C-terminal amino acids are essential for human heat shock protein 70 dimerization

The human inducible heat shock protein 70 (hHsp70), which is involved in several major pathologies, including neurodegenerative disorders and cancer, is a key molecular chaperone and contributes to the proper protein folding and maintenance of a large number of protein structures. Despite its role in disease, the current structural knowledge of hHsp70 is almost exclusively based on its Escherichia coli homolog, DnaK, even though these two proteins only share ~50 % amino acid identity. For the first time, we describe a complete heterologous production and purification strategy that allowed us to obtain a large amount of soluble, full-length, and non-tagged hHsp70. The protein displayed both an ATPase and a refolding activity when combined to the human Hsp40. Multi-angle light scattering and bio-layer interferometry analyses demonstrated the ability of hHsp70 to homodimerize. The role of the C-terminal part of hHsp70 was identified and confirmed by a study of a truncated version of hHsp70 that could neither dimerize nor present refolding activity

The metal ion-dependent adhesion site motif of the Enterococcus faecalis EbpA pilin mediates pilus function in catheter-associated urinary tract infection

Though the bacterial opportunist Enterococcus faecalis causes a myriad of hospital-acquired infections (HAIs), including catheter-associated urinary tract infections (CAUTIs), little is known about the virulence mechanisms that it employs. However, the endocarditis- and biofilm-associated pilus (Ebp), a member of the sortase-assembled pilus family, was shown to play a role in a mouse model of E. faecalis ascending UTI. The Ebp pilus comprises the major EbpC shaft subunit and the EbpA and EbpB minor subunits. We investigated the biogenesis and function of Ebp pili in an experimental model of CAUTI using a panel of chromosomal pilin deletion mutants. A nonpiliated pilus knockout mutant (EbpABC(−) strain) was severely attenuated compared to its isogenic parent OG1RF in experimental CAUTI. In contrast, a nonpiliated ebpC deletion mutant (EbpC(−) strain) behaved similarly to OG1RF in vivo because it expressed EbpA and EbpB. Deletion of the minor pilin gene ebpA or ebpB perturbed pilus biogenesis and led to defects in experimental CAUTI. We discovered that the function of Ebp pili in vivo depended on a predicted metal ion-dependent adhesion site (MIDAS) motif in EbpA’s von Willebrand factor A domain, a common protein domain among the tip subunits of sortase-assembled pili. Thus, this study identified the Ebp pilus as a virulence factor in E. faecalis CAUTI and also defined the molecular basis of this function, critical knowledge for the rational development of targeted therapeutics

Pilin and Sortase Residues Critical for Endocarditis- and Biofilm-Associated Pilus Biogenesis in Enterococcus faecalis

Enterococci commonly cause hospital-acquired infections, such as infective endocarditis and catheter-associated urinary tract infections. In animal models of these infections, a long hairlike extracellular protein fiber known as the endocarditis- and biofilm-associated (Ebp) pilus is an important virulence factor for Enterococcus faecalis. For Ebp and other sortase-assembled pili, the pilus-associated sortases are essential for fiber formation as they create covalent isopeptide bonds between the sortase recognition motif and the pilin-like motif of the pilus subunits. However, the molecular requirements governing the incorporation of the three pilus subunits (EbpA, EbpB, and EbpC) have not been investigated in E. faecalis. Here, we show that a Lys residue within the pilin-like motif of the EbpC subunit was necessary for EbpC polymerization. However, incorporation of EbpA into the pilus fiber only required its sortase recognition motif (LPXTG), while incorporation of EbpB only required its pilin-like motif. Only the sortase recognition motif would be required for incorporation of the pilus tip subunit, while incorporation of the base subunit would only require the pilin recognition motif. Thus, these data support a model with EbpA at the tip and EbpB at the base of an EbpC polymer. In addition, the housekeeping sortase, SrtA, was found to process EbpB and its predicted catalytic Cys residue was required for efficient cell wall anchoring of mature Ebp pili. Thus, we have defined molecular interactions involved in fiber polymerization, minor subunit organization, and pilus subcellular compartmentalization in the E. faecalis Ebp pilus system. These studies advance our understanding of unique molecular mechanisms of sortase-assembled pilus biogenesis

Mécanisme, catalyse et spécificité structurale des méthionine sulfoxyde réductases de classe B et la protéine PilB de Neisseria meningitidis

Texte intégral accessible uniquement aux membres de l'Université de LorraineUbiquitous enzyme methionine sulfoxide reductases (Msrs) are involved in oxidative stress resistance, aging process but also in bacteria pathogenicity like for Neisseria genius. The two Msrs classes : MsrA and MsrB structural-unrelated catalyze the reduction of the two stereoisomeric forms R and S of the sulfoxide function from the methionine sulfoxide. They share a similar three-step chemical mechanism including formation of a sulfenic acid intermediate following by intramolecular disulfide bond formation, reduced in the last step by the thioredoxin (Trx). The structure function studies are conduced to 1) characterization of active site amino acids involved in substrate recognition and reductase step catalysis leading to sulfenic acid formation in Neisseria meningitidis (N. meningitidis) MsrB, we have proposed a scenario for the reductase step with a major role of the acid / base catalyst His 103 2) characterization of different MsrB sub-classes mechanisms, Xanthomonas campestris MsrB present a Cys 31 located in a flexible loop compare to the Cys 63 from N. meningitidis MsrB also located in a flexible loop, the Mycoplasma pulmonis MsrB don't posses recycling Cys; and 3) characterization of the N. meningitidis PilB, a three domains protein located in the periplasm, PilB possess MsrA and MsrB activities, and a oxydoreductase activity carried by the N-terminal domain, moreover this domain can reduced the oxidized MsrA and MsrB domains.Les méthionine sulfoxyde réductases (Msr) sont des enzymes ubiquitaires impliquées dans la résistance au stress oxydant, les processus de vieillissement mais également dans la virulence de certaines bactéries pathogènes. Deux classes de Msr : MsrA et MsrB, structuralement distinctes, catalysent respectivement la réduction des stéréoisomères S et R de la fonction sulfoxyde de la méthionine sulfoxyde selon un même mécanisme catalytique en trois étapes impliquant la formation d?un intermédiaire acide sulfénique suivie de celle d'un pont disulfure intramoléculaire ensuite réduit par la thiorédoxine (Trx). Des études de relation structure-fonction, ont permis 1) de caractériser les résidus du site actif de la MsrB de Neisseria meningitidis (N. meningitidis) impliqués dans la reconnaissance du substrat, et dans la catalyse de l?étape réductase conduisant à la formation de l?intermédiaire acide sulfénique, et de proposer un scénario pour la catalyse de l?étape réductase dans lequel le résidu His 103 joue un rôle majeur de catalyseur acide / base ; 2) de caractériser le mécanisme des autres sous-classes de MsrB, qui diffèrent de la sous-classe représentée par la MsrB de N. meningitidis par l?absence de la Cys de régénération en position 63, notamment celui de la MsrB de Xanthomonas campestris, qui possède une Cys de régénération en position 31 située dans une boucle flexible ; et 3) de caractériser la protéine PilB de N. meningitidis, protéine à trois domaines, localisée dans le périplasme portant non seulement les activités MsrA et MsrB mais aussi une activité disulfure oxydoréductase au niveau de son domaine N-terminal dont le rôle est de régénérer les activités Msr

Mécanisme, catalyse et spécificité structurale des méthionine sulfoxyde réductases de classe B et la protéine PilB de Neisseria meningitidis

Texte intégral accessible uniquement aux membres de l'Université de LorraineUbiquitous enzyme methionine sulfoxide reductases (Msrs) are involved in oxidative stress resistance, aging process but also in bacteria pathogenicity like for Neisseria genius. The two Msrs classes : MsrA and MsrB structural-unrelated catalyze the reduction of the two stereoisomeric forms R and S of the sulfoxide function from the methionine sulfoxide. They share a similar three-step chemical mechanism including formation of a sulfenic acid intermediate following by intramolecular disulfide bond formation, reduced in the last step by the thioredoxin (Trx). The structure function studies are conduced to 1) characterization of active site amino acids involved in substrate recognition and reductase step catalysis leading to sulfenic acid formation in Neisseria meningitidis (N. meningitidis) MsrB, we have proposed a scenario for the reductase step with a major role of the acid / base catalyst His 103 2) characterization of different MsrB sub-classes mechanisms, Xanthomonas campestris MsrB present a Cys 31 located in a flexible loop compare to the Cys 63 from N. meningitidis MsrB also located in a flexible loop, the Mycoplasma pulmonis MsrB don't posses recycling Cys; and 3) characterization of the N. meningitidis PilB, a three domains protein located in the periplasm, PilB possess MsrA and MsrB activities, and a oxydoreductase activity carried by the N-terminal domain, moreover this domain can reduced the oxidized MsrA and MsrB domains.Les méthionine sulfoxyde réductases (Msr) sont des enzymes ubiquitaires impliquées dans la résistance au stress oxydant, les processus de vieillissement mais également dans la virulence de certaines bactéries pathogènes. Deux classes de Msr : MsrA et MsrB, structuralement distinctes, catalysent respectivement la réduction des stéréoisomères S et R de la fonction sulfoxyde de la méthionine sulfoxyde selon un même mécanisme catalytique en trois étapes impliquant la formation d?un intermédiaire acide sulfénique suivie de celle d'un pont disulfure intramoléculaire ensuite réduit par la thiorédoxine (Trx). Des études de relation structure-fonction, ont permis 1) de caractériser les résidus du site actif de la MsrB de Neisseria meningitidis (N. meningitidis) impliqués dans la reconnaissance du substrat, et dans la catalyse de l?étape réductase conduisant à la formation de l?intermédiaire acide sulfénique, et de proposer un scénario pour la catalyse de l?étape réductase dans lequel le résidu His 103 joue un rôle majeur de catalyseur acide / base ; 2) de caractériser le mécanisme des autres sous-classes de MsrB, qui diffèrent de la sous-classe représentée par la MsrB de N. meningitidis par l?absence de la Cys de régénération en position 63, notamment celui de la MsrB de Xanthomonas campestris, qui possède une Cys de régénération en position 31 située dans une boucle flexible ; et 3) de caractériser la protéine PilB de N. meningitidis, protéine à trois domaines, localisée dans le périplasme portant non seulement les activités MsrA et MsrB mais aussi une activité disulfure oxydoréductase au niveau de son domaine N-terminal dont le rôle est de régénérer les activités Msr

Mecanism, catalysis and structural specificity of the methionine sulfoxide reductase B and the PilB protein from Neisseria meningitidis

Les méthionine sulfoxyde réductases (Msr) sont des enzymes ubiquitaires impliquées dans la résistance au stress oxydant, les processus de vieillissement mais également dans la virulence de certaines bactéries pathogènes. Deux classes de Msr : MsrA et MsrB, structuralement distinctes, catalysent respectivement la réduction des stéréoisomères S et R de la fonction sulfoxyde de la méthionine sulfoxyde selon un même mécanisme catalytique en trois étapes impliquant la formation d’un intermédiaire acide sulfénique suivie de celle d’un pont disulfure intramoléculaire ensuite réduit par la thiorédoxine (Trx). Des études de relation structure-fonction, ont permis 1) de caractériser les résidus du site actif de la MsrB de Neisseria meningitidis (N. meningitidis) impliqués dans la reconnaissance du substrat, et dans la catalyse de l’étape réductase conduisant à la formation de l’intermédiaire acide sulfénique, et de proposer un scénario pour la catalyse de l’étape réductase dans lequel le résidu His 103 joue un rôle majeur de catalyseur acide / base; 2) de caractériser le mécanisme des autres sous-classes de MsrB, qui diffèrent de la sous-classe représentée par la MsrB de N. meningitidis par l’absence de la Cys de régénération en position 63, notamment celui de la MsrB de Xanthomonas campestris, qui possède une Cys de régénération en position 31 située dans une boucle flexible ; et 3) de caractériser la protéine PilB de N. meningitidis, protéine à trois domaines, localisée dans le périplasme portant non seulement les activités MsrA et MsrB mais aussi une activité disulfure oxydoréductase au niveau de son domaine N-terminal dont le rôle est de régénérer les activités Msr.Ubiquitous enzyme methionine sulfoxide reductases (Msrs) are involved in oxidative stress resistance, aging process but also in bacteria pathogenicity like for Neisseria genius. The two Msrs classes: MsrA and MsrB structural-unrelated catalyze the reduction of the two stereoisomeric forms R and S of the sulfoxide function from the methionine sulfoxide. They share a similar three-step chemical mechanism including formation of a sulfenic acid intermediate following by intramolecular disulfide bond formation, reduced in the last step by the thioredoxin (Trx). The structure function studies are conduced to 1) characterization of active site amino acids involved in substrate recognition and reductase step catalysis leading to sulfenic acid formation in Neisseria meningitidis (N. meningitidis) MsrB, we have proposed a scenario for the reductase step with a major role of the acid / base catalyst His 103 2) characterization of different MsrB sub-classes mechanisms, Xanthomonas campestris MsrB present a Cys 31 located in a flexible loop compare to the Cys 63 from N. meningitidis MsrB also located in a flexible loop, the Mycoplasma pulmonis MsrB don’t posses recycling Cys; and 3) characterization of the N. meningitidis PilB, a three domains protein located in the periplasm, PilB possess MsrA and MsrB activities, and a oxydoreductase activity carried by the N-terminal domain, moreover this domain can reduced the oxidized MsrA and MsrB domains

Structure-function relationships of brazzein, a sweet-tasting protein and its interactions with the human sweet taste receptor

International audienceBrazzein is a small heat- and pH-stable sweet-tasting protein isolated from the West African plant, Pentadiplandra brazzeana. Brazzein combines a highly sweet potency, a long history of human consumption, and a remarkable stability, giving it great potential as a natural sweetener. Due to the difficulties of obtaining brazzein from its natural source, several efforts have been made to express brazzein using various heterologous expression systems. Brazzein like all classes of sweet compounds (natural sugars, natural and artificial sweeteners) are perceived through the activation of the T1R2/T1R3 heterodimeric sweet taste receptor. T1R2 and T1R3 subunits are members of the small family of class C G-protein coupled receptors (GPCRs). Class C GPCRs possess a large N-terminal domain (NTD) linked to a heptahelical transmembrane domain by a cysteine rich domain (CRD). Cellular assays, molecular docking and site-directed mutagenesis studies have revealed that the NTD of T1R2 (T1R2-NTD) contain the primary binding site for most of the sweet ligands including natural sugars and, artificial and natural sweeteners including sweet-tasting proteins. We will summarize the structure-activity relationship of brazzein and will describe the current knowledge on the putative molecular mechanism of receptor activation, which remains to be elucidated