Benzene, among many small nonpolar molecules, stabilizes the L99A mutant of T4 Lysozyme (T4L) by binding to the buried hydrophobic cavity created by the L99A mutation. Although crystal structures of both free and benzene bound T4L L99A are available, an atomistic insight of how benzene reaches the solvent-inaccessible cavity from solvent are still elusive. We address this longstanding question by simulating at an atomistic detail, the entire binding process of benzene from solvent to the buried cavity of L99A T4L and augmenting the results by NMR spectroscopy.  In our multiple unbiased microsecond long molecular dynamics (MD) simulations, benzene starts from a random position in the solvent, diffuses across the solvent and the protein surface and ultimately spontaneously reach the solvent-inaccessible binding site, with the simulated binding pose matching the crystallographic pose at an angstrom-level accuracy. The simulation identifies a secondary binding site of benzene which are also augmented by NMR experiments. The resulting atomistic trajectories and underlying Markov state Model identify and quantify multiple novel ligand binding pathways of very low activation barriers, each involving the transient opening of a channel to the cavity by crucial fluctuations in the positions of helices in the C-terminal domain.  Additional Free energy simulations simulations  show that these channels opening events would have been free-energetically more unfavorable in the wild type protein rendering the approach of benzene towards the cavity improbable in wild type T4 Lysozyme.  Enhanced sampling techniques used to simulate the unbinding process complement and enhance the ensemble of pathways observed from the binding trajectories. The estimated binding and rate constants are in very good agreement with experiment. Our work unequivocally establishes that access to multiple potential pathways and low free energy barrier makes the benzene a rapid binder of T4 Lysozyme , which are facilitated by key fluctuation of certain C-terminal helices.   The current work  provide unprecedented  insights on the atomistic details of ligand-binding and escape  pathways to and from a solvent-inaccessible protein cavity that will trigger design of future experiments.

How does benzene recognize the buried cavity in T4 Lysozyme L99A?

254th National Meeting (2017)

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