Structural and Mechanistic Insights into the Doughnut‐Shaped Lytic Transglycosylase from Campylobacter jejuni

Abstract

The bacterial cell wall peptidoglycan (PG) layer undergoes constant remodeling. The breakdown of the peptidoglycan layer is, in part, carried out by lytic transglycosylases (LT). The inhibition of lytic transglycosylases has been shown to potentiate β‐lactam antibiotics: this is caused by either a direct effect on cell wall integrity, or indirectly, via downregulation of β‐lactamase expression by altering PG fragment concentrations. A sub‐family of LTs are soluble LTs which harbor an unusual doughnut‐shaped structure. To probe the structure and mechanism of a new member of this family, the Campylobacter jejuni Cj0843c LT, we determined its crystal structure in the absence and presence of the bulgecin A inhibitor, carried out activity measurements and biophysical experiments, and performed molecular dynamics (MD) studies with PG strands. The domain organization of Cj0843c reveals a new variation of the doughnut‐shaped architecture. Its structure is similar to that of the larger E. coli SLT70 yet Cj0843c's U‐shaped U‐domain is shorter and it accomplishes completion of its circular nature by having a separate NU‐domain that is tethered to the U‐domain via a flexible linker. The bulgecin A inhibitor binds to the active site of Cj0843c and causes a tightening of the active site groove concomitant with a 9.5°C increase in thermal stability of Cj0843c. We find that Bulgecin A inhibits Cj0843c activity, as measured using a turbidity assay, and potentiates ampicillin susceptibility of C. jejuni. The electrostatic surface of Cj0843c and similar LTs reveal that their inner ring surfaces are predominantly positively charged. This surface characteristic is likely to complement the charge of the tetrapeptide‐disaccharide units of the PG substrate as each tetrapeptide section harbors three carboxylate groups. Molecular dynamics (MD) simulations of a 4 or 5 disaccharide tetrapeptide unit PG strand in the Cj0843c active site either in a product or substrate binding mode, respectively, revealed key PG interactions. The GlcNAc‐2 and MurNAc‐1 occupy positions and interactions similar to that of bulgecin A's moieties when bound to Cj0843c. The terminal tetrapeptide moiety that is either attached to the 1,6‐anhydroMurNAc+2 in the substrate binding mode or to the 1,6‐anhydroMurNAc‐1 in the product binding mode forms salt bridges with a nearby positively charged pocket. When a PG strand is positioned in a random position mostly outside the central hole of Cj0843c, a 1 μs MD simulation revealed that the PG strand is able to enter the hole and that its terminal tetrapeptide is able to find the positively charged pocket forming stable anchoring interactions throughout the remainder of the simulation. The MD simulations reveal global fluctuations regarding the contracting and dilating of Cj0843c's circumference and key movements in the active site when the PG strand is present in the active site groove. Our results suggest that Cj0843c's doughnut shape favors non‐ or minimally‐crosslinked sections of PG strands and point to a mechanism for the mostly exolytic nature of this class of LTs and potential processivity in degrading PG strands.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.