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

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 (T m = 73.5 °C; ΔT m > 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.

Advancing Enzyme’s Stability and Catalytic Efficiency through Synergy of Force-Field Calculations, Evolutionary Analysis, and Machine Learning

“…73 °C (Δ T m app ∼ 23 °C, Figure a), making it the most stable haloalkane dehalogenase to date. Although the energy and structural basis of DhaA115 thermostability have been elucidated by the global analysis of thermal denaturation experiments and crystallographic analyses, respectively, , the specific structural regions most prone to unfolding are currently unknown. To this end, we have carried out hydrogen/deuterium exchange coupled with mass spectrometry detection (HDX-MS) of DhaA115 after one- and thirty-minute incubation at 67 °C to compare solvent accessibility of residues in the native and the denatured states, respectively (Figure c).…”

“…73 °C (ΔT m app ∼ 23 °C, Figure 2a), making it the most stable haloalkane dehalogenase to date. Although the energy and structural basis of DhaA115 thermostability have been elucidated by the global analysis of thermal denaturation experiments and crystallographic analyses, respectively, 21,29 the specific structural regions most prone to unfolding are currently unknown.…”

“…40 To retrieve insights into the catalytic mechanisms of the new variants, we carried out steady-state kinetic measurements at temperatures between 25 and 60 °C using isothermal titration calorimetry (ITC) and DBE as the substrate. Similar to DhaA115, the kinetics of DBE conversion by the newly designed variants showed clear signs of substrate inhibition, 29 thus, we accounted for this during the fitting (Supplementary Figure 6). The thermodynamic signatures of catalysis differed between the FireProt-based variants (DhaA115 and DhaA223) and the PROSS-based ones (DhaA222 and DhaA231).…”

Site‐specific crosslinking and assembly of tetrameric β‐glucuronidase improve glycyrrhizin hydrolysis

“…Although computational design strategy (King et al, 2012;Wargacki et al, 2021) and domain swapping (Lafita et al, 2019;Markova et al, 2021) for accurately tuning oligomerization have drawn increasing attention, considerable computational screening and laborious work are required, which hinder the industrial application of protein assembly.…”

CalFitter 2.0: Leveraging the power of singular value decomposition to analyse protein thermostability

“…To our best knowledge, it is the only tool that allows simultaneous fitting of data from temperature scanning experiments together with unfolding kinetics. The recent examples of CalFitter use include decoding the mechanism of domain-swapping of computationally stabilized haloalkane dehalogenase ( 14 ), explaining the kinetic stability of cold adapted subtilase ( 15 ), elucidating the aggregation propensity of polyketide cyclase ( 16 ), or study of dihydrofolate reductase evolution ( 17 ).…”