Capturing Invisible Motions in the Transition from Ground to Rare Excited States of T4 Lysozyme L99A

Abstract

Proteins commonly sample a number of conformational states to carry out their biological function, often requiring transitions from the ground state to higher-energy states. Characterizing the mechanisms that guide these transitions at the atomic level promises to impact our understanding of functional protein dynamics and energy landscapes. The leucine-99-toalanine (L99A) mutant of T4 lysozyme is a model system that has an experimentally well characterized excited sparsely populated state as well as a ground state. Despite the exhaustive study of L99A protein dynamics, the conformational changes that permit transitioning to the experimentally detected excited state (~3%, DG~2 kcal/mol) remain unclear. Here, we describe the transitions from the ground state to this sparsely populated excited state of L99A as observed through a single molecular dynamics (MD) trajectory on the Anton supercomputer. Aside from detailing the ground-to-excited-state transition, the trajectory samples multiple metastates and an intermediate state en route to the excited state. Dynamic motions between these states enable cavity surface openings large enough to admit benzene on timescales congruent with known rates for benzene binding. Thus, these fluctuations between rare protein states provide an atomic description of the concerted motions that illuminate potential path(s) for ligand binding. These results reveal, to our knowledge, a new level of complexity in the dynamics of buried cavities and their role in creating mobile defects that affect protein dynamics and ligand binding.

Atomic Resolution Mechanism of Ligand Binding to a Solvent Inaccessible Cavity in T4 Lysozyme

“…3). Similar tunnels from the protein surface to the cavity were also observed in a 30 µs MD simulation of T4L L99A using the AMBER force field [37]. The existence of multiple pathways coupled with a small net activation barrier explains how 20 benzene can bind the occluded cavity rapidly.…”

Absolute Binding Free Energies between T4 Lysozyme and 141 Small Molecules: Calculations Based on Multiple Rigid Receptor Configurations

“…Receptor sampling schemes may include biased sampling towards crystallographically-observed structures, Gaussian accelerated molecular dynamics, 73 and long molecular dynamics simulations with specialized supercomputers. 74 Representative snapshots may be selected based on structural features rather than equal simulation time intervals. While there are many ways to improve the calculations, our present results indicate that ILT-based free energy calculations are a promising way to bridge the gap between molecular docking and flexible-receptor alchemical pathway methods.…”

Discovering loop conformational flexibility in T4 lysozyme mutants through artificial intelligence aided molecular dynamics

“…The center of the ranges were set to the local minima closest to those reported in Ref. 23 and the range around these centers was set by hand to be a static range across mutants which covered the minima but overlap with the other state for any mutants. The RMSD cutoffs were then set to be the RMSD of the each mutant's equilibrated structure + .1Å.…”

“…Previous computational studies have attempted to detail underlying mechanisms of this transition, but many aspects are still not fully understood (23,24). For instance, in addition to the L99A mutant, two other point mutants of T4 lysozyme have been discovered which very significantly alter the population of the ground and excited states (20).…”

“…1) so drastically affect this protein's conformational landscape is still missing. While computational and experimental methods have clarified such structural fluctuations and ligand binding pathways in L99A T4L in recent years, this system still presents a challenge due to the high degree of flexibility and size of the surface-exposed loop near the binding pocket (23,25). This set of three mutants thus serves as an excellent benchmarking set for methods that seek to sample loop conformations including this current work.…”