Mutations in penicillin-binding protein 2 from cephalosporin-resistant Neisseria gonorrhoeae hinder ceftriaxone acylation by restricting protein dynamics

Avinash Singh,Jonathan M Turner,Joshua Tomberg,Alena Fedarovich,Magnus Unemo,Robert A Nicholas,Christopher Davies

doi:10.1074/jbc.ra120.012617

Abstract

The global incidence of the sexually transmitted disease gonorrhea is expected to rise due to the spread of Neisseria gonorrhoeae strains with decreased susceptibility to extended-spectrum cephalosporins (ESCs). ESC resistance is conferred by mosaic variants of penicillin-binding protein 2 (PBP2) that have diminished capacity to form acylated adducts with cephalosporins. To elucidate the molecular mechanisms of ESC resistance, we conducted a biochemical and high-resolution structural analysis of PBP2 variants derived from the decreased-susceptibility N. gonorrhoeae strain 35/02 and ESC-resistant strain H041. Our data reveal that mutations both lower affinity of PBP2 for ceftriaxone and restrict conformational changes that normally accompany acylation. Specifically, we observe that a G545S substitution hinders rotation of the β3 strand necessary to form the oxyanion hole for acylation and also traps ceftriaxone in a noncanonical configuration. In addition, F504L and N512Y substitutions appear to prevent bending of the β3-β4 loop that is required to contact the R1 group of ceftriaxone in the active site. Other mutations also appear to act by reducing flexibility in the protein. Overall, our findings reveal that restriction of protein dynamics in PBP2 underpins the ESC resistance of N. gonorrhoeae.

Highlights

The global incidence of the sexually transmitted disease gonorrhea is expected to rise due to the spread of Neisseria gonorrhoeae strains with decreased susceptibility to extended-spectrum cephalosporins (ESCs)
We find that structures of penicillin-binding protein 2 (PBP2) derived from the reduced susceptibility strain 35/02 and ESC-resistant strain H041 are nearly identical to each other, but differ from WT PBP2 in the ␤3–␤4 loop
We find that the ␤3–␤4 loop occupies the same position when tPBP2H041 is acylated by ceftriaxone, whereas this loop moves a considerable distance when tPBP2WT is acylated by ESCs

Summary

Introduction

The global incidence of the sexually transmitted disease gonorrhea is expected to rise due to the spread of Neisseria gonorrhoeae strains with decreased susceptibility to extended-spectrum cephalosporins (ESCs). ESC resistance is conferred by mosaic variants of penicillin-binding protein 2 (PBP2) that have diminished capacity to form acylated adducts with cephalosporins. A sexually-transmitted infection caused by Neisseria gonorrhoeae, is increasingly difficult to treat due to emergence of strains exhibiting decreased susceptibility or resistance to the extended-spectrum cephalosporins (ESCs) ceftri-. An important event that led to this change was the isolation in Japan of the first N. gonorrhoeae strain (H041) exhibiting high-level resistance to cefixime and ceftriaxone [5]. PBP2 is a two-domain protein composed of a TPase catalytic domain and an N-terminal domain of unknown function [22] This latter domain may act as a pedestal to project the active site toward peptidoglycan or could mediate protein–protein interactions with other components of the peptidoglycan machinery. Evidence has uncovered an allosteric site in the N-terminal domain of Staphylococcus

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