Water will find a way: transport through narrow tunnels in hydrolases

ABSTRACT An aqueous environment is necessary for life as we know it, and water is required for almost all biochemical processes at a molecular level. Proteins employ water molecules in many ways. Hence, proteins need to transport water molecules across their internal network of tunnels to reach the desired action sites, either inside them or acting as molecular pipes to control cellular osmotic pressure. Even though water is an essential player in enzymatic activity and stability, its transport has been mostly neglected, with water transport studies mainly focused on the transport across membrane proteins. The transport of molecules through a protein’s tunnel network is hard to study experimentally, rendering molecular dynamics simulations the most popular approach to study such events. In this study, we focused on the transport of water molecules across three different α/β-hydrolases: haloalkane dehalogenase, epoxide hydrolase, and lipase. Employing 5 μs adaptive simulation per system, we observed that only a few tunnels were responsible for the majority of water transport in dehalogenase, contrasting with a higher diversity of tunnels in other enzymes. Interestingly, water molecules could traverse narrow tunnels with even sub-angstrom bottlenecks, which is surprising given the commonly accepted water molecule radii of 1.4 Å. Our analysis of the transport events in such narrow tunnels showed a markedly increased number of hydrogen bonds formed between the water molecules and protein, likely compensating for the steric penalty of the process. Overall, these commonly disregarded narrow tunnels accounted for ∼20% of total water transport observed, highlighting the need to move past the standard geometrical limits on the functional tunnels to account for relevant transport processes properly. Finally, we showed how the obtained insights could be applied to explain the differences in a mutant of the human soluble epoxide hydrolase associated with a higher incidence of ischemic stroke.