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 catalyses 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.