Atomic resolution mechanism of ligand binding to a solvent inaccessible cavity in T4 lysozyme

Ligand binding sites in proteins are often localized to deeply buried cavities, inaccessible to bulk solvent. Yet, in many cases binding of cognate ligands occurs rapidly. An intriguing system is presented by the L99A cavity mutant of T4 Lysozyme (T4L L99A) that rapidly binds benzene (~106 M-1s-1). Although the protein has long served as a model system for protein thermodynamics and crystal structures of both free and benzene-bound T4L L99A are available, the kinetic pathways by which benzene reaches its solvent-inaccessible binding cavity remain elusive. The current work, using extensive molecular dynamics simulation, achieves this by capturing the complete process of spontaneous recognition of benzene by T4L L99A at atomistic resolution. A series of multi-microsecond unbiased molecular dynamics simulation trajectories unequivocally reveal how benzene, starting in bulk solvent, diffuses to the protein and spontaneously reaches the solvent inaccessible cavity of T4L L99A. The simulated and high-resolution X-ray derived bound structures are in excellent agreement. A robust four-state Markov model, developed using cumulative 60 μs trajectories, identifies and quantifies multiple ligand binding pathways with low activation barriers. Interestingly, none of these identified binding pathways required large conformational changes for ligand access to the buried cavity. Rather, these involve transient but crucial opening of a channel to the cavity via subtle displacements in the positions of key helices (helix4/helix6, helix7/helix9) leading to rapid binding. Free energy simulations further elucidate that these channel-opening events would have been unfavorable in wild type T4L. Taken together and via integrating with results from experiments, these simulations provide unprecedented mechanistic insights into the complete ligand recognition process in a buried cavity. By illustrating the power of subtle helix movements in opening up multiple pathways for ligand access, this work offers an alternate view of ligand recognition in a solvent-inaccessible cavity, contrary to the common perception of a single dominant pathway for ligand binding.Author summary: Proteins often bind ligands in buried cavities that appear to be inaccessible based on static structures. The mechanisms and pathways by which ligands reach their binding sites in such cases are, thus, often unknown. Yet, ligand recognition by occluded cavities can happen rapidly. A central question remains: How does such a process occur? Experiments that provide insight at atomic resolution are currently lacking. In the current work, we have used a computational approach to capture the process by which a ligand, benzene, binds to a buried cavity in the L99A cavity mutant of T4 Lysozyme. Using multiple long, unbiased atomistic simulations, we have discovered how benzene, starting from bulk solvent, finds and binds the solvent-inaccessible cavity. We find that there is no single dominant pathway. Rather, simulated trajectories discover multiple binding pathways with low activation barriers, facilitating a rapid recognition process. We highlight the role of subtle movements in helix positions in opening up multiple crucial paths for benzene to reach its binding cavity without the need for large-scale distortions of the protein structure, explaining the small activation energies.