Raczynska, A. | bioRxiv Science

bioRxiv2021

Evolution of tunnels in alpha/beta-hydrolase fold proteins - what can we learn from studying epoxide hydrolases?

The evolutionary variability of a proteins residues is highly dependent on protein region and function. Solvent-exposed residues, excluding those at interaction interfaces, are more variable than buried residues whereas active site residues are considered to be conserved. The abovementioned rules apply also to /{beta}-hydrolase fold proteins - one of the oldest and the biggest superfamily of enzymes with buried active sites equipped with tunnels linking the reaction site with the exterior. We selected soluble epoxide hydrolases as representative of this family to conduct the first systematic study on the evolution of tunnels. We hypothesised that tunnels are lined by mostly conserved residues, and are equipped with a number of specific variable residues that are able to respond to evolutionary pressure. The hypothesis was confirmed, and we suggested a general and detailed way of the tunnels evolution analysis based on entropy values calculated for tunnels residues. We also found three different cases of entropy distribution among tunnel-lining residues. These observations can be applied for protein reengineering mimicking the natural evolution process. We propose a perforation mechanism for new tunnels design via the merging of internal cavities or protein surface perforation. Based on the literature data, such a strategy of new tunnel design could significantly improve the enzymes performance and can be applied widely for enzymes with buried active sites. Author SummarySo far very little is known about proteins tunnels evolution. The goal of this study is to evaluate the evolution of tunnels in the family of soluble epoxide hydrolases - representatives of numerous /{beta}-hydrolase fold enzymes. As a result two types of tunnels evolution analysis were proposed (a general and a detailed approach), as well as a perforation mechanism which can mimic native evolution in proteins and can be used as an additional strategy for enzymes redesign.

bioinformatics↗

bioRxiv2020

Molecular Dynamics Simulations Indicate the COVID-19 Mpro Is Not a Viable Target for Small-Molecule Inhibitors Design

The novel coronavirus whose outbreak took place in December 2019 continues to spread at a rapid rate worldwide. In the absence of an effective vaccine, inhibitor repurposing or de novo drug design may offer a longer-term strategy to combat this and future infections due to similar viruses. Here, we report on detailed classical and mix-solvent molecular dynamics simulations of the main protease (Mpro) enriched by evolutionary and stability analysis of the protein. The results were compared with those for a highly similar SARS Mpro protein. In spite of a high level of sequence similarity, the active sites in both proteins show major differences in both shape and size indicating that repurposing SARS drugs for COVID-19 may be futile. Furthermore, analysis of the binding sites conformational changes during the simulation time indicates its flexibility and plasticity, which dashes hopes for rapid and reliable drug design. Conversely, structural stability of the protein with respect to flexible loop mutations indicates that the virus mutability will pose a further challenge to the rational design of small-molecule inhibitors. However, few residues contribute significantly to the protein stability and thus can be considered as key anchoring residues for Mpro inhibitor design.

molecular biology↗