Mechanistic insights into SteAB regulation of cell wall hydrolase RipA in <i>Mycobacterium tuberculosis</i>

D,L-endopeptidase RipA is the major peptidoglycan (PG) hydrolase required for cell separation in <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), as RipA defects severely hinder cell division and increase antibiotic vulnerability. Despite extensive studies, the mechanisms governing <i>Mtb</i> RipA regulation remain controversial and poorly understood. Here, we report an integrative structural and functional analysis of the <i>Mtb</i> SteAB system, a regulatory transmembrane septal complex encoded by adjacent genes <i>Rv1697</i> (<i>steA</i>) and <i>Rv1698/mctB</i> (<i>steB</i>) that are conserved across <i>Mycobacteriales</i>. The separate crystal structures of the cytoplasmic core of <i>Mt</i>SteA and the periplasmic core of <i>Mt</i>SteB in complex with the RipA coiled-coil domain (RipA_CC), along with biochemical evidence that these proteins form a stable transmembrane physical complex, define the function of the SteAB complex as a regulator of RipA-mediated cell separation in <i>Mtb</i>, arguing against a previously proposed role of MctB (Rv1698) as a putative mycobacterial outer membrane copper transporter. Our structural findings showed that, upon <i>Mt</i>SteB binding, RipA_CC is oriented perpendicular to the membrane, bringing its endopeptidase catalytic domain in physical contact with the PG layer, while the homodimeric structural core of SteA revealed a conserved functional pocket similar to the phosphonucleotide-binding site of thiamine pyrophosphokinase. These data, coupled with the <i>in vivo</i> phenotypic analysis of a <i>steAB</i> knockout mutant of <i>Cglu</i>, support a model in which the SteAB heterotetramer orchestrates the productive positioning of RipA leading to PG hydrolysis activation. These findings shed new light on the regulation of mycobacterial cell wall remodeling, with implications for understanding <i>Mtb</i> pathogenesis and identifying novel antimicrobial targets.IMPORTANCEPeptidoglycan (PG) is a major component of the bacterial cell wall. A flexible but strong PG mesh encloses the cell, conferring mechanical resistance and preventing cell lysis. This PG mesh is continually remodeled during the bacterial life by the coordinated action of tightly regulated PG-hydrolases and synthetases. Here, we report the structural characterization of the <i>M. tuberculosis</i> SteAB system, which regulates the action of the major enzyme responsible for disassembling the PG mesh to allow daughter cell separation at the end of cell division. The proposed model for the septal control of PG hydrolysis illustrates how the transmembrane SteAB complex can promote enzyme activation and provides structural information that may help target the activation mechanism for antibiotic development.