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 to and 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 timescales. 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 multi-step 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 potential for drug development, precision medicine, and rational protein engineering.