Reinforcing Tunnel Network Exploration in Proteins Using Gaussian Accelerated Molecular Dynamics.

Tunnels are structural conduits in biomolecules responsible for transporting chemical compounds and solvent molecules from the active site. They have been shown to be present in a wide variety of enzymes across all functional and structural classes. However, the study of such pathways is experimentally challenging, because they are typically transient. Computational methods, such as molecular dynamics (MD) simulations, have been successfully proposed to explore tunnels. Conventional MD (cMD) provides structural details to characterize tunnels but suffers from sampling limitations to capture rare tunnel openings on longer time scales. Therefore, in this study, we explored the potential of Gaussian accelerated MD (GaMD) simulations to improve the exploration of complex tunnel networks in enzymes. We used the haloalkane dehalogenase LinB and its two variants with engineered transport pathways, which are not only well-known for their application potential but have also been extensively studied experimentally and computationally regarding their tunnel networks and their importance in multistep catalytic reactions. Our study demonstrates that GaMD efficiently improves tunnel sampling and allows the identification of all known tunnels for LinB and its two mutants. Furthermore, the improved sampling provided insight into a previously unknown transient side tunnel (ST). The extensive conformational landscape explored by GaMD simulations allowed us to investigate in detail the mechanism of ST opening. We determined variant-specific dynamic properties of ST opening, which were previously inaccessible due to limited sampling of cMD. Our comprehensive analysis supports multiple indicators of the functional relevance of the ST, emphasizing its potential significance beyond structural considerations. In conclusion, our research proves that the GaMD method can overcome the sampling limitations of cMD for the effective study of tunnels in enzymes, providing further means for identifying rare tunnels in enzymes with the potential for drug development, precision medicine, and rational protein engineering.

TransportTools: a library for high-throughput analyses of internal voids in biomolecules and ligand transport through them.

SUMMARY: Information regarding pathways through voids in biomolecules and their roles in ligand transport is critical to our understanding of the function of many biomolecules. Recently, the advent of high-throughput molecular dynamics simulations has enabled the study of these pathways, and of rare transport events. However, the scale and intricacy of the data produced requires dedicated tools in order to conduct analyses efficiently and without excessive demand on users. To fill this gap, we developed the TransportTools, which allows the investigation of pathways and their utilization across large, simulated datasets. TransportTools also facilitates the development of custom-made analyses. AVAILABILITY AND IMPLEMENTATION: TransportTools is implemented in Python3 and distributed as pip and conda packages. The source code is available at https://github.com/labbit-eu/transport_tools. Data are available in a repository and can be accessed via a link: https://doi.org/10.5281/zenodo.5642954. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Mechanisms of influence of the microtubule over-stabilizing ligands on the structure and intrinsic dynamics of α,ß-Tubulin.

The intervention into the cell cycle progression by administering microtubule over-stabilizing ligands that arrest the mitotic cell division by preventing spindle dissociation, is a promising strategy to fight against cancers. The building blocks of the microtubules and the spindles, i.e. the α,ß-tubulin dimer, upon binding of such ligands, stay more comfortably in the microtubular multimeric form; the phenomenon of which is the key to the said over-stabilization. Using two such over-stabilizing ligands, Taxol and Taxotere, the present work reports the collective changes that these ligands induce on the structure and dynamics of the α,ß-tubulin dimer which could be reconciled as the molecular basis of the over-stabilization of the microtubules; the trends have been found to be statistically significant across all independent calculations on them. The ligand binding increases the coherence between the residue communities of the two opposite faces of the ß-subunit, which in a periodic arrangement in microtubule are knwon to form intermolecular contact with each other. This is likely to create an indirect cooperativity between those structural regions and this is a consequence of the reshuffling of the internal network of interactions upon ligand binding. Such reorganizations are also complemented by the increased contributions of the softer modes of the intrinsic dynamics more, which is likely to increase the plasticity of the system favourable for making structural adjustments in a multimer. Further, the ligands are able to compensate the drawback of lacking one phosphate group in protein-GDP interactions compared to the same for protein-GTP and this is in agreement with the hints form the earlier reports. The findings form a mechanistic basis of the enhanced capacity of the α,ß-tubulin dimer to get more favourably accommodated into the microtubule superstructure upon binding either of Taxol and Taxotere.