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

Yano M K Yoga, Daouda AK Traore, Mahjooba Sidiqi, Christopher Szeto, Nicole R Pendini, Andrew Barker, Peter Leedman, Jacqueline A Wilce, Matthew C Wilce

Research output: Contribution to journalArticleResearchpeer-review

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 alphaCP1 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 alphaCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.
Original languageEnglish
Pages (from-to)5101 - 5114
Number of pages14
JournalNucleic Acids Research
Volume40
Issue number11
DOIs
Publication statusPublished - 2012

Cite this

@article{9ff05754b77a4dcf8ff2c42898d26ded,
title = "Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides",
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 alphaCP1 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 alphaCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.",
author = "Yoga, {Yano M K} and Traore, {Daouda AK} and Mahjooba Sidiqi and Christopher Szeto and Pendini, {Nicole R} and Andrew Barker and Peter Leedman and Wilce, {Jacqueline A} and Wilce, {Matthew C}",
year = "2012",
doi = "10.1093/nar/gks058",
language = "English",
volume = "40",
pages = "5101 -- 5114",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "11",

}

Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides. / Yoga, Yano M K; Traore, Daouda AK; Sidiqi, Mahjooba; Szeto, Christopher; Pendini, Nicole R; Barker, Andrew; Leedman, Peter; Wilce, Jacqueline A; Wilce, Matthew C.

In: Nucleic Acids Research, Vol. 40, No. 11, 2012, p. 5101 - 5114.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides

AU - Yoga, Yano M K

AU - Traore, Daouda AK

AU - Sidiqi, Mahjooba

AU - Szeto, Christopher

AU - Pendini, Nicole R

AU - Barker, Andrew

AU - Leedman, Peter

AU - Wilce, Jacqueline A

AU - Wilce, Matthew C

PY - 2012

Y1 - 2012

N2 - 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 alphaCP1 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 alphaCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.

AB - 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 alphaCP1 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 alphaCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.

UR - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=22344691

U2 - 10.1093/nar/gks058

DO - 10.1093/nar/gks058

M3 - Article

VL - 40

SP - 5101

EP - 5114

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 11

ER -