A dry ligand-binding cavity in a solvated protein

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 β-lactoglobulin. Because spatially delocalized water molecules may escape detection by x-ray diffraction, we use water 17O and 2H magnetic relaxation dispersion (MRD), 13C 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 Å3 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 β-lactoglobulin pore and carbon nanotubes is caused by subtle differences in water–wall interactions and water entropy.

[1]  C. Verma,et al.  Hydration of a hydrophobic cavity and its functional role: A simulation study of human interleukin‐1β , 2007, Proteins.

[2]  Gerhard Hummer,et al.  Metastable water clusters in the nonpolar cavities of the thermostable protein tetrabrachion. , 2007, Journal of the American Chemical Society.

[3]  Robert Abel,et al.  Motifs for molecular recognition exploiting hydrophobic enclosure in protein–ligand binding , 2007, Proceedings of the National Academy of Sciences.

[4]  B. Matthews,et al.  Determination of solvent content in cavities in IL-1β using experimentally phased electron density , 2006, Proceedings of the National Academy of Sciences.

[5]  O. Steinhauser,et al.  Simulation studies of the protein-water interface. II. Properties at the mesoscopic resolution. , 2006, The Journal of chemical physics.

[6]  Laxmikant V. Kalé,et al.  Scalable molecular dynamics with NAMD , 2005, J. Comput. Chem..

[7]  Gerhard Hummer,et al.  Cooperative water filling of a nonpolar protein cavity observed by high-pressure crystallography and simulation. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Ruhong Zhou,et al.  Observation of a dewetting transition in the collapse of the melittin tetramer , 2005, Nature.

[9]  Sheldon Park,et al.  Statistical and molecular dynamics studies of buried waters in globular proteins , 2005, Proteins.

[10]  Charles A Laughton,et al.  Van der Waals interactions dominate ligand-protein association in a protein binding site occluded from solvent water. , 2005, Journal of the American Chemical Society.

[11]  A. Stuchebrukhov,et al.  Thermodynamic properties of internal water molecules in the hydrophobic cavity around the catalytic center of cytochrome c oxidase. , 2005, The journal of physical chemistry. B.

[12]  Gerhard Hummer,et al.  Water clusters in nonpolar cavities. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[13]  B. García-Moreno E.,et al.  Stabilization of internal charges in a protein: water penetration or conformational change? , 2004, Biophysical journal.

[14]  G. Hummer,et al.  Electric field and temperature effects on water in the narrow nonpolar pores of carbon nanotubes. , 2004, The Journal of chemical physics.

[15]  B. Halle,et al.  Protein hydration dynamics in solution: a critical survey. , 2004, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[16]  S. Rick,et al.  Hydration free energies and entropies for water in protein interiors. , 2004, Journal of the American Chemical Society.

[17]  Donald Hamelberg,et al.  Standard free energy of releasing a localized water molecule from the binding pockets of proteins: double-decoupling method. , 2004, Journal of the American Chemical Society.

[18]  Sergei Sukharev,et al.  Water dynamics and dewetting transitions in the small mechanosensitive channel MscS. , 2004, Biophysical journal.

[19]  B. Halle.  Biomolecular cryocrystallography: structural changes during flash-cooling. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[20]  B. Halle.  Cross-relaxation between macromolecular and solvent spins: The role of long-range dipole couplings , 2003 .

[21]  Kristian Rother,et al.  Inhomogeneous molecular density: reference packing densities and distribution of cavities within proteins , 2003, Bioinform..

[22]  B. Halle,et al.  Protein self‐association in solution: The bovine β ‐lactoglobulin dimer and octamer , 2003, Protein science : a publication of the Protein Society.

[23]  H. Molinari,et al.  EF Loop Conformational Change Triggers Ligand Binding in β-Lactoglobulins* , 2003, Journal of Biological Chemistry.

[24]  B. Halle,et al.  Biomolecular hydration: From water dynamics to hydrodynamics , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[25]  B. Halle,et al.  Water dynamics in the large cavity of three lipid-binding proteins monitored by (17)O magnetic relaxation dispersion. , 2003, Journal of molecular biology.

[26]  Oliver Beckstein,et al.  Liquid–vapor oscillations of water in hydrophobic nanopores , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[27]  G. Hummer,et al.  Filling and emptying kinetics of carbon nanotubes in water , 2002 .

[28]  M. Perduca,et al.  pH and ionic strength dependence of protein (un)folding and ligand binding to bovine beta-lactoglobulins A and B. , 2002, Biochemistry.

[29]  J Andrew McCammon,et al.  Structural and dynamic properties of water around acetylcholinesterase , 2002, Protein science : a publication of the Protein Society.

[30]  Y. Goto,et al.  Manipulating Monomer-Dimer Equilibrium of Bovine β-Lactoglobulin by Amino Acid Substitution* , 2002, The Journal of Biological Chemistry.

[31]  Lindsay Sawyer,et al.  The ligand-binding site of bovine beta-lactoglobulin: evidence for a function? , 2002, Journal of molecular biology.

[32]  I. Polikarpov,et al.  The X-ray structure of a recombinant major urinary protein at 1.75 A resolution. A comparative study of X-ray and NMR-derived structures. , 2001, Acta crystallographica. Section D, Biological crystallography.

[33]  G. Hummer,et al.  Water conduction through the hydrophobic channel of a carbon nanotube , 2001, Nature.

[34]  E. Liong,et al.  Waterproofing the Heme Pocket , 2001, The Journal of Biological Chemistry.

[35]  D R Flower,et al.  Lipocalins: unity in diversity. , 2000, Biochimica et biophysica acta.

[36]  Lawrence J. Marnett,et al.  Structural insights into the stereochemistry of the cyclooxygenase reaction , 2000, Nature.

[37]  D. Uhrín,et al.  Structural changes accompanying pH-induced dissociation of the beta-lactoglobulin dimer. , 2000, Biochemistry.

[38]  G. Hummer,et al.  Water penetration and escape in proteins , 2000, Proteins.

[39]  A M Gronenborn,et al.  Disordered water within a hydrophobic protein cavity visualized by x-ray crystallography. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[40]  E N Baker,et al.  Structural basis of the Tanford transition of bovine beta-lactoglobulin. , 1998, Biochemistry.

[41]  Gottfried Otting,et al.  NMR studies of water bound to biological molecules , 1997 .

[42]  G. Otting,et al.  NMR identification of hydrophobic cavities with ow water occupancies in protein structures using small gas molecules , 1997, Nature Structural Biology.

[43]  Darren R. Flower,et al.  Bovine β-lactoglobulin at 1.8 Å resolution — still an enigmatic lipocalin , 1997 .

[44]  M. Gilson,et al.  The statistical-thermodynamic basis for computation of binding affinities: a critical review. , 1997, Biophysical journal.

[45]  B. Halle,et al.  Using buried water molecules to explore the energy landscape of proteins , 1996, Nature Structural Biology.

[46]  William L. Jorgensen,et al.  Importance of Polarization for Dipolar Solutes in Low-Dielectric Media: 1,2-Dichloroethane and Water in Cyclohexane , 1995 .

[47]  P. Kollman,et al.  A Second Generation Force Field for the Simulation of Proteins, Nucleic Acids, and Organic Molecules , 1995 .

[48]  A M Gronenborn,et al.  Demonstration of positionally disordered water within a protein hydrophobic cavity by NMR. , 1995, Science.

[49]  A. Kleinfeld,et al.  Equilibrium constants for the binding of fatty acids with fatty acid-binding proteins from adipocyte, intestine, heart, and liver measured with the fluorescent probe ADIFAB. , 1994, The Journal of biological chemistry.

[50]  G J Kleywegt,et al.  Detection, delineation, measurement and display of cavities in macromolecular structures. , 1994, Acta crystallographica. Section D, Biological crystallography.

[51]  J. Gordon,et al.  Rat intestinal fatty acid binding protein. A model system for analyzing the forces that can bind fatty acids to proteins. , 1993, The Journal of biological chemistry.

[52]  Arieh Ben-Naim,et al.  Solvation thermodynamics of nonionic solutes , 1984 .

[53]  B. Halle,et al.  Multinuclear Relaxation Dispersion Studies of Protein Hydration , 2002 .

[54]  G. Jameson,et al.  Flexibility, functionality and hydrophobicity of bovine β-lactoglobulin ☆ , 2002 .

[55]  L. Sawyer,et al.  beta-lactoglobulin binds palmitate within its central cavity. , 1999, The Journal of biological chemistry.