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...
Bromodomains are epigenetic reader domains, which have come under increasing scrutiny both from a... more Bromodomains are epigenetic reader domains, which have come under increasing scrutiny both from academic and pharmaceutical research groups. Effective targeting of the BAZ2B bromodomain by small molecule inhibitors has been recently reported, but no structural information is yet available on the interaction with its natural binding partner, acetylated histone H3K14ac. We have assigned the BAZ2B bromodomain and studied its interaction with H3K14ac acetylated peptides by NMR spectroscopy using both chemical shift perturbation (CSP) data and clean chemical exchange (CLEANEX-PM) NMR experiments. The latter was used to characterize water molecules known to play an important role in mediating interactions. Besides the anticipated Kac binding site, we consistently found the bromodomain BC loop as hotspots for the interaction. This information was used to create a data-driven model for the complex using HADDOCK. Our findings provide both structure and dynamics characterization that will be useful in the quest for potent and selective inhibitors to probe the function of the BAZ2B bromodomain.
FEBS 1996 NMR studies of the free a subunit of human chorionic gonadotropin Structural influences... more FEBS 1996 NMR studies of the free a subunit of human chorionic gonadotropin Structural influences of N-glycosylation and the / 3 subunit on the conformation of the a subunit
The three-dimensional structure of the fMet-tRNA fMet-binding domain of translation initiation fa... more The three-dimensional structure of the fMet-tRNA fMet-binding domain of translation initiation factor IF2 from Bacillus stearothermophilus has been determined by heteronuclear NMR spectroscopy. Its structure consists of six antiparallel b-strands, connected via loops, and forms a closed b-barrel similar to domain II of elongation factors EF-Tu and EF-G, despite low sequence homology. Two structures of the ternary complexes of the EF-Tu´aminoacyl-tRNAǴ DP analogue have been reported and were used to propose and discuss the possible fMet-tRNA fMet-binding site of IF2.
prokaryotic translational apparatus and has been found Padualaan 8, 3584 CH Utrecht, The Netherla... more prokaryotic translational apparatus and has been found Padualaan 8, 3584 CH Utrecht, The Netherlands, 1 Laboratorio di also in the chloroplasts of several plants. The gene Genetica, Dipartimento di Biologia MCA, Università degli Studi di encoding IF1 in E.coli, infA, has been identified and Camerino, via Camerini, 62032 Camerino and 2 Dipartimento di cloned (Sands et al., 1987). The expression of infA in Scienze e Tecnologie Chimiche, Università degli Studi di Roma E.coli is under growth-rate control (Cummings et al.
The interaction between fMet-tRNA f Met and Bacillus stearothermophilus translation initiation fa... more The interaction between fMet-tRNA f Met and Bacillus stearothermophilus translation initiation factor IF2 has been characterized. We demonstrate that essentially all thermodynamic determinants governing the stability and the speci®city of this interaction are localized within the acceptor hexanucleotide fMet-3¢ACCAAC of the initiator tRNA and a fairly small area at the surface of the b-barrel structure of the 90amino acid C-terminal domain of IF2 (IF2 C-2). A weak but speci®c interaction between IF2 C-2 and formyl-methionyl was also demonstrated. The surface of IF2 C-2 interacting with fMet-tRNA f Met has been mapped using two independent approaches, sitedirected mutagenesis and NMR spectroscopy, which yielded consistent results. The binding site comprises C668 and G715 located in a groove accommodating the methionyl side-chain, R700, in the vicinity of the formyl group, Y701 and K702 close to the acyl bond between fMet and tRNA f Met , and the surface lined with residues K702-S660, along which the acceptor arm of the initiator tRNA spans in the direction 3¢ to 5¢.
Trimethylation of lysine residue K4 of histone H3 (H3K4me3) strongly correlates with active promo... more Trimethylation of lysine residue K4 of histone H3 (H3K4me3) strongly correlates with active promoters for RNA polymerase II-transcribed genes. Several reader proteins, including the basal transcription factor TFIID, for this nucleosomal mark have been identified. Its TAF3 subunit specifically binds the H3K4me3 mark via its conserved plant homeodomain (PHD) finger. Here, we report the solution structure of the TAF3-PHD finger and its complex with an H3K4me3 peptide. Using a combination of NMR, mutagenesis, and affinity measurements, we reveal the structural basis of binding affinity, methylation-state specificity, and crosstalk with asymmetric dimethylation of R2. A unique local structure rearrangement in the K4me3-binding pocket of TAF3 due to a conserved sequence insertion underscores the requirement for cation-p interactions by two aromatic residues. Interference by asymmetric dimethylation of arginine 2 suggests that a H3R2/K4 ''methyl-methyl'' switch in the histone code dynamically regulates TFIID-promoter association.
Proteins: Structure, Function, and Bioinformatics, 2014
Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a tran... more Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a transcriptional repressor of the basic helix-loop-helix (bHLH) family. Using nuclear magnetic resonance methods, we have determined the structure and dynamics of a recombinant protein, H1H, which includes an N-terminal segment, b1, containing functionally important phosphorylation sites, the basic region b2, required for binding to DNA, and the HLH domain. We show that a proline residue in the sequence divides the protein in two parts, a flexible and disordered N-terminal region including b1 and a structured, mainly helical region comprising b2 and the HLH domain. Binding of H1H to a double strand DNA oligonucleotide was monitored through the chemical shift perturbation of backbone amide resonances, and showed that the interaction surface involves not only the b2 segment but also several residues in the b1 and HLH regions.
Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using it... more Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65°C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical characterization. Combining thermal inactivation profiles, circular dichroism, X-ray structure analyses and NMR experiments revealed that mutations of surface amino acid residues counteract the tendency of Lipase A to undergo precipitation under thermal stress. Reduced precipitation of the unfolding intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention.
IF2 is one of three bacterial translation initiation factors that are conserved through all kingd... more IF2 is one of three bacterial translation initiation factors that are conserved through all kingdoms of life. It binds the 30S and 50S ribosomal subunits, as well as fMet-tRNA f Met. After these interactions, fMet-tRNA f Met is oriented to the ribosomal P-site where the first amino acid of the nascent polypeptide, formylmethionine, is presented. The C-terminal domain of Bacillus stearothermophilus IF2, which is responsible for recognition and binding of fMet-tRNA f Met , contains two structured modules. Previously, the solution structure of the most C-terminal module, IF2-C2, has been elucidated by NMR spectroscopy and direct interactions between this subdomain and fMet-tRNA f Met were reported. In the present NMR study we have obtained the spectral assignment of the other module of the C-terminal domain (IF2-C1) and determined its solution structure and backbone dynamics. The IF2-C1 core forms a flattened fold consisting of a central four-stranded parallel b-sheet flanked by three a-helices. Although its overall organization resembles that of subdomain III of the archaeal IF2-homolog eIF5B whose crystal structure had previously been reported, some differences of potential functional significance are evident.
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...
Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the... more Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N-and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges.
Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the... more Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N-and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges.
To maintain the integrity of the genome, multiple DNA repair systems exist to repair damaged DNA.... more To maintain the integrity of the genome, multiple DNA repair systems exist to repair damaged DNA. Recognition of altered DNA, including bulky adducts, pyrimidine dimers and interstrand crosslinks (ICL), partially depends on proteins containing helixhairpin-helix (HhH) domains. To understand how ICL is specifically recognized by the Fanconi anemia proteins FANCM and FAAP24, we determined the structure of the HhH domain of FAAP24. Although it resembles other HhH domains, the FAAP24 domain contains a canonical hairpin motif followed by distorted motif. The HhH domain can bind various DNA substrates; using nuclear magnetic resonance titration experiments, we demonstrate that the canonical HhH motif is required for double-stranded DNA (dsDNA) binding, whereas the unstructured N-terminus can interact with single-stranded DNA. Both DNA binding surfaces are used for binding to ICL-like single/double-strand junction-containing DNA substrates. A structural model for FAAP24 bound to dsDNA has been made based on homology with the translesion polymerase iota. Sitedirected mutagenesis, sequence conservation and charge distribution support the dsDNA-binding model. Analogous to other HhH domain-containing proteins, we suggest that multiple FAAP24 regions together contribute to binding to single/doublestrand junction, which could contribute to specificity in ICL DNA recognition.
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...
Bromodomains are epigenetic reader domains, which have come under increasing scrutiny both from a... more Bromodomains are epigenetic reader domains, which have come under increasing scrutiny both from academic and pharmaceutical research groups. Effective targeting of the BAZ2B bromodomain by small molecule inhibitors has been recently reported, but no structural information is yet available on the interaction with its natural binding partner, acetylated histone H3K14ac. We have assigned the BAZ2B bromodomain and studied its interaction with H3K14ac acetylated peptides by NMR spectroscopy using both chemical shift perturbation (CSP) data and clean chemical exchange (CLEANEX-PM) NMR experiments. The latter was used to characterize water molecules known to play an important role in mediating interactions. Besides the anticipated Kac binding site, we consistently found the bromodomain BC loop as hotspots for the interaction. This information was used to create a data-driven model for the complex using HADDOCK. Our findings provide both structure and dynamics characterization that will be useful in the quest for potent and selective inhibitors to probe the function of the BAZ2B bromodomain.
FEBS 1996 NMR studies of the free a subunit of human chorionic gonadotropin Structural influences... more FEBS 1996 NMR studies of the free a subunit of human chorionic gonadotropin Structural influences of N-glycosylation and the / 3 subunit on the conformation of the a subunit
The three-dimensional structure of the fMet-tRNA fMet-binding domain of translation initiation fa... more The three-dimensional structure of the fMet-tRNA fMet-binding domain of translation initiation factor IF2 from Bacillus stearothermophilus has been determined by heteronuclear NMR spectroscopy. Its structure consists of six antiparallel b-strands, connected via loops, and forms a closed b-barrel similar to domain II of elongation factors EF-Tu and EF-G, despite low sequence homology. Two structures of the ternary complexes of the EF-Tu´aminoacyl-tRNAǴ DP analogue have been reported and were used to propose and discuss the possible fMet-tRNA fMet-binding site of IF2.
prokaryotic translational apparatus and has been found Padualaan 8, 3584 CH Utrecht, The Netherla... more prokaryotic translational apparatus and has been found Padualaan 8, 3584 CH Utrecht, The Netherlands, 1 Laboratorio di also in the chloroplasts of several plants. The gene Genetica, Dipartimento di Biologia MCA, Università degli Studi di encoding IF1 in E.coli, infA, has been identified and Camerino, via Camerini, 62032 Camerino and 2 Dipartimento di cloned (Sands et al., 1987). The expression of infA in Scienze e Tecnologie Chimiche, Università degli Studi di Roma E.coli is under growth-rate control (Cummings et al.
The interaction between fMet-tRNA f Met and Bacillus stearothermophilus translation initiation fa... more The interaction between fMet-tRNA f Met and Bacillus stearothermophilus translation initiation factor IF2 has been characterized. We demonstrate that essentially all thermodynamic determinants governing the stability and the speci®city of this interaction are localized within the acceptor hexanucleotide fMet-3¢ACCAAC of the initiator tRNA and a fairly small area at the surface of the b-barrel structure of the 90amino acid C-terminal domain of IF2 (IF2 C-2). A weak but speci®c interaction between IF2 C-2 and formyl-methionyl was also demonstrated. The surface of IF2 C-2 interacting with fMet-tRNA f Met has been mapped using two independent approaches, sitedirected mutagenesis and NMR spectroscopy, which yielded consistent results. The binding site comprises C668 and G715 located in a groove accommodating the methionyl side-chain, R700, in the vicinity of the formyl group, Y701 and K702 close to the acyl bond between fMet and tRNA f Met , and the surface lined with residues K702-S660, along which the acceptor arm of the initiator tRNA spans in the direction 3¢ to 5¢.
Trimethylation of lysine residue K4 of histone H3 (H3K4me3) strongly correlates with active promo... more Trimethylation of lysine residue K4 of histone H3 (H3K4me3) strongly correlates with active promoters for RNA polymerase II-transcribed genes. Several reader proteins, including the basal transcription factor TFIID, for this nucleosomal mark have been identified. Its TAF3 subunit specifically binds the H3K4me3 mark via its conserved plant homeodomain (PHD) finger. Here, we report the solution structure of the TAF3-PHD finger and its complex with an H3K4me3 peptide. Using a combination of NMR, mutagenesis, and affinity measurements, we reveal the structural basis of binding affinity, methylation-state specificity, and crosstalk with asymmetric dimethylation of R2. A unique local structure rearrangement in the K4me3-binding pocket of TAF3 due to a conserved sequence insertion underscores the requirement for cation-p interactions by two aromatic residues. Interference by asymmetric dimethylation of arginine 2 suggests that a H3R2/K4 ''methyl-methyl'' switch in the histone code dynamically regulates TFIID-promoter association.
Proteins: Structure, Function, and Bioinformatics, 2014
Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a tran... more Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a transcriptional repressor of the basic helix-loop-helix (bHLH) family. Using nuclear magnetic resonance methods, we have determined the structure and dynamics of a recombinant protein, H1H, which includes an N-terminal segment, b1, containing functionally important phosphorylation sites, the basic region b2, required for binding to DNA, and the HLH domain. We show that a proline residue in the sequence divides the protein in two parts, a flexible and disordered N-terminal region including b1 and a structured, mainly helical region comprising b2 and the HLH domain. Binding of H1H to a double strand DNA oligonucleotide was monitored through the chemical shift perturbation of backbone amide resonances, and showed that the interaction surface involves not only the b2 segment but also several residues in the b1 and HLH regions.
Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using it... more Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65°C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical characterization. Combining thermal inactivation profiles, circular dichroism, X-ray structure analyses and NMR experiments revealed that mutations of surface amino acid residues counteract the tendency of Lipase A to undergo precipitation under thermal stress. Reduced precipitation of the unfolding intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention.
IF2 is one of three bacterial translation initiation factors that are conserved through all kingd... more IF2 is one of three bacterial translation initiation factors that are conserved through all kingdoms of life. It binds the 30S and 50S ribosomal subunits, as well as fMet-tRNA f Met. After these interactions, fMet-tRNA f Met is oriented to the ribosomal P-site where the first amino acid of the nascent polypeptide, formylmethionine, is presented. The C-terminal domain of Bacillus stearothermophilus IF2, which is responsible for recognition and binding of fMet-tRNA f Met , contains two structured modules. Previously, the solution structure of the most C-terminal module, IF2-C2, has been elucidated by NMR spectroscopy and direct interactions between this subdomain and fMet-tRNA f Met were reported. In the present NMR study we have obtained the spectral assignment of the other module of the C-terminal domain (IF2-C1) and determined its solution structure and backbone dynamics. The IF2-C1 core forms a flattened fold consisting of a central four-stranded parallel b-sheet flanked by three a-helices. Although its overall organization resembles that of subdomain III of the archaeal IF2-homolog eIF5B whose crystal structure had previously been reported, some differences of potential functional significance are evident.
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...
Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the... more Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N-and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges.
Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the... more Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N-and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges.
To maintain the integrity of the genome, multiple DNA repair systems exist to repair damaged DNA.... more To maintain the integrity of the genome, multiple DNA repair systems exist to repair damaged DNA. Recognition of altered DNA, including bulky adducts, pyrimidine dimers and interstrand crosslinks (ICL), partially depends on proteins containing helixhairpin-helix (HhH) domains. To understand how ICL is specifically recognized by the Fanconi anemia proteins FANCM and FAAP24, we determined the structure of the HhH domain of FAAP24. Although it resembles other HhH domains, the FAAP24 domain contains a canonical hairpin motif followed by distorted motif. The HhH domain can bind various DNA substrates; using nuclear magnetic resonance titration experiments, we demonstrate that the canonical HhH motif is required for double-stranded DNA (dsDNA) binding, whereas the unstructured N-terminus can interact with single-stranded DNA. Both DNA binding surfaces are used for binding to ICL-like single/double-strand junction-containing DNA substrates. A structural model for FAAP24 bound to dsDNA has been made based on homology with the translesion polymerase iota. Sitedirected mutagenesis, sequence conservation and charge distribution support the dsDNA-binding model. Analogous to other HhH domain-containing proteins, we suggest that multiple FAAP24 regions together contribute to binding to single/doublestrand junction, which could contribute to specificity in ICL DNA recognition.
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Papers by Rolf Boelens