Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico

Abstract

The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M−1s−1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M−1s−1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.

Macromolecular room temperature crystallography

“…Merging data from multiple isomorphous crystals allows discarding those segments of data from each crystal that are compromised by radiation damage and assemble data from several crystals into a complete, undamaged data set (see the section ‘collecting and merging data from multiple crystals’). Nonetheless, several examples demonstrate that structural models at RT provide new insights into protein dynamics underlying function (Fraser et al ., 2009), ligand binding and discovery (Fischer et al ., 2014, 2015), and water dynamics (Thomaston et al ., 2017; Darby et al ., 2019), with many opportunities to extend into other fields like protein engineering and design (Biel et al ., 2017; Broom et al ., 2020). Ongoing methodological advances will further bolster the ability to collect physiologically relevant data (Helliwell, 2020) at synchrotrons and XFELs.…”

“…During the initial directed evolution, using the increase in flexibility as a guide to escape local energy minima emerged as the most promising strategy to increase the binding affinity of redesigned ubiquitin variants to the deubiquitinase USP7 (Biel et al ., 2017). Other work on laboratory evolution of a designed Kemp eliminase used RTX to recapitulate shifts in the conformational ensemble towards catalytically-productive sub-states (Broom et al ., 2020). This suggests that RTX guided ensemble modeling can help inform directed evolution efforts.…”

Understanding the origins of loss of protein function by analyzing the effects of thousands of variants on activity and abundance

“…We find that these include the active sites in NUDT15 and PTEN, but also discover functionally important sites adjacent to these active sites. The importance of second shell positions for modulating the structure or dynamics of active site residues has for example also emerged in studies of ligand binding ( Tinberg et al, 2013 ) and enzyme evolution and design ( Campbell et al, 2016 ; Broom et al, 2020 ). In our analysis of functional residues, we mostly focused on general effects at each position, rather than specific effects of individual substitutions.…”

Unlocking Iminium Catalysis in Artificial Enzymes to Create a Friedel-Crafts Alkylase

“…Mutation of the S95 residue to glycine involves a significant shift in helical propensity 53 and thus may perturb the secondary structure in the particularly dynamic α4 helix, 36,54 thus shifting the conformational ensemble toward more catalytically productive states. 55,56…”