Symmetric Dimeric Structure and Ligand Recognition of CutR, a LysR-Type Transcriptional Regulator from Mycobacterium sp. Strain JC1

Mycobacteria possess carbon monoxide dehydrogenase (CO-DH) to utilize CO as an energy source and to resist host defense mechanisms. The expression of the CO-DH gene is regulated by CutR, a LysR-type transcriptional regulator (LTTR) that exhibits unique characteristics, suggesting that it functions as a dimer rather than the typical tetramer. Size-exclusion chromatography revealed that CutR forms a stable dimer. Electrophoretic mobility shift assays demonstrated that dimeric CutR specifically binds to an inverted repeat sequence (IR1) containing T-n12-A motifs located upstream of the <i>cutB</i> gene, which encodes the medium subunit of CO-DH. Crystal structure determination at 1.8 Å resolution revealed that CutR consists of an N-terminal DNA-binding domain with a winged helix-turn-helix motif and a C-terminal ligand-binding domain comprising two regulatory subdomains (RD1 and RD2), forming a unique two-fold symmetrical homodimer. This dimer is stabilized through four interfaces, including an extensive 12-stranded antiparallel β-sheet formed between RD1 subdomains via intertwining C-terminal β11 strands. This represents the first symmetric dimeric LTTR structure with tightly associated ligand-binding domains. The recognition helices are spaced closer together than they are in typical DNA-bound LTTRs, despite binding longer T-n12-A sequences, suggesting that a conformational change is required to enhance DNA-binding affinity. A putative ligand-binding site was identified between the RD1 and RD2 subdomains, where glycerol binding induced local conformational changes. Comparative genomic analysis revealed conservation of CutR and the IR1 sequence across <i>Mycobacterium</i> species, supporting the dimeric regulatory mechanism and providing new insights into LTTR diversity.