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

Authors

Markova, K., Kunka, A., Chmelova, K., Havlasek, M., Babkova, P., Marques, S. M., Vasina, M., Planas-Iglesias, J., Chaloupkova, R., Bednar, D., Prokop, Z., Damborsky, J., Marek, M.

Source

ACS CATALYSIS 11: 12864-12885 (2021)

Abstract

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 α/β-hydrolase enzyme (Tm = 73.5°C; ΔTm > 23°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 may (i)
activate cryptic hinge-loop regions and (ii) establish secondary interfaces, where they make
extensive non-covalent interactions between the intertwined protomers. The existence of
domain-swapped dimersin a solution isfurther confirmed experimentally by data obtained from
SAXS and crosslinking 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 increased catalytic rate and elimination of substrate
inhibition. These findings provide additional enzyme engineering avenues for next-generation
biocatalysts.

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Citation

Markova, K., Kunka, A., Chmelova, K., Havlasek, M., Babkova, P., Marques, S. M., Vasina, M., Planas-Iglesias, J., Chaloupkova, R., Bednar, D., Prokop, Z., Damborsky, J., Marek, M., 2021: Computational Enzyme Stabilization Can Affect Folding Energy Landscapes and Lead to Catalytically Enhanced Domain-Swapped Dimers. ACS Catalysis 11: 12864-12885.

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