MacP bypass variants of Streptococcus pneumoniae PBP2a suggest a conserved mechanism for the activation of bifunctional cell wall synthases.

The peptidoglycan (PG) layer protects bacteria from osmotic lysis and defines their shape. The class A penicillin-binding proteins (aPBPs) are PG synthases that possess both glycan polymerization and crosslinking activities needed for PG biogenesis. In Gram-negative bacteria, aPBPs require activation by outer membrane lipoproteins, which are thought to stimulate their cognate synthase by inducing conformational changes that promote polymerase function. How aPBPs are controlled in Gram-positive bacteria is less clear. One of the few known regulators is MacP in Streptococcus pneumoniae (Sp). MacP is required for the activity of Sp PBP2a, but its mode of action has been obscure. We therefore selected for PBP2a variants capable of functioning in the absence of MacP. Amino acid substitutions that bypassed the MacP requirement for PBP2a function in vivo also activated its polymerase activity in vitro. Many of these changes mapped to the interface between the transmembrane (TM) helix and polymerase domain in a model PBP2a structure. This region is conformationally flexible in the experimentally determined structures of aPBPs and undergoes a structural transition upon binding the substrate-mimicking drug moenomycin. Our findings suggest that MacP promotes PG polymerization by altering the TM-polymerase domain interface in PBP2a and that this mechanism for aPBP activation may be broadly conserved. Furthermore, Sp cells expressing an activated PBP2a variant displayed heterogeneous shapes, highlighting the importance of proper aPBP regulation in cell morphogenesis.IMPORTANCEClass A penicillin-binding proteins (aPBPs) play critical roles in bacterial cell wall biogenesis. As the targets of penicillin, they are among the most important drug targets in history. Although the biochemical activities of these enzymes have been well studied, little is known about how they are regulated in cells to control when and where peptidoglycan is made. In this report, we isolate variants of the Streptococcus pneumoniae enzyme PBP2a that function in cells without MacP, a partner normally required for its activity. The amino acid substitutions activate the cell wall synthase activity of PBP2a, and their location in a model structure suggests an activation mechanism for this enzyme that is shared with aPBPs from distantly related organisms with distinct activators.

Allosteric activation of cell wall synthesis during bacterial growth.

The peptidoglycan (PG) cell wall protects bacteria against osmotic lysis and determines cell shape, making this structure a key antibiotic target. Peptidoglycan is a polymer of glycan chains connected by peptide crosslinks, and its synthesis requires precise spatiotemporal coordination between glycan polymerization and crosslinking. However, the molecular mechanism by which these reactions are initiated and coupled is unclear. Here we use single-molecule FRET and cryo-EM to show that an essential PG synthase (RodA-PBP2) responsible for bacterial elongation undergoes dynamic exchange between closed and open states. Structural opening couples the activation of polymerization and crosslinking and is essential in vivo. Given the high conservation of this family of synthases, the opening motion that we uncovered likely represents a conserved regulatory mechanism that controls the activation of PG synthesis during other cellular processes, including cell division.

The prokaryotic Na+/Ca2+ exchanger NCX_Mj transports Na+ and Ca2+ in a 3:1 stoichiometry.

Intracellular Ca2+ signals control a wide array of cellular processes. These signals require spatial and temporal regulation of the intracellular Ca2+ concentration, which is achieved in part by a class of ubiquitous membrane proteins known as sodium-calcium exchangers (NCXs). NCXs are secondary-active antiporters that power the translocation of Ca2+ across the cell membrane by coupling it to the flux of Na+ in the opposite direction, down an electrochemical gradient. Na+ and Ca2+ are translocated in separate steps of the antiport cycle, each of which is thought to entail a mechanism whereby ion-binding sites within the protein become alternately exposed to either side of the membrane. The prokaryotic exchanger NCX_Mj, the only member of this family with known structure, has been proposed to be a good functional and structural model of mammalian NCXs; yet our understanding of the functional properties of this protein remains incomplete. Here, we study purified NCX_Mj reconstituted into liposomes under well-controlled experimental conditions and demonstrate that this homologue indeed shares key functional features of the NCX family. Transport assays and reversal-potential measurements enable us to delineate the essential characteristics of this antiporter and establish that its ion-exchange stoichiometry is 3Na+:1Ca2+ Together with previous studies, this work confirms that NCX_Mj is a valid model system to investigate the mechanism of ion recognition and membrane transport in sodium-calcium exchangers.