Evolution of tunnels in α/β-hydrolase fold proteins – what can we learn from studying epoxide hydrolases?

Abstract

The evolutionary variability of a protein's residues is highly dependent on protein region and protein function. Solvent-exposed residues, excluding those at interaction interfaces, are more variable than buried residues. Active site residues are considered to be conserved as they ensure an enzyme's activity and selectivity. The abovementioned rules apply also to α/β-hydrolase fold proteins - an example of enzymes with buried active sites equipped with tunnels linking the reaction site with the exterior. We hypothesised two scenarios: (1) tunnels are lined by mostly variable residues, allowing adaptation to the evolutionary pressures of a changeable environment; or (2) tunnels are lined by mostly conserved amino acids, and are equipped with a number of specific variable residues that are able to respond to evolutionary pressure. We also wanted to check if evolutionary analysis can help distinguish functional and non-functional tunnels. Soluble epoxide hydrolases (sEHs) represent a good case study for the analysis of the evolution of tunnels in an α/β-hydrolase fold family due to their size and architecture. Here, we propose methods for the comparison of tunnels detected in both crystal structures and molecular dynamics simulations, as well as the assignment of tunnel functionality, and we identify critical steps for careful tunnel inspection. We also compare the entropy values of the tunnel-lining residues and system-specific compartments in seven selected sEHs from different clades. We present three different cases of entropy distribution among tunnel-lining residues. As a result, we propose a 'perforation' model for tunnel evolution via the merging of internal cavities or surface perforations. We also report an approach for the identification of highly variable tunnel-lining residues as potential targets to be used for the fine-tuning of selected enzymes.

Structure-function relationship between soluble epoxide hydrolases structure and their tunnel network

“…Such structural arrangement was preserved in most of the EHs which supports the hypothesis regarding the origin of the positioning of the active site between both domains. However, according to other reports [ [15] , [21] , [37] , [44] , [45] ], this was not the only pathway leading to the active site. Other tunnels were located in the cap and main domains, as well as between those domains.…”

“…4 . The inlets were then clustered to represent tunnels entries reported elsewhere [ 15 ]. It should be noted that not all tunnels were represented in other structures.…”

“…To date, little is known about EHs, however, several studies have been conducted to investigate their structural features [ [15] , [16] , [17] , [18] ]. sEHs belong to the α/β-hydrolases family that display a modular structure with a central catalytic domain, the main domain, formed by eight superhelically twisted β-strands [ [17] , [36] , [38] ].…”

“…In our other study [ 15 ], sEHs were employed as a sample system in order to investigate the evolution of tunnels. It was determined that most tunnels should be considered as variable structural features of proteins.…”

“…Additionally, the amino acids forming the protein core are also more conserved [ [11] , [12] ] and tend to evolve slower than the surface residues, excluding those involved in protein–protein and/or protein–ligand interactions [ [13] , [14] ]. However, in our other study [ 15 ] we elucidate that in the case of the soluble epoxide hydrolases (sEHs) most of their tunnels should be considered as variable structural features with only one exception - the tunnel located at the border between the main and cap domains. These counterintuitive findings has inspired the investigation of the structure–function relationship of sEHs in more detail.…”