RESUMEN
Proteases play a major role in many vital physiological processes. Trypsin-like serine proteases (TLPs), in particular, are paramount in proteolytic cascade systems such as blood coagulation and complement activation. The structural topology of TLPs is highly conserved, with the trypsin fold comprising two ß-barrels connected by a number of variable surface-exposed loops that provide a surprising capacity for functional diversity and substrate specificity. To expand our understanding of the roles these loops play in substrate and co-factor interactions, we employ a systematic methodology akin to the natural truncations and insertions observed through evolution of TLPs. The approach explores a larger deletion space than classical random or directed mutagenesis. Using FVIIa as a model system, deletions of 1-7 amino acids through the surface exposed 170 loop, a vital allosteric regulator, was introduced. All variants were extensively evaluated by established functional assays and computational loop modelling with Rosetta. The approach revealed detailed structural and functional insights recapitulation and expanding on the main findings in relation to 170 loop functions elucidated over several decades using more cumbersome crystallization and single deletion/mutation methodologies. The larger deletion space was key in capturing the most active variant, which unexpectedly had a six-amino acid truncation. This variant would have remained undiscovered if only 2-3 deletions were considered, supporting the usefulness of the methodology in general protease engineering approaches. Our findings shed further light on the complex role that surface-exposed loops play in TLP function and supports the important role of loop length in the regulation and fine-tunning of enzymatic function throughout evolution.
Asunto(s)
Factor VIIa , Serina Endopeptidasas , Serina Endopeptidasas/metabolismo , Especificidad por Sustrato , Tripsina/metabolismoRESUMEN
Two decades of research have uncovered the mechanism by which the complex of tissue factor (TF) and the plasma serine protease factor VIIa (FVIIa) mediates the initiation of blood coagulation. Membrane-anchored TF directly interacts with substrates and induces allosteric effects in the protease domain of FVIIa. These properties are also recapitulated by the soluble ectodomain of TF (sTF). At least two interdependent allosteric activation pathways originate at the FVIIa:sTF interface are proposed to enhance FVIIa activity upon sTF binding. Here, we sought to engineer an sTF-independent FVIIa variant by stabilizing both proposed pathways, with one pathway terminating at segment 215-217 in the activation domain and the other pathway terminating at the N terminus insertion site. To stabilize segment 215-217, we replaced the flexible 170 loop of FVIIa with the more rigid 170 loop from trypsin and combined it with an L163V substitution (FVIIa-VYT). The FVIIa-VYT variant exhibited 60-fold higher amidolytic activity than FVIIa, and displayed similar FX activation and antithrombin inhibition kinetics to the FVIIa.sTF complex. The sTF-independent activity of FVIIa-VYT was partly mediated by an increase in the N terminus insertion and, as shown by X-ray crystallography, partly by Tyr-172 inserting into a cavity in the activation domain stabilizing the S1 substrate-binding pocket. The combination with L163V likely drove additional changes in a delicate hydrogen-bonding network that further stabilized S1-S3 sites. In summary, we report the first FVIIa variant that is catalytically independent of sTF and provide evidence supporting the existence of two TF-mediated allosteric activation pathways.
Asunto(s)
Coagulación Sanguínea , Factor VIIa/metabolismo , Ingeniería de Proteínas , Tromboplastina/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Cristalografía por Rayos X , Factor VIIa/química , Factor VIIa/genética , Humanos , Modelos Moleculares , Mutagénesis , Desplegamiento Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
A critical step in injury-induced initiation of blood coagulation is the formation of the complex between the trypsin-like protease coagulation factor VIIa (FVIIa) and its cofactor tissue factor (TF), which converts FVIIa from an intrinsically poor enzyme to an active protease capable of activating zymogens of downstream coagulation proteases. Unlike its constitutively active ancestor trypsin, FVIIa is allosterically activated (by TF). Here, ensemble refinement of crystallographic structures, which uses multiple copies of the entire structure as a means of representing structural flexibility, is applied to explore the impacts of inhibitor binding to trypsin and FVIIa, as well as cofactor binding to FVIIa. To assess the conformational flexibility and its role in allosteric pathways in these proteases, main-chain hydrogen bond networks are analyzed by calculating the hydrogen-bond propensity. Mapping pairwise propensity differences between relevant structures shows that binding of the inhibitor benzamidine to trypsin has a minor influence on the protease flexibility. For FVIIa, in contrast, the protease domain is "locked" into the catalytically competent trypsin-like configuration upon benzamidine binding as indicated by the stabilization of key structural features: the nonprime binding cleft and the oxyanion hole are stabilized, and the effect propagates from the active site region to the calcium-binding site and to the vicinity of the disulphide bridge connecting with the light chain. TF binding to FVIIa furthermore results in stabilization of the 170 loop, which in turn propagates an allosteric signal from the TF-binding region to the active site. Analyses of disulphide bridge energy and flexibility reflect the striking stability difference between the unregulated enzyme and the allosterically activated form after inhibitor or cofactor binding. The ensemble refinement analyses show directly, for the first time to our knowledge, whole-domain structural footprints of TF-induced allosteric networks present in x-ray crystallographic structures of FVIIa, which previously only have been hypothesized or indirectly inferred.
Asunto(s)
Factor VIIa/química , Factor VIIa/metabolismo , Regulación Alostérica , Apoenzimas/química , Apoenzimas/metabolismo , Benzamidinas/farmacología , Cristalografía por Rayos X , Disulfuros/química , Activación Enzimática/efectos de los fármacos , Modelos Moleculares , Dominios Proteicos , Pliegue de Proteína , Tripsina/química , Tripsina/metabolismo , Tripsinógeno/metabolismoRESUMEN
Recombinant factor VIIa (FVIIa) variants with increased activity offer the promise to improve the treatment of bleeding episodes in patients with inhibitor-complicated hemophilia. Here, an approach was adopted to enhance the activity of FVIIa by selectively optimizing substrate turnover at the membrane surface. Under physiological conditions, endogenous FVIIa engages its cell-localized cofactor tissue factor (TF), which stimulates activity through membrane-dependent substrate recognition and allosteric effects. To exploit these properties of TF, a covalent complex between FVIIa and the soluble ectodomain of TF (sTF) was engineered by introduction of a nonperturbing cystine bridge (FVIIa Q64C-sTF G109C) in the interface. Upon coexpression, FVIIa Q64C and sTF G109C spontaneously assembled into a covalent complex with functional properties similar to the noncovalent wild-type complex. Additional introduction of a FVIIa-M306D mutation to uncouple the sTF-mediated allosteric stimulation of FVIIa provided a final complex with FVIIa-like activity in solution, while exhibiting a two to three orders-of-magnitude increase in activity relative to FVIIa upon exposure to a procoagulant membrane. In a mouse model of hemophilia A, the complex normalized hemostasis upon vascular injury at a dose of 0.3 nmol/kg compared with 300 nmol/kg for FVIIa.
Asunto(s)
Terapia Biológica/métodos , Factor VIIa/química , Hemofilia A/terapia , Ingeniería de Proteínas/métodos , Tromboplastina/química , Regulación Alostérica , Animales , Coagulación Sanguínea/efectos de los fármacos , Modelos Animales de Enfermedad , Factor VIIa/genética , Factor VIIa/farmacología , Factor VIIa/uso terapéutico , Femenino , Hemofilia A/fisiopatología , Humanos , Cinética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Simulación de Dinámica Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Tromboplastina/genética , Tromboplastina/farmacología , Tromboplastina/uso terapéuticoRESUMEN
The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215-217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215-217 conformation is pivotal to the enhanced activity of the FVIIa variants. We speculate that the allosteric regulation of FVIIa activity by TF binding follows a similar path in conjunction with protease domain N terminus insertion, suggesting a more complete molecular basis of TF-mediated allosteric enhancement of FVIIa activity.
Asunto(s)
Factor VIIa/metabolismo , Modelos Moleculares , Tromboplastina/metabolismo , Tripsina/metabolismo , Regulación Alostérica , Dominio Catalítico , Cristalografía por Rayos X , Estabilidad de Enzimas , Factor VIIa/química , Factor VIIa/genética , Humanos , Cinética , Simulación de Dinámica Molecular , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Desplegamiento Proteico , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidad , Tromboplastina/química , Tromboplastina/genética , Tripsina/química , Tripsina/genéticaRESUMEN
Calmodulin (CaM) is the central mediator of intracellular Ca(2+) signalling in cardiomyocytes, where it conveys the intricate Ca(2+) transients to the proteins controlling cardiac contraction. We recently linked two separate mutations in CaM (N53I and N97S) to dominantly inherited catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmic disorder in which exercise or acute emotion can lead to syncope and sudden cardiac death. Given the ubiquitous presence of CaM in all eukaryote cells, it is particular intriguing that carriers of either mutation show no additional symptoms. Here, we investigated the effects of the CaM CPVT mutations in a zebrafish animal model. Three-day-old embryos injected with either CaM mRNA showed no detectable pathologies or developmental abnormalities. However, embryos injected with CPVT CaM mRNA displayed increased heart rate compared to wild-type CaM mRNA under ß-adrenergic stimulation, demonstrating a conserved dominant cardiac specific effect between zebrafish and human carriers of these mutations. Motivated by the highly similar physiological phenotypes, we compared the effects of the N53I and N97S mutations on the biophysical and functional properties of CaM. Surprisingly, the mutations have opposing effects on CaM C-lobe Ca(2+) binding affinity and kinetics, and changes to the CaM N-lobe Ca(2+) binding are minor and specific to the N53I mutation. Furthermore, both mutations induce differential perturbations to structure and stability towards unfolding. Our results suggest different molecular disease mechanisms for the CPVT (N53I and N97S mutations) and strongly support that cardiac contraction is the physiological process most sensitive to CaM integrity.
Asunto(s)
Calmodulina/química , Calmodulina/metabolismo , Taquicardia Ventricular/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismo , Animales , Señalización del Calcio/genética , Señalización del Calcio/fisiología , Calmodulina/genética , Mutación , Pliegue de Proteína , Taquicardia Ventricular/genética , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Calmodulin is the primary sensor of intracellular calcium (Ca(2+)) levels in eukaryotic cells playing a key role in the proper deciphering of Ca(2+) signalling. Given the versatility of Ca(2+) as a secondary messenger, it is not surprising that calmodulin interacts with a vast number of proteins. Calmodulin is an extraordinarily conserved protein, which has not evolved since the genesis of the vertebrate lineage, and further is encoded by three different non-allelic genes in the human genome. The protein displays a high degree of conformational plasticity, allowing for target proteins to evolve specific modes of calmodulin interaction and regulation during Ca(2+) sensing. The recent identification of two calmodulin mutations giving rise to a heart arrhythmia with catecholaminergic polymorphic ventricular tachycardia-like symptoms and sudden cardiac death in young individuals, and the following identification of another three calmodulin mutations linked to recurrent cardiac arrest in infants, is in many ways intriguing. How can mutations result in cardiac-specific phenotypes when calmodulin is fundamental for correct Ca(2+) signal interpretation in virtually all cells in vertebrate organisms? Are there specific cardiac target protein interactions that are affected by these mutations? Another challenge is to elucidate how one mutated allele out of six encoding an identical calmodulin protein results in a dominant phenotype. Here we aim to give an overview of components in the cardiac contraction cycle whose function is modulated by calmodulin. In principle, these may all be implicated in the pathogenic molecular mechanism linking calmodulin mutations to cardiac arrhythmia and sudden cardiac death.
Asunto(s)
Arritmias Cardíacas/etiología , Calcio/metabolismo , Calmodulina/genética , Mutación/genética , Animales , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patología , Calmodulina/metabolismo , Humanos , Contracción MuscularRESUMEN
We demonstrate the distribution of the important extracellular matrix protein laminin in a novel biomaterial consisting of a hydrogel underpinned by nanofibrillar networks. These are formed by the immobilised enzyme mediated self-assembly of fmoc-L(3) (9-fluorenylmethoxycarbonyl-tri-leucine). The peptide assembly yields nanofibrils formed of ß-sheets that are locked together via π-stacking interactions. This ordering allows the localisation of the peptide sidechains on the surface, creating a hydrophobic environment. This induces the formation of bundles of these nanofibrils producing a clear hydrogel. This mechanism enables the three dimensional distribution of laminin throughout the network via supramolecular interactions. These forces favour the formation and improve the order of the network itself, as observed by spectroscopic and mechanical testing. In order to test the stability and suitability of this class of material for in vivo applications, we utilise microinjection to deliver the biomaterial under fine spatial control into a dystrophic zebrafish model organism, which lacks laminin as a result of a genetic mutation. Using confocal and transmission electron microscopy, we confirm that the biomaterial remains stable structurally, and is confined spatially to the site of injection.
Asunto(s)
Hidrogeles/química , Péptidos/química , Conformación Proteica , Proteínas/química , Animales , Animales Modificados Genéticamente , Fluorenos/química , Hidrogeles/síntesis química , Laminina/genética , Laminina/metabolismo , Leucina/química , Ensayo de Materiales , Estructura Molecular , Nanofibras/química , Nanofibras/ultraestructura , Pez Cebra/anatomía & histología , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Biotransformation of the sesquiterpenoid trans-nerolidol by Aspergillus niger has previously been investigated as a method for the formation of 12-hydroxy-trans-nerolidol, a precursor in the synthesis of the industrially interesting flavor alpha-sinensal. We characterized biotransformations of cis-nerolidol, trans-nerolidol, and a commercially available cis/trans-nerolidol mixture in repeated batch cultures of A. niger grown in computer-controlled bioreactors. On-line quantification of titrant addition in pH control allowed characterization of (1) maximal specific growth rate in exponential growth phases, (2) exponential induction of acid formation in postexponential phases, (3) inhibition of organic acid formation after nerolidol addition, and (4) exponential recovery from this inhibition. Addition of a (+/-)-cis/trans-nerolidol mixture during exponential or postexponential phase to cultures grown in minimal medium at high dissolved oxygen tension (above 50% air saturation), to cultures at low dissolved oxygen tension (5% air saturation), or to cultures grown in rich medium demonstrated that the physiological state before nerolidol addition had a major influence on biotransformation. The maximal molar yield of 12-hydroxy-trans-nerolidol (9%) was obtained by addition of a (+/-)-cis/trans-nerolidol mixture to the culture in the postexponential phase at high dissolved oxygen tension in minimal medium. Similar yields were obtained in rich medium, where the rate of biotransformation was doubled.