RESUMEN
Plasminogen (Plg) is the inactive form of plasmin (Plm) that exists in two major glycoforms, referred to as glycoforms I and II (GI and GII). In the circulation, Plg assumes an activation-resistant "closed" conformation via interdomain interactions and is mediated by the lysine binding site (LBS) on the kringle (KR) domains. These inter-domain interactions can be readily disrupted when Plg binds to lysine/arginine residues on protein targets or free L-lysine and analogues. This causes Plg to convert into an "open" form, which is crucial for activation by host activators. In this study, we investigated how various ligands affect the kinetics of Plg conformational change using small-angle X-ray scattering (SAXS). We began by examining the open and closed conformations of Plg using size-exclusion chromatography (SEC) coupled with SAXS. Next, we developed a high-throughput (HTP) 96-well SAXS assay to study the conformational change of Plg. This method enables us to determine the Kopen value, which is used to directly compare the effect of different ligands on Plg conformation. Based on our analysis using Plg GII, we have found that the Kopen of ε-aminocaproic acid (EACA) is approximately three times greater than that of tranexamic acid (TXA), which is widely recognized as a highly effective ligand. We demonstrated further that Plg undergoes a conformational change when it binds to the C-terminal peptides of the inhibitor α2-antiplasmin (α2AP) and receptor Plg-RKT. Our findings suggest that in addition to the C-terminal lysine, internal lysine(s) are also necessary for the formation of open Plg. Finally, we compared the conformational changes of Plg GI and GII directly and found that the closed form of GI, which has an N-linked glycosylation, is less stable. To summarize, we have successfully determined the response of Plg to various ligand/receptor peptides by directly measuring the kinetics of its conformational changes.
Asunto(s)
Lisina , Plasminógeno , Ligandos , Dispersión del Ángulo Pequeño , Rayos X , Difracción de Rayos X , Serina Proteasas , AnticuerposRESUMEN
Apoptosis signal-regulating kinases (ASK1-3) are apical kinases of the p38 and JNK MAP kinase pathways. They are activated by diverse stress stimuli, including reactive oxygen species, cytokines, and osmotic stress; however, a molecular understanding of how ASK proteins are controlled remains obscure. Here, we report a biochemical analysis of the ASK1 kinase domain in conjunction with its N-terminal thioredoxin-binding domain, along with a central regulatory region that links the two. We show that in solution the central regulatory region mediates a compact arrangement of the kinase and thioredoxin-binding domains and the central regulatory region actively primes MKK6, a key ASK1 substrate, for phosphorylation. The crystal structure of the central regulatory region reveals an unusually compact tetratricopeptide repeat (TPR) region capped by a cryptic pleckstrin homology domain. Biochemical assays show that both a conserved surface on the pleckstrin homology domain and an intact TPR region are required for ASK1 activity. We propose a model in which the central regulatory region promotes ASK1 activity via its pleckstrin homology domain but also facilitates ASK1 autoinhibition by bringing the thioredoxin-binding and kinase domains into close proximity. Such an architecture provides a mechanism for control of ASK-type kinases by diverse activators and inhibitors and demonstrates an unexpected level of autoregulatory scaffolding in mammalian stress-activated MAP kinase signaling.
Asunto(s)
MAP Quinasa Quinasa Quinasa 5/química , MAP Quinasa Quinasa Quinasa 5/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Humanos , MAP Quinasa Quinasa 6/química , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/metabolismo , MAP Quinasa Quinasa Quinasa 5/genética , Modelos Biológicos , Modelos Moleculares , Mutación , Fosforilación , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Plasminogen (Plg) is the zymogen form of the serine protease plasmin (Plm), and it plays a crucial role in fibrinolysis as well as wound healing, immunity, tissue remodeling and inflammation. Binding to the targets via the lysine-binding sites allows for Plg activation by plasminogen activators (PAs) present on the same target. Cellular uptake of fibrin degradation products leads to apoptosis, which represents one of the pathways for cross-talk between fibrinolysis and tissue remodeling. Therapeutic manipulation of Plm activity plays a vital role in the treatments of a range of diseases, whereas Plm inhibitors are used in trauma and surgeries as antifibrinolytic agents. Plm inhibitors are also used in conditions such as angioedema, menorrhagia and melasma. Here, we review the rationale for the further development of new Plm inhibitors, with a particular focus on the structural studies of the active site inhibitors of Plm. We compare the binding mode of different classes of inhibitors and comment on how it relates to their efficacy, as well as possible future developments.
Asunto(s)
Plasminógeno/metabolismo , Animales , Antifibrinolíticos/farmacología , Apoptosis/genética , Apoptosis/fisiología , Humanos , Plasminógeno/genética , Activadores Plasminogénicos/farmacología , Inhibidores de Proteasas/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a â¼70° opening of the bent and distorted central ß-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane ß-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of ß-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into ß-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted ß-barrel. The intermediate structures of the MACPF domain during refolding into the ß-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function.
Asunto(s)
Membrana Celular/química , Complejo de Ataque a Membrana del Sistema Complemento/química , Proteínas Fúngicas/química , Proteínas Hemolisinas/química , Pleurotus/química , Proteínas Recombinantes de Fusión/química , Animales , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Eritrocitos/química , Eritrocitos/citología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expresión Génica , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Modelos Moleculares , Unión Proteica , Pliegue de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , OvinosRESUMEN
Antibodies represent a highly successful class of molecules that bind a wide-range of targets in therapeutic-, diagnostic- and research-based applications. The antibody repertoire is composed of the building blocks required to develop an effective adaptive immune response against foreign insults. A number of species have developed novel genetic and structural mechanisms from which they derive these antibody repertoires, however, traditionally antibodies are isolated from human, and rodent sources. Due to their high-value therapeutic, diagnostic, biotechnological and research applications, much innovation has resulted in techniques and approaches to isolate novel antibodies. These approaches are bolstered by advances in our understanding of species immune repertoires, next generation sequencing capacity, combinatorial antibody discovery and high-throughput screening. Structural determination of antibodies and antibody-antigen complexes has proven to be pivotal to our current understanding of the immune repertoire for a range of species leading to advances in man-made libraries and fine tuning approaches to develop antibodies from immune-repertoires. Furthermore, the isolation of antibodies directed against antigens of importance in health, disease and developmental processes, has yielded a plethora of structural and functional insights. This review highlights the significant contribution of antibody-based crystallography to our understanding of adaptive immunity and its application to providing critical information on a range of human-health related indications.
Asunto(s)
Inmunización Pasiva/métodos , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Inmunoglobulina G/ultraestructura , Anticuerpos de Cadena Única/ultraestructura , Inmunidad Adaptativa , Animales , Antígenos/inmunología , Cristalografía por Rayos X , Humanos , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/química , Modelos Moleculares , Chaperonas Moleculares/biosíntesis , Chaperonas Moleculares/química , Chaperonas Moleculares/ultraestructura , Conformación Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Anticuerpos de Cadena Única/biosíntesis , Anticuerpos de Cadena Única/química , Especificidad de la EspecieRESUMEN
Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein perforin and granzyme proteases from cytoplasmic granules into the cleft formed between the abutting killer and target cell membranes. Perforin, a 67-kilodalton multidomain protein, oligomerizes to form pores that deliver the pro-apoptopic granzymes into the cytosol of the target cell. The importance of perforin is highlighted by the fatal consequences of congenital perforin deficiency, with more than 50 different perforin mutations linked to familial haemophagocytic lymphohistiocytosis (type 2 FHL). Here we elucidate the mechanism of perforin pore formation by determining the X-ray crystal structure of monomeric murine perforin, together with a cryo-electron microscopy reconstruction of the entire perforin pore. Perforin is a thin 'key-shaped' molecule, comprising an amino-terminal membrane attack complex perforin-like (MACPF)/cholesterol dependent cytolysin (CDC) domain followed by an epidermal growth factor (EGF) domain that, together with the extreme carboxy-terminal sequence, forms a central shelf-like structure. A C-terminal C2 domain mediates initial, Ca(2+)-dependent membrane binding. Most unexpectedly, however, electron microscopy reveals that the orientation of the perforin MACPF domain in the pore is inside-out relative to the subunit arrangement in CDCs. These data reveal remarkable flexibility in the mechanism of action of the conserved MACPF/CDC fold and provide new insights into how related immune defence molecules such as complement proteins assemble into pores.
Asunto(s)
Membrana Celular/metabolismo , Linfocitos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Animales , Colesterol/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Factor de Crecimiento Epidérmico/química , Granzimas/metabolismo , Humanos , Ratones , Modelos Moleculares , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/ultraestructura , Estructura Terciaria de ProteínaRESUMEN
Antibodies are high value therapeutic, diagnostic, biotechnological, and research tools. Combinatorial approaches to antibody discovery have facilitated access to unique antibodies by surpassing the diversity limitations of the natural repertoire, exploitation of immune repertoires from multiple species, and tailoring selections to isolate antibodies with desirable biophysical attributes. The V-gene repertoire of the chicken does not utilize highly diverse sequence and structures, which is in stark contrast to the mechanism employed by humans, mice, and primates. Recent exploitation of the avian immune system has generated high quality, high affinity antibodies to a wide range of antigens for a number of therapeutic, diagnostic and biotechnological applications. Furthermore, extensive examination of the amino acid characteristics of the chicken repertoire has provided significant insight into mechanisms employed by the avian immune system. A paucity of avian antibody crystal structures has limited our understanding of the structural consequences of these uniquely chicken features. This paper presents the crystal structure of two chicken single chain fragment variable (scFv) antibodies generated from large libraries by phage display against important human antigen targets, which capture two unique CDRL1 canonical classes in the presence and absence of a non-canonical disulfide constrained CDRH3. These structures cast light on the unique structural features of chicken antibodies and contribute further to our collective understanding of the unique mechanisms of diversity and biochemical attributes that render the chicken repertoire of particular value for antibody generation.
Asunto(s)
Anticuerpos/química , Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo/inmunología , Pollos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos/genética , Pollos/genética , Cristalización , Cristalografía por Rayos X , Humanos , Cinética , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología , Relación Estructura-ActividadRESUMEN
The Store.Synchrotron service, a fully functional, cloud computing-based solution to raw X-ray data archiving and dissemination at the Australian Synchrotron, is described. The service automatically receives and archives raw diffraction data, related metadata and preliminary results of automated data-processing workflows. Data are able to be shared with collaborators and opened to the public. In the nine months since its deployment in August 2013, the service has handled over 22.4â TB of raw data (â¼1.7â million diffraction images). Several real examples from the Australian crystallographic community are described that illustrate the advantages of the approach, which include real-time online data access and fully redundant, secure storage. Discoveries in biological sciences increasingly require multidisciplinary approaches. With this in mind, Store.Synchrotron has been developed as a component within a greater service that can combine data from other instruments at the Australian Synchrotron, as well as instruments at the Australian neutron source ANSTO. It is therefore envisaged that this will serve as a model implementation of raw data archiving and dissemination within the structural biology research community.
Asunto(s)
Cristalografía por Rayos X , Curaduría de Datos/métodos , Australia , Sincrotrones , Flujo de TrabajoRESUMEN
The caspase recruitment domain (CARD) is present in death-domain superfamily proteins involved in inflammation and apoptosis. BinCARD is named for its ability to interact with Bcl10 and inhibit downstream signalling. Human BinCARD is expressed as two isoforms that encode the same N-terminal CARD region but which differ considerably in their C-termini. Both isoforms are expressed in immune cells, although BinCARD-2 is much more highly expressed. Crystals of the CARD fold common to both had low symmetry (space group P1). Molecular replacement was unsuccessful in this low-symmetry space group and, as the construct contains no methionines, first one and then two residues were engineered to methionine for MAD phasing. The double-methionine variant was produced as a selenomethionine derivative, which was crystallized and the structure was solved using data measured at two wavelengths. The crystal structures of the native and selenomethionine double mutant were refined to high resolution (1.58 and 1.40â Å resolution, respectively), revealing the presence of a cis-peptide bond between Tyr39 and Pro40. Unexpectedly, the native crystal structure revealed that all three cysteines were oxidized. The mitochondrial localization of BinCARD-2 and the susceptibility of its CARD region to redox modification points to the intriguing possibility of a redox-regulatory role.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/química , Proteínas/química , Proteínas/metabolismo , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , Cristalografía por Rayos X , Cisteína/metabolismo , Células HeLa , Humanos , Mitocondrias/metabolismo , Modelos Moleculares , Mutación , Oxidación-Reducción , Prolina/química , Conformación Proteica , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas/genética , SelenometioninaRESUMEN
Current therapeutics and prophylactics for malaria are under severe challenge as a result of the rapid emergence of drug-resistant parasites. The human malaria parasite Plasmodium falciparum expresses two neutral aminopeptidases, PfA-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for growth and development inside the red blood cell. These enzymes are essential for parasite viability and are validated therapeutic targets. We previously reported the X-ray crystal structure of the monomeric PfA-M1 and proposed a mechanism for substrate entry and free amino acid release from the active site. Here, we present the X-ray crystal structure of the hexameric leucine aminopeptidase, PfA-M17, alone and in complex with two inhibitors with antimalarial activity. The six active sites of the PfA-M17 hexamer are arranged in a disc-like fashion so that they are orientated inwards to form a central catalytic cavity; flexible loops that sit at each of the six entrances to the catalytic cavern function to regulate substrate access. In stark contrast to PfA-M1, PfA-M17 has a narrow and hydrophobic primary specificity pocket which accounts for its highly restricted substrate specificity. We also explicate the essential roles for the metal-binding centers in these enzymes (two in PfA-M17 and one in PfA-M1) in both substrate and drug binding. Our detailed understanding of the PfA-M1 and PfA-M17 active sites now permits a rational approach in the development of a unique class of two-target and/or combination antimalarial therapy.
Asunto(s)
Aminopeptidasas/química , Diseño de Fármacos , Plasmodium falciparum/enzimología , Proteínas Protozoarias/química , Aminopeptidasas/antagonistas & inhibidores , Aminopeptidasas/metabolismo , Antimaláricos/química , Antimaláricos/metabolismo , Antimaláricos/farmacología , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Metales/química , Metales/metabolismo , Modelos Moleculares , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Especificidad por SustratoRESUMEN
Perforin is a pore-forming protein whose normal function enables cytotoxic T and natural killer (NK) cells to kill virus-infected and transformed cells. Conversely, unwanted perforin activity can also result in auto-immune attack, graft rejection and aberrant responses to pathogens. Perforin is critical for the function of the granule exocytosis cell death pathway and is therefore a target for drug development. In this study, by screening a fragment library using NMR and surface plasmon resonance, we identified 4,4-diaminodiphenyl sulfone (dapsone) as a perforin ligand. We also found that dapsone has modest (mM) inhibitory activity of perforin lytic activity in a red blood cell lysis assay in vitro. Sequential modification of this lead fragment, guided by structural knowledge of the ligand binding site and binding pose, and supported by SPR and ligand-detected 19F NMR, enabled the design of nanomolar inhibitors of the cytolytic activity of intact NK cells against various tumour cell targets. Interestingly, the ligands we developed were largely inert with respect to direct perforin-mediated red blood cell lysis but were very potent in the context of perforin's action on delivering granzymes in the immune synapse, the context in which it functions physiologically. Our work indicates that a fragment-based, structure-guided drug discovery strategy can be used to identify novel ligands that bind perforin. Moreover, these molecules have superior physicochemical properties and solubility compared to previous generations of perforin ligands.
Asunto(s)
Dapsona , Células Asesinas Naturales , Perforina/metabolismo , Ligandos , Células Asesinas Naturales/metabolismo , Muerte Celular , Dapsona/metabolismoRESUMEN
In mammals, the enzyme D-xylulokinase (XK; EC 2.7.1.17) catalyses the last step of the glucuronate-xylulose pathway, in which the ketopentose sugar D-xylulose is phosphorylated to yield D-xylulose 5-phosphate (Xu5P). Xu5P is also a metabolite of the pentose phosphate pathway and acts as a signalling molecule that regulates lipogenesis and glycolysis in the liver. To date, no eukaryotic XK has been structurally characterized. A putative human XK was expressed in Escherichia coli aided by molecular chaperones, purified and crystallized. A seeding procedure involving repeated rounds of seeding was developed and proved to be essential for obtaining diffraction-quality crystals. Preliminary X-ray diffraction analysis was performed using synchrotron radiation. This resulted in the collection of a complete diffraction data set to 2.7â Å resolution from a crystal belonging to the trigonal space group P3(1) or P3(2) with unit-cell parameters a = b = 101.87, c = 158.85â Å.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Cristalografía por Rayos X , HumanosRESUMEN
Cell surface pili are polymeric protein assemblies that enable bacteria to adhere to surfaces and to specific host tissues. The pili expressed by Gram-positive bacteria constitute a unique paradigm in which sortase-mediated covalent linkages join successive pilin subunits like beads on a string. These pili are formed from two or three distinct types of pilin subunit, typically encoded in small gene clusters, often with their cognate sortases. In Group A streptococci (GAS), a major pilin forms the polymeric backbone, whereas two minor pilins are located at the tip and the base. Here, we report the 1.9-A resolution crystal structure of the GAS basal pilin FctB, revealing an immunoglobulin (Ig)-like N-terminal domain with an extended proline-rich tail. Unexpected structural homology between the FctB Ig-like domain and the N-terminal domain of the GAS shaft pilin helps explain the use of the same sortase for polymerization of the shaft and its attachment to FctB. It also enabled the identification, from mass spectral data, of the lysine residue involved in the covalent linkage of FctB to the shaft. The proline-rich tail forms a polyproline-II helix that appears to be a common feature of the basal (cell wall-anchoring) pilins. Together, our results indicate distinct structural elements in the pilin proteins that play a role in selecting for the appropriate sortases and thereby help orchestrate the ordered assembly of the pilus.
Asunto(s)
Adhesión Bacteriana , Proteínas Fimbrias/química , Fimbrias Bacterianas/metabolismo , Streptococcus pyogenes/metabolismo , Secuencia de Aminoácidos , Aminoaciltransferasas/genética , Proteínas Bacterianas/genética , Sitios de Unión/genética , Dicroismo Circular , Cristalografía por Rayos X , Cisteína Endopeptidasas/genética , Electroforesis en Gel de Poliacrilamida , Proteínas Fimbrias/genética , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Streptococcus pyogenes/clasificación , Streptococcus pyogenes/genéticaRESUMEN
The common pathogen Streptococcus pyogenes colonizes the human skin and tonsils and can invade underlying tissues. This requires the adhesion of S. pyogenes to host surface receptors mediated through adhesins. The laminin-binding protein Lbp has been suggested as an adhesin, specific for the human extracellular matrix protein laminin. Sequence alignments, however, indicate a relationship between Lbp and a family of bacterial metal-binding receptors. To further analyze the role of Lbp in S. pyogenes and its potential role in pathogenicity, Lbp has been crystallized, and its structure has been solved at a resolution of 2.45 A (R = 0.186; R(free) = 0.251). Lbp has the typical metal-binding receptor fold, comprising two globular (beta/alpha)(4) domains connected by a helical backbone. The two domains enclose the metal-binding site, which contains a zinc ion. The interaction of Lbp with laminin was further investigated and shown to be specific in vitro. Localization studies with antibodies specific for Lbp show that the protein is attached to the membrane. The data suggest that Lbp is primarily a zinc-binding protein, and we suggest that its interaction with laminin in vivo may be mediated via zinc bound to laminin.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Laminina/metabolismo , Receptores de Laminina/metabolismo , Streptococcus pyogenes/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biotinilación , Ensayo de Inmunoadsorción Enzimática , Humanos , Immunoblotting , Inmunoprecipitación , Datos de Secuencia Molecular , Conejos , Receptores de Laminina/química , Receptores de Laminina/genética , Streptococcus pyogenes/genética , Zinc/metabolismoRESUMEN
Antifibrinolytic drugs provide important pharmacological interventions to reduce morbidity and mortality from excessive bleeding during surgery and after trauma. Current drugs used for inhibiting the dissolution of fibrin, the main structural component of blood clots, are associated with adverse events due to lack of potency, high doses, and nonselective inhibition mechanisms. These drawbacks warrant the development of a new generation of highly potent and selective fibrinolysis inhibitors. Here, we use the 14-amino acid backbone-cyclic sunflower trypsin inhibitor-1 scaffold to design a highly potent ( Ki = 0.05 nM) inhibitor of the primary serine protease in fibrinolysis, plasmin. This compound displays a million-fold selectivity over other serine proteases in blood, inhibits fibrinolysis in plasma more effectively than the gold-standard therapeutic inhibitor aprotinin, and is a promising candidate for development of highly specific fibrinolysis inhibitors with reduced side effects.
Asunto(s)
Fibrinolisina/antagonistas & inhibidores , Péptidos Cíclicos/química , Inhibidores de Serina Proteinasa/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Fibrinolisina/metabolismo , Fibrinólisis/efectos de los fármacos , Humanos , Simulación de Dinámica Molecular , Péptidos/síntesis química , Péptidos/química , Péptidos/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Inhibidores de Serina Proteinasa/farmacologíaRESUMEN
Plasminogen (Plg)-binding M protein (PAM) is a group A streptococcal cell surface receptor that is crucial for bacterial virulence. Previous studies revealed that, by binding to the kringle 2 (KR2) domain of host Plg, the pathogen attains a proteolytic microenvironment on the cell surface that facilitates its dissemination from the primary infection site. Each of the PAM molecules in their dimeric assembly consists of two Plg binding motifs (called the a1 and a2 repeats). To date, the molecular interactions between the a1 repeat and KR2 have been structurally characterized, whereas the role of the a2 repeat is less well defined. Here, we report the 1.7-Å x-ray crystal structure of KR2 in complex with a monomeric PAM peptide that contains both the a1 and a2 motifs. The structure reveals how the PAM peptide forms key interactions simultaneously with two KR2 via the high-affinity lysine isosteres within the a1a2 motifs. Further studies, through combined mutagenesis and functional characterization, show that a2 is a stronger KR2 binder than a1, suggesting that these two motifs may play discrete roles in mediating the final PAM-Plg assembly.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Plasminógeno/metabolismo , Streptococcus pyogenes/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Unión Proteica , Dominios Proteicos , Estabilidad Proteica , Relación Estructura-ActividadRESUMEN
The laminin-binding protein Lbp (Spy2007) from Streptococcus pyogenes (a group A streptococcus) mediates adhesion to the human basal lamina glycoprotein laminin. Accordingly, Lbp is essential in in vitro models of cell adhesion and invasion. However, the molecular and structural basis of laminin binding by bacteria remains unknown. Therefore, the lbp gene has been cloned for recombinant expression in Escherichia coli. Lbp has been purified and crystallized from 30%(w/v) PEG 1500 by the sitting-drop vapour-diffusion method. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 42.62, b = 92.16, c = 70.61 A, beta = 106.27 degrees, and diffracted to 2.5 A resolution.
Asunto(s)
Proteínas Portadoras/química , Laminina/metabolismo , Streptococcus pyogenes/química , Secuencia de Bases , Proteínas Portadoras/metabolismo , Cristalización , Cristalografía por Rayos X , Cartilla de ADN , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
SPRY domain- and SOCS box-containing proteins SPSB1, SPSB2, and SPSB4 interact with inducible nitric oxide synthase (iNOS), causing the iNOS to be polyubiquitinated and targeted for degradation. Inhibition of this interaction increases iNOS levels, and consequently cellular nitric oxide (NO) concentrations, and has been proposed as a potential strategy for killing intracellular pathogens. We previously described two DINNN-containing cyclic peptides (CP1 and CP2) as potent inhibitors of the murine SPSB-iNOS interaction. In this study, we report the crystal structures of human SPSB4 bound to CP1 and CP2 and human SPSB2 bound to CP2. We then used these structures to design a new inhibitor in which an intramolecular hydrogen bond was replaced with a hydrocarbon linkage to form a smaller macrocycle while maintaining the bound geometry of CP2 observed in the crystal structures. This resulting pentapeptide SPSB-iNOS inhibitor (CP3) has a reduced macrocycle ring size, fewer nonbinding residues, and includes additional conformational constraints. CP3 has a greater affinity for SBSB2 ( KD = 7 nM as determined by surface plasmon resonance) and strongly inhibits the SPSB2-iNOS interaction in macrophage cell lysates. We have also determined the crystal structure of CP3 in complex with human SPSB2, which reveals the structural basis for the increased potency of CP3 and validates the original design.
Asunto(s)
Antiinfecciosos/química , Péptidos y Proteínas de Señalización Intracelular/química , Óxido Nítrico Sintasa de Tipo II/metabolismo , Oligopéptidos/química , Péptidos Cíclicos/química , Proteínas Supresoras de la Señalización de Citocinas/química , Animales , Antiinfecciosos/farmacología , Diseño de Fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Unión Proteica , Células RAW 264.7 , Proteínas Supresoras de la Señalización de Citocinas/metabolismoRESUMEN
Complement component 9 (C9) functions as the pore-forming component of the Membrane Attack Complex (MAC). During MAC assembly, multiple copies of C9 are sequentially recruited to membrane associated C5b8 to form a pore. Here we determined the 2.2 Å crystal structure of monomeric murine C9 and the 3.9 Å resolution cryo EM structure of C9 in a polymeric assembly. Comparison with other MAC proteins reveals that the first transmembrane region (TMH1) in monomeric C9 is uniquely positioned and functions to inhibit its self-assembly in the absence of C5b8. We further show that following C9 recruitment to C5b8, a conformational change in TMH1 permits unidirectional and sequential binding of additional C9 monomers to the growing MAC. This mechanism of pore formation contrasts with related proteins, such as perforin and the cholesterol dependent cytolysins, where it is believed that pre-pore assembly occurs prior to the simultaneous release of the transmembrane regions.
Asunto(s)
Complemento C9/química , Complejo de Ataque a Membrana del Sistema Complemento/química , Proteínas de la Membrana/química , Dominios Proteicos , Animales , Complemento C9/genética , Complemento C9/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/ultraestructura , Proteínas del Sistema Complemento/química , Proteínas del Sistema Complemento/genética , Proteínas del Sistema Complemento/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Modelos Moleculares , Unión ProteicaRESUMEN
The zymogen protease plasminogen and its active form plasmin perform key roles in blood clot dissolution, tissue remodeling, cell migration, and bacterial pathogenesis. Dysregulation of the plasminogen/plasmin system results in life-threatening hemorrhagic disorders or thrombotic vascular occlusion. Accordingly, inhibitors of this system are clinically important. Currently, tranexamic acid (TXA), a molecule that prevents plasminogen activation through blocking recruitment to target substrates, is the most widely used inhibitor for the plasminogen/plasmin system in therapeutics. However, TXA lacks efficacy on the active form of plasmin. Thus, there is a need to develop specific inhibitors that target the protease active site. Here we report the crystal structures of plasmin in complex with the novel YO (trans-4-aminomethylcyclohexanecarbonyl-l-tyrosine-n-octylamide) class of small molecule inhibitors. We found that these inhibitors form key interactions with the S1 and S3' subsites of the catalytic cleft. Here, the TXA moiety of the YO compounds inserts into the primary (S1) specificity pocket, suggesting that TXA itself may function as a weak plasmin inhibitor, a hypothesis supported by subsequent biochemical and biophysical analyses. Mutational studies reveal that F587 of the S' subsite plays a key role in mediating the inhibitor interaction. Taken together, these data provide a foundation for the future development of small molecule inhibitors to specifically regulate plasmin function in a range of diseases and disorders.