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Computational enzyme stabilization can affect folding energy landscapes and lead to catalytically enhanced domain-swapped dimers

DOI: 10.1021/acscatal.1c03343 DOI Help

Authors: Klara Markova (Masaryk University; St. Anne’s University Hospital Brno) , Antonin Kunka (Masaryk University; St. Anne’s University Hospital Brno) , Klaudia Chmelova (Masaryk University) , Martin Havlasek (Masaryk University) , Petra Babkova (Masaryk University) , Sérgio M. Marques (Masaryk University; St. Anne’s University Hospital Brno) , Michal Vasina (Masaryk University; St. Anne’s University Hospital Brno) , Joan Planas-Iglesias (Masaryk University) , Radka Chaloupkova (Masaryk University) , David Bednar (Masaryk University; St. Anne’s University Hospital Brno) , Zbynek Prokop (Masaryk University; St. Anne’s University Hospital Brno) , Jiri Damborsky (Masaryk University; St. Anne’s University Hospital Brno) , Martin Marek (Masaryk University; St. Anne’s University Hospital Brno)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Catalysis , VOL 562 , PAGES 12864 - 12885

State: Published (Approved)
Published: October 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 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.

Journal Keywords: protein folding; protein design; α/β-hydrolase; haloalkane dehalogenase; domain swapping; energy landscape; oligomerization

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I03-Macromolecular Crystallography

Other Facilities: ID23-1 at ESRF; PXIII at Swiss Light Source

Added On: 11/10/2021 08:26

Discipline Tags:

Catalysis Life Sciences & Biotech Biophysics Structural biology Chemistry Biochemistry

Technical Tags:

Diffraction Macromolecular Crystallography (MX)