Abstract
The evolutionary variability of a protein’s 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 α/β-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 enzyme’s performance and can be applied widely for enzymes with buried active sites.
Author Summary So 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 α/β-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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
We noticed that the multiple sequence alignment (MSA) used for calculating the tunnels' variability was not adequately post-processed. The use of such an MSA for statistical analysis caused the previously obtained results concerning the variability of tunnels to be not correct. After verifying all the results, the variability of the tunnels changed significantly - almost all identified tunnels can be described as conserved (less variable), which confirms the hypothesis presented in the manuscript. Moreover, the obtained results are in line with the proposed perforation model.