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

Abstract

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.T he peptidoglycan (PG) layer forms a protective shell that surrounds the cytoplasmic membrane of bacteria to prevent osmotic rupture and provide cells with their characteristic shape (1). This complex macromolecule is composed of glycan strands crosslinked to one another by attached peptide chains to form the exoskeletal matrix. Because of its essentiality and uniqueness to bacteria, the PG layer is an important therapeutic target. Many antibiotics in our current arsenal block PG assembly, with penicillin and related beta-lactam drugs being the most wellknown and widely used. These molecules target major PG synthase enzymes called penicillin-binding proteins (PBPs) (1).The PBPs function in the final stage of the three-part pathway for PG biogenesis (1). Precursor synthesis begins in the cytoplasm with the production of the activated sugar molecules uridine diphosphate (UDP)-N-acetylmuramic acid pentapeptide (UDP-MurNAc-pep 5 ) and UDP-N-acetylglucosamine (UDPGlcNAc). In the second, membrane-associated phase, UDPMurNAc-pep 5 is converted to the precursor lipid-I by MraY, which transfers phospho-MurNAc-pep to the lipid carrier undecaprenol-phosphate (Und-P). Lipid-II is formed by MurG via the addition of GlcNAc to lipid-I from UDP-GlcNAc. This final precursor contains the basic monomeric unit of PG, the disaccharide-pentapeptide. After its production, lipid-II must be flipped by MurJ (2, 3) ...

Induced conformational changes activate the peptidoglycan synthase PBP1B

“…While the majority of the molecules are in an inactive or off state, a minority exist in the active state able to perform catalysis. This notion is consistent with previous observations (Markovski et al, ). The activity observed would be proportional to the longevity of this active state.…”

“…Precisely how these changes boost the respective functions is unknown as NMR with the full length PBP1B‐LpoB complex is not feasible because of the molecular weight of >110 kDa and obtaining a co‐crystal of PBP1B in complex with LpoB is problematic, likely due to the highly dynamic nature of the interaction. The activation signal proposed here is consistent with previously identified versions of PBP1B, isolated through suppressor selection, which are able to partially bypass the need for activation by LpoB (Markovski et al, ). Two of these, I202F and Q411R, cluster in the inter‐domain region of PBP1B proximal to both α‐helix 14 and Loop 1.…”

“…The question nevertheless remained, how does interaction with the non‐catalytic UB2H domain activate both catalytic domains of PBP1B? Interestingly, a genetic screen isolated three PBP1B versions with amino acid substitutions clustering in the inter‐domain region of the synthase, and one in the GTase domain, which partially bypass the need for activation by LpoB (Markovski et al, ). This supports a model in which activation of the synthase proceeds via a conformational change initiated by LpoB binding to its UB2H domain (Egan et al, ; Markovski et al, ).…”

“…Interestingly, a genetic screen isolated three PBP1B versions with amino acid substitutions clustering in the inter‐domain region of the synthase, and one in the GTase domain, which partially bypass the need for activation by LpoB (Markovski et al, ). This supports a model in which activation of the synthase proceeds via a conformational change initiated by LpoB binding to its UB2H domain (Egan et al, ; Markovski et al, ). This hypothesis was further supported by the isolation of P. aeruginosa PBP1B versions with similar Lpo bypass mutations (Greene et al, ).…”

Factors essential for L,D-transpeptidase-mediated peptidoglycan cross-linking and β-lactam resistance in Escherichia coli

“…Bypass of the D,D-transpeptidase activity of PBPs by YcbB is potentially useful to explore the function of members of the polymerization complexes. For example, the fact that LpoB remains essential in the L,D-transpeptidation context indicates that it directly stimulates the glycosyltransferase activity of PBP1b, as indicated in a recent study (Markovski et al, 2016).

10.7554/eLife.19469.015Figure 10.Peptidoglycan polymerization in mutant M1.The lipid intermediate II (Lipid II) consists in the disaccharide-pentapeptide subunit linked to the undecaprenyl lipid transporter (C 55 ) by a pyrophosphate bond.

…”