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Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides.

Yoga YM, Traore DA, Sidiqi M, Szeto C, Pendini NR, Barker A, Leedman PJ, Wilce JA, Wilce MC - Nucleic Acids Res. (2012)

Bottom Line: The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites.SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet.The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC Australia.

ABSTRACT
Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.

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Related in: MedlinePlus

Schematic representations of the αCP1–KH1/11-mer DNA complex. (A) αCP1–KH1 formed as a dimer with two protein molecules bound to a single 11-nt strand of DNA, resulting in a continuous network throughout the crystal. (B) A cartoon representation of the KH domain bound to the target DNA. The 5′-tetrad of the target DNA that form contacts with the first KH domain is shown, illustrating the positioning of the critical bases about α-helix 1 and between the GXXG and variable loops. (C) The electrostatic potential emanating from the αCP1–KH1 (in the same orientation as cartoon alongside) structure calculated using the APBS software package (46). Potential contours are shown at +1 kT/e (blue) and −1 kT/e (red) and were obtained by solution of the linearized Poisson–Boltzmann equation at 150 mM ionic strength with a solute dielectric of 2 and a solvent dielectric of 78.5. The blue contour represents a positive potential directing oligonucleotides to the binding cleft. (D) Summary of the contacts between αCP1–KH1 and bound DNA tetrad of sequence 5′-CCCT-3′. Van der Waals contacts are coloured orange, and hydrogen bond interactions are coloured blue. The residues making important contacts with the oligonucleotide sugar–phosphate backbone are listed on the left, and the residues making contacts with the pyrimidine rings, and thus underling base specificity, are listed on the right.
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gks058-F3: Schematic representations of the αCP1–KH1/11-mer DNA complex. (A) αCP1–KH1 formed as a dimer with two protein molecules bound to a single 11-nt strand of DNA, resulting in a continuous network throughout the crystal. (B) A cartoon representation of the KH domain bound to the target DNA. The 5′-tetrad of the target DNA that form contacts with the first KH domain is shown, illustrating the positioning of the critical bases about α-helix 1 and between the GXXG and variable loops. (C) The electrostatic potential emanating from the αCP1–KH1 (in the same orientation as cartoon alongside) structure calculated using the APBS software package (46). Potential contours are shown at +1 kT/e (blue) and −1 kT/e (red) and were obtained by solution of the linearized Poisson–Boltzmann equation at 150 mM ionic strength with a solute dielectric of 2 and a solvent dielectric of 78.5. The blue contour represents a positive potential directing oligonucleotides to the binding cleft. (D) Summary of the contacts between αCP1–KH1 and bound DNA tetrad of sequence 5′-CCCT-3′. Van der Waals contacts are coloured orange, and hydrogen bond interactions are coloured blue. The residues making important contacts with the oligonucleotide sugar–phosphate backbone are listed on the left, and the residues making contacts with the pyrimidine rings, and thus underling base specificity, are listed on the right.

Mentions: The αCP1–KH1/11-mer structure reveals two αCP1–KH1 domains bound at adjacent CCCT sequences (Figure 3A). The final model defined the position of residues 13–83 of αCP1–KH1 and the four bound bases in each of the binding clefts. Although the two KH domains bound to the same oligonucleotide are held very closely, they do not make contact with one another (Figure 3A). Interestingly, in this crystal form, each KH1 domain is covalently linked to an adjacent KH1 domain by a disulphide bond formed through their C54 residues. Furthermore, each KH domain exists as a dimer with a KH domain from an adjacent unit as previously observed for other KH domain structures (26,27,30,31).Figure 3.


Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides.

Yoga YM, Traore DA, Sidiqi M, Szeto C, Pendini NR, Barker A, Leedman PJ, Wilce JA, Wilce MC - Nucleic Acids Res. (2012)

Schematic representations of the αCP1–KH1/11-mer DNA complex. (A) αCP1–KH1 formed as a dimer with two protein molecules bound to a single 11-nt strand of DNA, resulting in a continuous network throughout the crystal. (B) A cartoon representation of the KH domain bound to the target DNA. The 5′-tetrad of the target DNA that form contacts with the first KH domain is shown, illustrating the positioning of the critical bases about α-helix 1 and between the GXXG and variable loops. (C) The electrostatic potential emanating from the αCP1–KH1 (in the same orientation as cartoon alongside) structure calculated using the APBS software package (46). Potential contours are shown at +1 kT/e (blue) and −1 kT/e (red) and were obtained by solution of the linearized Poisson–Boltzmann equation at 150 mM ionic strength with a solute dielectric of 2 and a solvent dielectric of 78.5. The blue contour represents a positive potential directing oligonucleotides to the binding cleft. (D) Summary of the contacts between αCP1–KH1 and bound DNA tetrad of sequence 5′-CCCT-3′. Van der Waals contacts are coloured orange, and hydrogen bond interactions are coloured blue. The residues making important contacts with the oligonucleotide sugar–phosphate backbone are listed on the left, and the residues making contacts with the pyrimidine rings, and thus underling base specificity, are listed on the right.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367169&req=5

gks058-F3: Schematic representations of the αCP1–KH1/11-mer DNA complex. (A) αCP1–KH1 formed as a dimer with two protein molecules bound to a single 11-nt strand of DNA, resulting in a continuous network throughout the crystal. (B) A cartoon representation of the KH domain bound to the target DNA. The 5′-tetrad of the target DNA that form contacts with the first KH domain is shown, illustrating the positioning of the critical bases about α-helix 1 and between the GXXG and variable loops. (C) The electrostatic potential emanating from the αCP1–KH1 (in the same orientation as cartoon alongside) structure calculated using the APBS software package (46). Potential contours are shown at +1 kT/e (blue) and −1 kT/e (red) and were obtained by solution of the linearized Poisson–Boltzmann equation at 150 mM ionic strength with a solute dielectric of 2 and a solvent dielectric of 78.5. The blue contour represents a positive potential directing oligonucleotides to the binding cleft. (D) Summary of the contacts between αCP1–KH1 and bound DNA tetrad of sequence 5′-CCCT-3′. Van der Waals contacts are coloured orange, and hydrogen bond interactions are coloured blue. The residues making important contacts with the oligonucleotide sugar–phosphate backbone are listed on the left, and the residues making contacts with the pyrimidine rings, and thus underling base specificity, are listed on the right.
Mentions: The αCP1–KH1/11-mer structure reveals two αCP1–KH1 domains bound at adjacent CCCT sequences (Figure 3A). The final model defined the position of residues 13–83 of αCP1–KH1 and the four bound bases in each of the binding clefts. Although the two KH domains bound to the same oligonucleotide are held very closely, they do not make contact with one another (Figure 3A). Interestingly, in this crystal form, each KH1 domain is covalently linked to an adjacent KH1 domain by a disulphide bond formed through their C54 residues. Furthermore, each KH domain exists as a dimer with a KH domain from an adjacent unit as previously observed for other KH domain structures (26,27,30,31).Figure 3.

Bottom Line: The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites.SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet.The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC Australia.

ABSTRACT
Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.

Show MeSH
Related in: MedlinePlus