UV oxidation of cyclic AMP receptor protein, a global bacterial gene regulator, decreases DNA binding and cleaves DNA at specific sites

Abstract

UV light is a widely-employed, and environmentally-sensitive bactericide but its mechanism of action is not fully defined. Proteins are major chromophores and targets for damage due to their abundance, but the role of proteins in inducing damage to bound DNA, and the effects on DNA-protein interactions is less well characterized. in E. coli (and other Gram-negative bacteria) the cyclic AMp receptor protein (CRP/CAP) regulates more than 500 genes. In this study we show that exposure of isolated dimeric CRP-cAMP to UV modifies specific Met, Trp, Tyr, and Pro side-chains, induces inter-protein Tyr63-Tyr41 cross-links, and decreases DNA binding via oxidation of Met114/Pro110 residues in close proximity at the CRP dimer interface. UV exposure also modifies DNA-bound cAMP-CRP, with this resulting in DNA cleavage at specific G/C residues within the sequence bound to CRP, but not at other G/C sites. Oxidation also increases CRP dissociation from DNA. The modifications at the CRP dimer interface, and the sitespecific DNA strand cleavage are proposed to occur via oxidation of two species Met residues (Met114 and Met189, respectively) to reactive persulfoxides that damage neighbouring amino acids and DNA bases. These data suggest that modification to CRP, and bound DNA, contributes to UV sensitivity.The cyclic AMP receptor protein (CRP) (also known as the catabolite activator protein, CAP) is a global regulator of gene expression in E. coli and other Gram negative bacteria. CRP regulates more than 500 genes in E. coli by binding to approximately 300 high-affinity sites 1 . CRP also binds to more than 10000 low-affinity sites in the E. coli genome, indicating that CRP is not only a specific transcriptional regulator, but also a chromosome shaping protein 2,3 . CRP binds as a dimer to DNA sites, and subsequently with RNA polymerase to activate transcription. This activity is cAMP dependent, with complexation resulting in a conformational transition that converts apoCRP from a low-to a high-affinity DNA binding protein which binds at specific sequences upstream of the promoter 4 . The structural basis of CRP interactions with cAMP is well understood 5 . CRP is a homo-dimeric protein with each subunit able to bind one cAMP in a large N-terminal domain that is also responsible for subunit-subunit interactions 6,7 . A smaller C-terminal domain contains a helix-turn-helix motif involved in DNA attachment, with binding generating a strong kink in the DNA chain 8 . This CRP-DNA binding domain is highly conserved across many bacterial regulatory proteins 9,10 .Cell killing can also be readily induced by high energy radiation, UV and visible light in the presence of a sensitizer 11,12 . Proteins are both major UV absorbing species [13][14][15] , and major targets for oxidation due to their high abundance 16 . Trp, Tyr, Met, Cys, cystine and His side-chains are particularly prone to modification by UV light, or oxidants (e.g. singlet oxygen, 1 O 2 ) generated by energy transfer from other excited states 13,14,16 .We have therefore exam...

Oxidative Crosslinking of Peptides and Proteins: Mechanisms of Formation, Detection, Characterization and Quantification

“…Thus, it is possible to distinguish crosslinked peptides from non-crosslinked peptides based on their isotope labeling pattern [ 167 , 203 ]. This strategy has been successfully applied to validate oxidation-induced crosslinks in a wide range of proteins, including glucose-6-phosphate dehydrogenase, lysozyme, RNase A, elastin, fibronectin, cyclic AMP receptor protein, C-reactive protein and β-2-microglobulin (B2M) [ 53 , 54 , 55 , 56 , 57 , 59 , 86 , 93 , 94 , 203 ].…”

Oxidative Modification of Proteins: From Damage to Catalysis, Signaling, and Beyond

“…Hence, the proposed mechanism of protein protection against widespread oxidation by Met residues and the catalytic recycling provides efficient antioxidant protection to the specific protein and its environment. This early idea of Met residues functioning as oxidant scavengers has been challenged by new data (72,369). We also direct the reader to the discussion below regarding Met residue oxidation and loss of protein activity.…”

The Response Regulator VC1795 of Vibrio Pathogenicity Island-2 Contributes to Intestinal Colonization by Vibrio cholerae

“…In V. cholerae, CRP (VC2614) was composed of 633 bases and 210 amino acids, with a protein size of 23.64 KDa and a GC content of 47.23%. After cAMP forms the cAMP-CRP polymer with its receptor protein CRP, it binds to TGTGA-N6-TCACA, the upstream conserved sequence of the promoter, and interacts with the RNA polymerase, thus regulating gene transcription [19][20][21]. In addition, the concentration of cAMP is very important for the transcriptional activity of CRP.…”