Computational Enzyme Stabilization Can Affect Folding Energy Landscapes and Lead to Catalytically Enhanced Domain-Swapped Dimers

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Authors

MARKOVÁ Klára KUNKA Antonín CHMELOVÁ Klaudia HAVLÁSEK Martin BABKOVÁ Petra MARQUES Sérgio Manuel VAŠINA Michal PLANAS IGLESIAS Joan CHALOUPKOVÁ Radka BEDNÁŘ David PROKOP Zbyněk DAMBORSKÝ Jiří MAREK Martin

Year of publication 2021
Type Article in Periodical
Magazine / Source ACS Catalysis
MU Faculty or unit

Faculty of Science

Citation
Web https://pubs.acs.org/doi/10.1021/acscatal.1c03343
Doi http://dx.doi.org/10.1021/acscatal.1c03343
Keywords protein folding; protein design; alpha/beta-hydrolase; haloalkane dehalogenase; domain swapping; energy landscape; oligonicrization; catalytic efficiency; substrate inhibition
Description The functionality of an enzyme depends on its unique three-dimensional structure, which is a result of the folding process when the nascent polypeptide follows a funnel-like energy landscape to reach a global energy minimum. Computer-encoded algorithms are increasingly employed to stabilize native proteins for use in research and biotechnology applications. Here, we reveal a unique example where the computational stabilization of a monomeric alpha/beta-hydrolase enzyme (T-m = 73.5 degrees C; Delta T-m > 23 degrees C) affected the protein folding energy landscape. The introduction of eleven single-point stabilizing mutations based on force field calculations and evolutionary analysis yielded soluble domain-swapped intermediates trapped in local energy minima. Crystallographic structures revealed that these stabilizing mutations might (i) activate cryptic hinge-loop regions and (ii) establish secondary interfaces, where they make extensive noncovalent interactions between the intertwined protomers. The existence of domain-swapped dimers in a solution is further confirmed experimentally by data obtained from small-angle X-ray scattering (SAXS) and cross-linking mass spectrometry. Unfolding experiments showed that the domain-swapped dimers can be irreversibly converted into native-like monomers, suggesting that the domain swapping occurs exclusively in vivo. Crucially, the swapped-dimers exhibited advantageous catalytic properties such as an increased catalytic rate and elimination of substrate inhibition. These findings provide additional enzyme engineering avenues for next-generation biocatalysts.
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