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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 separate KH domains of αCP1 to target RNA and DNA using SPR. Sensorgrams of αCP1 KH1, KH2 and KH3 binding to biotinylated mRNA (5′-CUCUCCUUUCUUUUUCUUCUUCCCUCCCUA-3′) representing nucleotides 3296–3325 of androgen receptor mRNA (flow cell 2) and biotinylated DNA (5′-CTCTCCTTTCTTTTTCTTCTTCCCTCCCTA-3′) analogous to the above RNA sequence (flow cell 3) captured on SA-coated sensor chips at a range of protein concentrations are shown. 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-F2: Binding analysis of separate KH domains of αCP1 to target RNA and DNA using SPR. Sensorgrams of αCP1 KH1, KH2 and KH3 binding to biotinylated mRNA (5′-CUCUCCUUUCUUUUUCUUCUUCCCUCCCUA-3′) representing nucleotides 3296–3325 of androgen receptor mRNA (flow cell 2) and biotinylated DNA (5′-CTCTCCTTTCTTTTTCTTCTTCCCTCCCTA-3′) analogous to the above RNA sequence (flow cell 3) captured on SA-coated sensor chips at a range of protein concentrations are shown. 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: Having shown that αCP1–KH1 binds to both DNA and RNA using EMSA, we utilized SPR for a more sensitive comparison of KH domain binding affinities and binding kinetics to target RNA and the analogous DNA sequence. SPR binding curves showing a series of αCP1–KH interactions (at concentrations of between 0.312 μM and 10 μM) with a 30-nt oligonucleotide representing AR mRNA (nucleotides 3296–3325) are shown in Figure 2. This RNA sequence includes 19-nt of U-rich sequence preceding the 11-nt C-rich target sequence (as used in the EMSA above) as a spacer between the Biacore chip and the binding site. Alongside these curves are the sensorgrams obtained simultaneously using the analogous sequence of DNA. It is to be noted that the binding curves exhibited complex binding kinetics, most likely due to the lengthy oligonucleotides immobilized on the chip and the presence of two cytosine triplets at the 3′-ends that constitute two αCP1–KH domain target sites. Where binding was observed, an approximated steady state binding analysis is presented.Figure 2.


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 separate KH domains of αCP1 to target RNA and DNA using SPR. Sensorgrams of αCP1 KH1, KH2 and KH3 binding to biotinylated mRNA (5′-CUCUCCUUUCUUUUUCUUCUUCCCUCCCUA-3′) representing nucleotides 3296–3325 of androgen receptor mRNA (flow cell 2) and biotinylated DNA (5′-CTCTCCTTTCTTTTTCTTCTTCCCTCCCTA-3′) analogous to the above RNA sequence (flow cell 3) captured on SA-coated sensor chips at a range of protein concentrations are shown. 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-F2: Binding analysis of separate KH domains of αCP1 to target RNA and DNA using SPR. Sensorgrams of αCP1 KH1, KH2 and KH3 binding to biotinylated mRNA (5′-CUCUCCUUUCUUUUUCUUCUUCCCUCCCUA-3′) representing nucleotides 3296–3325 of androgen receptor mRNA (flow cell 2) and biotinylated DNA (5′-CTCTCCTTTCTTTTTCTTCTTCCCTCCCTA-3′) analogous to the above RNA sequence (flow cell 3) captured on SA-coated sensor chips at a range of protein concentrations are shown. 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: Having shown that αCP1–KH1 binds to both DNA and RNA using EMSA, we utilized SPR for a more sensitive comparison of KH domain binding affinities and binding kinetics to target RNA and the analogous DNA sequence. SPR binding curves showing a series of αCP1–KH interactions (at concentrations of between 0.312 μM and 10 μM) with a 30-nt oligonucleotide representing AR mRNA (nucleotides 3296–3325) are shown in Figure 2. This RNA sequence includes 19-nt of U-rich sequence preceding the 11-nt C-rich target sequence (as used in the EMSA above) as a spacer between the Biacore chip and the binding site. Alongside these curves are the sensorgrams obtained simultaneously using the analogous sequence of DNA. It is to be noted that the binding curves exhibited complex binding kinetics, most likely due to the lengthy oligonucleotides immobilized on the chip and the presence of two cytosine triplets at the 3′-ends that constitute two αCP1–KH domain target sites. Where binding was observed, an approximated steady state binding analysis is presented.Figure 2.

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