Sarkar DK, Surpeta B, Brezovsky J, 2024: Incorporating prior knowledge in the seeds of adaptive sampling molecular dynamics simulations of ligand transport in enzymes with buried active sites. Journal of Chemical Theory and Computation, DOI: 10.1021/acs.jctc.4c00452. full text dataset

Because most proteins have buried active sites, protein tunnels or channels play a crucial role in the transport of small molecules into buried cavities for enzymatic catalysis. Tunnels can critically modulate the biological process of protein–ligand recognition. Various molecular dynamics methods have been developed for exploring and exploiting the protein–ligand conformational space to extract high-resolution details of the binding processes, a recent example being energetically unbiased high-throughput adaptive sampling simulations. The current study systematically contrasted the role of integrating prior knowledge while generating useful initial protein–ligand configurations, called seeds, for these simulations. Using a nontrivial system of a haloalkane dehalogenase mutant with multiple transport tunnels leading to a deeply buried active site, simulations were employed to derive kinetic models describing the process of association and dissociation of the substrate molecule. The most knowledge-based seed generation enabled high-throughput simulations that could more consistently capture the entire transport process, explore the complex network of transport tunnels, and predict equilibrium dissociation constants, k_off/k_on, on the same order of magnitude as experimental measurements. Overall, the infusion of more knowledge into the initial seeds of adaptive sampling simulations could render analyses of transport mechanisms in enzymes more consistent even for very complex biomolecular systems, thereby promoting drug development efforts and the rational design of enzymes with buried active sites.

Sequeiros-Borja C, Bartlomiej Surpeta, Thirunavukarasu AS, Dongmo Foumthuim CJ, Igor Marchlewski, Brezovsky J, 2024: Water will find its way: transport through narrow tunnels in hydrolases. Journal of Chemical Information and Modeling, DOI: 10.1021/acs.jcim.4c00094. full text dataset-Hal dataset-Epx dataset-Lip dataset-hEpx dataset-E470G dataset-interactions dataset-Hal with different MD settings

An aqueous environment is vital for life as we know it, and water is essential for nearly all biochemical processes at the molecular level. Proteins utilize water molecules in various ways. Consequently, proteins must transport water molecules across their internal network of tunnels to reach the desired action sites, either within them or by functioning as molecular pipes to control cellular osmotic pressure. Despite water playing a crucial role in enzymatic activity and stability, its transport has been largely overlooked, with studies primarily focusing on water transport across membrane proteins. The transport of molecules through a protein’s tunnel network is challenging to study experimentally, making molecular dynamics simulations the most popular approach for investigating 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. Using a 5 μs adaptive simulation per system, we observed that only a few tunnels were responsible for the majority of water transport in dehalogenase, in contrast to a higher diversity of tunnels in other enzymes. Interestingly, water molecules could traverse narrow tunnels with subangstrom bottlenecks, which is surprising given the commonly accepted water molecule radius of 1.4 Å. Our analysis of the transport events in such narrow tunnels revealed 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 the total water transport observed, emphasizing the need to surpass the standard geometrical limits on the functional tunnels to properly account for the relevant transport processes. Finally, we demonstrated 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.