Abstract

The 317 residue PBCV-1 mRNA capping enzyme catalyzes the second enzymatic reaction in the formation of an N-7-methyl-GMP cap on the 5'-end of the nascent mRNA. It is composed of two globular domains bound by a short flexible peptide linker, which have been shown to undergo opening and closing events. The small size and experimentally demonstrated domain mobility make the PBCV-1 capping enzyme an ideally suited model system to explore domain mobility in context of substrate binding. Here, we specifically address the following four questions: (1) How does substrate binding affect relative domain mobility: is the system better described by an induced fit or population shift mechanism? (2) What are the gross characteristics of a conformation capable of binding substrate? (3) Does "domain gating" of the active site affect the rate of substrate binding? (4) Does the magnitude of receptor conformational fluctuations confer substrate specificity by sterically occluding molecules of a particular size or geometry? We answer these questions using a combination of theory, Brownian dynamics, and molecular dynamics. Our results show that binding efficiency is a function of conformation but that isomerization between efficient and inefficient binding conformations does not impact the substrate association rate. Additionally, we show that conformational flexibility alone is insufficient to explain single stranded mRNA specificity. While our results are specific to the PBCV-1 mRNA capping enzyme, they provide a useful context within which the substrate binding behavior of similarly structured enzymes or proteins may be considered.