Dataset Information

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.

ABSTRACT: Poly(C)-binding proteins (PCBPs) are KH (hnRNP K homology) domain-containing proteins that recognize poly(C) DNA and RNA sequences in mammalian cells. Binding poly(C) sequences via the KH domains is critical for PCBP functions. To reveal the mechanisms of KH domain-D/RNA recognition and its functional importance, we have determined the crystal structures of PCBP2 KH1 domain in complex with a 12-nucleotide DNA corresponding to two repeats of the human C-rich strand telomeric DNA and its RNA equivalent. The crystal structures reveal molecular details for not only KH1-DNA/RNA interaction but also protein-protein interaction between two KH1 domains. NMR studies on a protein construct containing two KH domains (KH1 + KH2) of PCBP2 indicate that KH1 interacts with KH2 in a way similar to the KH1-KH1 interaction. The crystal structures and NMR data suggest possible ways by which binding certain nucleic acid targets containing tandem poly(C) motifs may induce structural rearrangement of the KH domains in PCBPs; such structural rearrangement may be crucial for some PCBP functions.

SUBMITTER: Du Z

PROVIDER: S-EPMC1894928 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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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.

Du Zhihua Z Lee John K JK Fenn Sebastian S Tjhen Richard R Stroud Robert M RM James Thomas L TL

RNA (New York, N.Y.) 20070525 7

Poly(C)-binding proteins (PCBPs) are KH (hnRNP K homology) domain-containing proteins that recognize poly(C) DNA and RNA sequences in mammalian cells. Binding poly(C) sequences via the KH domains is critical for PCBP functions. To reveal the mechanisms of KH domain-D/RNA recognition and its functional importance, we have determined the crystal structures of PCBP2 KH1 domain in complex with a 12-nucleotide DNA corresponding to two repeats of the human C-rich strand telomeric DNA and its RNA equiv ...[more]

PMID: 17526645

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Project description:APOBEC3G (A3G) is a single-stranded DNA-specific cytidine deaminase that preferentially converts cytidine to uridine at the third position of triplet cytosine (CCC) hotspots. A3G restricts the infectivity of viruses, such as HIV-1, by targeting CCC hotspots scattered through minus DNA strands, reverse-transcribed from genomic RNA. Previously, we developed a real-time NMR method and elucidated the origin of the 3'→5' polarity of deamination of DNA by the C-terminal domain of A3G (CD2), which is a phenomenon by which a hotspot located closer to the 5'-end is deaminated more effectively than one less close to the 5'-end, through quantitative analysis involving nonspecific binding to and sliding along DNA. In the present study we applied the real-time NMR method to analyze the catalytic activity of CD2 toward DNA oligonucleotides containing a nucleotide analog at a single or multiple positions. Analyses revealed the importance of the sugar and base moieties throughout the consecutive 5 nucleotides, the CCC hotspot being positioned at the center. It was also shown that the sugar or base moieties of the nucleotides outside this 5 nucleotide recognition sequence are also relevant as to CD2's activity. Analyses involving DNA oligonucleotides having two CCC hotspots linked by a long sequence of either deoxyribonucleotides, ribonucleotides or abasic deoxyribonucleotides suggested that the phosphate backbone is required for CD2 to slide along the DNA strand and to exert the 3'→5' polarity. Examination of the effects of different salt concentrations on the 3'→5' polarity indicated that the higher the salt concentration, the less prominent the 3'→5' polarity. This is most likely the result of alleviation of sliding due to a decrease in the affinity of CD2 with the phosphate backbone at high salt concentrations. We also investigated the reactivity of substrates containing 5-methylcytidine (5mC) or 5-hydroxymethylcytidine, and found that A3G exhibited low activity toward 5mC.

Dataset Information

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.

Publications

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.

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