The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function ... more The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the Nucleotide Excision Repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wildtype and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface where, in contrast to F231, L231 lacks interactions stabilizing the XPF-ERCC1 complex. One of the two anchor points is severely distorted and this results in a more dynamic complex causing reduced stability and an increased dissociation rate of the mutant complex as compared to wildtype...
Protein science : a publication of the Protein Society, 2016
Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit... more Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit, promotes ribosomal binding of fMet-tRNA, and favors the joining of the small and large ribosomal subunits yielding a 70S initiation complex ready to enter the translation elongation phase. Within the IF2 molecule subdomain G3, which is believed to play an important role in the IF2-30S interaction, is positioned between the GTP-binding G2 and the fMet-tRNA binding C-terminal subdomains. In this study the solution structure of subdomain G3 of Geobacillus stearothermophilus IF2 has been elucidated. G3 forms a core structure consisting of two β-sheets with each four anti-parallel strands, followed by a C-terminal α-helix. In line with its role as linker between G3 and subdomain C1, this helix has no well-defined orientation but is endowed with a dynamic nature. The structure of the G3 core is that of a typical OB-fold module, similar to that of the corresponding subdomain of Thermus thermo...
H-NS is an abundant transcriptional regulator present in Gram negative bacteria whose functional ... more H-NS is an abundant transcriptional regulator present in Gram negative bacteria whose functional significance and biological role are fairly well understood (Pon et al., 2005). In contrast, the mechanism of interaction of H-NS with its target DNA is still poorly understood at the molecular level. From a structural point of view H-NS is constituted by an N-terminal domain (residues 1-64), a linker region (residues 65-89) and a C-terminal domain (residues 90-136). The N-terminal domain is responsible for protein oligomerization while the C-terminal domain is responsible for binding to DNA (Ueguchi et al., 1996; Spurio et al. 1997). The linker region is not structured in solution; this allows the C-terminal domain to have a high mobility in solution and to behave as an independent domain (Smith et al., 2000). The structure of the C-domain of H-NS was solved many years ago by NMR spectroscopy, but the way in which this domain interacts with the specific target DNA is not known at the molecular level (Shindo et al., 1995; Shindo et al., 1999). In the present study we have used a combined approach utilizing both molecular biology and NMR techniques to investigate the interaction of H-NS with its specific target DNA at the molecular level. Our results show that: i) H-NS binds to the target DNA with a specific, positively charged, region; ii) a short DNA fragment, (10-15 bp) is the target of H-NS binding. This sequence seems to be at least in part conserved among the different promoter regions recognized by the protein and has structural features different from a canonical B-DNA. References Pon C.L., Stella S., Gualerzi C.O. (2005) in DNA conformation and transcription , edited by Takashi Ohyama, pp 1-14. Shindo H., Iwaki T., Ieda R., Kurumizaka H., Ueguchi C., Mizuno T., Morikawa S., Nakamura H., Kuboniwa H. (1995) FEBS Lett. 360, 125-131. Shindo H., Ohnuki A., Ginba H., Katoh E., Ueguchi C., Mizuno T., Yamazaki T. (1999) FEBS Lett. 455, 63-9. Smyth C.P., Lundback T., Renzoni D., Siligardi G., Beavil R., Layton M., Sidebotham J.M., Hinton J.C., Driscoll P.C., Higgins C.F., Ladbury J.E. (2000) Mol. Microbiol. 36, 962–972. Spurio R., Falconi M., Brandi A., Pon C.L., Gualerzi C.O. (1997) EMBO J. 16, 1795-1805. Ueguchi C., Suzuki T., Yoshida T., Tanaka K., Mizuno T. (1996) J.Mol.Biol. 263, 149-162.
Previously, we evolved Lipase A from Bacillus subtilis towards increased thermostability. The res... more Previously, we evolved Lipase A from Bacillus subtilis towards increased thermostability. The resulting mutant retains significant catalytic activity upon heating above 60 °C (and up to 100 °C) and cooling down, whereas wild-type lipase precipitates irreversibly and does not show significant activity in these conditions. Kinetic thermostability of proteins has not been characterized well on the molecular structure level so far, therefore our aim is to study it using NMR spectroscopy. Here, nearly complete (1)H, (13)C and (15)N resonance assignments are reported for wild-type and mutant Lipase A. Chemical shifts were used to predict secondary structure elements of both Lipase A variants.
Knowledge of the structural properties of allergenic proteins is a necessary prerequisite to bett... more Knowledge of the structural properties of allergenic proteins is a necessary prerequisite to better understand the molecular bases of their action, and also to design targeted structural/functional modifications. Peamaclein is a recently identified 7 kDa peach allergen that has been associated with severe allergic reactions in sensitive subjects. This protein represents the first component of a new allergen family, which has no 3D structure available yet. Here, we report the first experimental data on the 3D-structure of Peamaclein. Almost 75% of the backbone resonances, including two helical stretches in the N-terminal region, and four out of six cysteine pairs have been assigned by 2D-NMR using a natural protein sample. Simulated gastrointestinal digestion experiments have highlighted that Peamaclein is even more resistant to digestion than the peach major allergen Pru p 3. Only the heat-denatured protein becomes sensitive to intestinal proteases. Similar to Pru p 3, Peamaclein ke...
Proceedings of the National Academy of Sciences, 2006
Translational initiation factor 2 (IF2) is a guanine nucleotide-binding protein that can bind gua... more Translational initiation factor 2 (IF2) is a guanine nucleotide-binding protein that can bind guanosine 3′,5′-(bis) diphosphate (ppGpp), an alarmone involved in stringent response in bacteria. In cells growing under optimal conditions, the GTP concentration is very high, and that of ppGpp very low. However, under stress conditions, the GTP concentration may decline by as much as 50%, and that of ppGpp can attain levels comparable to those of GTP. Here we show that IF2 binds ppGpp at the same nucleotide-binding site and with similar affinity as GTP. Thus, GTP and the alarmone ppGpp can be considered two alternative physiologically relevant IF2 ligands. ppGpp interferes with IF2-dependent initiation complex formation, severely inhibits initiation dipeptide formation, and blocks the initiation step of translation. Our data suggest that IF2 has the properties of a cellular metabolic sensor and regulator that oscillates between an active GTP-bound form under conditions allowing active pr...
The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function ... more The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the Nucleotide Excision Repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wildtype and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface where, in contrast to F231, L231 lacks interactions stabilizing the XPF-ERCC1 complex. One of the two anchor points is severely distorted and this results in a more dynamic complex causing reduced stability and an increased dissociation rate of the mutant complex as compared to wildtype...
Protein science : a publication of the Protein Society, 2016
Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit... more Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit, promotes ribosomal binding of fMet-tRNA, and favors the joining of the small and large ribosomal subunits yielding a 70S initiation complex ready to enter the translation elongation phase. Within the IF2 molecule subdomain G3, which is believed to play an important role in the IF2-30S interaction, is positioned between the GTP-binding G2 and the fMet-tRNA binding C-terminal subdomains. In this study the solution structure of subdomain G3 of Geobacillus stearothermophilus IF2 has been elucidated. G3 forms a core structure consisting of two β-sheets with each four anti-parallel strands, followed by a C-terminal α-helix. In line with its role as linker between G3 and subdomain C1, this helix has no well-defined orientation but is endowed with a dynamic nature. The structure of the G3 core is that of a typical OB-fold module, similar to that of the corresponding subdomain of Thermus thermo...
H-NS is an abundant transcriptional regulator present in Gram negative bacteria whose functional ... more H-NS is an abundant transcriptional regulator present in Gram negative bacteria whose functional significance and biological role are fairly well understood (Pon et al., 2005). In contrast, the mechanism of interaction of H-NS with its target DNA is still poorly understood at the molecular level. From a structural point of view H-NS is constituted by an N-terminal domain (residues 1-64), a linker region (residues 65-89) and a C-terminal domain (residues 90-136). The N-terminal domain is responsible for protein oligomerization while the C-terminal domain is responsible for binding to DNA (Ueguchi et al., 1996; Spurio et al. 1997). The linker region is not structured in solution; this allows the C-terminal domain to have a high mobility in solution and to behave as an independent domain (Smith et al., 2000). The structure of the C-domain of H-NS was solved many years ago by NMR spectroscopy, but the way in which this domain interacts with the specific target DNA is not known at the molecular level (Shindo et al., 1995; Shindo et al., 1999). In the present study we have used a combined approach utilizing both molecular biology and NMR techniques to investigate the interaction of H-NS with its specific target DNA at the molecular level. Our results show that: i) H-NS binds to the target DNA with a specific, positively charged, region; ii) a short DNA fragment, (10-15 bp) is the target of H-NS binding. This sequence seems to be at least in part conserved among the different promoter regions recognized by the protein and has structural features different from a canonical B-DNA. References Pon C.L., Stella S., Gualerzi C.O. (2005) in DNA conformation and transcription , edited by Takashi Ohyama, pp 1-14. Shindo H., Iwaki T., Ieda R., Kurumizaka H., Ueguchi C., Mizuno T., Morikawa S., Nakamura H., Kuboniwa H. (1995) FEBS Lett. 360, 125-131. Shindo H., Ohnuki A., Ginba H., Katoh E., Ueguchi C., Mizuno T., Yamazaki T. (1999) FEBS Lett. 455, 63-9. Smyth C.P., Lundback T., Renzoni D., Siligardi G., Beavil R., Layton M., Sidebotham J.M., Hinton J.C., Driscoll P.C., Higgins C.F., Ladbury J.E. (2000) Mol. Microbiol. 36, 962–972. Spurio R., Falconi M., Brandi A., Pon C.L., Gualerzi C.O. (1997) EMBO J. 16, 1795-1805. Ueguchi C., Suzuki T., Yoshida T., Tanaka K., Mizuno T. (1996) J.Mol.Biol. 263, 149-162.
Previously, we evolved Lipase A from Bacillus subtilis towards increased thermostability. The res... more Previously, we evolved Lipase A from Bacillus subtilis towards increased thermostability. The resulting mutant retains significant catalytic activity upon heating above 60 °C (and up to 100 °C) and cooling down, whereas wild-type lipase precipitates irreversibly and does not show significant activity in these conditions. Kinetic thermostability of proteins has not been characterized well on the molecular structure level so far, therefore our aim is to study it using NMR spectroscopy. Here, nearly complete (1)H, (13)C and (15)N resonance assignments are reported for wild-type and mutant Lipase A. Chemical shifts were used to predict secondary structure elements of both Lipase A variants.
Knowledge of the structural properties of allergenic proteins is a necessary prerequisite to bett... more Knowledge of the structural properties of allergenic proteins is a necessary prerequisite to better understand the molecular bases of their action, and also to design targeted structural/functional modifications. Peamaclein is a recently identified 7 kDa peach allergen that has been associated with severe allergic reactions in sensitive subjects. This protein represents the first component of a new allergen family, which has no 3D structure available yet. Here, we report the first experimental data on the 3D-structure of Peamaclein. Almost 75% of the backbone resonances, including two helical stretches in the N-terminal region, and four out of six cysteine pairs have been assigned by 2D-NMR using a natural protein sample. Simulated gastrointestinal digestion experiments have highlighted that Peamaclein is even more resistant to digestion than the peach major allergen Pru p 3. Only the heat-denatured protein becomes sensitive to intestinal proteases. Similar to Pru p 3, Peamaclein ke...
Proceedings of the National Academy of Sciences, 2006
Translational initiation factor 2 (IF2) is a guanine nucleotide-binding protein that can bind gua... more Translational initiation factor 2 (IF2) is a guanine nucleotide-binding protein that can bind guanosine 3′,5′-(bis) diphosphate (ppGpp), an alarmone involved in stringent response in bacteria. In cells growing under optimal conditions, the GTP concentration is very high, and that of ppGpp very low. However, under stress conditions, the GTP concentration may decline by as much as 50%, and that of ppGpp can attain levels comparable to those of GTP. Here we show that IF2 binds ppGpp at the same nucleotide-binding site and with similar affinity as GTP. Thus, GTP and the alarmone ppGpp can be considered two alternative physiologically relevant IF2 ligands. ppGpp interferes with IF2-dependent initiation complex formation, severely inhibits initiation dipeptide formation, and blocks the initiation step of translation. Our data suggest that IF2 has the properties of a cellular metabolic sensor and regulator that oscillates between an active GTP-bound form under conditions allowing active pr...
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