Journal Article

Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity

Bernhardt, Thomas G., Lupoli, Tania J., Uehara, Tsuyoshi, Walker, Suzanne, Markovski, Monica, Kahne, Daniel E., Bohrhunter, Jessica L.

2016

Overview

To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a of Escherichia coli require the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b–LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed.

• Publisher: National Academy of Sciences
• Subject: Amino acids / Antibiotics / Bacterial Outer Membrane Proteins - chemistry / Bacterial Outer Membrane Proteins - ultrastructure / Binding Sites / Biological Sciences / Cell Wall - chemistry / Cell Wall - metabolism / Cell Wall - ultrastructure / Coenzymes - chemistry / Coenzymes - ultrastructure / Computer Simulation / Cytoplasm / E coli / Enzyme Activation / Escherichia coli Proteins - chemistry / Escherichia coli Proteins - ultrastructure / Genotype & phenotype / Microbiology / Models, Chemical / Models, Molecular / Penicillin-Binding Proteins - chemistry / Penicillin-Binding Proteins - metabolism / Penicillin-Binding Proteins - ultrastructure / Polymerization / Polymers / Protein Binding / Protein Conformation / Proteins / Structure-Activity Relationship