Mutation and crystallization of the first KH domain of human polycytosine-binding protein 1 (PCBP1) in complex with DNA

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Polycytosine-binding proteins (PCBPs) are triple KH-domain proteins that play an important role in the regulation of translation of eukaryotic mRNA. They are also utilized by viral RNA and have been shown to interact with ssDNA. Underlying their function is the specific recognition of C-rich nucleotides by their KH domains. However, the structural basis of this recognition is only partially understood. Here, the preparation of a His-tagged KH domain is described, representing the first domain of PCBP1 that incorporates a C54S mutation as well as the addition of a C-terminal tryptophan. This construct has facilitated the preparation of highly diffracting crystals in complex with C-rich DNA (sequence ACCCCA). Crystals of the KH1-DNA complex were grown using the hanging-drop vapour-diffusion method in 0.1 M phosphate-citrate pH 4.2, 40 (v/v) PEG 300. X-ray diffraction data were collected to 1.77 A resolution and the diffraction was consistent with space group P2(1), with unit-cell parameters a = 38.59, b = 111.88, c = 43.42 A, alpha = gamma = 90.0, beta = 93.37 degrees . The structure of the KH1-DNA complex will further our insight into the basis of cytosine specificity by PCBPs.
Original languageEnglish
Pages (from-to)1257 - 1261
Number of pages5
JournalActa Crystallographica. Section F: Structural Biology Communications
Volume67
Issue numberPt 10
DOIs
Publication statusPublished - 2011

Cite this

@article{dccf423782b8488787d267eefaf41d8a,
title = "Mutation and crystallization of the first KH domain of human polycytosine-binding protein 1 (PCBP1) in complex with DNA",
abstract = "Polycytosine-binding proteins (PCBPs) are triple KH-domain proteins that play an important role in the regulation of translation of eukaryotic mRNA. They are also utilized by viral RNA and have been shown to interact with ssDNA. Underlying their function is the specific recognition of C-rich nucleotides by their KH domains. However, the structural basis of this recognition is only partially understood. Here, the preparation of a His-tagged KH domain is described, representing the first domain of PCBP1 that incorporates a C54S mutation as well as the addition of a C-terminal tryptophan. This construct has facilitated the preparation of highly diffracting crystals in complex with C-rich DNA (sequence ACCCCA). Crystals of the KH1-DNA complex were grown using the hanging-drop vapour-diffusion method in 0.1 M phosphate-citrate pH 4.2, 40 (v/v) PEG 300. X-ray diffraction data were collected to 1.77 A resolution and the diffraction was consistent with space group P2(1), with unit-cell parameters a = 38.59, b = 111.88, c = 43.42 A, alpha = gamma = 90.0, beta = 93.37 degrees . The structure of the KH1-DNA complex will further our insight into the basis of cytosine specificity by PCBPs.",
author = "Yoga, {Yano M K} and Traore, {Daouda A K} and Wilce, {Jacqueline Anne} and Wilce, {Matthew Charles}",
year = "2011",
doi = "10.1107/S1744309111028004",
language = "English",
volume = "67",
pages = "1257 -- 1261",
journal = "Acta Crystallographica. Section F: Structural Biology Communications",
issn = "1744-3091",
publisher = "International Union of Crystallography",
number = "Pt 10",

}

TY - JOUR

T1 - Mutation and crystallization of the first KH domain of human polycytosine-binding protein 1 (PCBP1) in complex with DNA

AU - Yoga, Yano M K

AU - Traore, Daouda A K

AU - Wilce, Jacqueline Anne

AU - Wilce, Matthew Charles

PY - 2011

Y1 - 2011

N2 - Polycytosine-binding proteins (PCBPs) are triple KH-domain proteins that play an important role in the regulation of translation of eukaryotic mRNA. They are also utilized by viral RNA and have been shown to interact with ssDNA. Underlying their function is the specific recognition of C-rich nucleotides by their KH domains. However, the structural basis of this recognition is only partially understood. Here, the preparation of a His-tagged KH domain is described, representing the first domain of PCBP1 that incorporates a C54S mutation as well as the addition of a C-terminal tryptophan. This construct has facilitated the preparation of highly diffracting crystals in complex with C-rich DNA (sequence ACCCCA). Crystals of the KH1-DNA complex were grown using the hanging-drop vapour-diffusion method in 0.1 M phosphate-citrate pH 4.2, 40 (v/v) PEG 300. X-ray diffraction data were collected to 1.77 A resolution and the diffraction was consistent with space group P2(1), with unit-cell parameters a = 38.59, b = 111.88, c = 43.42 A, alpha = gamma = 90.0, beta = 93.37 degrees . The structure of the KH1-DNA complex will further our insight into the basis of cytosine specificity by PCBPs.

AB - Polycytosine-binding proteins (PCBPs) are triple KH-domain proteins that play an important role in the regulation of translation of eukaryotic mRNA. They are also utilized by viral RNA and have been shown to interact with ssDNA. Underlying their function is the specific recognition of C-rich nucleotides by their KH domains. However, the structural basis of this recognition is only partially understood. Here, the preparation of a His-tagged KH domain is described, representing the first domain of PCBP1 that incorporates a C54S mutation as well as the addition of a C-terminal tryptophan. This construct has facilitated the preparation of highly diffracting crystals in complex with C-rich DNA (sequence ACCCCA). Crystals of the KH1-DNA complex were grown using the hanging-drop vapour-diffusion method in 0.1 M phosphate-citrate pH 4.2, 40 (v/v) PEG 300. X-ray diffraction data were collected to 1.77 A resolution and the diffraction was consistent with space group P2(1), with unit-cell parameters a = 38.59, b = 111.88, c = 43.42 A, alpha = gamma = 90.0, beta = 93.37 degrees . The structure of the KH1-DNA complex will further our insight into the basis of cytosine specificity by PCBPs.

UR - http://www.ncbi.nlm.nih.gov/pubmed/22102042

U2 - 10.1107/S1744309111028004

DO - 10.1107/S1744309111028004

M3 - Article

VL - 67

SP - 1257

EP - 1261

JO - Acta Crystallographica. Section F: Structural Biology Communications

JF - Acta Crystallographica. Section F: Structural Biology Communications

SN - 1744-3091

IS - Pt 10

ER -