Specificity determinants for autoproteolysis of LexA, a key regulator of bacterial SOS mutagenesis.
Bacteria utilize the tightly regulated stress response (SOS) pathway to respond to a variety of genotoxic agents, including antimicrobials. Activation of the SOS response is regulated by a key repressor-protease, LexA, which undergoes autoproteolysis in the setting of stress, resulting in derepression of SOS genes. Remarkably, genetic inactivation of LexA's self-cleavage activity significantly decreases acquired antibiotic resistance in infection models and renders bacteria hypersensitive to traditional antibiotics, suggesting that a mechanistic study of LexA could help inform its viability as a novel target for combating acquired drug resistance. Despite structural insights into LexA, a detailed knowledge of the enzyme's protease specificity is lacking. Here, we employ saturation and positional scanning mutagenesis on LexA's internal cleavage region to analyze >140 mutants and generate a comprehensive specificity profile of LexA from the human pathogen Pseudomonas aeruginosa (LexAPa). We find that the LexAPa active site possesses a unique mode of substrate recognition. Positions P1-P3 prefer small hydrophobic residues that suggest specific contacts with the active site, while positions P5 and P1' show a preference for flexible glycine residues that may facilitate the conformational change that permits autoproteolysis. We further show that stabilizing the β-turn within the cleavage region enhances LexA autoproteolytic activity. Finally, we identify permissive positions flanking the scissile bond (P4 and P2') that are tolerant to extensive mutagenesis. Our studies shed light on the active site architecture of the LexA autoprotease and provide insights that may inform the design of probes of the SOS pathway.
Biochemistry and Molecular Biophysics Graduate Group, Perelman School of Medicine, University of Pennsylvania , 3610 Hamilton Walk, Philadelphia, Pennsylvania 19014, United States.
Charlie Y
CY
Mo
L Dillon
LD
Birdwell
Rahul M
RM
Kohli
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D016296:Mutagenesis; D059748:Proteolysis; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D013014:SOS Response, Genetics; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity / Q000502:physiology; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein