RESUMEN
BACKGROUND: Prior studies have shown worse outcomes in patients with cardiogenic shock (CS) who have reduced left ventricular ejection fraction (LVEF), but the association between other transthoracic echocardiogram (TTE) findings and mortality in CS patients remains uncertain. We hypothesized that Doppler TTE measurements would outperform LVEF for risk stratification. METHODS: Retrospective analysis of cardiac intensive care unit patients with an admission diagnosis of CS and a TTE within 1 day of admission. Hospital survivors and inpatient deaths were compared, and multivariable logistic regression was used to analyze the associations between TTE variables and hospital mortality. RESULTS: We included 1,085 patients, with a median age of 69.5 (59.6, 77.5) years; 37% were females and 62% had an acute coronary syndrome. Most patients (66%) had moderate or severe left ventricular (LV) systolic dysfunction, and 48% had moderate or severe right ventricular (RV) systolic dysfunction. Hospital mortality occurred in 31%, and inpatient deaths had a lower median LVEF (29% vs. 35%, Pâ<â0.001). Patients with mild or no LV or RV dysfunction were at lower risk of adjusted hospital mortality (Pâ<â0.01). The LV outflow tract (LVOT) velocity-time integral (VTI) was the single best predictor of hospital mortality. After multivariable adjustment, both the LVEF and LVOT VTI remained strongly associated with hospital mortality (Pâ<â0.001). CONCLUSIONS: Early comprehensive Doppler TTE can provide important prognostic insights in CS patients, highlighting its potential utility in clinical practice. The LVOT VTI, reflecting forward flow, is an important measurement to obtain on bedside TTE.
Asunto(s)
Cuidados Críticos , Ecocardiografía Doppler , Choque Cardiogénico/mortalidad , Choque Cardiogénico/fisiopatología , Función Ventricular Izquierda/fisiología , Función Ventricular Derecha/fisiología , Anciano , Femenino , Mortalidad Hospitalaria , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Choque Cardiogénico/diagnóstico por imagen , Volumen Sistólico/fisiología , Tasa de SupervivenciaRESUMEN
The assembly of pili on the cell wall of Gram-positive bacteria requires transpeptidase enzymes called sortases. In Streptococcus agalactiae, the PI-1 pilus island of strain 2603V/R encodes two pilus-specific sortases (SrtC1 and SrtC2) and three pilins (GBS80, GBS52 and GBS104). Although either pilus-specific sortase is sufficient for the polymerization of the major pilin, GBS80, incorporation of the minor pilins GBS52 and GBS104 into the pilus structure requires SrtC1 and SrtC2, respectively. The S. agalactiae housekeeping sortase, SrtA, whose gene is present at a different location and does not catalyze pilus polymerization, was shown to be involved in cell wall anchoring of pilus polymers. To understand the structural basis of sortases involved in such diverse functions, we determined the crystal structures of S. agalactiae SrtC1 and SrtA. Both enzymes are made of an eight-stranded beta-barrel core with variations in their active site architecture. SrtA exhibits a catalytic triad arrangement similar to that in Streptococcus pyogenes SrtA but different from that in Staphylococcus aureus SrtA. In contrast, the SrtC1 enzyme contains an N-terminal helical domain and a 'lid' in its putative active site, which is similar to that seen in Streptococcus pneumoniae pilus-specific sortases, although with subtle differences in positioning and composition. To understand the effect of such differences on substrate recognition, we have also determined the crystal structure of a SrtC1 mutant, in which the conserved DP(W/F/Y) motif was replaced with the sorting signal motif of GBS80, IPNTG. By comparing the structures of WT wild type SrtA and SrtC1 and the 'lid' mutant of SrtC1, we propose that structural elements within the active site and the lid may be important for defining the role of specific sortase in pili biogenesis.
Asunto(s)
Aminoaciltransferasas/química , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Fimbrias Bacterianas/enzimología , Peptidil Transferasas/química , Peptidil Transferasas/metabolismo , Streptococcus agalactiae/enzimología , Secuencia de Aminoácidos , Aminoaciltransferasas/genética , Proteínas Bacterianas/genética , Dominio Catalítico , Cristalografía por Rayos X , Cisteína Endopeptidasas/genética , Estabilidad de Enzimas , Fimbrias Bacterianas/genética , Genes Esenciales , Metionina , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Especificidad de Órganos , Peptidil Transferasas/genética , Streptococcus agalactiae/citología , Streptococcus agalactiae/genética , Relación Estructura-ActividadRESUMEN
The clumping factor B (ClfB) of Staphylococcus aureus is a surface protein that binds to fibrinogen (Ni Eidhin, D., Perkins, S., Francois, P., Vaudaux, P., Hook, M., and Foster, T. J., 1998 Mol. Microbiol. 30, 245-257). The ligand-binding activity is located in the approximately 500-residue A-region (residues 44-542), which represents the N-terminal half of the MSCRAMM protein. We now hypothesize that the ClfB A-region is composed of three subdomains, which we have named N1, N2, and N3, respectively. To examine this hypothesis, we expressed recombinant forms of the individual putative subdomains, the tandem motifs N12 and N23, and the full-length A-region N123. Far UV circular dichroism spectra showed that each subdomain is composed mainly of beta-sheets with little or no discernible alpha-helices. Heat-induced unfolding of individual subdomains occurred with a single state transition and was reversible, indicating that the subdomains can fold as discreet units. Gel permeation chromatography indicated that N2, N3, and N23 are globular. In contrast, domain N1 appeared to be elongated and conferred a somewhat elongated structure on segments containing this subdomain (i.e. N12 or N123). N123, N12, and N23 all bound to fibrinogen, but N23 had a higher affinity for fibrinogen than that observed for the full-length A-region; N123 or for N12. However, an extended N terminus of N23 was required for ligand binding. A form of N23 that was generated by proteolytic processing and lacked the N-terminal extension was unable to bind fibrinogen. Recombinant forms of individual subdomains did not bind fibrinogen. The addition of recombinant N23 effectively inhibited ClfB-mediated bacterial adherence to fibrinogen, and N123 caused some reduction in bacterial attachment, whereas N12 was essentially inactive. Antibodies raised against the central N2 domain of the A-region were the most effective at inhibiting bacterial adhesion to immobilized fibrinogen, although anti-N3 or anti-N1 antibodies also caused some reduction in ClfB-mediated adherence to fibrinogen.
Asunto(s)
Adhesinas Bacterianas , Proteínas Bacterianas/química , Fibrinógeno/metabolismo , Staphylococcus aureus/química , Secuencias de Aminoácidos , Adhesión Celular , Dicroismo Circular , Colágeno/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/metabolismo , Fibrinógeno/química , Inmunoglobulina G/metabolismo , Ligandos , Plásmidos/metabolismo , Unión Proteica , Desnaturalización Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Temperatura , Rayos UltravioletaRESUMEN
Complement convertases are bimolecular complexes expressing protease activity only against C3 and C5. Their action is necessary for production of the biological activities of the complement system. Formation of these complexes proceeds through sequential protein-protein interactions and proteolytic cleavages of high specificity. Recent structural, mutational and functional data on factors D and B have significantly enhanced our understanding of the assembly, action, and regulation of the alternative pathway convertase. These processes were shown to depend critically on conformational changes, only some of which are reversible. The need for such changes is dictated by the zymogen-like configurations of the active centers of these unique serine proteases. The structural determinants of some of these changes have been defined from structural and mutational analyses of the two enzymes. Transition of factor D from the zymogen-like to the catalytically active conformation is completely reversible, while the active conformation of the catalytic center of the Bb fragment of factor B is irreversibly attenuated to a great extent on dissociation of the convertase complex. Both mechanisms contribute to the regulation of the proteolytic activity of these enzymes. Additional studies are necessary for a complete description of the elegant mechanisms mediating these processes.
Asunto(s)
Convertasas de Complemento C3-C5/biosíntesis , Convertasas de Complemento C3-C5/química , Factor D del Complemento/química , Vía Alternativa del Complemento , Animales , Sitios de Unión , Dominio Catalítico , C3 Convertasa de la Vía Alternativa del Complemento , Convertasas de Complemento C3-C5/fisiología , Complemento C3b/inmunología , Complemento C3b/metabolismo , Factor B del Complemento/metabolismo , Factor D del Complemento/fisiología , Activación Enzimática , Precursores Enzimáticos/química , Ratones , Modelos Moleculares , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Serina Endopeptidasas/química , Especificidad por Sustrato , Factor de von Willebrand/químicaRESUMEN
FKBP12.6 is a novel isoform of FKBP12, which selectively binds to the cardiac ryanodine receptor (RyR2). The crystal structure of FKBP12.6 in complex with rapamycin has now been determined at 2.0 A resolution. The structures of FKBP12.6 and FKBP12 are nearly identical, except for a displacement observed in the helical region of FKBP12.6 toward the hydrophobic pocket. This displacement was not predicted by homology modeling studies. Analyses of the residues that are likely to confer the RyR2-binding specificity are presented.
Asunto(s)
Inmunofilinas/química , Sirolimus/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Humanos , Inmunofilinas/metabolismo , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sirolimus/metabolismo , Proteínas de Unión a TacrolimusRESUMEN
BACKGROUND: [corrected] The Staphylococcus aureus collagen-binding protein Cna mediates bacterial adherence to collagen. The primary sequence of Cna has a non-repetitive collagen-binding A region, followed by the repetitive B region. The B region has one to four 23 kDa repeat units (B(1)-B(4)), depending on the strain of origin. The affinity of the A region for collagen is independent of the B region. However, the B repeat units have been suggested to serve as a 'stalk' that projects the A region from the bacterial surface and thus facilitate bacterial adherence to collagen. To understand the biological role of these B-region repeats we determined their three-dimensional structure. RESULTS: B(1) has two domains (D(1) and D(2)) placed side-by-side. D(1) and D(2) have similar secondary structure and exhibit a unique fold that resembles but is the inverse of the immunoglobulin-like (IgG-like) domains. Comparison with similar immunoglobulin superfamily (IgSF) structures shows novel packing arrangements between the D(1) and D(2) domains. In the B(1)B(2) crystal structure, an omission of a single glycine residue in the D(2)-D(3) linker loop, compared to the D(1)-D(2) and D(3)-D(4) linker loops, resulted in projection of the D(3) and D(4) in a spatially new orientation. We also present a model for B(1)B(2)B(3)B(4). CONCLUSIONS: The B region of the Cna collagen adhesin has a novel fold that is reminiscent of but is inverse in nature to the IgG fold. This B region assembly could effectively provide the needed flexibility and stability for presenting the ligand binding A region away from the bacterial cell surface.
Asunto(s)
Adhesinas Bacterianas , Proteínas Bacterianas/química , Colágeno/metabolismo , Staphylococcus aureus/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Inmunoglobulina G/química , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Secuencias Repetitivas de Aminoácido , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Staphylococcus aureus/genética , Electricidad EstáticaRESUMEN
Factor B is a serine protease, which despite its trypsin-like specificity has Asn instead of the typical Asp at the bottom of the S(1) pocket (position 189, chymotrypsinogen numbering). Asp residues are present at positions 187 and 226 and either one could conceivably provide the negative charge for binding the P(1)-Arg of the substrate. Determination of the crystal structure of the factor B serine protease domain has revealed that the side chain of Asp(226) is within the S(1) pocket, whereas Asp(187) is located outside the pocket. To investigate the possible role of these atypical structural features in substrate binding and catalysis, we constructed a panel of mutants of these residues. Replacement of Asp(187) caused moderate (50-60%) decrease in hemolytic activity, compared with wild type factor B, whereas replacement of Asn(189) resulted in more profound reductions (71-95%). Substitutions at these two positions did not significantly affect assembly of the alternative pathway C3 convertase. In contrast, elimination of the negative charge from Asp(226) completely abrogated hemolytic activity and also affected formation of the C3 convertase. Kinetic analyses of the hydrolysis of a P(1)-Arg containing thioester by selected mutants confirmed that residue Asp(226) is a primary structural determinant for P(1)-Arg binding and catalysis.
Asunto(s)
Factor B del Complemento/metabolismo , Secuencia de Aminoácidos , Asparagina , Ácido Aspártico , Dominio Catalítico , Convertasas de Complemento C3-C5/metabolismo , Factor B del Complemento/genética , Factor D del Complemento/metabolismo , Venenos Elapídicos/metabolismo , Hemólisis , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Factor B and C2 are two central enzymes for complement activation. They are multidomain serine proteases and require cofactor binding for full expression of proteolytic activities. We present a 2.1 A crystal structure of the serine protease domain of factor B. It shows a number of structural motifs novel to the chymotrypsin fold, which by sequence homology are probably present in C2 as well. These motifs distribute characteristically on the protein surface. Six loops surround the active site, four of which shape substrate-binding pockets. Three loops next to the oxyanion hole, which typically mediate zymogen activation, are much shorter or absent. Three insertions including the linker to the preceding domain bulge from the side opposite to the active site. The catalytic triad and non-specific substrate-binding site display active conformations, but the oxyanion hole displays a zymogen-like conformation. The bottom of the S1 pocket has a negative charge at residue 226 instead of the typical 189 position. These unique structural features may play different roles in domain-domain interaction, cofactor binding and substrate binding.
Asunto(s)
Quimotripsina/química , Factor B del Complemento/química , Serina Endopeptidasas/química , Secuencia de Aminoácidos , Sitios de Unión , Quimotripsina/metabolismo , Quimotripsinógeno/química , Complemento C2/química , Factor B del Complemento/metabolismo , Secuencia Conservada , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
A putative collagen-binding MSCRAMM, Ace, of Enterococcus faecalis was identified by searching bacterial genome data bases for proteins containing domains homologous to the ligand-binding region of Cna, the collagen-binding MSCRAMM from Staphylococcus aureus. Ace was predicted to have a molecular mass of 71 kDa and contains features characteristic of cell surface proteins on Gram-positive bacteria, including a LPXTG motif for cross-linking to the cell wall. The N-terminal region of Ace contained a region (residues 174-319) in which 56% of the residues are identical or similar when compared with the minimal ligand-binding region of Cna (Cna 151-318); the remainder of the Ace A domain has 46% similarity with the corresponding region of the Cna A domain. Antibodies raised against recombinant Ace A domain were used to verify the cell surface expression of Ace on E. faecalis. These antibodies also effectively inhibited the adhesion of enterococcal cells to a collagen substrate, suggesting that Ace is a functional collagen-binding MSCRAMM. Structural modeling of the conserved region in Ace (residues 174-319) suggested a structure very similar to that reported for residues 151-318 of the Cna collagen-binding domain in which the ligand-binding site was identified as a trench transversing a beta-sheet face (Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 10, 833-838). Biochemical analyses of recombinant Ace and Cna A domains supported the modeling data in that the secondary structures were similar as determined by CD spectroscopy and both proteins bound at multiple sites in type I collagen with micromolar affinities, but with different apparent kinetics. We conclude that Ace is a collagen-binding MSCRAMM on enterococci and is structurally and functionally related to the staphylococcal Cna protein.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/aislamiento & purificación , Colágeno/metabolismo , Enterococcus faecalis/metabolismo , Secuencia de Aminoácidos , Western Blotting , Proteínas Portadoras/química , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Most mammalian cells and some pathogenic bacteria are capable of adhering to collagenous substrates in processes mediated by specific cell surface adherence molecules. Crystal structures of collagen-binding regions of the human integrin alpha(2)beta(1) and a Staphylococcus aureus adhesin reveal a "trench" on the surface of both of these proteins. This trench can accommodate a collagen triple-helical structure and presumably represents the ligand-binding site (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517; Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., Schneider, A., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 4, 833-838). We report here the crystal structure of the alpha subunit I domain from the alpha(1)beta(1) integrin. This collagen-binding protein also contains a trench on one face in which the collagen triple helix may be docked. Furthermore, we compare the collagen-binding mechanisms of the human alpha(1) integrin I domain and the A domain from the S. aureus collagen adhesin, Cna. Although the S. aureus and human proteins have unrelated amino acid sequences, secondary structure composition, and cation requirements for effective ligand binding, both proteins bind at multiple sites within one collagen molecule, with the sites in collagen varying in their affinity for the adherence molecule. We propose that (i) these evolutionarily dissimilar adherence proteins recognize collagen via similar mechanisms, (ii) the multisite, multiclass protein/ligand interactions observed in these two systems result from a binding-site trench, and (iii) this unusual binding mechanism may be thematic for proteins binding extended, rigid ligands that contain repeating structural motifs.
Asunto(s)
Proteínas Bacterianas/química , Colágeno/química , Integrinas/química , Staphylococcus aureus/metabolismo , Adhesinas Bacterianas/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , Integrina alfa1beta1 , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Staphylococcus aureus/patogenicidad , Resonancia por Plasmón de SuperficieRESUMEN
Recombinant proteins of monomeric and dimeric B-domain repeats of a Staphylococcus aureus FDA 574 collagen-binding adhesin have been crystallized. The single repeat unit (B1) was crystallized in a body-centered orthorhombic lattice with a = 96.9, b = 101.3, c = 120. 8 A in either the I222 or I212121 space group. These crystals diffracted to 2.5 A resolution and the calculated Vm values of 3.2 and 2.2 A3 Da-1 suggest the possibility of a dimer or a trimer in the asymmetric unit. The two-repeat fragment (B1B2) crystallized in the orthorhombic space group P212121 with cell dimensions a = 42.4, b = 79.4, c = 130.4 A and diffracted to 2.3 A resolution. For this species, the calculated Vm value of 2.2 A3 Da-1 indicates the presence of a monomer in the asymmetric unit.
Asunto(s)
Integrinas/química , Staphylococcus aureus/química , Proteínas Portadoras/química , Cristalización , Cristalografía por Rayos X , Conformación Proteica , Receptores de Colágeno , Proteínas Recombinantes/químicaRESUMEN
Recombinant constructs encoding the fibrinogen-binding domains of ClfA and ClfB from Staphylococcus aureus have been crystallized. ClfA was crystallized in the orthorhombic space group P212121 with unit-cell parameters a = 39.58, b = 81.39 and c = 112.65 A. A complete data set was recorded to 2.1 A resolution and had a Vm of 2. 3 A3 Da-1 with 46.5% solvent, suggesting one molecule per asymmetric unit. Co-crystals of ClfA with the 17 amino-acid C-terminal peptide of fibrinogen gamma-chain diffracted to 2.1 A resolution and had unit-cell parameters a = 39.11, b = 81.39 and c = 109.51 A in the space group P212121. ClfB was crystallized in the tetragonal space group P41212 or P43212 with unit-cell parameters a = 96.31, b = 96. 31 and c = 84.13 A and diffracted to 2.45 A resolution. The estimated Vm of 2.6 A3 Da-1 with 53% solvent indicated one molecule in the asymmetric unit.
Asunto(s)
Proteínas Bacterianas/química , Fibrinógeno/metabolismo , Proteínas de la Membrana/química , Fragmentos de Péptidos/química , Staphylococcus aureus/química , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Cristalización , Cristalografía por Rayos X , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación ProteicaRESUMEN
Recombinant uracil phosphoribosyltransferase (UPRT) enzyme of Toxoplasma gondii was expressed in Escherichia coli and purified from the cell-free extract by a combination of chromatographic steps. The recombinant protein was enzymatically active when tested in an in vitro UPRT assay. The purified protein was crystallized using the hanging-drop vapor-diffusion technique with ammonium phosphate as precipitant. The crystallized protein also exhibited UPRT activity. Crystals diffract to 2.4 A resolution and belong to space group P3121 or P3221 with unit-cell dimensions a = b = 119.9, c = 70.8 A and two molecules per asymmetric unit.
Asunto(s)
Pentosiltransferasa/química , Pentosiltransferasa/aislamiento & purificación , Toxoplasma/enzimología , Animales , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Expresión Génica , Pentosiltransferasa/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Toxoplasma/genéticaRESUMEN
The crystal structure of profactor D, determined at 2.1 A resolution with an Rfree and an R-factor of 25.1 and 20.4%, respectively, displays highly flexible or disordered conformation for five regions: N-22, 71-76, 143-152, 187-193 and 215-223. A comparison with the structure of its mature serine protease, complement factor D, revealed major conformational changes in the similar regions. Comparisons with the zymogen-active enzyme pairs of chymotrypsinogen, trypsinogen and prethrombin-2 showed a similar distribution of the flexible regions. However, profactor D is the most flexible of the four, and its mature enzyme displays inactive, self-inhibited active site conformation. Examination of the surface properties of the N-terminus-binding pocket indicates that Ile16 may play the initial positioning role for the N-terminus, and Leu17 probably also helps in inducing the required conformational changes. This process, perhaps shared by most chymotrypsinogen-like zymogens, is followed by a factor D-unique step, the re-orientation of an external Arg218 to an internal position for salt-bridging with Asp189, leading to the generation of the self-inhibited factor D.
Asunto(s)
Factor D del Complemento/química , Precursores Enzimáticos/química , Precursores de Proteínas/química , Serina Endopeptidasas/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Activación Enzimática , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Homología de Secuencia de AminoácidoRESUMEN
Sequence analysis of surface proteins from Gram-positive bacteria indicates a composite organization consisting of unique and repeated segments. Thus, these proteins may contain discrete domains that could fold independently. In this paper, we have used a panel of biophysical methods, including gel permeation chromatography, analytical ultracentrifugation, circular dichroism, and fluorescence spectroscopy, to analyze the structural organization of the Staphylococcus aureus collagen adhesin, CNA. Our results indicate that the structure, function, and folding of the ligand-binding domain (A) are not affected by the presence or absence of the other major domain (B). In addition, little or no interaction is observed between the nearly identical repeat units within the B domain. We propose that CNA is indeed a mosaic protein in which the different domains previously indicated by sequence analysis operate independently.
Asunto(s)
Adhesinas Bacterianas/química , Proteínas Bacterianas/química , Colágeno/química , Estructura Terciaria de Proteína , Staphylococcus aureus/química , Adhesinas Bacterianas/genética , Proteínas Bacterianas/genética , Dicroismo Circular , Colágeno/genética , Dimerización , Modelos Moleculares , Imitación Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Secuencias Repetitivas de AminoácidoRESUMEN
Quinolinate phosphoribosyltransferase (QPRT), purified from hog liver, has been crystallized using PEG 8000 as the precipitant. The crystals form long hexagonal rods in the space group P6322 with cell dimensions a = b = 121.7, c = 94.5 A. Based on the unit-cell dimensions and the calculated molecular mass of 33 500 Da, the Matthews coefficient suggests one molecule per asymmetric unit (Vm = 3.45 A3 Da-1; 64% solvent). Three native data sets were collected to a resolution of 2.5 A and merged to provide a set that is 94.7% complete, with an Rsym value of 9.6%.
Asunto(s)
Pentosiltransferasa/química , Animales , Cristalización , Cristalografía por Rayos X , PorcinosRESUMEN
Factor D is a serine protease essential for the activation of the alternative pathway of complement. The structures of native factor D and a complex formed with isatoic anhydride inhibitor were determined at resolution of 2.3 and 1.5 A, respectively, in an isomorphous monoclinic crystal form containing one molecule per asymmetric unit. The native structure was compared with structures determined previously in a triclinic cell containing two molecules with different active site conformations. The current structure shows greater similarity with molecule B in the triclinic cell, suggesting that this may be the dominant factor D conformation in solution. The major conformational differences with molecule A in the triclinic cell are located in four regions, three of which are close to the active site and include some of the residues shown to be critical for factor D catalytic activity. The conformational flexibility associated with these regions is proposed to provide a structural basis for the previously proposed substrate-induced reversible conformational changes in factor D. The high-resolution structure of the factor D/isatoic anhydride complex reveals the binding mode of the mechanism-based inhibitor. The higher specificity towards factor D over trypsin and thrombin is based on hydrophobic interactions between the inhibitor benzyl ring and the aliphatic side-chain of Arg218 that is salt bridged with Asp189 at the bottom of the primary specificity (S1) pocket. Comparison of factor D structural variants with other serine protease structures revealed the presence of a unique "self-inhibitory loop". This loop (214-218) dictates the resting-state conformation of factor D by (1) preventing His57 from adopting active tautomer conformation, (2) preventing the P1 to P3 residues of the substrate from forming anti-parallel beta-sheets with the non-specific substrate binding loop, and (3) blocking the accessibility of Asp189 to the positive1y charged P1 residue of the substrate. The conformational switch from resting-state to active-state can only be induced by the single macromolecular substrate, C3b-bound factor B. This self-inhibitory mechanism is highly correlated with the unique functional properties of factor D, which include high specificity toward factor B, low esterolytic activity toward synthetic substrates, and absence of regulation by zymogen and serpin-like or other natural inhibitors in blood.
Asunto(s)
Factor D del Complemento/química , Factor D del Complemento/metabolismo , Oxazinas/química , Sitios de Unión , Cumarinas/química , Cumarinas/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Histidina , Isocumarinas , Modelos Moleculares , Oxazinas/metabolismo , Conformación Proteica , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/metabolismo , AguaRESUMEN
The crystal structure of the recombinant 19,000 M(r) binding domain from the Staphylococcus aureus collagen adhesin has been determined at 2 A resolution. The domain fold is a jelly-roll, composed of two antiparallel beta-sheets and two short alpha-helices. Triple-helical collagen model probes were used in a systematic docking search to identify the collagen-binding site. A groove on beta-sheet I exhibited the best surface complementarity to the collagen probes. This site partially overlaps with the peptide sequence previously shown to be critical for collagen binding. Recombinant proteins containing single amino acid mutations designed to disrupt the surface of the putative binding site exhibited significantly lower affinities for collagen. Here we present a structural perspective for the mode of collagen binding by a bacterial surface protein.
Asunto(s)
Adhesinas Bacterianas/química , Adhesinas Bacterianas/metabolismo , Colágeno/metabolismo , Pliegue de Proteína , Estructura Secundaria de Proteína , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli , Modelos Moleculares , Modelos Estructurales , Señales de Clasificación de Proteína/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/metabolismoRESUMEN
The three dimensional structure of calcium-bound domain VI of porcine calpain has been determined to 1.9 A resolution. The crystal structure reveals five EF-hands, one more than previously suggested. There are two EF-hand pairs, one pair (EF1-EF2) displays an 'open' conformation and the other (EF3-EF4) a 'closed' conformation. Unusually, a calcium atom is found at the C-terminal end of the calcium binding loop of EF4. With two additional residues in the calcium binding loop, the fifth EF-hand (EF5) is in a 'closed' conformation. EF5 pairs up with the corresponding fifth EF-hand of a non-crystallographically related molecule. Considering the EF5's role in a homodimer formation of domain VI, we suggest a model for the assembly of heterodimeric calpain. The crystal structure of a Ca2+ bound domain VI-inhibitor (PD150606) complex has been refined to 2.1 A resolution. A possible mode for calpain inhibition is discussed.