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

Klara Markova,Jiri Damborsky,Martin Marek,Klaudia Chmelova,Philippe Carpentier,Sérgio M Marques,David Bednar

doi:10.1039/d0sc03367g

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.

Highlights

Enzymes have evolved for billions of years, and will continue to do so as long as life on earth exists.[1]
To obtain precise structural information about how the DhaA enzyme is thermostabilized, we focused our efforts on crystallization of the most stabilized enzyme variant, DhaA115
We found that the tunnels in DhaA115 were still very narrow during the molecular dynamics (MD) simulations (Table S2 and Fig. S8 and S9†), with no tunnels detected for the selected probe size (0.7 A) in a large fraction of the trajectories

Summary

Introduction

Enzymes have evolved for billions of years, and will continue to do so as long as life on earth exists.[1]. Enhancing protein thermostability involves changes that shi the folding–unfolding balance toward the folded form. Proteins will continue to unfold anyway, these stronger interactions will either slow down unfolding or speed up refolding processes.[4] A structured form can be stabilized through non-covalent interactions including hydrophobic interactions, hydrogen bonds, salt bridges and van der Waals forces.[5] Increasing the number of stabilizing electrostatic interactions between residues of opposite charge reinforces proteins' thermal stability.[6] Hydrophobic interactions have been shown to contribute proportionally more effectively to protein stability than hydrogen bonds.[7] The hydrophobic effect is the dominant driving force in protein folding, and designing a well-packed hydrophobic core is usually an efficient strategy for engineering stable proteins.[4]

Methods

Results

Conclusion