Structural analyses of the Haemophilus influenzae peptidoglycan synthase activator LpoA suggest multiple conformations in solution

Abstract

In many Gram-negative bacteria, the peptidoglycan synthase PBP1A requires the outer membrane lipoprotein LpoA for constructing a functional peptidoglycan required for bacterial viability. Previously, we have shown that the C-terminal domain of LpoA (LpoA) has a highly conserved, putative substrate-binding cleft between two α/β lobes. Here, we report a 2.0 Å resolution crystal structure of the LpoA N-terminal domain. Two subdomains contain tetratricopeptide-like motifs that form a concave groove, but their relative orientation differs by ∼45° from that observed in an NMR structure of the LpoA N domain. We also determined three 2.0-2.8 Å resolution crystal structures containing four independent full-length LpoA molecules. In contrast to an elongated model previously suggested for LpoA, each LpoA formed a U-shaped structure with a different C-domain orientation. This resulted from both N-domain twisting and rotation of the C domain (up to 30°) at the end of the relatively immobile interdomain linker. Moreover, a previously predicted hinge between the lobes of the LpoA C domain exhibited variations of up to 12°. Small-angle X-ray scattering data revealed excellent agreement with a model calculated by normal mode analysis from one of the full-lengthLpoA molecules but even better agreement with an ensemble of this molecule and two of the partially extended normal mode analysis-predicted models. The different LpoA structures helped explain how an outer membrane-anchored LpoA can either withdraw from or extend toward the inner membrane-bound PBP1A through peptidoglycan gaps and hence regulate the synthesis of peptidoglycan necessary for bacterial viability.

Conserved mechanism of cell-wall synthase regulation revealed by the identification of a new PBP activator in Pseudomonas aeruginosa

“…The TPR domains are the most likely candidates for the portion of Pa LpoP that directly interfaces with Pa PBP1b, and this region probably does so by binding the UB2H domain like Ec LpoB. Consistent with this possibility, TPR domains are broadly distributed in biology to mediate protein-protein interactions (15) and are found in many regulators of cell-wall biogenesis, including LpoA (5)(6)(7)18), NlpI (19,20), and CpoB (21). Interestingly, the periplasmic protein CpoB in E. coli is thought to use its TPR domain to associate with Ec PBP1b via its UB2H domain during cell division to coordinate Ec PBP1b activity with that of the Tol-Pal system during cell division (21).…”

Crystal structures of the amino-terminal domain of LpoA from Escherichia coli and Haemophilus influenzae

“…The predicted sequence of the mature EcLpoA N-domain protein was serine followed by residues 31-252 of EcLpoA (formula weight 25 154). The cloning, expression and purification of residues 33-253 of the HiLpoA N domain were identical to the procedures described previously (Sathiyamoorthy et al, 2017). The gene fragment with NcoI and SalI restriction sites corresponding to residues 33-253 of HiLpoA was amplified using PCR from genomic DNA isolated from the H. influenzae Rd strain (ATCC catalog No.…”

“…LpoA is composed of two domains: a primarily helical amino-terminal (N) domain and a carboxyl-terminal domain involved in interactions with PBP1A. Analyses of multiple crystal structures of full-length LpoA from Haemophilus influenzae (HiLpoA), together with small-angle X-ray scattering results, suggested that LpoA is a flexible molecule that is capable of extending through gaps or holes in PG in order to interact with the inner-membranebound PBP1A (Sathiyamoorthy et al, 2017). Although the interdomain linker is responsible for much of the flexibility of the molecule, twists and bends of up to 5 have been observed within the N domain and are likely to affect the overall length of the molecule.…”

“…The structure of the N domain of E. coli LpoA was first determined using NMR (EcNMR) and, although composed of TPR-like helix-turn-helix motifs, was observed to be relatively flat without a significant superhelical twist (Jean et al, 2014). When the orthorhombic crystal structure of the N domain of H. influenzae LpoA (HiOrt) was reported (Sathiyamoorthy et al, 2017), its subdomains 1 and 2 were noted to each be very similar to the corresponding subdomains in EcNMR. However, when the two intact N-domain structures were superimposed, based solely on least-squares fitting of their subdomains 1 (specifically residues 33-148), their subdomains 2 were related by a 45 rotation (see Fig.…”

“…However, when the two intact N-domain structures were superimposed, based solely on least-squares fitting of their subdomains 1 (specifically residues 33-148), their subdomains 2 were related by a 45 rotation (see Fig. 2 in Sathiyamoorthy et al, 2017).…”