Trapping Conformational States Along Ligand-Binding Dynamics of Peptide Deformylase: The Impact of Induced Fit on Enzyme Catalysis

Abstract

For several decades, molecular recognition has been considered one of the most fundamental processes in biochemistry. For enzymes, substrate binding is often coupled to conformational changes that alter the local environment of the active site to align the reactive groups for efficient catalysis and to reach the transition state. Adaptive substrate recognition is a well-known concept; however, it has been poorly characterized at a structural level because of its dynamic nature. Here, we provide a detailed mechanism for an induced-fit process at atomic resolution. We take advantage of a slow, tight binding inhibitor-enzyme system, actinonin-peptide deformylase. Crystal structures of the initial open state and final closed state were solved, as well as those of several intermediate mimics captured during the process. Ligand-induced reshaping of a hydrophobic pocket drives closure of the active site, which is finally “zipped up” by additional binding interactions. Together with biochemical analyses, these data allow a coherent reconstruction of the sequence of events leading from the encounter complex to the key-lock binding state of the enzyme. A “movie” that reconstructs this entire process can be further extrapolated to catalysis. The notion of induced fit when a protein binds its ligand—like a glove adapting to the shape of a hand—is a central concept of structural biochemistry introduced over 50 years ago. A detailed molecular demonstration of this phenomenon has eluded biochemists, however, largely due to the difficulty of capturing the steps of this very transient process: the “conformational change.” In this study, we were able to see this process by using X-ray diffraction to determine more than 10 distinct structures adopted by a single enzyme when it binds a ligand. To do this, we took advantage of the “slow, tight-binding” of a potent inhibitor to its specific target enzyme to trap intermediates in the binding process, which allowed us to monitor the action of an enzyme in real-time at atomic resolution. We showed the kinetics of the conformational change from an initial open state, including the encounter complex, to the final closed state of the enzyme. From these data and other biochemical and biophysical analyses, we make a coherent causal reconstruction of the sequence of events leading to inhibition of the enzyme's activity. We also generated a movie that reconstructs the sequence of events during the encounter. Our data provide new insights into how enzymes achieve a catalytically competent conformation in which the reactive groups are brought into close proximity, resulting in catalysis.