Trapping conformational states along ligand-binding dynamics of peptide deformylase: the impact of induced fit on enzyme catalysis.
Bottom Line: 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.
Affiliation: CNRS, ISV, UPR2355, Gif-sur-Yvette, France.
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.
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Mentions: To unravel the dynamics of the recognition process, we surmised that it should be possible to freeze the conformational change along the pathway by introducing selected, minor variations within the above-mentioned crucial residues involved in the collective motion. In this respect, site-directed mutagenesis of AtPDF was performed on Gly41, Ile42, and Ile130. Single substitutions were made at Gly41 (G41A/Q/M), Ile42 (I42A/F/N/W), and Ile130 (I130A/F), and the variants were purified and characterized. These mutant proteins showed no change in overall stability, as evidenced by DSC experiments (unpublished data). However, two variants of G41, G41Q and G41M, showed dramatic effects; the kcat/Km values were reduced by three orders of magnitude due to large decreases in the kcat values compared to the WT enzyme (Figure 5A and Table S1). The reduced kcat/Km values suggest an altered ability of these variants to attain the final enzyme-transition state complex and, as a result, to give rise to possible states different from the final E:I* complex. Substitutions at positions 42 and 130 only caused small reductions in the kcat values (Figure 5A, Figure S2C, and Table S1). The actinonin-binding potency of both G41 variants was also greatly reduced (Table S1 and Figure S2B). The time-dependent inhibition by actinonin of the most active variants was then studied (Table S3). The half-lives of the final complexes—as assessed by comparison of the 1/k6 values—were always significantly smaller (Table S3), suggesting that the conformational change induced by actinonin binding still occurred, but the C state is destabilized relative to the O state in the mutants compared to the WT. Accordingly, actinonin strongly stabilized almost all of the variants; Tm was increased by more than 20°C. This differs from the G41M and G41Q variants, which both showed increases in the Tm of only 12°C, consistent with reduced binding potency (Table S1).