Structural characterizations of thioredoxins (Trxs) are important for their involvement in severe... more Structural characterizations of thioredoxins (Trxs) are important for their involvement in severe pathologies and for their stable scaffold. Here we report a combined structural and spectroscopic characterization of a Trx isolated from the hyperthermophilic archaeon Sulfolobus solfataricus (SsTrxA1). Thermal denaturation unveils that SsTrxA1 is endowed with a remarkable stability in the explored temperature range 50-105°C. The structure of the oxidized form of SsTrxA1 determined at 1.9Å resolution presents a number of peculiar features. Although the protein was crystallized in a slightly acid medium (pH 6.5) as many as ten intramolecular/intermolecular carboxyl-carboxylate interactions involving glutamic and aspartic acid side chains are found in three independent SsTrxA1 molecules present in the asymmetric unit. Surprisingly for a hyperthermostable protein, the structure of SsTrxA1 is characterized by the presence (a) of a very limited number of intramolecular salt bridges and (b) of a cavity nearby Cys52, a residue that is frequently a phenylananine in other members of the family. Chemical denaturation investigations carried out on SsTrxA1 and SsTrxA2 show that both proteins present a significant stability against guanidine hydrochloride, thus indicating that ionic interactions play a minor role in their stabilization. Compared to Trxs from mesophilic sources, SsTrxA1 displays a longer α-helix 1 and a shorter loop connecting this α-helix with β-strand 2. As these features are shared with Trxs isolated from thermophilic sources, the shortening of this loop may be a general strategy adopted to stabilize this fold. This feature may be exploited for the design of hyperthermostable Trx scaffolds.
Protein science : a publication of the Protein Society, Jan 15, 2017
Adherence, colonization, and survival of mycobacteria in host cells require surface adhesins, whi... more Adherence, colonization, and survival of mycobacteria in host cells require surface adhesins, which are attractive pharmacotherapeutic targets. A large arsenal of pilus and non-pilus adhesins have been identified in mycobacteria. These adhesins are capable of interacting with host cells, including macrophages and epithelial cells and are essential to microbial pathogenesis. In the last decade, several structures of mycobacterial adhesins responsible for adhesion to either macrophages or extra cellular matrix proteins have been elucidated. In addition, key structural and functional information have emerged for the process of mycobacterial adhesion to epithelial cells, mediated by the Heparin Binding Hemagglutinin HBHA. In this review, we provide an overview of the structural and functional features of mycobacterial adhesins and discuss their role as important biomarkers for diagnostics and therapeutics. Based on the reported data, it appears clear that adhesins are endowed with a var...
ABSTRACT The elongation factor 1a from the hyperthermophilic archaeon Sulfolobus solfataricus (Ss... more ABSTRACT The elongation factor 1a from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-1a) belongs to the family of GTPbinding proteins (GNBP). In the fulfilment of its biological function SsEF-1a switches between an active GTP-bound and an inactive GDP-bound form and elicits an intrinsic GTPase activity. Surprisingly, the 3D structure of SsEF-1A in complex with GDP showed that, in contrast with most of the other GNBP, no magnesium ions have been found in the nucleotide binding site, despite the presence of the cation in the crystallization medium. Furthermore, a conserved aspartic acid (D60 in SsEF-1a), although present in the substrate binding site of the factor, is located more far away with respect to its position occupied in all the other GNBPs with known structure. To study the involvement of this amino acid position on the functional properties of SsEF-1a, a mutagenic analysis on this position has been started. In this communication, the results on the biochemical characterization of a first mutated form of SsEF-1a is reported.The D60A mutant of SsEF-1a, was obtained as heterologous expressed purified protein and characterised. The expression system used produced a folded protein able to catalyse, although to a slightly lower extent with respect to the wild type enzyme, protein synthesis in vitro and NaCl-dependent intrinsic GTPase activity. The affinity for guanosine nucleotides and heterologous aa-tRNA was almost identical to that exhibited by wild type SsEF-1a; vice versa, the GDP exchange rate was one order of magnitude faster on the mutated elongation factor, a property partially restored when the exchange reaction was analysed in the presence of the magnesium ions chelating agent EDTA. Finally, the D60A substitution only a little affected the high thermal stability of the elongation factor. From a structural point of view, the analysis of the data reported confirmed that this conserved carboxyl group belongs to a protein region differentiating the GDP binding mode among elongation factors from different organisms.
Heparin-binding hemagglutinin (HBHA) is a virulence factor of tuberculosis which is responsible f... more Heparin-binding hemagglutinin (HBHA) is a virulence factor of tuberculosis which is responsible for extrapulmonary dissemination of this disease. A thorough biochemical characterization of HBHA has provided experimental evidence of a coiled-coil nature of HBHA. These data, together with the low-resolution structures of a full-length form and a truncated form of HBHA obtained by small-angle X-ray scattering, have unambiguously indicated that HBHA has a dimeric structure with an elongated shape.
Structural characterizations of thioredoxins (Trxs) are important for their involvement in severe... more Structural characterizations of thioredoxins (Trxs) are important for their involvement in severe pathologies and for their stable scaffold. Here we report a combined structural and spectroscopic characterization of a Trx isolated from the hyperthermophilic archaeon Sulfolobus solfataricus (SsTrxA1). Thermal denaturation unveils that SsTrxA1 is endowed with a remarkable stability in the explored temperature range 50-105°C. The structure of the oxidized form of SsTrxA1 determined at 1.9Å resolution presents a number of peculiar features. Although the protein was crystallized in a slightly acid medium (pH 6.5) as many as ten intramolecular/intermolecular carboxyl-carboxylate interactions involving glutamic and aspartic acid side chains are found in three independent SsTrxA1 molecules present in the asymmetric unit. Surprisingly for a hyperthermostable protein, the structure of SsTrxA1 is characterized by the presence (a) of a very limited number of intramolecular salt bridges and (b) of a cavity nearby Cys52, a residue that is frequently a phenylananine in other members of the family. Chemical denaturation investigations carried out on SsTrxA1 and SsTrxA2 show that both proteins present a significant stability against guanidine hydrochloride, thus indicating that ionic interactions play a minor role in their stabilization. Compared to Trxs from mesophilic sources, SsTrxA1 displays a longer α-helix 1 and a shorter loop connecting this α-helix with β-strand 2. As these features are shared with Trxs isolated from thermophilic sources, the shortening of this loop may be a general strategy adopted to stabilize this fold. This feature may be exploited for the design of hyperthermostable Trx scaffolds.
Protein science : a publication of the Protein Society, Jan 15, 2017
Adherence, colonization, and survival of mycobacteria in host cells require surface adhesins, whi... more Adherence, colonization, and survival of mycobacteria in host cells require surface adhesins, which are attractive pharmacotherapeutic targets. A large arsenal of pilus and non-pilus adhesins have been identified in mycobacteria. These adhesins are capable of interacting with host cells, including macrophages and epithelial cells and are essential to microbial pathogenesis. In the last decade, several structures of mycobacterial adhesins responsible for adhesion to either macrophages or extra cellular matrix proteins have been elucidated. In addition, key structural and functional information have emerged for the process of mycobacterial adhesion to epithelial cells, mediated by the Heparin Binding Hemagglutinin HBHA. In this review, we provide an overview of the structural and functional features of mycobacterial adhesins and discuss their role as important biomarkers for diagnostics and therapeutics. Based on the reported data, it appears clear that adhesins are endowed with a var...
ABSTRACT The elongation factor 1a from the hyperthermophilic archaeon Sulfolobus solfataricus (Ss... more ABSTRACT The elongation factor 1a from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-1a) belongs to the family of GTPbinding proteins (GNBP). In the fulfilment of its biological function SsEF-1a switches between an active GTP-bound and an inactive GDP-bound form and elicits an intrinsic GTPase activity. Surprisingly, the 3D structure of SsEF-1A in complex with GDP showed that, in contrast with most of the other GNBP, no magnesium ions have been found in the nucleotide binding site, despite the presence of the cation in the crystallization medium. Furthermore, a conserved aspartic acid (D60 in SsEF-1a), although present in the substrate binding site of the factor, is located more far away with respect to its position occupied in all the other GNBPs with known structure. To study the involvement of this amino acid position on the functional properties of SsEF-1a, a mutagenic analysis on this position has been started. In this communication, the results on the biochemical characterization of a first mutated form of SsEF-1a is reported.The D60A mutant of SsEF-1a, was obtained as heterologous expressed purified protein and characterised. The expression system used produced a folded protein able to catalyse, although to a slightly lower extent with respect to the wild type enzyme, protein synthesis in vitro and NaCl-dependent intrinsic GTPase activity. The affinity for guanosine nucleotides and heterologous aa-tRNA was almost identical to that exhibited by wild type SsEF-1a; vice versa, the GDP exchange rate was one order of magnitude faster on the mutated elongation factor, a property partially restored when the exchange reaction was analysed in the presence of the magnesium ions chelating agent EDTA. Finally, the D60A substitution only a little affected the high thermal stability of the elongation factor. From a structural point of view, the analysis of the data reported confirmed that this conserved carboxyl group belongs to a protein region differentiating the GDP binding mode among elongation factors from different organisms.
Heparin-binding hemagglutinin (HBHA) is a virulence factor of tuberculosis which is responsible f... more Heparin-binding hemagglutinin (HBHA) is a virulence factor of tuberculosis which is responsible for extrapulmonary dissemination of this disease. A thorough biochemical characterization of HBHA has provided experimental evidence of a coiled-coil nature of HBHA. These data, together with the low-resolution structures of a full-length form and a truncated form of HBHA obtained by small-angle X-ray scattering, have unambiguously indicated that HBHA has a dimeric structure with an elongated shape.
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Papers by Alessia Ruggiero