Molecular architecture of the PBP2-MreC core bacterial cell wall synthesis complex

Abstract

Bacterial cell wall biosynthesis is an essential process that requires the coordinated activity of peptidoglycan biosynthesis enzymes within multi-protein complexes involved in cell division (the "divisome") and lateral wall growth (the "elongasome"). MreC is a structural protein that serves as a platform during wall elongation, scaffolding other essential peptidoglycan biosynthesis macromolecules, such as penicillin-binding proteins. Despite the importance of these multi-partite complexes, details of their architecture have remained elusive due to the transitory nature of their interactions. Here, we present the crystal structures of the soluble PBP2:MreC core elongasome complex from Helicobacter pylori, and of uncomplexed PBP2. PBP2 recognizes the two-winged MreC molecule upon opening of its N-terminal region, revealing a hydrophobic zipper that serves as binding platform. The PBP2:MreC interface is essential both for protein recognition in vitro and maintenance of bacterial shape and growth. This work allows visualization as to how peptidoglycan machinery proteins are scaffolded, revealing interaction regions that could be targeted by tailored inhibitors.Bacterial wall biosynthesis is a complex process that requires the coordination of multiple enzymes. Here, the authors structurally characterize the PBP2:MreC complex involved in peptidoglycan elongation and cross-linking, and demonstrate that its disruption leads to loss of H. pylori shape and inability to sustain growth.

Fig. 1

Architecture and structural arrangement of PBP2 and the PBP2:MreC complex from H. pylori. a Schematic diagram of PBP2 and the limitations of different domains. b PBP2 folds into anchor, head, linker, and transpeptidase domain. The anchor is “clasped” against the helical head. c The PBP2 anchor region sways away from the head through movement of a hinge region, exposing a previously hidden hydrophobic region that allows binding of MreC1

Fig. 2

MreC binding engenders opening of the N-terminus of PBP2. a Overlay of MreC-bound and unbound PBP2 structures, indicating movement of the anchor region (in red). Opening of the N-terminal region of PBP2 exposes a hydrophobic region on the head that is complemented by a non-polar face on the surface of MreC (b), forming a hydrophobic zipper whose disruption hinders complex formation. c Structure-based sequence alignment between class B PBPs from H. pylori (this work), P. aeruginosa PAO1 G3XCV7, E. coli O157:H7 P0AD67, and K. pneumoniae A0A0W8ASI8 (UniProt codes). Head, anchor, linker, and TP are indicated in color above the sequence. Residues involved in interaction between head and anchor are indicated with black asterisks. Active site residues are indicated with hash tags. Figure generated with ESPRIPT

Fig. 3

Class B PBPs from Gram-negative and Gram-positive display similarities at key interfaces. a PBP2 from H. pylori, this work, b PBP2b from S. pneumoniae (2WAD), c PBP2x from S. pneumoniae (1RP5), d PBP3 from P. aeruginosa (3PBN) harbor anchor (red) and head (blue) regions stabilized by hydrophobic interactions, which could be disrupted and reoriented upon binding to a partner PG-biosynthesis molecule (such as MreC). An Arg-Arg-Glu constellation, indicated here in magenta, is present in the structures of all class B PBPs solved to date, and sits at the interface between anchor and head

Fig. 4

Determination of the effects of the 3A and 3D mutations on MreC’s hydrophobic zipper on H. pylori. a The mreC3A and mreC3D conditional mutants (N6 mreC3A pMEG4 and N6 mreC3D pMEG4) were grown in the presence or absence of IPTG (1 mM). Cell shape was monitored by scanning electron microscopy (representative images of bacteria after 24 h of growth are illustrated) and compared to the wild-type strain N6 pILL2150. b The control strain N6 pILL2150, the conditional mreC mutant (N6 ∆mreC pMEG4), and the 3A mutant (N6 mreC3A pMEG4) were grown in liquid culture in the presence or absence of IPTG. Viable bacteria were monitored by measuring the number of colony forming units (CFU/mL) as a function of time of growth (h); c, d Changes in cell shape in the presence or absence of IPTG (1 mM) were monitored by measuring bacterial cell diameter using the ImageJ software. Each dot represents the measured diameter (c) and length (d) of one individual bacterium. The difference in diameter (and length) in the presence or absence of IPTG was statistically significant for strains N6 ∆mreC pMEG4 and N6 mreC3A pMEG4 (unpaired two-tailed student t-test; ns = not significant; ***P < 0.0001 with a minimum of 100 bacteria being measured per strain and per growth condition). All other strains behaved similarly to the control strain N6 pILL2150

Fig. 5

MreC employs different surfaces in order to maximize interactions with partner molecules. MreC (blue) interacts both with PBP2 (gray ribbon) and with another MreC butterfly (orange). The latter recognition region is composed mostly of hydrophobic interactions, with few exceptions (yellow sticks). Residues Phe169 and Phe182, localized at the center of the MreC–MreC interaction platform that is provided by a neighboring molecule (with apostrophes), are also involved in the formation of the hydrophobic zipper that interacts with PBP2 (purple sticks). This suggests that MreC can modify the protein partner with which it interacts through a slight adjustment of the residues available for binding