RESUMEN
ABSTRACT: Activation of von Willebrand factor (VWF) is a tightly controlled process governed primarily by local elements around its A1 domain. Recent studies suggest that the O-glycosylated sequences flanking the A1 domain constitute a discontinuous and force-sensitive autoinhibitory module (AIM), although its extent and conformation remains controversial. Here, we used a targeted screening strategy to identify 2 groups of nanobodies. One group, represented by clone 6D12, is conformation insensitive and binds the N-terminal AIM (NAIM) sequence that is distal from A1; 6D12 activates human VWF and induces aggregation of platelet-rich plasma at submicromolar concentrations. The other group, represented by clones Nd4 and Nd6, is conformation sensitive and targets the C-terminal AIM (CAIM). Nd4 and Nd6 inhibit ristocetin-induced platelet aggregation and reduce VWF-mediated platelet adhesion under flow. A crystal structure of Nd6 in complex with AIM-A1 shows a novel conformation of both CAIM and NAIM that are primed to interact, providing a model of steric hindrance stabilized by the AIM as the mechanism for regulating GPIbα binding to VWF. Hydrogen-deuterium exchange mass spectrometry analysis shows that binding of 6D12 induces the exposure of the GPIbα-binding site in the A1 domain, but binding of inhibitory nanobodies reduces it. Overall, these results suggest that the distal portion of NAIM is involved in specific interactions with CAIM, and binding of nanobodies to the AIM could either disrupt its conformation to activate VWF or stabilize its conformation to upkeep VWF autoinhibition. These reported nanobodies could facilitate future studies of VWF functions and related pathologies.
Asunto(s)
Anticuerpos de Dominio Único , Factor de von Willebrand , Factor de von Willebrand/metabolismo , Factor de von Willebrand/química , Humanos , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Conformación Proteica , Dominios Proteicos , Unión Proteica , Adhesividad Plaquetaria/efectos de los fármacos , Cristalografía por Rayos X , Animales , Plaquetas/metabolismoRESUMEN
Rondaptivan pegol (previously BT200) is a PEGylated RNA aptamer that binds to the A1 domain of VWF. Recent clinical trials demonstrated that BT200 significantly increased plasma VWF-FVIII levels by attenuating VWF clearance. The biological mechanism(s) through which BT200 attenuates in vivo clearance of VWF have not been defined. We hypothesized that BT200 interaction with the VWF-A1 domain may increase plasma VWF levels by attenuating macrophage-mediated clearance. We observed that full length- and VWF-A1A2A3 binding to macrophages, and VWF-A1 domain binding to LRP1 cluster II and cluster IV, were concentration-dependently inhibited by BT200. Additionally, full length VWF binding to LRP1 expressed on HEK293T (HEK-LRP1) cells was also inhibited by BT200. Importantly, BT200 interacts with the VWF-A1 domain in proximity to a conserved cluster of four lysine residues (K1405, K1406, K1407 and K1408). Alanine mutagenesis of this K1405-K1408 cluster (VWF-4A) significantly (p<0.001) attenuated binding of VWF to both LRP1 clusters II and IV. Furthermore, in vivo clearance of VWF-4A was significantly (p<0.001) reduced compared to wild type VWF. BT200 did not significantly inhibit binding of VWF-4A to LRP1 cluster IV or HEK-LRP1 cells. Finally, BT200 interaction with the VWF-A1 domain also inhibited binding to macrophage galactose lectin (MGL) and the SR-AI scavenger receptor. Collectively, our findings demonstrate that BT200 prolongs VWF half-life by attenuating macrophage-mediated clearance and specifically the interaction of K1405-1408 in the VWF-A1 domain with macrophage LRP1. These data support the concept that targeted inhibition of VWF clearance pathways represent a novel therapeutic approach for VWD and hemophilia A.
RESUMEN
Ubiquitin-specific proteases (USPs) are crucial for controlling cellular proteostasis and signaling pathways but how deubiquitination is selective remains poorly understood, in particular between paralogues. Here, we developed a fusion tag method by mining the Protein Data Bank and trapped USP11, a key regulator of DNA double-strand break repair, in complex with a novel engineered substrate mimetic. Together, this enabled structure determination of USP11 as a Michaelis-like complex that revealed key S1 and S1' binding site interactions with a substrate. Combined mutational, enzymatic, and binding experiments identified Met77 in linear diubiquitin as a significant residue that leads to substrate discrimination. We identified an aspartate "gatekeeper" residue in the S1' site of USP11 as a contributing feature for discriminating against linear diubiquitin. When mutated to a glycine, the corresponding residue in paralog USP15, USP11 acquired elevated activity toward linear diubiquitin in-gel shift assays, but not controls. The reverse mutation in USP15 confirmed that this position confers paralog-specific differences impacting diubiquitin cleavage rates. The results advance our understanding of the molecular basis for the higher selectivity of USP11 compared to USP15 and may aid targeted inhibitor development. Moreover, the reported carrier-based crystallization strategy may be applicable to other challenging targets.
Asunto(s)
Modelos Moleculares , Proteasas Ubiquitina-Específicas , Sitios de Unión , Proteasas Ubiquitina-Específicas/química , Proteasas Ubiquitina-Específicas/metabolismo , Humanos , Ubiquitinación/genética , Estructura Terciaria de Proteína , Cristalografía por Rayos X , Especificidad por Sustrato/genéticaRESUMEN
For decades, it was considered that plasma kallikrein's (PKa) sole function within the coagulation cascade is the activation of factor (F)XII. Until recently, the two key known activators of FIX within the coagulation cascade were activated FXI(a) and the tissue factor-FVII(a) complex. Simultaneously, and using independent experimental approaches, three groups identified a new branch of the coagulation cascade, whereby PKa can directly activate FIX. These key studies identified that (1) FIX or FIXa can bind with high affinity to either prekallikrein (PK) or PKa; (2) in human plasma, PKa can dose dependently trigger thrombin generation and clot formation independent of FXI; (3) in FXI knockout murine models treated with intrinsic pathway agonists, PKa activity results in increased formation of FIXa:AT complexes, indicating direct activation of FIX by PKa in vivo. These findings suggest that there is both a canonical (FXIa-dependent) and non-canonical (PKa-dependent) pathway of FIX activation. These three recent studies are described within this review, alongside historical data that hinted at the existence of this novel role of PKa as a coagulation clotting factor. The implications of direct PKa cleavage of FIX remain to be determined physiologically, pathophysiologically, and in the context of next-generation anticoagulants in development.
RESUMEN
Glycoprotein VI (GPVI) is the major signaling receptor for collagen on platelets. We have raised 54 nanobodies (Nb), grouped into 33 structural classes based on their complementary determining region 3 loops, against recombinant GPVI-Fc (dimeric GPVI) and have characterized their ability to bind recombinant GPVI, resting and activated platelets, and to inhibit platelet activation by collagen. Nbs from 6 different binding classes showed the strongest binding to recombinant GPVI-Fc, suggesting that there was not a single dominant class. The most potent 3, Nb2, 21, and 35, inhibited collagen-induced platelet aggregation with nanomolar half maximal inhibitory concentration (IC50) values and inhibited platelet aggregation under flow. The binding KD of the most potent Nb, Nb2, against recombinant monomeric and dimeric GPVI was 0.6 and 0.7 nM, respectively. The crystal structure of monomeric GPVI in complex with Nb2 revealed a binding epitope adjacent to the collagen-related peptide (CRP) binding groove within the D1 domain. In addition, a novel conformation of GPVI involving a domain swap between the D2 domains was observed. The domain swap is facilitated by the outward extension of the C-C' loop, which forms the domain swap hinge. The functional significance of this conformation was tested by truncating the hinge region so that the domain swap cannot occur. Nb2 was still able to displace collagen and CRP binding to the mutant, but signaling was abolished in a cell-based NFAT reporter assay. This demonstrates that the C-C' loop region is important for GPVI signaling but not ligand binding and suggests the domain-swapped structure may represent an active GPVI conformation.
Asunto(s)
Complejo Antígeno-Anticuerpo , Plaquetas , Glicoproteínas de Membrana Plaquetaria , Multimerización de Proteína , Anticuerpos de Dominio Único , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/metabolismo , Plaquetas/química , Plaquetas/metabolismo , Humanos , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Dominios Proteicos , Multimerización de Proteína/efectos de los fármacos , Multimerización de Proteína/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/farmacologíaRESUMEN
ADAMTS13 is a plasma metalloprotease with the primary function of cleaving VWF to maintain hemostasis. Circulating ADAMTS13 is in the closed conformation until blood vessel injury triggers a VWF-dependant activation to the open active form of the protein. ADAMTS13 is a multi-domain protein with the domains broadly functioning to interact and cleave VWF or maintain global latency of ADAMTS13. Thrombotic Thrombocytopenic Purpura is a disease characterized by excessive thrombi formation in the microvasculature, diagnosis is made when ADAMTS13 activity is <10%. In the hereditary form, a variety of mutations are found throughout all domains of ADAMTS13, examples are given alongside details of each domain in this article. ADAMTS13 mutations can inhibit the binding and cleavage of VWF directly or indirectly through reduced secretion, leading to increased size of VWF multimers and platelet recruitment. Molecular characterization of ADAMTS13 may provide insight into the mechanisms of TTP to aid in both scientific and clinical research.
Asunto(s)
Púrpura Trombocitopénica Trombótica , Humanos , Púrpura Trombocitopénica Trombótica/genética , Factor de von Willebrand/genética , Factor de von Willebrand/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAMTS13/genética , Mutación , Mutación de Línea GerminalRESUMEN
The contact system is composed of factor XII (FXII), prekallikrein (PK), and cofactor high-molecular-weight kininogen (HK). The globular C1q receptor (gC1qR) has been shown to interact with FXII and HK. We reveal the FXII fibronectin type II domain (FnII) binds gC1qR in a Zn2+-dependent fashion and determined the complex crystal structure. FXIIFnII binds the gC1qR trimer in an asymmetric fashion, with residues Arg36 and Arg65 forming contacts with 2 distinct negatively charged pockets. gC1qR residues Asp185 and His187 coordinate a Zn2+ adjacent to the FXII-binding site, and a comparison with the ligand-free gC1qR crystal structure reveals the anionic G1-loop becomes ordered upon FXIIFnII binding. Additional conformational changes in the region of the Zn2+-binding site reveal an allosteric basis for Zn2+ modulation of FXII binding. Mutagenesis coupled with surface plasmon resonance demonstrate the gC1qR Zn2+ site contributes to FXII binding, and plasma-based assays reveal gC1qR stimulates coagulation in a FXII-dependent manner. Analysis of the binding of HK domain 5 (HKD5) to gC1qR shows only 1 high-affinity binding site per trimer. Mutagenesis studies identify a critical G3-loop located at the center of the gC1qR trimer, suggesting steric occlusion as the mechanism for HKD5 asymmetric binding. Gel filtration experiments reveal that gC1qR clusters FXII and HK into a higher-order 500-kDa ternary complex. These results support the conclusion that extracellular gC1qR can act as a chaperone to cluster contact factors, which may be a prelude for initiating the cascades that drive bradykinin generation and the intrinsic pathway of coagulation.
Asunto(s)
Sitio Alostérico , Sitios de Unión , Proteínas Portadoras/química , Factor XII/química , Quininógenos/química , Glicoproteínas de Membrana/química , Proteínas Mitocondriales/química , Modelos Moleculares , Receptores de Complemento/química , Anciano , Proteínas Portadoras/metabolismo , Factor XII/metabolismo , Femenino , Humanos , Cinética , Quininógenos/metabolismo , Ligandos , Glicoproteínas de Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Modelos Biológicos , Simulación de Dinámica Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica , Conformación Proteica , Receptores de Complemento/metabolismo , Proteínas Recombinantes , Relación Estructura-Actividad , Zinc/química , Zinc/metabolismoRESUMEN
A careful balance between active-site and exosite contributions is critically important for the specificity of many proteases, but this balance is not yet defined for some of the serine proteases that serve as coagulation factors. Basavaraj and Krishnaswamy have closed an important gap in our knowledge of coagulation factor X activation by the intrinsic Xase complex by showing that exosite binding plays a critical role in this process, which they describe as a "dock and lock." This finding not only significantly enhances our understanding of this step in the coagulation cascade and highlights parallels with the prothrombinase complex, but will also provide a novel rationale for inhibitor development in the future.
Asunto(s)
Coagulación Sanguínea , Factor X , Cisteína Endopeptidasas , Proteínas de NeoplasiasRESUMEN
Leishmania mexicana is an obligate intracellular protozoan parasite that causes the cutaneous form of leishmaniasis affecting South America and Mexico. The cysteine protease LmCPB is essential for the virulence of the parasite and therefore, it is an appealing target for antiparasitic therapy. A library of nitrile-based cysteine protease inhibitors was screened against LmCPB to develop a treatment of cutaneous leishmaniasis. Several compounds are sufficiently high-affinity LmCPB inhibitors to serve both as starting points for drug discovery projects and as probes for target validation. A 1.4 Å X ray crystal structure, the first to be reported for LmCPB, was determined for the complex of this enzyme covalently bound to an azadipeptide nitrile ligand. Mapping the structure-activity relationships for LmCPB inhibition revealed superadditive effects for two pairs of structural transformations. Therefore, this work advances our understanding of azadipeptidyl and dipeptidyl nitrile structure-activity relationships for LmCPB structure-based inhibitor design. We also tested the same series of inhibitors on related cysteine proteases cathepsin L and Trypanosoma cruzi cruzain. The modulation of these mammalian and protozoan proteases represents a new framework for targeting papain-like cysteine proteases.
Asunto(s)
Compuestos Aza/farmacología , Catepsina B/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/farmacología , Leishmania mexicana/efectos de los fármacos , Tripanocidas/farmacología , Compuestos Aza/síntesis química , Compuestos Aza/química , Catepsina B/metabolismo , Cristalografía por Rayos X , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/química , Dipéptidos/síntesis química , Dipéptidos/química , Dipéptidos/farmacología , Relación Dosis-Respuesta a Droga , Leishmania mexicana/enzimología , Simulación de Dinámica Molecular , Estructura Molecular , Nitrilos/síntesis química , Nitrilos/química , Nitrilos/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Relación Estructura-Actividad , Tripanocidas/síntesis química , Tripanocidas/químicaRESUMEN
Ubiquitin-specific protease 15 (USP15) regulates important cellular processes, including transforming growth factor ß (TGF-ß) signaling, mitophagy, mRNA processing, and innate immune responses; however, structural information on USP15's catalytic domain is currently unavailable. Here, we determined crystal structures of the USP15 catalytic core domain, revealing a canonical USP fold, including a finger, palm, and thumb region. Unlike for the structure of paralog USP4, the catalytic triad is in an inactive configuration with the catalytic cysteine â¼10 Å apart from the catalytic histidine. This conformation is atypical, and a similar misaligned catalytic triad has so far been observed only for USP7, although USP15 and USP7 are differently regulated. Moreover, we found that the active-site loops are flexible, resulting in a largely open ubiquitin tail-binding channel. Comparison of the USP15 and USP4 structures points to a possible activation mechanism. Sequence differences between these two USPs mainly map to the S1' region likely to confer specificity, whereas the S1 ubiquitin-binding pocket is highly conserved. Isothermal titration calorimetry monoubiquitin- and linear diubiquitin-binding experiments showed significant differences in their thermodynamic profiles, with USP15 displaying a lower affinity for monoubiquitin than USP4. Moreover, we report that USP15 is weakly inhibited by the antineoplastic agent mitoxantrone in vitro A USP15-mitoxantrone complex structure disclosed that the anthracenedione interacts with the S1' binding site. Our results reveal first insights into USP15's catalytic domain structure, conformational changes, differences between paralogs, and small-molecule interactions and establish a framework for cellular probe and inhibitor development.
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Dominio Catalítico , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteasas Ubiquitina-Específicas/química , Humanos , Unión Proteica , Homología de Secuencia de Aminoácido , Ubiquitina/metabolismo , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
When blood is exposed to variety of artificial surfaces and biologic substances, the plasma proteins factor XII (FXII) and prekallikrein undergo reciprocal proteolytic conversion to the proteases αFXIIa and α-kallikrein by a process called contact activation. These enzymes contribute to host-defense responses including coagulation, inflammation, and fibrinolysis. The initiating event in contact activation is debated. To test the hypothesis that single-chain FXII expresses activity that could initiate contact activation, we prepared human FXII variants lacking the Arg353 cleavage site required for conversion to αFXIIa (FXII-R353A), or lacking the 3 known cleavage sites at Arg334, Arg343, and Arg353 (FXII-T, for "triple" mutant), and compared their properties to wild-type αFXIIa. In the absence of a surface, FXII-R353A and FXII-T activate prekallikrein and cleave the tripeptide S-2302, demonstrating proteolytic activity. The activity is several orders of magnitude weaker than that of αFXIIa. Polyphosphate, an inducer of contact activation, enhances PK activation by FXII-T, and facilitates FXII-T activation of FXII and FXI. In plasma, FXII-T and FXII-R353A, but not FXII lacking the active site serine residue (FXII-S544A), shortened the clotting time of FXII-deficient plasma and enhanced thrombin generation in a surface-dependent manner. The effect was not as strong as for wild-type FXII. Our results support a model for induction of contact activation in which activity intrinsic to single-chain FXII initiates αFXIIa and α-kallikrein formation on a surface. αFXIIa, with support from α-kallikrein, subsequently accelerates contact activation and is responsible for the full procoagulant activity of FXII.
Asunto(s)
Coagulación Sanguínea , Factor XII/metabolismo , Proteolisis , Dominio Catalítico/genética , Factor XIIa/metabolismo , Humanos , Calicreínas/metabolismo , Propiedades de SuperficieRESUMEN
The purpose of antithrombotic therapy is the prevention of thrombus formation and/or its extension with a minimum risk of bleeding. The inhibition of a variety of proteolytic processes, particularly those of the coagulation cascade, has been reported as a property of plant protease inhibitors. The role of trypsin inhibitors (TIs) from Delonix regia (Dr) and Acacia schweinfurthii (As), members of the Kunitz family of protease inhibitors, was investigated on blood coagulation, platelet aggregation, and thrombus formation. Different from Acacia schweinfurthii trypsin inhibitor (AsTI), Delonix regia trypsin inhibitor (DrTI) is a potent inhibitor of FXIa with a Kiapp of 1.3 × 10-9 M. In vitro, both inhibitors at 100 µg corresponding to the concentrations of 21 µM and 15.4 µM of DrTI and AsTI, respectively, increased approximately 2.0 times the activated partial thromboplastin time (aPTT) in human plasma compared to the control, likely due to the inhibition of human plasma kallikrein (huPK) or activated factor XI (FXIa), in the case of DrTI. Investigating in vivo models of arterial thrombus formation and bleeding time, DrTI and AsTI, 1.3 µM and 0.96 µM, respectively, prolonged approximately 50% the time for total carotid artery occlusion in mice compared to the control. In contrast to heparin, the bleeding time in mice treated with the two inhibitors did not differ from that of the control group. DrTI and AsTI inhibited 49.3% and 63.8%, respectively, ex vivo murine platelet aggregation induced by adenosine diphosphate (ADP), indicating that these protein inhibitors prevent arterial thrombus formation possibly by interfering with the plasma kallikrein (PK) proteolytic action on the intrinsic coagulation pathway and its ability to enhance the platelet aggregation activity on the intravascular compartment leading to the improvement of a thrombus.
Asunto(s)
Plantas/química , Calicreína Plasmática/metabolismo , Inhibidores de Proteasas/uso terapéutico , Trombosis/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Inhibidores de Proteasas/farmacologíaRESUMEN
Factor XI (FXI) is the zymogen of FXIa, which cleaves FIX in the intrinsic pathway of coagulation. FXI is known to exist as a dimer and interacts with multiple proteins via its 4 apple domains in the "saucer section" of the enzyme; however, to date, no complex crystal structure has been described. To investigate protein interactions of FXI, a large random peptide library consisting of 10(6) to 10(7) peptides was screened for FXI binding, which identified a series of FXI binding motifs containing the signature Asp-Phe-Pro (DFP) tripeptide. Motifs containing this core tripeptide were found in diverse proteins, including the known ligand high-molecular-weight kininogen (HK), as well as the extracellular matrix proteins laminin and collagen V. To define the binding site on FXI, we determined the crystal structure of FXI in complex with the HK-derived peptide NPISDFPDT. This revealed the location of the DFP peptide bound to the FXI apple 2 domain, and central to the interaction, the DFP phenylalanine side-chain inserts into a major hydrophobic pocket in the apple 2 domain and the isoleucine occupies a flanking minor pocket. Two further structures of FXI in complex with the laminin-derived peptide EFPDFP and a DFP peptide from the random screen demonstrated binding in the same pocket, although in a slightly different conformation, thus revealing some flexibility in the molecular interactions of the FXI apple 2 domain.
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Factor XI/química , Factor XI/metabolismo , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Asparagina/química , Sitios de Unión , Células CHO , Cricetinae , Cricetulus , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Fragmentos de Péptidos/química , Fenilalanina/química , Prolina/química , Unión Proteica , Dominios Proteicos , Estructura Cuaternaria de ProteínaRESUMEN
Pseudomonas aeruginosaproduces a number of alkylquinolone-type secondary metabolites best known for their antimicrobial effects and involvement in cell-cell communication. In the alkylquinolone biosynthetic pathway, the ß-ketoacyl-(acyl carrier protein) synthase III (FabH)-like enzyme PqsBC catalyzes the condensation of octanoyl-coenzyme A and 2-aminobenzoylacetate (2-ABA) to form the signal molecule 2-heptyl-4(1H)-quinolone. PqsBC, a potential drug target, is unique for its heterodimeric arrangement and an active site different from that of canonical FabH-like enzymes. Considering the sequence dissimilarity between the subunits, a key question was how the two subunits are organized with respect to the active site. In this study, the PqsBC structure was determined to a 2 Å resolution, revealing that PqsB and PqsC have a pseudo-2-fold symmetry that unexpectedly mimics the FabH homodimer. PqsC has an active site composed of Cys-129 and His-269, and the surrounding active site cleft is hydrophobic in character and approximately twice the volume of related FabH enzymes that may be a requirement to accommodate the aromatic substrate 2-ABA. From physiological and kinetic studies, we identified 2-aminoacetophenone as a pathway-inherent competitive inhibitor of PqsBC, whose fluorescence properties could be used forin vitrobinding studies. In a time-resolved setup, we demonstrated that the catalytic histidine is not involved in acyl-enzyme formation, but contributes to an acylation-dependent increase in affinity for the second substrate 2-ABA. Introduction of Asn into the PqsC active site led to significant activity toward the desamino substrate analog benzoylacetate, suggesting that the substrate 2-ABA itself supplies the asparagine-equivalent amino function that assists in catalysis.
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3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , 4-Quinolonas/química , Acilcoenzima A/química , Aminobenzoatos/química , Proteínas Bacterianas/química , Pseudomonas aeruginosa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/antagonistas & inhibidores , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , 4-Quinolonas/metabolismo , Acetofenonas/química , Acilcoenzima A/metabolismo , Secuencia de Aminoácidos , Aminobenzoatos/metabolismo , Antibacterianos/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Sitios de Unión , Unión Competitiva , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Pseudomonas aeruginosa/enzimología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Platelet-von Willebrand factor (VWF) interactions must be tightly regulated in order to promote effective hemostasis and prevent occlusive thrombus formation. However, it is unclear what role the inherent properties of the bond formed between the platelet receptor glycoprotein Ibα and the A1 domain of VWF play in these processes. Using VWF-A1 knock-in mice with mutations that enhance (I1309V) or disrupt (R1326H) platelet receptor glycoprotein Ibα binding, we now demonstrate that the kinetic interplay between two distinct contact surfaces influences the site and extent to which platelets bind VWF. Incorporation of R1326H mutation into the major site shortened bond lifetime, yielding defects in hemostasis and thrombosis comparable to VWF-deficient animals. Similarly, disrupting this region of contact with an allosteric inhibitor impaired human platelet accrual in damaged arterioles. In contrast, the I1309V mutation near the minor site prolonged bond lifetime, which was essential for the development of a type 2B-like VWD phenotype. However, combining the R1326H and I1309V mutations normalized both bond kinetics and the hemostatic and thrombotic properties of VWF. These findings broaden our understanding of mechanisms governing platelet-VWF interactions in health and disease, and underscore the importance of combined biophysical and genetic approaches in identifying potential therapeutic avenues for treating bleeding and thrombotic disorders.
Asunto(s)
Hemostasis , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Trombosis/metabolismo , Factor de von Willebrand/metabolismo , Animales , Sitios de Unión/genética , Plaquetas/metabolismo , Humanos , Cinética , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Mutación , Adhesividad Plaquetaria/genética , Complejo GPIb-IX de Glicoproteína Plaquetaria/química , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Unión Proteica , Estructura Terciaria de Proteína , Trombosis/sangre , Trombosis/genética , Factor de von Willebrand/química , Factor de von Willebrand/genéticaRESUMEN
Bacterial populations co-ordinate gene expression collectively through quorum sensing (QS), a cell-to-cell communication mechanism employing diffusible signal molecules. The LysR-type transcriptional regulator (LTTR) protein PqsR (MvfR) is a key component of alkyl-quinolone (AQ)-dependent QS in Pseudomonas aeruginosa. PqsR is activated by 2-alkyl-4-quinolones including the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone), its precursor 2-heptyl-4-hydroxyquinoline (HHQ) and their C9 congeners, 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS) and 2-nonyl-4-hydroxyquinoline (NHQ). These drive the autoinduction of AQ biosynthesis and the up-regulation of key virulence determinants as a function of bacterial population density. Consequently, PqsR constitutes a potential target for novel antibacterial agents which attenuate infection through the blockade of virulence. Here we present the crystal structures of the PqsR co-inducer binding domain (CBD) and a complex with the native agonist NHQ. We show that the structure of the PqsR CBD has an unusually large ligand-binding pocket in which a native AQ agonist is stabilized entirely by hydrophobic interactions. Through a ligand-based design strategy we synthesized and evaluated a series of 50 AQ and novel quinazolinone (QZN) analogues and measured the impact on AQ biosynthesis, virulence gene expression and biofilm development. The simple exchange of two isosteres (OH for NH2) switches a QZN agonist to an antagonist with a concomitant impact on the induction of bacterial virulence factor production. We also determined the complex crystal structure of a QZN antagonist bound to PqsR revealing a similar orientation in the ligand binding pocket to the native agonist NHQ. This structure represents the first description of an LTTR-antagonist complex. Overall these studies present novel insights into LTTR ligand binding and ligand-based drug design and provide a chemical scaffold for further anti-P. aeruginosa virulence drug development by targeting the AQ receptor PqsR.
Asunto(s)
Proteínas Bacterianas/metabolismo , Pseudomonas aeruginosa/fisiología , Quinolonas/metabolismo , Percepción de Quorum , Transducción de Señal , Factores de Transcripción/metabolismo , Alquilación , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/agonistas , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Sitios de Unión , Biopelículas/efectos de los fármacos , Diseño de Fármacos , Regulación Bacteriana de la Expresión Génica , Ligandos , Conformación Molecular , Proteínas Mutantes/agonistas , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/agonistas , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/patogenicidad , Quinolonas/química , Quinolonas/farmacología , Percepción de Quorum/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Factores de Transcripción/agonistas , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/química , Virulencia/efectos de los fármacosAsunto(s)
Hemostáticos , Enfermedades de von Willebrand , Plaquetas , Humanos , Factor de von WillebrandRESUMEN
The CUG-BP, Elav-like family (CELF) of RNA-binding proteins control gene expression at a number of different levels by regulating pre-mRNA splicing, deadenylation and mRNA stability. We present structural insights into the binding selectivity of CELF member 1 (CELF1) for GU-rich mRNA target sequences of the general form 5'-UGUNxUGUNyUGU and identify a high affinity interaction (Kd â¼ 100 nM for x = 2 and y = 4) with simultaneous binding of all three RNA recognition motifs within a single 15-nt binding element. RNA substrates spin-labelled at either the 3' or 5' terminus result in differential nuclear magnetic resonance paramagnetic relaxation enhancement effects, which are consistent with a non-sequential 2-1-3 arrangement of the three RNA recognition motifs on UGU sites in a 5' to 3' orientation along the RNA target. We further demonstrate that CELF1 binds to dispersed single-stranded UGU sites at the base of an RNA hairpin providing a structural rationale for recognition of CUG expansion repeats and splice site junctions in the regulation of alternative splicing.