Increased Diels-Alderase activity through backbone remodeling guided by Foldit players

Abstract

Computational enzyme design holds promise for the production of renewable fuels, drugs and chemicals. De novo enzyme design has generated catalysts for several reactions, but with lower catalytic efficiencies than naturally occurring enzymes. Here we report the use of game-driven crowdsourcing to enhance the activity of a computationally designed enzyme through the functional remodeling of its structure. Players of the online game Foldit were challenged to remodel the backbone of a computationally designed bimolecular Diels-Alderase to enable additional interactions with substrates. Several iterations of design and characterization generated a 24-residue helix-turn-helix motif, including a 13-residue insertion, that increased enzyme activity >18-fold. X-ray crystallography showed that the large insertion adopts a helix-turn-helix structure positioned as in the Foldit model. These results demonstrate that human creativity can extend beyond the macroscopic challenges encountered in everyday life to molecular-scale design problems.

Figure 1

Crystal structure of Foldit design is similar to player model. (a) Crystal structure of starting designed Diels Alderase. The structure of DA_20_10 has not been solved, shown in the Figure is the structure of a precursor (3I1C, yellow) which differs by only three amino acid substitutions. The modeled transition state of the Diels-Alder reaction is depicted in cyan. (b) Structure of starting Diels Alderase (yellow) overlaid on CE6 player predicted structure (purple). CE6 has a 24 amino acid loop remodel, including a 13-residue insertion, converting an unstructured loop into a helix-turn-helix motif. (c) Crystal structure of CE6 (3U0S, light blue) overlaid with player predicted structure of CE6 (purple). The helix turn helix motif is clearly present and has a position close to that in the predicted structure. (d) Correlation between CE6 remodeled helices and corresponding experimental electron density. The electron density map is contoured to 1.0 σ. Sidechain alanine 45 is near the center of the picture on the left, with Helix 1 above and Helix 2 below. (e) The player designed side chains in CE6 that form the interface between Helix 1 (purple) and the transition state model (cyan) are found in the same rotameric conformation as in the crystal structure of CE6 (3U0S, light blue). Figure 1d generated in Coot . All other Figures generated in PyMol .

Figure 2

Kinetic characterization for DA_20_10 (▲), CE0 (■), CE4 (◢), and CE6 (◆). (a) Product observed after three hours with the dieneophile held constant at 50mM and the diene at 3mM, 1.5mM, 0.8mM, 0.4mM, 0.2mM, and 0.1mM. (b) Product observed after three hours with the diene held constant at 3mM and the dienophile at 50mM, 25mM, 12mM, 6mM, 3mM, and 1.5mM. The kinetic constants were determined using 10μM purified protein (Supplementary Fig. 8) and a standard Michaelis–Menten assay as described in the supplemental material.