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 Å resolution and the diffraction was consistent with space group P2(1), with unit-cell parameters a = 38.59, b = 111.88, c = 43.42 Å, α = γ = 90.0, β = 93.37°. The structure of the KH1-DNA complex will further our insight into the basis of cytosine specificity by PCBPs.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, VIC 3800, Australia.
authors.forename: Yano M K
authors.initials: YM
authors.lastname: Yoga
authors.forename: Daouda A K
authors.initials: DA
authors.lastname: Traore
authors.forename: Jacqueline A
authors.initials: JA
authors.lastname: Wilce
authors.forename: Matthew C J
authors.initials: MC
authors.lastname: Wilce
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins
chemical_list: D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C094604:PCBP1 protein, human; D016601:RNA-Binding Proteins; D004247:DNA
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