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Substitution of tryptophan 89 with tyrosine switches the DNA binding mode of PC4.

ABSTRACT: PC4, a well-known general transcription cofactor, has multiple functions in transcription and DNA repair. Residue W89, is engaged in stacking interactions with DNA in PC4, but substituted by tyrosine in some PC4 orthologous proteins. In order to understand the consequences and reveal the molecular details of this substitution we have determined the crystal structures of the PC4 orthologue MoSub1 and a PC4 W89Y mutant in complex with DNA. In the structure of MoSub1-DNA complex, Y74 interacts directly with a single nucleotide of oligo DNA. By comparison, the equivalent residue, W89 in wild type PC4 interacts with two nucleotides and the base of the second nucleotide has distinct orientation relative to that of the first one. A hydrophobic patch around W89 that favours interaction with two nucleotides is not formed in the PC4 W89Y mutant. Therefore, the change of the surface hydrophobicity around residue 89 results in a difference between the modes of DNA interaction. These results indicate that the conserved Y74 in MoSub1 or W89 in PC4, are not only key residues in making specific interactions with DNA but also required to determine the DNA binding mode of PC4 proteins.

SUBMITTER: Huang J

PROVIDER: S-EPMC4350104 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Substitution of tryptophan 89 with tyrosine switches the DNA binding mode of PC4.

Huang Jinguang J Zhao Yanxiang Y Liu Huaian H Huang Dan D Cheng Xiankun X Zhao Wensheng W Taylor Ian A IA Liu Junfeng J Peng You-Liang YL

Scientific reports 20150305

PC4, a well-known general transcription cofactor, has multiple functions in transcription and DNA repair. Residue W89, is engaged in stacking interactions with DNA in PC4, but substituted by tyrosine in some PC4 orthologous proteins. In order to understand the consequences and reveal the molecular details of this substitution we have determined the crystal structures of the PC4 orthologue MoSub1 and a PC4 W89Y mutant in complex with DNA. In the structure of MoSub1-DNA complex, Y74 interacts dire ...[more]

PMID: 25739870

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Project description:BACKGROUND: Protein-DNA interactions are essential for fundamental biological activities including DNA transcription, replication, packaging, repair and rearrangement. Proteins interacting with DNA can be classified into two categories of binding mechanisms - sequence-specific and non-specific binding. Protein-DNA specific binding provides a mechanism to recognize correct nucleotide base pairs for sequence-specific identification. Protein-DNA non-specific binding shows sequence independent interaction for accelerated targeting by interacting with DNA backbone. Both sequence-specific and non-specific binding residues contribute to their roles for interaction. RESULTS: The proposed framework has two stage predictors: DNA-binding residues prediction and binding mode prediction. In the first stage - DNA-binding residues prediction, the predictor for DNA specific binding residues achieves 96.45% accuracy with 50.14% sensitivity, 99.31% specificity, 81.70% precision, and 62.15% F-measure. The predictor for DNA non-specific binding residues achieves 89.14% accuracy with 53.06% sensitivity, 95.25% specificity, 65.47% precision, and 58.62% F-measure. While combining prediction results of sequence-specific and non-specific binding residues with OR operation, the predictor achieves 89.26% accuracy with 56.86% sensitivity, 95.63% specificity, 71.92% precision, and 63.51% F-measure. In the second stage, protein-DNA binding mode prediction achieves 75.83% accuracy while using support vector machine with multi-class prediction. CONCLUSION: This article presents the design of a sequence based predictor aiming to identify sequence-specific and non-specific binding residues in a transcription factor with DNA binding-mechanism concerned. The protein-DNA binding mode prediction was introduced to help improve DNA-binding residues prediction. In addition, the results of this study will help with the design of binding-mechanism concerned predictors for other families of proteins interacting with DNA.

Project description:Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of structurally diverse alkylated and oxidized purine lesions from DNA to initiate the base excision repair pathway. Recognition of a base lesion requires flipping of the damaged nucleotide into a relatively open active site pocket between two conserved tyrosine residues, Y127 and Y159. We have mutated each of these amino acids to tryptophan and measured the kinetic effects on the nucleotide flipping and base excision steps. The Y127W and Y159W mutant proteins have robust glycosylase activity toward DNA containing 1,N(6)-ethenoadenine (?A), within 4-fold of that of the wild-type enzyme, raising the possibility that tryptophan fluorescence could be used to probe the DNA binding and nucleotide flipping steps. Stopped-flow fluorescence was used to compare the time-dependent changes in tryptophan fluorescence and ?A fluorescence. For both mutants, the tryptophan fluorescence exhibited two-step binding with essentially identical rate constants as were observed for the ?A fluorescence changes. These results provide evidence that AAG forms an initial recognition complex in which the active site pocket is perturbed and the stacking of the damaged base is disrupted. Upon complete nucleotide flipping, there is further quenching of the tryptophan fluorescence with coincident quenching of the ?A fluorescence. Although these mutations do not have large effects on the rate constant for excision of ?A, there are dramatic effects on the rate constants for nucleotide flipping that result in 40-100-fold decreases in the flipping equilibrium relative to wild-type. Most of this effect is due to an increased rate of unflipping, but surprisingly the Y159W mutation causes a 5-fold increase in the rate constant for flipping. The large effect on the equilibrium for nucleotide flipping explains the greater deleterious effects that these mutations have on the glycosylase activity toward base lesions that are in more stable base pairs.

Dataset Information

Substitution of tryptophan 89 with tyrosine switches the DNA binding mode of PC4.

Publications

Substitution of tryptophan 89 with tyrosine switches the DNA binding mode of PC4.

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