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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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Binding analysis of αCP1–KH domains of αCP1 to target RNA and DNA using SPR. Sensorgrams of αCP1–KH1 binding to a series of biotinylated DNA sequences, designed to test the preferential binding of cytosine at each of the four nucleotide binding positions. The sequences are shown above each set of sensorgrams with the binding tetrad underlined. Five adenines were used as a spacer between the biotin and the oligonucleotide binding site. Oligonucletides were captured on SA-coated sensor chips at a range of protein concentrations. Binding curves, derived from the approximated steady state binding of the proteins, were used to determine equilibrum dissociation constants (KDs). Errors are standard errors arising from fits.
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gks058-F4: Binding analysis of αCP1–KH domains of αCP1 to target RNA and DNA using SPR. Sensorgrams of αCP1–KH1 binding to a series of biotinylated DNA sequences, designed to test the preferential binding of cytosine at each of the four nucleotide binding positions. The sequences are shown above each set of sensorgrams with the binding tetrad underlined. Five adenines were used as a spacer between the biotin and the oligonucleotide binding site. Oligonucletides were captured on SA-coated sensor chips at a range of protein concentrations. Binding curves, derived from the approximated steady state binding of the proteins, were used to determine equilibrum dissociation constants (KDs). Errors are standard errors arising from fits.

Mentions: To investigate the position-specific binding preferences of αCP1–KH1, we used SPR to monitor binding to a series of oligonuceotides in which cytosines were systematically replaced by adenine or thymine. Figure 4 shows sensorgrams of αCP1–KH1 binding to different 10-mer DNA containing oligonucelotide target tetrads and the steady state binding analysis in the cases where quantifiable binding was measured. A 5-nt A-rich spacer was included at the 5′-end of the oligonucleotide to distance the binding site from the matrix surface and another adenine was added at the 3′-end to restrict the possible binding mode to the target tetrad since purines have not been observed to bind at the fourth position. By this reasoning, we could assume that the target tetrad (underlined in each of the sequences) would be bound in the same register at the αCP1–KH1 binding site.Figure 4.


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)

Binding analysis of αCP1–KH domains of αCP1 to target RNA and DNA using SPR. Sensorgrams of αCP1–KH1 binding to a series of biotinylated DNA sequences, designed to test the preferential binding of cytosine at each of the four nucleotide binding positions. The sequences are shown above each set of sensorgrams with the binding tetrad underlined. Five adenines were used as a spacer between the biotin and the oligonucleotide binding site. Oligonucletides were captured on SA-coated sensor chips at a range of protein concentrations. Binding curves, derived from the approximated steady state binding of the proteins, were used to determine equilibrum dissociation constants (KDs). Errors are standard errors arising from fits.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks058-F4: Binding analysis of αCP1–KH domains of αCP1 to target RNA and DNA using SPR. Sensorgrams of αCP1–KH1 binding to a series of biotinylated DNA sequences, designed to test the preferential binding of cytosine at each of the four nucleotide binding positions. The sequences are shown above each set of sensorgrams with the binding tetrad underlined. Five adenines were used as a spacer between the biotin and the oligonucleotide binding site. Oligonucletides were captured on SA-coated sensor chips at a range of protein concentrations. Binding curves, derived from the approximated steady state binding of the proteins, were used to determine equilibrum dissociation constants (KDs). Errors are standard errors arising from fits.
Mentions: To investigate the position-specific binding preferences of αCP1–KH1, we used SPR to monitor binding to a series of oligonuceotides in which cytosines were systematically replaced by adenine or thymine. Figure 4 shows sensorgrams of αCP1–KH1 binding to different 10-mer DNA containing oligonucelotide target tetrads and the steady state binding analysis in the cases where quantifiable binding was measured. A 5-nt A-rich spacer was included at the 5′-end of the oligonucleotide to distance the binding site from the matrix surface and another adenine was added at the 3′-end to restrict the possible binding mode to the target tetrad since purines have not been observed to bind at the fourth position. By this reasoning, we could assume that the target tetrad (underlined in each of the sequences) would be bound in the same register at the αCP1–KH1 binding site.Figure 4.

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