Slow Protein Dynamics Elicits New Enzymatic Functions by Means of Epistatic Interactions

Protein evolution depends on the adaptation of these molecules to different functional challenges. This occurs by tuning their biochemical, biophysical, and structural traits through the accumulation of mutations. While the role of protein dynamics in biochemistry is well recognized, there are limited examples providing experimental evidence of the optimization of protein dynamics during evolution. Here we report an NMR study of four variants of the CTX-M beta-lactamases, in which the interplay of two mutations outside the active site enhances the activity against a cephalosporin substrate, ceftazidime. The crystal structures of these enzymes do not account for this activity enhancement. By using NMR, here we show that the combination of these two mutations increases the backbone dynamics in a slow timescale and the exposure to the solvent of an otherwise buried beta-sheet. The two mutations located in this beta-sheet trigger conformational changes in loops located at the opposite side of the active site. We postulate that the most active variant explores alternative conformations that enable binding of the more challenging substrate ceftazidime. The impact of the mutations in the dynamics is context-dependent, in line with the epistatic effect observed in the catalytic activity of the different variants. These results reveal the existence of a dynamic network in CTX-M beta-lactamases that has been exploited in evolution to provide a net gain-of-function, highlighting the role of alternative conformations in protein evolution.

Automated summary

Protein evolution involves the adaptation of molecules to different functional challenges through the accumulation of mutations. This study focuses on CTX-M beta-lactamases and demonstrates the optimization of protein dynamics through two mutations outside the active site, which enhance the enzyme's activity against a specific substrate. NMR analysis reveals that these mutations increase backbone dynamics and the exposure to solvent of a buried beta-sheet. The conformational changes induced by the mutations in this beta-sheet trigger further changes in loops at the opposite side of the active site. These findings highlight the importance of alternative conformations in protein evolution.