bi500026e_si_001.pdf (1.66 MB)
Specificity Determinants for Autoproteolysis of LexA, a Key Regulator of Bacterial SOS Mutagenesis
journal contribution
posted on 2015-12-17, 02:14 authored by Charlie
Y. Mo, L. Dillon Birdwell, Rahul M. KohliBacteria 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.