Functional Irreplaceability of Escherichia coli and Shewanella oneidensis OxyRs Is Critically Determined by Intrinsic...

LysR-type transcriptional regulators (LTTRs), which function in diverse biological processes in prokaryotes, are composed of a conserved structure with an N-terminal DNA-binding domain (DBD) and a C-terminal signal-sensing regulatory domain (RD). LTTRs that sense and respond to the same signal are often functionally exchangeable in bacterial species across wide phyla, but this phenomenon has not been demonstrated for the H₂O₂-sensing and -responding OxyRs. Here, we systematically examined the biochemical and structural determinants differentiating activator-only OxyRs from dual-activity ones by comparing OxyRs from two Gammaproteobacteria, Escherichia coli and Shewanella oneidensis. Our data show that EcOxyR could function as neither an activator nor a repressor in S. oneidensis. Using SoOxyR-based OxyR chimeras and mutants, we demonstrated that residues 283 to 289, which form the first half of the last C-terminal α-helix (α10), are critical for the proper function of SoOxyR and cannot be replaced with the EcOxyR counterpart. Crystal structural analysis reveals that α10 is important for the oligomerization of SoOxyR, which, unlike EcOxyR, forms several high-order oligomers upon DNA binding. As the mechanisms of OxyR oligomerization vary substantially among bacterial species, our findings underscore the importance of subtle structural features in determining regulatory activities of structurally similar proteins descending from a common ancestor. IMPORTANCE Evolution may drive homologous proteins to be functionally nonexchangeable in different organisms. However, much is unknown about the mechanisms underlying this phenomenon beyond amino acid substitutions. Here, we systematically examined the biochemical and structural determinants differentiating functionally nonexchangeable OxyRs, H₂O₂-responding transcriptional regulators from two Gammaproteobacteria, Escherichia coli and Shewanella oneidensis. Using SoOxyR-based OxyR chimeras and mutants, we demonstrated that residues 283 to 289, which form the first half of the last C-terminal α-helix (α10), are critical for the proper function of SoOxyR and cannot be replaced with the EcOxyR counterpart. Crystal structural analysis reveals that this last helix is critical for formation of high-order oligomers upon DNA binding, a phenomenon not observed with EcOxyR. Our findings provide a new dimension to differences in sequence and structural features among bacterial species in determining regulatory activities of homologous regulators.