Engineering the Protein Dynamics of an Ancestral Luciferase

Authors

Schenkmayerova, A., Pinto, G. P., Toul, M., Marek, M., Hernychova, L., Planas-Iglesias, J., Daniel Liskova, V., Pluskal, D., Vasina, M., Emond, S., Dörr, M., Chaloupkova, R., Bednar, D., Prokop, Z., Hollfelder, F., Bornscheuer, U. T., Damborsky, J.

Source

NATURE COMMUNICATIONS 12: 3616 (2021)

Abstract

Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins.

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Citation

Schenkmayerova, A., Pinto, G. P., Toul, M., Marek, M., Hernychova, L., Planas-Iglesias, J., Daniel Liskova, V., Pluskal, D., Vasina, M., Emond, S., Dörr, M., Chaloupkova, R., Bednar, D., Prokop, Z., Hollfelder, F., Bornscheuer, U. T., Damborsky, J., 2021: Engineering the Protein Dynamics of an Ancestral Luciferase. Nature Communications 12: 3616.

Dmitri and Jan to Present at XXVIth Biochemistry Congress
Poster Award for Martin
TA ČR GAMA 2 Grant for Martin
Martin to Present at XXV IUCr 2021
Open Position: Postdoc in Enzymology & Protein Engineering
Open Position: Postdoc in Protein Structural Biology
2nd Hands-on Computational Enzyme Design Course Successfully Behind Us