Outer-membrane lipoprotein LpoB spans the periplasm to stimulate the peptidoglycan synthase PBP1B

Abstract

Bacteria surround their cytoplasmic membrane with an essential, stress-bearing peptidoglycan (PG) layer. Growing and dividing cells expand their PG layer by using membrane-anchored PG synthases, which are guided by dynamic cytoskeletal elements. In Escherichia coli, growth of the mainly single-layered PG is also regulated by outer membrane-anchored lipoproteins. The lipoprotein LpoB is required for the activation of penicillin-binding protein (PBP) 1B, which is a major, bifunctional PG synthase with glycan chain polymerizing (glycosyltransferase) and peptide cross-linking (transpeptidase) activities. Here, we report the structure of LpoB, determined by NMR spectroscopy, showing an N-terminal, 54-aalong flexible stretch followed by a globular domain with similarity to the N-terminal domain of the prevalent periplasmic protein TolB. We have identified the interaction interface between the globular domain of LpoB and the noncatalytic UvrB domain 2 homolog domain of PBP1B and modeled the complex. Amino acid exchanges within this interface weaken the PBP1B-LpoB interaction, decrease the PBP1B stimulation in vitro, and impair its function in vivo. On the contrary, the N-terminal flexible stretch of LpoB is required to stimulate PBP1B in vivo, but is dispensable in vitro. This supports a model in which LpoB spans the periplasm to interact with PBP1B and stimulate PG synthesis. P eptidoglycan (PG) is an essential component of the bacterial cell envelope, required for cell shape and stability. It is composed of glycan chains that are connected by short peptides, and forms a net-like, elastic structure, called the sacculus, which encases the cytoplasmic/inner membrane (IM) (1). In Gramnegative bacteria, such as Escherichia coli, the sacculus is mainly single-layered and is firmly attached to the outer membrane (OM) by abundant OM proteins. Some of the most effective antibiotic agents, such as the β-lactams and glycopeptides, inhibit PG biosynthesis, resulting in cell lysis.Bacteria enlarge their sacculus by polymerizing new PG from lipid II precursor at the outer face of the IM and incorporating the newly made material into the existing PG layer. At the same time, a significant amount of old material is released. For synthesis and hydrolysis to be coupled, the corresponding enzymes have to be tightly regulated and coordinate their actions (2). How does this happen? The current view is that PG synthases [penicillin-binding proteins (PBPs)] and hydrolases form membrane-anchored multienzyme complexes, which are driven by cytoskeletal elements. More recently, it was established that dedicated regulators tightly control the activities of PG synthases and hydrolases and/or couple it to other cell envelope processes (3-6).PG synthesis requires glycosyltransferases (GTases) to polymerize the glycan chains and transpeptidases (TPases) to form peptide cross-links. Most bacteria carry several PG synthases, which can perform one or both enzymatic reactions. In E. coli, the bifunctional GTase/TPases PBP1A and PBP1B provide the main PG...

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

“…An important step forward was the identification of outer membrane lipoprotein cofactors required for the in vivo activity of the E. coli aPBPs, PBP1a and PBP1b (9, 10). However, although these factors are known to stimulate aPBP activity in vitro, and their 3D structures have been solved (11)(12)(13)18), very little is understood about the mechanisms by which the Lpo proteins promote PBP activity and whether the activation observed in vitro is physiologically relevant. Here, we addressed these issues by identifying a new class of PBP variants that bypass their normal cofactor requirement for in vivo function.…”

“…Three changes (I202F, Q411R, and Q447K) are clustered in the region between the major catalytic domains just behind the UB2H domain, where LpoB associates with PBP1b (10,12) (Fig. 1).…”

“…LpoA is specifically required for PBP1a function, whereas LpoB is specifically required for PBP1b function (9,10). Although they are unrelated, LpoA and LpoB both span the periplasm to interact directly with their cognate PBP (11)(12)(13), and when added to in vitro reactions, they modulate the PGT and TP activities of their target synthases via different mechanisms (9,10,14). LpoA activates the TP activity of PBP1a, which indirectly stimulates its PGT activity, indicating a coupling between the two active sites (10,14).…”

“…LpoA activates the TP activity of PBP1a, which indirectly stimulates its PGT activity, indicating a coupling between the two active sites (10,14). Similarly, LpoB stimulates the PGT activity of PBP1b, which in turn provides more material for crosslinking by the TP domain (9,12). At present, the mechanism of PBP activation by the Lpo factors remains unclear.…”

Structural Insights into Inhibition of Escherichia coli Penicillin-binding Protein 1B

“…Evidence suggests that PBP1a and PBP1b are part of multicomponent synthase complexes in which the spatiotemporal control of the GTase and TPase activities are tightly regulated by accessory proteins (1). E. coli PBP1b consists of five domains: (i) a transmembrane (TM) ␣-helix at the N terminus (residues 64 -87); (ii) a UvrB domain 2 homolog (UB2H) domain (residues 109 -200), which is unique to PBP1b and presumably interacts with periplasmic binding partners including the outer membrane-tethered regulatory protein LpoB (1,(3)(4)(5); (iii) a membrane-associated GTase domain (residues 203-367); (iv) a linker region connecting the GTase and TPase domains (residues 391-443); and (v) a C-terminal TPase domain (residues 444 -736) (Fig. 1).…”