Biology subjects

Mitusinska, K.

Publications and source records attributed to Mitusinska, K..

4 recordsLinked to original sources

bioRxiv2026

Glutamine Tautomerization Drives RhoGAP-Aided GTP Hydrolysis in Small Rho GTPases

Rho GTPases promote GTP hydrolysis aided by specific GTPase-activating proteins (GAPs). By alternating between an active GTP-bound and an inactive GDP-bound state, Rho GTPases function as molecular switches regulating cytoskeletal dynamics and cell motility. Despite their biological relevance, the detailed molecular mechanism underlying Rho GTPases catalysis remains contentious. Here, using classical and hybrid quantum-classical molecular dynamics, we resolve the mechanism of GTP hydrolysis in the RhoGAP-RhoA complex. We reveal that GTP hydrolysis proceeds through a dissociative nucleophilic substitution mechanism, driven by an amide [->] imide tautomerization of Gln63, which aids in delivering a proton from the nucleophilic water to the leaving phosphate group. The Gln63 imide tautomer also loosens RhoGAP-RhoA interfacial contacts, allowing solvent molecules to enter and drive a water-mediated reverse tautomerization of Gln63 that restores the catalytically-competent configuration of the RhoA active site. Conservation of key interface residues across Rho/Rho GAP family members suggests that this mechanism may be shared by most Rho GTPases.

biophysics↗

bioRxiv2023

Water migration through enzyme tunnels is sensitive to choice of explicit water model

Understanding the utilization of tunnels and water transport within enzymes is crucial for the catalytic function of enzymes, as water molecules can stabilize bound substrates and help with unbinding processes of products and inhibitors. Since the choice of water models for molecular dynamics simulations was shown to determine the accuracy of various calculated properties of the bulk solvent and solvated proteins, we have investigated if and to what extent the water transport through the enzyme tunnels depends on the selection of the water model. Here, we have focused on simulating enzymes with various well-defined tunnel geometries. In a systematic investigation using haloalkane dehalogenase as a model system, we focused on the well-established TIP3P, OPC, and TIP4P-Ew water models to explore their impact on using tunnels for water molecules transport. The TIP3P water model showed significantly faster migration, resulting in the transport of approximately 2.5 times more water molecules in comparison to OPC and 2.0 times greater than the TIP4P-Ew. The increase in migration of TIP3P water molecules was mainly due to faster transit times, and in the case of narrower tunnels, greater concurrent transport was evident as well. We have observed similar behavior in two different enzymes with buried active sites and different tunnel network topologies, indicating that our findings are likely not restricted to a particular enzyme family. Our study emphasizes the critical importance of water models in comprehending the use of enzyme tunnels for small molecule transport. Given the significant role of water availability in various stages of the catalytic cycle and solvation of substrates, products, and drugs, choosing an appropriate water model might be crucial for accurate simulations of complex enzymatic reactions, rational enzyme design, and predicting drug residence times.

bioinformatics↗

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↗