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The Cold Shock Domain of YB-1 Segregates RNA from DNA by Non-Bonded Interactions.

Kljashtorny V, Nikonov S, Ovchinnikov L, Lyabin D, Vodovar N, Curmi P, Manivet P - PLoS ONE (2015)

Bottom Line: Using molecular dynamics simulation approaches validated by experimental assays, the YB1 CSD was found to interact with nucleic acids in a sequence-dependent manner and with a higher affinity for RNA than DNA.The binding properties of the YB1 CSD were close to those observed for the related bacterial Cold Shock Proteins (CSP), albeit some differences in sequence specificity.The results provide insights in the molecular mechanisms whereby YB-1 interacts with nucleic acids.

View Article: PubMed Central - PubMed

Affiliation: Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia; Institut National de la Santé et de la Recherche Médicale (INSERM), UMR 829, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Bd François Mitterrand, 91025 Evry Cedex, France; Institut National de la Santé et de la Recherche Médicale (INSERM), UMRS 942, Hôpital Lariboisière, 41 boulevard de la Chapelle, 75475 Paris cedex 10, France; Assistance Publique-Hôpitaux de paris (APHP), Hôpital Lariboisière, Service de Biochimie et de Biologie Moléculaire, Paris, France.

ABSTRACT
The human YB-1 protein plays multiple cellular roles, of which many are dictated by its binding to RNA and DNA through its Cold Shock Domain (CSD). Using molecular dynamics simulation approaches validated by experimental assays, the YB1 CSD was found to interact with nucleic acids in a sequence-dependent manner and with a higher affinity for RNA than DNA. The binding properties of the YB1 CSD were close to those observed for the related bacterial Cold Shock Proteins (CSP), albeit some differences in sequence specificity. The results provide insights in the molecular mechanisms whereby YB-1 interacts with nucleic acids.

No MeSH data available.


Related in: MedlinePlus

Superposition between the bacterial CSP and the CSDYB-1 nucleic acid-binding domains.A. Key interactions between the bacterial CSP (green) and an oligo(dT) (orange) obtained from the CSP:oligo(dT) crystal structure.[22] The intermolecular H-bond formed between the protein and the ssDNA are shown as blue dotted lines. B. Superposition of the CSDYB-1 (gray) with the CSP:dT6 complex (green and orange, respectively). The side chains of amino acids involved in stacking with nucleotide bases are highlighted in blue. These three binding sites, that are structurally equivalent between the bacterial CSP and the CSDYB-1 are labelled N1, N2 and N3. The nucleotide shown in magenta represents the symmetrically related molecule of the complex and shows the additional nucleotide binding site formed by Phe30 and Phe38 (binding site 1, N1), which is only present in the bacterial CSP.
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pone.0130318.g002: Superposition between the bacterial CSP and the CSDYB-1 nucleic acid-binding domains.A. Key interactions between the bacterial CSP (green) and an oligo(dT) (orange) obtained from the CSP:oligo(dT) crystal structure.[22] The intermolecular H-bond formed between the protein and the ssDNA are shown as blue dotted lines. B. Superposition of the CSDYB-1 (gray) with the CSP:dT6 complex (green and orange, respectively). The side chains of amino acids involved in stacking with nucleotide bases are highlighted in blue. These three binding sites, that are structurally equivalent between the bacterial CSP and the CSDYB-1 are labelled N1, N2 and N3. The nucleotide shown in magenta represents the symmetrically related molecule of the complex and shows the additional nucleotide binding site formed by Phe30 and Phe38 (binding site 1, N1), which is only present in the bacterial CSP.

Mentions: The NMR structure of the human CSDYB-1 (PDB-entry 1H95) relaxed after 50 ns of MDS (see below the MDS protocol) was used as a starting model. This structure does not contain any nucleic acid as the interaction between the CSDYB-1:NA complex is not stable enough to be solved by NMR [22]. The NMR structure of CSDYB-1 [22] exhibits a β-barrel fold that is highly similar to the structures of the Bacillus caldolyticus Bc-CspB [26] and the Bacillus Subtilis Bs-CspB [27] CPSs, solved in the presence of single-stranded dT6 oligonucleotides (PDB-entries 2HAX and 2ES2, respectively). Indeed, from a structural standpoint, the nucleic binding sites of the CSDYB-1 and the aforementioned CSPs are highly conserved as illustrated by an RMSD value of 0.49Å for the 12 Cα-atoms of the binding site (amino acids 15–18, 26–30, and 59–61 based on Bs-CspB numbering; Fig 1A and 1B). Given the structural similarities observed between the CSDYB-1 and the bacterial CSP, the coordinates of the oligonucleotide from the Bs-CspB:dT6 were used to build the CSDYB-1:DNA complexes used in this study. Of note, the 6th residue of the dT6 oligonucleotide from the Bs-CspB:dT6 crystal was bound to symmetrically related Bs-CspB molecule (Fig 2, [26]). Therefore an additional T residue was added at 5'-terminus of the dT6 oligonucleotide to obtain the Bs-CspB:dT7 structure. Using the sugar-phosphate backbone of the oligo(dT) from the Bs-CspB:dT7, each nucleotide was substituted by A, C or G to obtain oligo(dA), oligo(dG) and oligo(dC). One additional nucleotide was added to each extremity, ending up with the CSDYB-1 bound to nonameric oligonucleotides. The polarity of oligonucleotide obtained in such a way and corresponding to the orientation of dT6 in the bacterial Bs-CspB:dT7 structure is referred as standard or direct orientation. In contrast to non-standard or reversed orientation where the strand is oriented upside down. To generate such conformation the atoms of the nucleotide bases were aligned to keep the stacking with the amino acid residues of CSDYB-1 whereas the atoms of sugar and phosphate groups were ignored. Thus, the reversed orientated oligonucleotide preserved the main contacts between nucleotide bases and amino acid residues and only had the flipped polarity of sugar-phosphate backbone.


The Cold Shock Domain of YB-1 Segregates RNA from DNA by Non-Bonded Interactions.

Kljashtorny V, Nikonov S, Ovchinnikov L, Lyabin D, Vodovar N, Curmi P, Manivet P - PLoS ONE (2015)

Superposition between the bacterial CSP and the CSDYB-1 nucleic acid-binding domains.A. Key interactions between the bacterial CSP (green) and an oligo(dT) (orange) obtained from the CSP:oligo(dT) crystal structure.[22] The intermolecular H-bond formed between the protein and the ssDNA are shown as blue dotted lines. B. Superposition of the CSDYB-1 (gray) with the CSP:dT6 complex (green and orange, respectively). The side chains of amino acids involved in stacking with nucleotide bases are highlighted in blue. These three binding sites, that are structurally equivalent between the bacterial CSP and the CSDYB-1 are labelled N1, N2 and N3. The nucleotide shown in magenta represents the symmetrically related molecule of the complex and shows the additional nucleotide binding site formed by Phe30 and Phe38 (binding site 1, N1), which is only present in the bacterial CSP.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493011&req=5

pone.0130318.g002: Superposition between the bacterial CSP and the CSDYB-1 nucleic acid-binding domains.A. Key interactions between the bacterial CSP (green) and an oligo(dT) (orange) obtained from the CSP:oligo(dT) crystal structure.[22] The intermolecular H-bond formed between the protein and the ssDNA are shown as blue dotted lines. B. Superposition of the CSDYB-1 (gray) with the CSP:dT6 complex (green and orange, respectively). The side chains of amino acids involved in stacking with nucleotide bases are highlighted in blue. These three binding sites, that are structurally equivalent between the bacterial CSP and the CSDYB-1 are labelled N1, N2 and N3. The nucleotide shown in magenta represents the symmetrically related molecule of the complex and shows the additional nucleotide binding site formed by Phe30 and Phe38 (binding site 1, N1), which is only present in the bacterial CSP.
Mentions: The NMR structure of the human CSDYB-1 (PDB-entry 1H95) relaxed after 50 ns of MDS (see below the MDS protocol) was used as a starting model. This structure does not contain any nucleic acid as the interaction between the CSDYB-1:NA complex is not stable enough to be solved by NMR [22]. The NMR structure of CSDYB-1 [22] exhibits a β-barrel fold that is highly similar to the structures of the Bacillus caldolyticus Bc-CspB [26] and the Bacillus Subtilis Bs-CspB [27] CPSs, solved in the presence of single-stranded dT6 oligonucleotides (PDB-entries 2HAX and 2ES2, respectively). Indeed, from a structural standpoint, the nucleic binding sites of the CSDYB-1 and the aforementioned CSPs are highly conserved as illustrated by an RMSD value of 0.49Å for the 12 Cα-atoms of the binding site (amino acids 15–18, 26–30, and 59–61 based on Bs-CspB numbering; Fig 1A and 1B). Given the structural similarities observed between the CSDYB-1 and the bacterial CSP, the coordinates of the oligonucleotide from the Bs-CspB:dT6 were used to build the CSDYB-1:DNA complexes used in this study. Of note, the 6th residue of the dT6 oligonucleotide from the Bs-CspB:dT6 crystal was bound to symmetrically related Bs-CspB molecule (Fig 2, [26]). Therefore an additional T residue was added at 5'-terminus of the dT6 oligonucleotide to obtain the Bs-CspB:dT7 structure. Using the sugar-phosphate backbone of the oligo(dT) from the Bs-CspB:dT7, each nucleotide was substituted by A, C or G to obtain oligo(dA), oligo(dG) and oligo(dC). One additional nucleotide was added to each extremity, ending up with the CSDYB-1 bound to nonameric oligonucleotides. The polarity of oligonucleotide obtained in such a way and corresponding to the orientation of dT6 in the bacterial Bs-CspB:dT7 structure is referred as standard or direct orientation. In contrast to non-standard or reversed orientation where the strand is oriented upside down. To generate such conformation the atoms of the nucleotide bases were aligned to keep the stacking with the amino acid residues of CSDYB-1 whereas the atoms of sugar and phosphate groups were ignored. Thus, the reversed orientated oligonucleotide preserved the main contacts between nucleotide bases and amino acid residues and only had the flipped polarity of sugar-phosphate backbone.

Bottom Line: Using molecular dynamics simulation approaches validated by experimental assays, the YB1 CSD was found to interact with nucleic acids in a sequence-dependent manner and with a higher affinity for RNA than DNA.The binding properties of the YB1 CSD were close to those observed for the related bacterial Cold Shock Proteins (CSP), albeit some differences in sequence specificity.The results provide insights in the molecular mechanisms whereby YB-1 interacts with nucleic acids.

View Article: PubMed Central - PubMed

Affiliation: Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia; Institut National de la Santé et de la Recherche Médicale (INSERM), UMR 829, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Bd François Mitterrand, 91025 Evry Cedex, France; Institut National de la Santé et de la Recherche Médicale (INSERM), UMRS 942, Hôpital Lariboisière, 41 boulevard de la Chapelle, 75475 Paris cedex 10, France; Assistance Publique-Hôpitaux de paris (APHP), Hôpital Lariboisière, Service de Biochimie et de Biologie Moléculaire, Paris, France.

ABSTRACT
The human YB-1 protein plays multiple cellular roles, of which many are dictated by its binding to RNA and DNA through its Cold Shock Domain (CSD). Using molecular dynamics simulation approaches validated by experimental assays, the YB1 CSD was found to interact with nucleic acids in a sequence-dependent manner and with a higher affinity for RNA than DNA. The binding properties of the YB1 CSD were close to those observed for the related bacterial Cold Shock Proteins (CSP), albeit some differences in sequence specificity. The results provide insights in the molecular mechanisms whereby YB-1 interacts with nucleic acids.

No MeSH data available.


Related in: MedlinePlus