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A dry ligand-binding cavity in a solvated protein.

ABSTRACT: Ligands usually bind to proteins by displacing water from the binding site. The affinity and kinetics of binding therefore depend on the hydration characteristics of the site. Here, we show that the extreme case of a completely dehydrated free binding site is realized for the large nonpolar binding cavity in bovine beta-lactoglobulin. Because spatially delocalized water molecules may escape detection by x-ray diffraction, we use water (17)O and (2)H magnetic relaxation dispersion (MRD), (13)C NMR spectroscopy, molecular dynamics simulations, and free energy calculations to establish the absence of water from the binding cavity. Whereas carbon nanotubes of the same diameter are filled by a hydrogen-bonded water chain, the MRD data show that the binding pore in the apo protein is either empty or contains water molecules with subnanosecond residence times. However, the latter possibility is ruled out by the computed hydration free energies, so we conclude that the 315 A(3) binding pore is completely empty. The apo protein is thus poised for efficient binding of fatty acids and other nonpolar ligands. The qualitatively different hydration of the beta-lactoglobulin pore and carbon nanotubes is caused by subtle differences in water-wall interactions and water entropy.

SUBMITTER: Qvist J

PROVIDER: S-EPMC2359778 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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A dry ligand-binding cavity in a solvated protein.

Qvist Johan J Davidovic Monika M Hamelberg Donald D Halle Bertil B

Proceedings of the National Academy of Sciences of the United States of America 20080421 17

Ligands usually bind to proteins by displacing water from the binding site. The affinity and kinetics of binding therefore depend on the hydration characteristics of the site. Here, we show that the extreme case of a completely dehydrated free binding site is realized for the large nonpolar binding cavity in bovine beta-lactoglobulin. Because spatially delocalized water molecules may escape detection by x-ray diffraction, we use water (17)O and (2)H magnetic relaxation dispersion (MRD), (13)C NM ...[more]

PMID: 18427121

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Project description: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.

Dataset Information

A dry ligand-binding cavity in a solvated protein.

Publications

A dry ligand-binding cavity in a solvated protein.

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