Inhibitory mechanism of the Qβ lysis protein A₂

Abstract

Summary The lysis protein A2, present as a single copy on the surface of Qβ virion particles, was previously shown to inhibit the activity of MurA, an enzyme that catalyzes the first committed step of murein biosynthesis. Here we report experiments with a two-hybrid study that indicates A2 and MurA interact directly. Moreover, experiments with a soluble MBP-A2 fusion indicate that the interaction between MurA and A2 is dependent on a substrate-induced conformational change featured in the UDP-NAG-liganded state of MurA but not the tetrahedral intermediate state. Moreover, based on the location of L138Q, the original dominant A2-resistant mutant that identified MurA as the target, a directed mutagenesis strategy has identified a continuous surface required for A2 binding. This surface spans the catalytic loop/cleft and encompasses both the catalytic and C-terminal domains. These data support a model in which A2 preferentially binds MurA liganded with UDP-NAG, thereby preventing catalysis by occluding PEP from accessing the active site.

Phage single-gene lysis: Finding the weak spot in the bacterial cell wall

“…However, in what seems to be the only instance of using virions for enzyme inhibition, it was shown that the catalytic activity of MurA, but not MurA L138Q , in crude extracts could be blocked by the addition of highly purified Q␤ virions. Later experiments done with purified MurA and Q␤ particles confirmed these results (68). Based on the turnover number of MurA and the number of purified virions needed to block its enzymatic activity, the MurA-A 2 dissociation constant was determined to be ϳ10 nM (67).…”

Asymmetric cryo-EM structure of the canonical Allolevivirus Qβ reveals a single maturation protein and the genomic ssRNA in situ

“…All of the Leviviridae have the same core genome, spanning 3.4-4.3 kb, encoding the maturation protein, the coat protein, and a subunit of RNAdependent RNA replicase (SI Appendix, Fig. S1) (10). The MS2-like phages, designated true leviviruses, have a fourth gene that encodes the lysis protein, whereas the Qβ-like phages, designated alloleviviruses, have the lysis function as an additional feature of the maturation protein (called A 2 in Qβ).…”

“…One of the beststudied ssRNA virus systems is the Leviviridae, which infects Gram-negative bacteria via a variety of retractile pili (5); extensive genetic and biochemical studies have been performed on two of these phages: MS2 and Qβ (5)(6)(7)(8)(9)(10)(11). All of the Leviviridae have the same core genome, spanning 3.4-4.3 kb, encoding the maturation protein, the coat protein, and a subunit of RNAdependent RNA replicase (SI Appendix, Fig.…”

A2 expression and assembly regulates lysis in Qβ infections

“…In support of this perspective, we note that the por mutants, selected for their ability to overcome the rat phenotype of murA L138Q , produce A 2 at two-to three-fold higher levels than the parental phage, presumably due to the disruption of local RNA secondary structures. This is necessary, because the L138Q mutation in MurA reduces the affinity for A 2 (Reed et al, 2012). As a consequence, lysis occurs much earlier in the Qb por infections and the average yield of virions is drastically curtailed (Fig.…”

“…Approximately 4610 11 p.f.u. of Qb was mixed with 1 mg purified MurAHis protein, as previously described (Reed et al, 2012). A Qb-only control as well as a sample including 1 mg BSA (Sigma-Aldrich) was prepared in parallel.…”