RESUMO
A revised version of Table 2 of Martin-Garcia et al. [J. Synchrotron Rad. (2022). 29, 896-907] is provided.
RESUMO
The increase in successful adaptations of serial crystallography at synchrotron radiation sources continues. To date, the number of serial synchrotron crystallography (SSX) experiments has grown exponentially, with over 40 experiments reported so far. In this work, we report the first SSX experiments with viscous jets conducted at ALBA beamline BL13-XALOC. Small crystals (15-30â µm) of five soluble proteins (lysozyme, proteinase K, phycocyanin, insulin and α-spectrin-SH3 domain) were suspended in lipidic cubic phase (LCP) and delivered to the X-ray beam with a high-viscosity injector developed at Arizona State University. Complete data sets were collected from all proteins and their high-resolution structures determined. The high quality of the diffraction data collected from all five samples, and the lack of specific radiation damage in the structures obtained in this study, confirm that the current capabilities at the beamline enables atomic resolution determination of protein structures from microcrystals as small as 15â µm using viscous jets at room temperature. Thus, BL13-XALOC can provide a feasible alternative to X-ray free-electron lasers when determining snapshots of macromolecular structures.
Assuntos
Lasers , Síncrotrons , Cristalografia por Raios X , Humanos , Substâncias Macromoleculares , Proteínas , ViscosidadeRESUMO
In the current study, we have evaluated the protective efficacy of the 'insertion domain' which is commonly found in the capsid penton base protein of many adenoviruses. Using the 'insertion domain' of the penton base protein of a representative fowl adenovirus, fowl adenovirus serotype 4 (FAdV-4), we find that the 'insertion domain' can readily be expressed in a soluble form in the bacterial system, and can be purified in sufficient quantities through simple chromatographic methods. We demonstrate that the 'insertion domain', when employed as a subunit vaccine candidate, provides complete protection against hydropericardium syndrome, caused by FAdV-4, in chickens. The data presented here indicate that the protein, adjuvanted with Montanide™ ISA71 VG, provides complete protection in chickens against a lethal FAdV-4 challenge after administration of two doses (100 µg of the protein per dose) two weeks apart (the first dose at the 7th day of life and a booster dose at the age of 21 days). Furthermore, the purified protein can be stored at low temperatures without any observable loss in the protein integrity up to one year, tested so far. Due to the conserved nature of the 'insertion domain' across the penton base protein of fowl adenoviruses, it is suggested that homologous insertion domains could be employed as highly stable and cost-effective subunit vaccine candidates against infections caused by respective fowl adenoviruses.
Assuntos
Infecções por Adenoviridae , Aviadenovirus , Doenças das Aves Domésticas , Animais , Proteínas do Capsídeo , Infecções por Adenoviridae/prevenção & controle , Infecções por Adenoviridae/veterinária , Galinhas , Capsídeo , Aviadenovirus/genética , Adenoviridae/genética , Vacinas de Subunidades Antigênicas , SorogrupoRESUMO
The emergent human pathogen Mycoplasma genitalium, with one of the smallest genomes among cells capable of growing in axenic cultures, presents a flask-shaped morphology due to a protrusion of the cell membrane, known as the terminal organelle, that is involved in cell adhesion and motility and is an important virulence factor of this microorganism. The terminal organelle is supported by a cytoskeleton complex of about 300 nm in length that includes three substructures: the terminal button, the rod and the wheel complex. The crystal structure of the MG491 protein, a proposed component of the wheel complex, has been determined at ~3 Å resolution. MG491 subunits are composed of a 60-residue N-terminus, a central three-helix-bundle spanning about 150 residues and a C-terminal region that appears to be quite flexible and contains the region that interacts with MG200, another key protein of the terminal organelle. The MG491 molecule is a tetramer presenting a unique organization as a dimer of asymmetric pairs of subunits. The asymmetric arrangement results in two very different intersubunit interfaces between the central three-helix-bundle domains, which correlates with the formation of only ~50% of the intersubunit disulfide bridges of the single cysteine residue found in MG491 (Cys87). Moreover, M. genitalium cells with a point mutation in the MG491 gene causing the change of Cys87 to Ser present a drastic reduction in motility (as determined by microcinematography) and important alterations in morphology (as determined by electron microscopy), while preserving normal levels of the terminal organelle proteins. Other variants of MG491, designed also according to the structural information, altered significantly the motility and/or the cell morphology. Together, these results indicate that MG491 plays a key role in the functioning, organization and stabilization of the terminal organelle.
Assuntos
Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/genética , Movimento Celular/fisiologia , Mycoplasma genitalium/citologia , Organelas/metabolismo , Aderência Bacteriana/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Adesão Celular , Citoesqueleto/metabolismo , Mutação/genética , Mycoplasma genitalium/genética , Mycoplasma genitalium/metabolismoRESUMO
Several mycoplasmas, such as the emergent human pathogen Mycoplasma genitalium, developed a complex polar structure, known as the terminal organelle (TO), responsible for a new type of cellular motility, which is involved in a variety of cell functions: cell division, adherence to host cells, and virulence. The TO cytoskeleton is organized as a multisubunit dynamic motor, including three main ultrastructures: the terminal button, the electrodense core, and the wheel complex. Here, we describe the interaction between MG200 and MG491, two of the main components of the TO wheel complex that connects the TO with the cell body and the cell membrane. The interaction between MG200 and MG491 has a KD in the 80 nm range, as determined by surface plasmon resonance. The interface between the two partners was confined to the "enriched in aromatic and glycine residues" (EAGR) box of MG200, previously described as a protein-protein interaction domain, and to a 25-residue-long peptide from the C-terminal region of MG491 by surface plasmon resonance and NMR spectroscopy studies. An atomic description of the MG200 EAGR box binding surface was also provided by solution NMR. An M. genitalium mutant lacking the MG491 segment corresponding to the peptide reveals specific alterations in cell motility and cell morphology indicating that the MG200-MG491 interaction plays a key role in the stability and functioning of the TO.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Motores Moleculares/metabolismo , Mycoplasma genitalium/citologia , Sequência de Aminoácidos , Movimento Celular , Dicroísmo Circular , Escherichia coli/metabolismo , Teste de Complementação Genética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mutação , Organelas/metabolismo , Peptídeos/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de SuperfícieRESUMO
Most bacteria use the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the synthesis of their essential isoprenoid precursors. The absence of the MEP pathway in humans makes it a promising new target for the development of much needed new and safe antimicrobial drugs. However, bacteria show a remarkable metabolic plasticity for isoprenoid production. For example, the NADPH-dependent production of MEP from 1-deoxy-D-xylulose 5-phosphate in the first committed step of the MEP pathway is catalyzed by 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) in most bacteria, whereas an unrelated DXR-like (DRL) protein was recently found to catalyze the same reaction in some organisms, including the emerging human and animal pathogens Bartonella and Brucella. Here, we report the x-ray crystal structures of the Brucella abortus DRL enzyme in its apo form and in complex with the broad-spectrum antibiotic fosmidomycin solved to 1.5 and 1.8 Å resolution, respectively. DRL is a dimer, with each polypeptide folding into three distinct domains starting with the NADPH-binding domain, in resemblance to the structure of bacterial DXR enzymes. Other than that, DRL and DXR show a low structural relationship, with a different disposition of the domains and a topologically unrelated C-terminal domain. In particular, the active site of DRL presents a unique arrangement, suggesting that the design of drugs that would selectively inhibit DRL-harboring pathogens without affecting beneficial or innocuous bacteria harboring DXR should be feasible. As a proof of concept, we identified two strong DXR inhibitors that have virtually no effect on DRL activity.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Proteínas de Bactérias/metabolismo , Brucella abortus/enzimologia , Complexos Multienzimáticos/metabolismo , Oxirredutases/metabolismo , Terpenos/metabolismo , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Biocatálise/efeitos dos fármacos , Brucella abortus/genética , Brucella abortus/metabolismo , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Fosfomicina/análogos & derivados , Fosfomicina/química , Fosfomicina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Oxirredutases/química , Oxirredutases/genética , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Mycoplasma genitalium is an emerging human pathogen with the smallest genome found among self-replicating organisms. M. genitalium presents a complex cytoskeleton with a differentiated protrusion known as the terminal organelle. This polar structure plays a central role in functions essential for the virulence of the microorganism, such as motility and cell-host adhesion. A well-conserved Enriched in Aromatic and Glycine Residues motif, the EAGR box, is present in many of the proteins found in the terminal organelle. We determined the crystal structure of the globular domain from M. genitalium MG200 that contains an EAGR box. This structural information is the first at near atomic resolution for the components of the terminal organelle. The structure revealed a dimer stabilized by a compact hydrophobic core that extends throughout the dimer interface. Monomers present a new fold that contains an accurate intra-subunit symmetry relating two conspicuous hairpins. Some features, such as the domain plasticity and the presence and organization of the intra- and inter-subunit symmetry axes, support a role for the EAGR box in protein-protein interactions. Genetic, biochemical and microcinematography analyses of MG200 variants lacking the EAGR box containing domain confirm the relevant and specific association of this domain with cell motility.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/citologia , Mycoplasma genitalium/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Humanos , Dados de Sequência Molecular , Mycoplasma genitalium/química , Mycoplasma genitalium/genética , Alinhamento de SequênciaRESUMO
Bacterial conjugation is an important route for horizontal gene transfer. The initial step in this process involves a macromolecular protein-DNA complex called the relaxosome, which in plasmids consists of the origin of transfer (oriT) and several proteins that prepare the transfer. The relaxosome protein named relaxase introduces a nick in one of the strands of the oriT to initiate the process. Additional relaxosome proteins can exist. Recently, several relaxosome proteins encoded on the Bacillus subtilis plasmid pLS20 were identified, including the relaxase, named RelpLS20, and two auxiliary DNA-binding factors, named Aux1pLS20 and Aux2pLS20. Here, we extend this characterization in order to define their function. We present the low-resolution SAXS envelope of the Aux1pLS20 and the atomic X-ray structure of the C-terminal domain of Aux2pLS20. We also study the interactions between the auxiliary proteins and the full-length RelpLS20, as well as its separate domains. The results show that the quaternary structure of the auxiliary protein Aux1pLS20 involves a tetramer, as previously determined. The crystal structure of the C-terminal domain of Aux2pLS20 shows that it forms a tetramer and suggests that it is an analog of TraMpF of plasmid F. This is the first evidence of the existence of a TraMpF analog in gram positive conjugative systems, although, unlike other TraMpF analogs, Aux2pLS20 does not interact with the relaxase. Aux1pLS20 interacts with the C-terminal domain, but not the N-terminal domain, of the relaxase RelpLS20. Thus, the pLS20 relaxosome exhibits some unique features despite the apparent similarity to some well-studied G- conjugation systems.
RESUMO
Family 16 carbohydrate active enzyme members Bacillus licheniformis 1,3-1,4-ß-glucanase and Populus tremula x tremuloides xyloglucan endotransglycosylase (XET16-34) are highly structurally related but display different substrate specificities. Although the first binds linear gluco-oligosaccharides, the second binds branched xylogluco-oligosaccharides. Prior engineered nucleophile mutants of both enzymes are glycosynthases that catalyze the condensation between a glycosyl fluoride donor and a glycoside acceptor. With the aim of expanding the glycosynthase technology to produce designer oligosaccharides consisting of hybrids between branched xylogluco- and linear gluco-oligosaccharides, enzyme engineering on the negative subsites of 1,3-1,4-ß-glucanase to accept branched substrates has been undertaken. Removal of the 1,3-1,4-ß-glucanase major loop and replacement with that of XET16-34 to open the binding cleft resulted in a folded protein, which still maintained some ß-glucan hydrolase activity, but the corresponding nucleophile mutant did not display glycosynthase activity with either linear or branched glycosyl donors. Next, point mutations of the 1,3-1,4-ß-glucanase ß-sheets forming the binding site cleft were mutated to resemble XET16-34 residues. The final chimeric protein acquired binding affinity for xyloglucan and did not bind ß-glucan. Therefore, binding specificity has been re-engineered, but affinity was low and the nucleophile mutant of the chimeric enzyme did not show glycosynthase activity to produce the target hybrid oligosaccharides. Structural analysis by X-ray crystallography explains these results in terms of changes in the protein structure and highlights further engineering approaches toward introducing the desired activity.
Assuntos
Glucanos/química , Glicosídeo Hidrolases/genética , Glicosiltransferases/genética , Proteínas Recombinantes de Fusão/química , Xilanos/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Bacillus , Cristalografia por Raios X , Ensaios Enzimáticos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Populus , Ligação Proteica , Engenharia de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , beta-Glucanas/químicaRESUMO
Tyrosine sulfation is an important post-translational modification of peptides and proteins which underpins and modulates many protein-protein interactions. In order to overcome the inherent instability of the native modification, we report the synthesis of two sulfonate analogues and their incorporation into two thrombin-inhibiting sulfopeptides. The effective mimicry of these sulfonate analogues for native sulfotyrosine was validated in the context of their thrombin inhibitory activity and binding mode, as determined by X-ray crystallography.
Assuntos
Antitrombinas/química , Peptídeos/química , Trombina/antagonistas & inibidores , Tirosina/análogos & derivados , Antitrombinas/síntese química , Antitrombinas/metabolismo , Cristalografia por Raios X , Ensaios Enzimáticos , Humanos , Peptídeos/síntese química , Peptídeos/metabolismo , Ligação Proteica , Trombina/metabolismo , Tirosina/químicaRESUMO
Viruses are obligate parasites that depend on a host cell for replication and survival. Consequently, to fully understand the viral processes involved in infection and replication, it is fundamental to study them in the cellular context. Often, viral infections induce significant changes in the subcellular organization of the host cell due to the formation of viral factories, alteration of cell cytoskeleton and/or budding of newly formed particles. Accurate 3D mapping of organelle reorganization in infected cells can thus provide valuable information for both basic virus research and antiviral drug development. Among the available techniques for 3D cell imaging, cryo-soft X-ray tomography stands out for its large depth of view (allowing for 10 µm thick biological samples to be imaged without further thinning), its resolution (about 50 nm for tomographies, sufficient to detect viral particles), the minimal requirements for sample manipulation (can be used on frozen, unfixed and unstained whole cells) and the potential to be combined with other techniques (i.e., correlative fluorescence microscopy). In this review we describe the fundamentals of cryo-soft X-ray tomography, its sample requirements, its advantages and its limitations. To highlight the potential of this technique, examples of virus research performed at BL09-MISTRAL beamline in ALBA synchrotron are also presented.
Assuntos
Tomografia por Raios X/métodos , Viroses/virologia , Fenômenos Fisiológicos Virais , Animais , Antivirais/farmacologia , Humanos , Tomografia por Raios X/instrumentação , Viroses/diagnóstico por imagem , Viroses/tratamento farmacológico , Vírus/química , Vírus/efeitos dos fármacosRESUMO
Despite possessing only 32 residues, the tsetse thrombin inhibitor (TTI) is among the most potent anticoagulants described, with sub-picomolar inhibitory activity against thrombin. Unexpectedly, TTI isolated from the fly is 2000-fold more active and 180 Da heavier than synthetic and recombinant variants. We predicted the presence of a tyrosine O-sulfate post-translational modification of TTI, prompting us to investigate the effect of the modification on anticoagulant activity. A combination of chemical synthesis and functional assays was used to reveal that sulfation significantly improved the inhibitory activity of TTI against thrombin. Using X-ray crystallography, we show that the N-terminal sulfated segment of TTI binds the basic exosite II of thrombin, establishing interactions similar to those of physiologic substrates, while the C-terminal segment abolishes the catalytic activity of thrombin. This non-canonical mode of inhibition, coupled with its potency and small size, makes TTI an attractive scaffold for the design of novel antithrombotics.
Assuntos
Anticoagulantes/farmacologia , Proteínas Antitrombina/farmacologia , Proteínas de Insetos/farmacologia , Trombina/antagonistas & inibidores , Tirosina/análogos & derivados , Animais , Anticoagulantes/síntese química , Anticoagulantes/química , Proteínas Antitrombina/síntese química , Proteínas Antitrombina/química , Linhagem Celular , Humanos , Proteínas de Insetos/síntese química , Proteínas de Insetos/química , Estrutura Molecular , Trombina/metabolismo , Moscas Tsé-Tsé , Tirosina/síntese química , Tirosina/química , Tirosina/farmacologiaRESUMO
In heme enzymes belonging to the peroxidase-cyclooxygenase superfamily the proximal histidine is in close interaction with a fully conserved asparagine. The crystal structure of a mixture of glycoforms of myeloperoxidase (MPO) purified from granules of human leukocytes prompted us to revise the orientation of this asparagine and the protonation status of the proximal histidine. The data we present contrast with previous MPO structures, but are strongly supported by molecular dynamics simulations. Moreover, comprehensive analysis of published lactoperoxidase structures suggest that the described proximal heme architecture is a general structural feature of animal heme peroxidases. Its importance is underlined by the fact that the MPO variant N421D, recombinantly expressed in mammalian cell lines, exhibited modified spectral properties and diminished catalytic activity compared with wild-type recombinant MPO. It completely lost its ability to oxidize chloride to hypochlorous acid, which is a characteristic feature of MPO and essential for its role in host defense. The presented crystal structure of MPO revealed further important differences compared with the published structures including the extent of glycosylation, interaction between light and heavy polypeptides, as well as heme to protein covalent bonds. These data are discussed with respect to biosynthesis and post-translational maturation of MPO as well as to its peculiar biochemical and biophysical properties.
Assuntos
Asparagina/química , Histidina/química , Leucócitos/enzimologia , Peroxidase/química , Asparagina/genética , Asparagina/metabolismo , Linhagem Celular , Cloretos/metabolismo , Cristalografia por Raios X , Glicosilação , Heme/química , Heme/genética , Heme/metabolismo , Histidina/genética , Histidina/metabolismo , Humanos , Ácido Hipocloroso/metabolismo , Lactoperoxidase/química , Lactoperoxidase/genética , Lactoperoxidase/metabolismo , Mutação de Sentido Incorreto , Oxirredução , Peroxidase/genética , Peroxidase/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Estrutura Terciária de Proteína/fisiologiaRESUMO
Hsp70 chaperones keep protein homeostasis facilitating the response of organisms to changes in external and internal conditions. Hsp70s have two domains-nucleotide binding domain (NBD) and substrate binding domain (SBD)-connected by a conserved hydrophobic linker. Functioning of Hsp70s depend on tightly regulated cycles of ATP hydrolysis allosterically coupled, often together with cochaperones, to the binding/release of peptide substrates. Here we describe the crystal structure of the Mycoplasma genitalium DnaK (MgDnaK) protein, an Hsp70 homolog, in the noncompact, nucleotide-bound/substrate-bound conformation. The MgDnaK structure resembles the one from the thermophilic eubacteria DnaK trapped in the same state. However, in MgDnaK the NBD and SBD domains remain close to each other despite the lack of direct interaction between them and with the linker contacting the two subdomains of SBD. These observations suggest that the structures might represent an intermediate of the protein where the conserved linker binds to the SBD to favor the noncompact state of the protein by stabilizing the SBDß-SBDα subdomains interaction, promoting the capacity of the protein to sample different conformations, which is critical for proper functioning of the molecular chaperone allosteric mechanism. Comparison of the solved structures indicates that the NBD remains essentially invariant in presence or absence of nucleotide.
Assuntos
Proteínas de Choque Térmico HSP70/química , Mycoplasma genitalium/química , Nucleotídeos/química , Clonagem Molecular , Cristalografia por Raios X , Proteínas de Choque Térmico HSP70/biossíntese , Modelos Moleculares , Mycoplasma genitalium/metabolismo , Conformação ProteicaRESUMO
The X-ray crystal structure of the 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS) from Arabidopsis thaliana has been solved at 2.3 A resolution in complex with a cytidine-5-monophosphate (CMP) molecule. This is the first structure determined of an MCS enzyme from a plant. Major differences between the A. thaliana and bacterial MCS structures are found in the large molecular cavity that forms between subunits and involve residues that are highly conserved among plants. In some bacterial enzymes, the corresponding cavity has been shown to be an isoprenoid diphosphate-like binding pocket, with a proposed feedback-regulatory role. Instead, in the structure from A. thaliana the cavity is unsuited for binding a diphosphate moiety, which suggests a different regulatory mechanism of MCS enzymes between bacteria and plants.
Assuntos
Arabidopsis/enzimologia , Bactérias/enzimologia , Enzimas/química , Eritritol/análogos & derivados , Terpenos/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Enzimas/isolamento & purificação , Eritritol/biossíntese , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
The crystal structure of the eubacteria Mycoplasma genitalium ORF MG438 polypeptide, determined by multiple anomalous dispersion and refined at 2.3 A resolution, reveals the organization of S subunits from the Type I restriction and modification system. The structure consists of two globular domains, with about 150 residues each, separated by a pair of 40 residue long antiparallel alpha-helices. The globular domains correspond to the variable target recognition domains (TRDs), as previously defined for S subunits on sequence analysis, while the two helices correspond to the central (CR1) and C-terminal (CR2) conserved regions, respectively. The structure of the MG438 subunit presents an overall cyclic topology with an intramolecular 2-fold axis that superimposes the N and the C-half parts, each half containing a globular domain and a conserved helix. TRDs are found to be structurally related with the small domain of the Type II N6-adenine DNA MTase TaqI. These relationships together with the structural peculiarities of MG438, in particular the presence of the intramolecular quasi-symmetry, allow the proposal of a model for S subunits recognition of their DNA targets in agreement with previous experimental results. In the crystal, two subunits of MG438 related by a crystallographic 2-fold axis present a large contact area mainly involving the symmetric interactions of a cluster of exposed hydrophobic residues. Comparison with the recently reported structure of an S subunit from the archaea Methanococcus jannaschii highlights the structural features preserved despite a sequence identity below 20%, but also reveals important differences in the globular domains and in their disposition with respect to the conserved regions.
Assuntos
Proteínas de Bactérias/química , Enzimas de Restrição-Modificação do DNA/química , Mycoplasma genitalium/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Cristalografia por Raios X , Enzimas de Restrição-Modificação do DNA/classificação , Enzimas de Restrição-Modificação do DNA/genética , DNA Bacteriano/genética , Modelos Moleculares , Dados de Sequência Molecular , Mycoplasma genitalium/genética , Estrutura Quaternária de Proteína , Subunidades Proteicas , Homologia de Sequência de Aminoácidos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Eletricidade EstáticaRESUMO
Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine alpha-thrombin.boophilin complex, refined at 2.35 A resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S(1) pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9 degrees and is displaced by 6 A, while the C-terminal domain rotates almost 6 degrees accompanied by a 3 A displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P(1) residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin.boophilin.trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.