Issue 41, 2020

Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst

Abstract

Computational design of protein catalysts with enhanced stabilities for use in research and enzyme technologies is a challenging task. Using force-field calculations and phylogenetic analysis, we previously designed the haloalkane dehalogenase DhaA115 which contains 11 mutations that confer upon it outstanding thermostability (Tm = 73.5 °C; ΔTm > 23 °C). An understanding of the structural basis of this hyperstabilization is required in order to develop computer algorithms and predictive tools. Here, we report X-ray structures of DhaA115 at 1.55 Å and 1.6 Å resolutions and their molecular dynamics trajectories, which unravel the intricate network of interactions that reinforce the αβα-sandwich architecture. Unexpectedly, mutations toward bulky aromatic amino acids at the protein surface triggered long-distance (∼27 Å) backbone changes due to cooperative effects. These cooperative interactions produced an unprecedented double-lock system that: (i) induced backbone changes, (ii) closed the molecular gates to the active site, (iii) reduced the volumes of the main and slot access tunnels, and (iv) occluded the active site. Despite these spatial restrictions, experimental tracing of the access tunnels using krypton derivative crystals demonstrates that transport of ligands is still effective. Our findings highlight key thermostabilization effects and provide a structural basis for designing new thermostable protein catalysts.

Graphical abstract: Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst

Supplementary files

Article information

Article type
Edge Article
Submitted
17 Jun 2020
Accepted
10 Sep 2020
First published
11 Sep 2020
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2020,11, 11162-11178

Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst

K. Markova, K. Chmelova, S. M. Marques, P. Carpentier, D. Bednar, J. Damborsky and M. Marek, Chem. Sci., 2020, 11, 11162 DOI: 10.1039/D0SC03367G

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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