Crystal structure of the third KH domain of human poly(C)-binding protein-2 in complex with a C-rich strand of human telomeric DNA at 1.6 A resolution

Abstract

KH (hnRNP K homology) domains, consisting of ∼70 amino acid residues, are present in a variety of nucleic-acid-binding proteins. Among these are poly(C)-binding proteins (PCBPs), which are important regulators of mRNA stability and posttranscriptional regulation in general. All PCBPs contain three different KH domains and recognize poly(C)-sequences with high affinity and specificity. To reveal the molecular basis of poly(C)-sequence recognition, we have determined the crystal structure, at 1.6 Å resolution, of PCBP2 KH3 domain in complex with a 7-nt DNA sequence (5′-AACCCTA-3′) corresponding to one repeat of the C-rich strand of human telomeric DNA. The domain assumes a type-I KH fold in a βααββα configuration. The protein–DNA interface could be studied in unprecedented detail and is made up of a series of direct and water-mediated hydrogen bonds between the protein and the DNA, revealing an especially dense network involving several structural water molecules for the last 2 nt in the core recognition sequence. Unlike published KH domain structures, the protein crystallizes without protein–protein contacts, yielding new insights into the dimerization properties of different KH domains. A nucleotide platform, an interesting feature found in some RNA molecules, was identified, evidently for the first time in DNA.

X-ray crystallographic and NMR studies of protein–protein and protein–nucleic acid interactions involving the KH domains from human poly(C)-binding protein-2

“…Crystal structures of the KH3 domain from hnRNP K in its free and DNAbound form and the KH3 domain from PCBP1 in its free form have also been published (Backe et al2005;Sidiqi et al 2005). We have also determined the crystal structure of PCBP2 KH3 in complex with a 7-nt human telomeric DNA (Fenn et al 2007). Interestingly, the kind of dimerization we observed in PCBP2 KH1 structures was not observed in the KH3 domain structures.…”

“…The KH1 + KH2 construct yielded excellent quality NMR spectra (Fig. 5A) (Fenn et al 2007). Positively charged, negatively charged, and uncharged hydrophilic and hydrophobic residues are colored in blue, red, yellow, and green, respectively; glycines are in white.…”

Insights into the biomedical effects of carboxylated single-wall carbon nanotubes on telomerase and telomeres

“…PCBP1 protein has been identified as the most specific binding protein for telomeric C-rich DNA and may be presented in the human telomere-telomerase complex 42,43 . We also observed removal of PCBP1 protein from telomere after treatment with SWNTs.…”

“…To determine the cause of telomere uncapping, we investigated the effect of SWNTs on the localization of TRF2 and POT1, two telomeric proteins that can induce telomere dysfunction and evoke DNAdamage signaling when their levels are reduced at telomeres 1,34,35 . Furthermore, we also studied the effect of SWNTs on the localization of PCBP1 (poly-(C) binding protein 1) at telomere, which has been identified as a specific telomere C-rich strand binding protein, and may have an important role in telomere maintenance 42,43 . Confocal microscopy showed that SWNTs specifically delocalized TRF2, POT1 and PCBP1 from TRF1 foci in HeLa cells after 2 weeks of treatment (Fig.…”

Structure of a Construct of a Human Poly(C)-binding Protein Containing the First and Second KH Domains Reveals Insights into Its Regulatory Mechanisms

“…For most samples, the final NMR buffer contains 50 mM deuterated sodium acetate (pH 5.4), 2 mM dithiothreitol, 0.1 M EDTA, in either 90% H 2 O/10% D 2 O or 100% D 2 O. Preparation of the KH3 construct has also been described previously (27). For the full-length construct, protein expression was induced with 0.1 mM isopropyl-␤-D-thiogalactopyranoside at A 600 ϭ 0.6 -0.8 at 10°C for 48 h before harvest.…”