Abstract
Poly(C)-binding proteins (PCBPs) constitute a family of nucleic acid-binding proteins that play important roles in a wide spectrum of regulatory mechanisms. The diverse functions of PCBPs are dependent on the ability of the PCBPs to recognize poly(C) sequences with high affinity and specificity. PCBPs contain three copies of KH (hnRNP K homology) domains, which are responsible for binding nucleic acids. We have determined the NMR structure of the first KH domain (KH1) from PCBP2. The PCBP2 KH1 domain adopts a structure with three alpha-helices packed against one side of a three-stranded antiparallel beta-sheet. Specific binding of PCBP2 KH1 to a number of poly(C) RNA and DNA sequences, including the C-rich strand of the human telomeric DNA repeat, the RNA template region of human telomerase, and regulatory recognition motifs in the poliovirus-1 5'-untranslated region, was established by monitoring chemical shift changes in protein (15)N-HSQC spectra. The nucleic acid binding groove was further mapped by chemical shift perturbation upon binding to a six-nucleotide human telomeric DNA. The binding groove is an alpha/beta platform formed by the juxtaposition of two alpha-helices, one beta-strand, and two flanking loops. Whereas there is a groove in common with all of the DNA and RNA binders with a hydrophobic floor accommodating a three-residue stretch of C residues, nuances in recognizing flanking residues are provided by hydrogen bonding partners in the KH domain. Specific interactions of PCBP2 KH1 with telomeric DNA and telomerase RNA suggest that PCBPs may participate in mechanisms involved in the regulation of telomere/telomerase functions.
Highlights
Poly(C)-binding proteins (PCBPs) constitute a family of nucleic acid-binding proteins that play important roles in a wide spectrum of regulatory mechanisms
Structure of the PCBP2 KH1 Domain—The secondary structure of the PCBP2 KH1 domain was defined by established NMR methods based on chemical shift indexing (CSI), backbone torsion angles derived from the HNHA experiment, and TALOS prediction, as well as characteristic NOE patterns
By monitoring chemical shift changes in 15N-HSQC spectra, we showed that both RNAs induced dramatic chemical shift changes in the PCBP2 KH1 spectra (Fig. 3c), proving that the PCBP2 KH1 domain, as an independent entity, can achieve specific recognition of the stem-loop B and loop B RNAs
Summary
Sample Preparation—The gene that encodes the first KH domain (KH1) of human PCBP2 was amplified by PCR using appropriate primers and a plasmid containing the gene for full-length PCBP2. Elution of the His-tagged protein was achieved by incubating the resin in a buffer containing 25 mM sodium phosphate (pH 5.5), 25 mM sodium chloride, and 0.5 M imidazole for 10 min at 4 °C. The protein solution was dialyzed extensively against NMR buffer containing 25 mM sodium phosphate (pH 5.5) and 25 mM sodium chloride, and it was concentrated by centrifugation on Centricon 3 (Amicon) to 1–2 mM. Structure and Nucleic Acid Binding of PCBP2 KH1 Domain angle restraints were derived from 3JHNHA values measured in a quantitative J correlation HNHA experiment [35], and backbone chemical shifts were derived using the program TALOS [36]. To monitor complex formation between the PCBP2 KH1 domain and various DNA/RNA molecules, we compared 15N-HSQC or TROSY-15NHSQC spectra of the 15N-labeled protein before and after nucleic acid titration. Structure figures were generated using MidasPlus [39] or Chimera [40] from the Computer Graphics Laboratory, University of California, San Francisco
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