RESUMO
The prothrombinase complex processes prothrombin to thrombin through sequential cleavage at Arg320 followed by Arg271 when cofactor, factor (f) Va, protease, fXa, and substrate, prothrombin, are all bound to the same membrane surface. In the absence of the membrane or cofactor, cleavage occurs in the opposite order. For the less favorable cleavage site at Arg320 to be cleaved first, it is thought that prothrombin docks on fVa in a way that presents Arg320 and hides Arg271 from the active site of fXa. Based on the crystal structure of the prothrombinase complex from the venom of the Australian eastern brown snake, pseutarin C, we modeled an initial prothrombin docking mode, which involved an interaction with discrete portions of the A1 and A2 domains of fV and the loop connecting the 2 domains, known as the a1-loop. We interrogated the proposed interface by site-directed PEGylation and by swapping the a1-loop in pseutarin C with that of human fV and fVIII and measuring the effect on rate and pathway of thrombin generation. PEGylation of residues within our proposed binding site greatly reduced the rate of thrombin generation, without affecting the pathway, whereas those outside the proposed interface had no effect. PEGylation of residues within the a1-loop also reduced the rate of thrombin generation. The sequence of the a1-loop was found to play a critical role in prothrombin binding and in the presentation of Arg320 for initial cleavage.
Assuntos
Venenos Elapídicos , Protrombina , Trombina , Austrália , Sítios de Ligação , Fator Va/metabolismo , Fator Xa/metabolismo , Humanos , Protrombina/metabolismo , Trombina/metabolismo , Tromboplastina/metabolismoRESUMO
Tick saliva is a rich source of antihemostatic, anti-inflammatory, and immunomodulatory molecules that actively help the tick to finish its blood meal. Moreover, these molecules facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-8, a salivary serpin from the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Iripin-8 displayed blood-meal-induced mRNA expression that peaked in nymphs and the salivary glands of adult females. Iripin-8 inhibited multiple proteases involved in blood coagulation and blocked the intrinsic and common pathways of the coagulation cascade in vitro. Moreover, Iripin-8 inhibited erythrocyte lysis by complement, and Iripin-8 knockdown by RNA interference in tick nymphs delayed the feeding time. Finally, we resolved the crystal structure of Iripin-8 at 1.89 Å resolution to reveal an unusually long and rigid reactive center loop that is conserved in several tick species. The P1 Arg residue is held in place distant from the serpin body by a conserved poly-Pro element on the P' side. Several PEG molecules bind to Iripin-8, including one in a deep cavity, perhaps indicating the presence of a small-molecule binding site. This is the first crystal structure of a tick serpin in the native state, and Iripin-8 is a tick serpin with a conserved reactive center loop that possesses antihemostatic activity that may mediate interference with host innate immunity.
Assuntos
Coagulação Sanguínea/fisiologia , Ativação do Complemento/fisiologia , Ixodes/metabolismo , Serpinas/metabolismo , Animais , Proteínas de Artrópodes/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Ativação do Complemento/efeitos dos fármacos , Ativação do Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Eritrócitos/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Ixodes/enzimologia , Ixodes/genética , Doença de Lyme , Ninfa , Saliva/química , Glândulas Salivares/metabolismo , Serpinas/ultraestruturaRESUMO
The aims of the study were to determine the long-term effects of dietary supplementation with microalgae (SCIM) on milk and blood fatty acid (FA) composition and reproductive hormones in early lactation dairy cows. Sixty Holstein-Friesian dairy cows (30 per treatment) were unsupplemented (Control) or supplemented with 100 g of SCIM (Schizochytrium limacinum sp.) per cow per day from 25 ± 0.5 d post-partum for 98 d. Intake and milk yield were recorded daily, with milk samples collected at weeks 0, 1, 2, 4, 8 and 14, and blood samples collected from 12 representative pairs per treatment at weeks 0, 2, 4, 8, and 14 for subsequent analysis of FA, ß-hydroxybutyrate, non-esterified fatty acids and glucose. At 33 ± 0.9 d postpartum the oestrus cycle of 24 cows (12 per treatment) were synchronized and plasma 13,14-dihydro-15-keto PGF2α (PGFM) concentrations determined following an oxytocin challenge. Data were analysed by repeated measures analysis of variance. There was no effect of treatment on dry matter intake, milk yield or milk fat content, with mean values across treatments of 22.1 and 40.6, and 37.2 g/kg respectively. Milk fat concentration of C22:6 n-3 increased rapidly in cows receiving SCIM, reaching a maximum of 0.38 g/100 g FA by week 14. Similarly, blood concentration of C22:6 n-3 increased to 1.6 g/100 g FA by week 14 in cows fed SCIM. There was no effect of treatment on plasma metabolites, but plasma glucose was lower in cows fed SCIM compared to the Control at week 2, and higher in week 8. There was no effect of treatment on peak plasma PGFM concentration or area under the curve. It is concluded that feeding SCIM rapidly increases blood and milk concentrations of C22:6 n-3 which are maintained over time, but does not improve plasma PGFM in dairy cows.
Assuntos
Suplementos Nutricionais , Dinoprosta/análogos & derivados , Ácidos Docosa-Hexaenoicos/análise , Microalgas , Leite/química , Animais , Bovinos/sangue , Bovinos/metabolismo , Dinoprosta/sangue , Ácidos Docosa-Hexaenoicos/sangue , Ácidos Graxos/análise , Ácidos Graxos/sangue , Feminino , Lactação , Microalgas/químicaRESUMO
Hemophilia is a bleeding disorder caused by deficiency in factors VIII or IX, the two components of the intrinsic Xase complex. Treatment with replacement factor can lead to the development of inhibitory antibodies, requiring the use of bypassing agents such as factor VIIa and factor concentrates. An alternative approach to bypass the Xase complex is to inhibit endogenous anticoagulant activities. Activated protein C (APC) breaks down the complex that produces thrombin by proteolytically inactivating factor Va. Defects in this mechanism (eg, factor V Leiden) are associated with thrombosis but result in less severe bleeding when co-inherited with hemophilia. Selective inhibition of APC might therefore be effective for the treatment of hemophilia. The endogenous inhibitors of APC are members of the serpin family: protein C inhibitor (PCI) and α1-antitrypsin (α1AT); however, both exhibit poor reactivity and selectivity for APC. We mutated residues in and around the scissile P1-P1' bond in PCI and α1AT, resulting in serpins with the desired specificity profile. The lead candidate was shown to promote thrombin generation in vitro and to restore fibrin and platelet deposition in an intravital laser injury model in hemophilia B mice. The power of targeting APC was further demonstrated by the complete normalization of bleeding after a severe tail clip injury in these mice. These results demonstrate that the protein C anticoagulant system can be successfully targeted by engineered serpins and that administration of such agents is effective at restoring hemostasis in vivo.
Assuntos
Hemofilia B/tratamento farmacológico , Inibidor da Proteína C/farmacologia , Proteína C/antagonistas & inibidores , Serpinas/farmacologia , Animais , Modelos Animais de Doenças , Desenho de Fármacos , Eletroforese em Gel de Poliacrilamida , Humanos , CamundongosRESUMO
Skewing of the human oral microbiome causes dysbiosis and preponderance of bacteria such as Porphyromonas gingivalis, the main etiological agent of periodontitis. P. gingivalis secretes proteolytic gingipains (Kgp and RgpA/B) as zymogens inhibited by a pro-domain that is removed during extracellular activation. Unraveling the molecular mechanism of Kgp zymogenicity is essential to design inhibitors blocking its activity. Here, we found that the isolated 209-residue Kgp pro-domain is a boomerang-shaped all-ß protein similar to the RgpB pro-domain. Using composite structural information of Kgp and RgpB, we derived a plausible homology model and mechanism of Kgp-regulating zymogenicity. Accordingly, the pro-domain would laterally attach to the catalytic moiety in Kgp and block the active site through an exposed inhibitory loop. This loop features a lysine (Lys129) likely occupying the S1 specificity pocket and exerting latency. Lys129 mutation to glutamate or arginine led to misfolded protein that was degraded in vivo Mutation to alanine gave milder effects but still strongly diminished proteolytic activity, without affecting the subcellular location of the enzyme. Accordingly, the interactions of Lys129 within the S1 pocket are also essential for correct folding. Uniquely for gingipains, the isolated Kgp pro-domain dimerized through an interface, which partially overlapped with that between the catalytic moiety and the pro-domain within the zymogen, i.e. both complexes are mutually exclusive. Thus, pro-domain dimerization, together with partial rearrangement of the active site upon activation, explains the lack of inhibition of the pro-domain in trans. Our results reveal that the specific latency mechanism of Kgp differs from those of Rgps.
Assuntos
Adesinas Bacterianas/química , Cisteína Endopeptidases/química , Precursores Enzimáticos/química , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/patogenicidade , Fatores de Virulência/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Infecções por Bacteroidaceae/enzimologia , Infecções por Bacteroidaceae/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Cisteína Endopeptidases Gingipaínas , Gengivite/enzimologia , Gengivite/genética , Humanos , Microbiota , Boca/microbiologia , Porphyromonas gingivalis/genética , Domínios Proteicos , Multimerização Proteica , Relação Estrutura-Atividade , Fatores de Virulência/metabolismoRESUMO
PURPOSE OF REVIEW: Hemophilia is a debilitating disease, marked by frequent, painful bleeding events, joint deterioration and early death. All current treatments consist of i.v. infusions of replacement factor or other procoagulant factors, and are incompletely effective, due in part to the short half-lives of the proteins. An alternative approach is to rebalance hemostasis by inhibiting natural anticoagulant mechanisms. In this article, we explain why activated protein C (APC) is an appropriate and safe target for the treatment of hemophilia. RECENT FINDINGS: A serpin (serine protease inhibitor) was engineered to specifically inhibit APC and was found to rescue hemostasis in a hemophilia mouse model, even after a severe tail clip injury. However, APC is also anti-inflammatory and has cytoprotective activities, raising safety concerns over the use of an APC inhibitor to treat hemophilia. We summarize the molecular basis of the anticoagulant and signaling activities of APC to assess the potential impact of targeting APC. SUMMARY: We conclude that the signaling and anticoagulant functions of APC are in spatially and kinetically distinct compartments, and that it is possible to specifically inhibit the anticoagulant activity of APC. Targeting APC with a serpin is remarkably effective and may be safe for long-term prophylactic use in the treatment of hemophilia.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Hemofilia A/tratamento farmacológico , Proteína C/antagonistas & inibidores , Serpinas/uso terapêutico , Animais , Modelos Animais de Doenças , Hemofilia A/sangue , Humanos , Camundongos , Proteína C/metabolismoRESUMO
Existing evidence suggests that in most cases antithrombin deficiency can be explained by mutations in its gene, SERPINC1. We investigated the molecular background of antithrombin deficiency in a single centre family cohort study. We included a total of 21 families comprising 15 original probands and sixty-six relatives, 6 of who were surrogate probands for the genetic analysis. Antithrombin activity and antigen levels were measured. The heparin-antithrombin binding ratio assay was used to distinguish between the different subtypes of type II antithrombin deficiency. SERPINC1 mutations were detected by direct sequencing of all 7 exons and regulatory regions, and multiplex ligation-dependent probe amplification. Eighty-six per cent of the families had a detrimental SERPINC1 gene mutation that segregated in the family. We detected 13 different SERPINC1 gene mutations of which 5 were novel. Among all these mutations, 44% was associated with type I deficiency, whereas the remainder was associated with type II heparin binding site (11%), type II pleiotropic effect (33%), type II reactive site (6%) or had the antithrombin Cambridge II mutation (6%). The current study reports several novel SERPINC1 mutations, thereby adding to our knowledge of the molecular background of antithrombin deficiency. Finally, our results point out the importance of future research outside the conventional SERPINC1 gene approach.
Assuntos
Deficiência de Antitrombina III/genética , Antitrombina III/genética , Mutação/genética , Adolescente , Adulto , Idoso , Proteínas Antitrombina/genética , Pré-Escolar , DNA Recombinante/genética , Éxons/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto/genética , Linhagem , Adulto JovemRESUMO
In this study, we describe a novel thrombomodulin (TM) mutation (c.1611C>A) that codes for a change from cysteine 537 to a premature stop codon (p.Cys537Stop). Three members of a family with a history of posttraumatic bleeding were identified to be heterozygous for this TM mutation. All coagulation screening tests, coagulation factor assays, and platelet function test results were within normal limits. However, the endogenous thrombin potential was markedly reduced at low-tissue factor concentration, and failure to correct with normal plasma indicated the presence of a coagulation inhibitor. Plasma TM levels were highly elevated (433-845 ng/ml, normal range 2-8 ng/ml, equating to 5 to 10 nM), and the addition of exogenous protein C further decreased thrombin generation. The mutation, p.Cys537Stop, results in a truncation within the carboxyl-terminal transmembrane helix. We predict that as a consequence of the truncation, the variant TM is shed from the endothelial surface into the blood plasma. This would promote systemic protein C activation and early cessation of thrombin generation within a developing hemostatic clot, thereby explaining the phenotype of posttraumatic bleeding observed within this family.
Assuntos
Transtornos Herdados da Coagulação Sanguínea/genética , Códon sem Sentido , Proteínas Mutantes/genética , Trombomodulina/genética , Adulto , Transtornos Herdados da Coagulação Sanguínea/sangue , Transtornos Herdados da Coagulação Sanguínea/terapia , Fatores de Coagulação Sanguínea/uso terapêutico , Testes de Coagulação Sanguínea , Análise Mutacional de DNA , Feminino , Humanos , Transplante de Rim , Masculino , Proteínas Mutantes/sangue , Proteínas Mutantes/química , Transplante de Pâncreas , Estrutura Terciária de Proteína , Trombomodulina/sangue , Trombomodulina/químicaRESUMO
Cysteine peptidases are key proteolytic virulence factors of the periodontopathogen Porphyromonas gingivalis, which causes chronic periodontitis, the most prevalent dysbiosis-driven disease in humans. Two peptidases, gingipain K (Kgp) and R (RgpA and RgpB), which differ in their selectivity after lysines and arginines, respectively, collectively account for 85% of the extracellular proteolytic activity of P. gingivalis at the site of infection. Therefore, they are promising targets for the design of specific inhibitors. Although the structure of the catalytic domain of RgpB is known, little is known about Kgp, which shares only 27% sequence identity. We report the high resolution crystal structure of a competent fragment of Kgp encompassing the catalytic cysteine peptidase domain and a downstream immunoglobulin superfamily-like domain, which is required for folding and secretion of Kgp in vivo. The structure, which strikingly resembles a tooth, was serendipitously trapped with a fragment of a covalent inhibitor targeting the catalytic cysteine. This provided accurate insight into the active site and suggested that catalysis may require a catalytic triad, Cys(477)-His(444)-Asp(388), rather than the cysteine-histidine dyad normally found in cysteine peptidases. In addition, a 20-Å-long solvent-filled interior channel traverses the molecule and links the bottom of the specificity pocket with the molecular surface opposite the active site cleft. This channel, absent in RgpB, may enhance the plasticity of the enzyme, which would explain the much lower activity in vitro toward comparable specific synthetic substrates. Overall, the present results report the architecture and molecular determinants of the working mechanism of Kgp, including interaction with its substrates.
Assuntos
Adesinas Bacterianas/química , Cisteína Endopeptidases/química , Periodontite/enzimologia , Periodontite/microbiologia , Porphyromonas gingivalis/enzimologia , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Cristalografia por Raios X , Cisteína Endopeptidases Gingipaínas , Humanos , Imunoglobulinas/química , Lisina/química , Modelos Moleculares , Dados de Sequência Molecular , Porphyromonas gingivalis/patogenicidade , Homologia de Sequência de Aminoácidos , Solventes/química , Fatores de VirulênciaRESUMO
The prothrombinase complex, composed of the protease factor (f)Xa and cofactor fVa, efficiently converts prothrombin to thrombin by specific sequential cleavage at 2 sites. How the complex assembles and its mechanism of prothrombin processing are of central importance to human health and disease, because insufficient thrombin generation is the root cause of hemophilia, and excessive thrombin production results in thrombosis. Efforts to determine the crystal structure of the prothrombinase complex have been thwarted by the dependence of complex formation on phospholipid membrane association. Pseutarin C is an intrinsically stable prothrombinase complex preassembled in the venom gland of the Australian Eastern Brown Snake (Pseudonaja textilis). Here we report the crystal structures of the fX-fV complex and of activated fXa from P textilis venom and the derived model of active pseutarin C. Structural analysis supports a single substrate binding channel on fVa, to which prothrombin and the intermediate meizothrombin bind in 2 different orientations, providing insight into the architecture and mechanism of the prothrombinase complex-the molecular engine of blood coagulation.
Assuntos
Fator V/química , Fator Xa/química , Venenos de Serpentes/enzimologia , Tromboplastina/química , Animais , Sítios de Ligação , Coagulação Sanguínea , Cristalografia por Raios X , Venenos Elapídicos/química , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , SerpentesAssuntos
Protrombina , Trombose , Ligação Genética , Humanos , Mutação , Linhagem , Protrombina/genética , Trombose/genética , Sequenciamento do ExomaRESUMO
Emphysema and liver cirrhosis can be caused by the Z mutation (Glu342Lys) in the serine protease inhibitor α1-antitrypsin (α1AT), which is found in more than 4% of the Northern European population. Homozygotes experience deficiency in the lung concomitantly with a massive accumulation of polymers within hepatocytes, causing their destruction. Recently, it was proposed that Z-α1AT polymerizes by a C-terminal domain swap. In this study, small-angle x-ray scattering (SAXS) was used to characterize Z-α1AT polymers in solution. The data show that the Z-α1AT trimer, tetramer, and pentamer all form ring-like structures in strong support of a common domain-swap polymerization mechanism that can lead to self-terminating polymers.
Assuntos
Simulação de Dinâmica Molecular , Multimerização Proteica , alfa 1-Antitripsina/química , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios X , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismoRESUMO
Thrombin is generated from prothrombin through cleavage at two sites by the prothrombinase complex. Prothrombinase is composed of a protease, factor (f) Xa, and a cofactor, fVa, which interact on negatively charged phospholipid surfaces and cleave prothrombin into thrombin 300 000 times faster than fXa alone. The balance between bleeding and thrombosis depends on the amount of thrombin produced, and this in turn depends on the function of the prothrombinase complex. How fXa and fVa interact and how improved prothrombin processing is conferred are of critical importance for understanding healthy and pathological blood clotting. Until recently, little structural information was available, and molecular models were built on partial structures with assembly guided by biochemical data. Last year our group published a crystal structure of a prothrombinase complex from the venom of the Australian Eastern Brown snake (known as Pseutarin C). Here we use the crystal structure of Pseutarin C as a starting point for homology modelling and assembly of the full human prothrombinase complex. The interface is complementary in shape and charge, and is consistent with much of the published biochemical data. The model of human prothrombinase presented here provides a powerful resource for contextualizing previous data and for designing future experiments.
Assuntos
Venenos Elapídicos/química , Fator V/química , Fator Xa/química , Animais , Cristalografia por Raios X , Fator Va/química , Humanos , Modelos Moleculares , SerpentesRESUMO
Protease nexin-1 (PN1) is a specific and extremely efficient inhibitor of thrombin. However, unlike other thrombin inhibitors belonging to the serpin family, PN1 is not synthesized in the liver and does not circulate in the blood. Rather, PN1 is expressed by multiple cell types, including macrophages, smooth muscle cells, and platelets, and it is on the surface of these cells, bound to glycosaminoglycans, that PN1 inhibits the signaling functions of thrombin. PN1 sets the threshold for thrombin-induced platelet activation and has been implicated in atherosclerosis. However, in spite of the emerging importance of PN1 in thrombosis and atherosclerosis, little is know about how it associates to cells and how it inhibits thrombin at rates that surpass the diffusion limit. To address these issues, we determined the crystal structures of PN1 in complex with heparin, and in complex with catalytically inert thrombin. The crystal structures suggest a unique 2-step mechanism of thrombin recognition involving rapid electrostatics-driven association to form an initial glycosaminoglycan-bridged complex, followed by a large conformational rearrangement to form the productive Michaelis complex.
Assuntos
Heparina/química , Serpina E2/química , Trombina/química , Sítios de Ligação , Configuração de Carboidratos , Cristalografia por Raios X , Humanos , Substâncias Macromoleculares/química , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Serpinas/química , Eletricidade EstáticaRESUMO
The classical pathway of complement is crucial to the immune system, but it also contributes to inflammatory diseases when dysregulated. Binding of the C1 complex to ligands activates the pathway by inducing autoactivation of associated C1r, after which C1r activates C1s. C1s cleaves complement component C4 and then C2 to cause full activation of the system. The interaction between C1s and C4 involves active site and exosite-mediated events, but the molecular details are unknown. In this study, we identified four positively charged amino acids on the serine protease domain that appear to form a catalytic exosite that is required for efficient cleavage of C4. These residues are coincidentally involved in coordinating a sulfate ion in the crystal structure of the protease. Together with other evidence, this pointed to the involvement of sulfate ions in the interaction with the C4 substrate, and we showed that the protease interacts with a peptide from C4 containing three sulfotyrosine residues. We present a molecular model for the interaction between C1s and C4 that provides support for the above data and poses questions for future research into this aspect of complement activation.
Assuntos
Domínio Catalítico/imunologia , Ativação do Complemento/imunologia , Complemento C1s/metabolismo , Complemento C4/metabolismo , Via Clássica do Complemento/imunologia , Serina Proteases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação de Anticorpos/imunologia , Complemento C4/imunologia , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismoRESUMO
Repeating intermolecular protein association by means of beta-sheet expansion is the mechanism underlying a multitude of diseases including Alzheimer's, Huntington's and Parkinson's and the prion encephalopathies. A family of proteins, known as the serpins, also forms large stable multimers by ordered beta-sheet linkages leading to intracellular accretion and disease. These 'serpinopathies' include early-onset dementia caused by mutations in neuroserpin, liver cirrhosis and emphysema caused by mutations in alpha(1)-antitrypsin (alpha(1)AT), and thrombosis caused by mutations in antithrombin. Serpin structure and function are quite well understood, and the family has therefore become a model system for understanding the beta-sheet expansion disorders collectively known as the conformational diseases. To develop strategies to prevent and reverse these disorders, it is necessary to determine the structural basis of the intermolecular linkage and of the pathogenic monomeric state. Here we report the crystallographic structure of a stable serpin dimer which reveals a domain swap of more than 50 residues, including two long antiparallel beta-strands inserting in the centre of the principal beta-sheet of the neighbouring monomer. This structure explains the extreme stability of serpin polymers, the molecular basis of their rapid propagation, and provides critical new insights into the structural changes which initiate irreversible beta-sheet expansion.
Assuntos
Antitrombina III/química , Antitrombina III/metabolismo , Biopolímeros/química , Biopolímeros/metabolismo , Cristalografia por Raios X , Dimerização , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
BACKGROUND: Thrombin is produced by the prothrombinase complex, composed of factor (f) Xa and fVa on a phospholipid (PL) membrane surface. Snakes of the Elapidae family have venom versions of these factors that cause coagulopathy in prey. Group C venoms contain both fXa and fVa orthologues. Group D venoms only contain a fXa orthologue and hijack fV(a) of the prey. Hopsarin D (HopD) is the venom fXa of the Stephen's Banded snake (Hoplocephalus stephensii). OBJECTIVES: We set out to address the following: Does HopD bind to human fVa with high affinity in the absence of PL? Does it process prothrombin through the meizothrombin pathway? Is the order of cleavage PL-dependent? Can HopD activate fV? METHODS: We produced and characterized full-length and truncated HopD. RESULTS: HopD is only able to clot plasma that contains fV, and competes with human fXa for fVa binding. HopD binds to both human fVa and fV with high affinity (Kdâ¼10nM), in contrast to fXa. HopD processes prothrombin down the meizothrombin route in the absence and presence of PL. Although HopD can bind to fV, conversion to fVa is necessary for prothrombin processing. HopD initiates clotting in the blood of prey by activating fV. CONCLUSIONS: HopD binds to fVa with high affinity and rapidly activates prothrombin in the absence of PL, exclusively through the meizothrombin intermediate. HopD binds with high affinity to both fVa and fV, suggesting that the B-domain does not sterically block fXa binding, but inhibits productive interaction in another way, and additionally prevents prothrombin binding.
RESUMO
Thrombin is the final protease generated in the blood coagulation cascade. It has multiple substrates and cofactors, and serves both pro- and anti-coagulant functions. How thrombin activity is directed throughout the evolution of a clot and the role of conformational change in determining thrombin specificity are issues that lie at the heart of the haemostatic balance. Over the last 20 years there have been a great number of studies supporting the idea that thrombin is an allosteric enzyme that can exist in two conformations differing in activity and specificity. However, recent work has shown that thrombin in its unliganded state is inherently flexible in regions that are important for activity. The effect of flexibility on activity is discussed in this review in context of the zymogen-to-protease conformational transition. Understanding thrombin function in terms of 'plasticity' provides a new conceptual framework for understanding regulation of enzyme activity in general. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.
Assuntos
Trombina/metabolismo , Trombina/fisiologia , Regulação Alostérica , Animais , Coagulação Sanguínea , Ativação Enzimática/fisiologia , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Proteólise , Especificidade por Substrato , Trombina/química , Trombina/genéticaRESUMO
α(1)-Antitrypsin (α1AT) deficiency is a disease with multiple manifestations, including cirrhosis and emphysema, caused by the accumulation of stable polymers of mutant protein in the endoplasmic reticulum of hepatocytes. However, the molecular basis of misfolding and polymerization remain unknown. We produced and crystallized a trimeric form of α1AT that is recognized by an antibody specific for the pathological polymer. Unexpectedly, this structure reveals a polymeric linkage mediated by domain swapping the carboxy-terminal 34 residues. Disulphide-trapping and antibody-binding studies further demonstrate that runaway C-terminal domain swapping, rather than the s4A/s5A domain swap previously proposed, underlies polymerization of the common Z-mutant of α1AT in vivo.
Assuntos
Deficiência de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , Animais , Células COS , Chlorocebus aethiops , Humanos , Modelos Moleculares , Mutação/genética , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Estrutura Terciária de Proteína , Deficiência de alfa 1-Antitripsina/metabolismoRESUMO
Factor (f) IXa is a critical enzyme for the formation of stable blood clots, and its deficiency results in hemophilia. The enzyme functions at the confluence of the intrinsic and extrinsic pathways by binding to fVIIIa and rapidly generating fXa. In spite of its importance, little is known about how fIXa recognizes its cofactor, its substrate, or its only known inhibitor, antithrombin (AT). However, it is clear that fIXa requires extensive exosite interactions to present substrates for efficient cleavage. Here we describe the 1.7-A crystal structure of fIXa in its recognition (Michaelis) complex with heparin-activated AT. It represents the highest resolution structure of both proteins and allows us to address several outstanding issues. The structure reveals why the heparin-induced conformational change in AT is required to permit simultaneous active-site and exosite interactions with fIXa and the nature of these interactions. The reactive center loop of AT has evolved to specifically inhibit fIXa, with a P2 Gly so as not to clash with Tyr99 on fIXa, a P4 Ile to fit snugly into the S4 pocket, and a C-terminal extension to exploit a unique wall-like feature of the active-site cleft. Arg150 is at the center of the exosite interface, interacting with AT residues on beta-sheet C. A surprising crystal contact is observed between the heparin pentasaccharide and fIXa, revealing a plausible mode of binding that would allow longer heparin chains to bridge the complex.