a substrate and inhibitor profile similar to that of other OTCases. The CP binding affinity of ROTCase is diminished when compared with that observed from ARGI, and inhibitors that compete with the CP binding site have K(i) values at least 10-fold higher for ROTCase than for ARGI. Arsenate did not inhibit ROTCase, and bisubstrate and dead-end inhibitors are less effective inhibitors of ROTCase than ARGI. These data suggest that PSorn is unable to bind tightly to either the apo or activated forms of ROTCase at the expense of CP binding and reduced k(cat).
Assuntos
Farmacorresistência Bacteriana , Ornitina Carbamoiltransferase/isolamento & purificação , Ornitina Carbamoiltransferase/metabolismo , Ornitina/análogos & derivados , Ornitina/farmacologia , Pseudomonas/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Ligação Competitiva , Medição da Troca de Deutério , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Cinética , Modelos Químicos , Dados de Sequência Molecular , Ornitina/química , Ornitina/metabolismo , Ornitina Carbamoiltransferase/antagonistas & inibidores , Ornitina Carbamoiltransferase/biossíntese , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The role of Porphyromonas gingivalis cysteine proteinases (gingipains) in the evasion of host cell-mediated immunity has not been fully determined. In this study, modulation by gingipains of accessory and co-stimulatory molecule expression on human CD4(+) T cells was evaluated. Arg-gingipain rather than Lys-gingipain binds to resting CD4(+) T cells in the presence of serum. The constitutive expression of CD28 on T cells was slightly up-regulated following challenge with gingipains, whereas CD45 and CD3 were not affected. Binding of anti-CD2 and anti-CD4 monoclonal antibodies (mAbs) was reduced after challenge of T cells with gingipains, but restored to 50 and 100%, respectively, of control levels, after 48h of incubation in medium depleted of gingipains. The induced expression, by anti-CD3 mAb, of CTLA-4, CD25, and CD40 ligand (CD40L) was decreased following incubation of T cells with gingipains which also led to decreased response to anti-CD3 and anti-CD28 mAbs as shown by reduction of interleukin-2 (IL-2) production. Cumulatively, these results indicate that activated gingipains attach to T cells and preferentially cleave CD2 and CD4 molecules, with potential to impair T cell responses at periodontal sites.
Assuntos
Antígenos CD2/metabolismo , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Cisteína Endopeptidases/metabolismo , Regulação da Expressão Gênica , Hemaglutininas/metabolismo , Porphyromonas gingivalis/imunologia , Adesinas Bacterianas , Animais , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Cisteína Endopeptidases Gingipaínas , Humanos , Interleucina-2/análise , Antígenos Comuns de Leucócito/metabolismo , CamundongosRESUMO
The extracellular Ca(2+)-sensing receptor is activated allosterically by l-amino acids, and recent molecular analysis indicates that amino acids are likely to bind in the receptor's Venus flytrap domain. In the current study we set out to identify residues in the VFT domain that specifically support amino acid binding and/or amino acid-dependent receptor activation. Herein we describe two mutations of the Ca(2+)-sensing receptor (CaR) Venus Flytrap domain, T145A and S170T, that specifically impair amino acid sensing, leaving Ca2+ sensing intact, as determined by receptor-dependent activation of intracellular Ca2+ mobilization in fura-2-loaded HEK293 cells. With respect to the wild-type CaR, T145A and S170T exhibited reduced sensitivity to l-Phe, and T145A also exhibited markedly impaired l/d selectivity. When combined, the double mutant T145A/S170T exhibited normal or near-normal sensitivity to extracellular Ca2+ but was resistant to l-Phe at concentrations up to 100 mm. We conclude that T145A/S170T selectively disables l-amino acid sensing and that the Ca2+ and l-amino acid-sensing functions of the CaR can be dissociated.
Assuntos
Aminoácidos/química , Mutação , Receptores de Detecção de Cálcio/química , Receptores de Detecção de Cálcio/genética , Sítio Alostérico , Aminoacilação , Western Blotting , Cálcio/metabolismo , Linhagem Celular , Citoplasma/metabolismo , Análise Mutacional de DNA , Humanos , Cinética , Mutagênese Sítio-Dirigida , Fenilalanina/química , Ligação Proteica , Estrutura Terciária de Proteína , Sensibilidade e Especificidade , Fatores de Tempo , TransfecçãoRESUMO
BACKGROUND: The hemoglobin-binding domain (HA2) of the Porphyromonasgingivalis gingipains and hemagglutinins strongly binds hemoglobin and hemin and is thought to play a key role in acquisition of this essential metabolite by the microorganism. METHODS: In this report, we partially characterized human anti-HA2 humoral antibodies and their relationship to periodontal disease in an analysis of titer and function. RESULTS: Overall, serum anti-HA2 antibodies were relatively low and dominated by the immunoglobulin M (IgM) isotype. Pre-therapy titers had a direct association with periodontal health. Levels of P. gingivalis in the plaque were directly related to pre-therapy anti-HA2 IgG levels, and were an important covariant in a significant direct relationship between pre- and post-therapy anti-HA2 titers. Post-therapy anti-HA2 IgG antibody titers were directly related to the capacity of serum IgG fractions to neutralize hemoglobin binding by Lys-gingipain (Kgp). Further, lower levels of neutralizing activity post-therapy were directly related to severe periodontitis within the patient cohort. CONCLUSIONS: These data suggest that anti-HA2 IgG antibodies correspond directly with periodontal health, possibly through their ability to neutralize P. gingivalis hemoglobin capture. The data also suggest that inadvertent or therapeutic inoculation of P. gingivalis in the plaque may contribute to generation of neutralizing anti-HA2 IgG and improvement of periodontal prognosis.