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.

Fig. 1.

View of the crystal structure 3BLG (9) of β-lactoglobulin, showing two water molecules (red) buried in small polar cavities (labeled 175 and 177 in 3BLG, with 177 closer to the calyx) and five water molecules (blue) modeled in the nonpolar calyx. We label the calyx waters 1–5 starting from the bottom; the corresponding 3BLG labels are 178, 190, 179, 180, and 233. The cavity surface in the calyx is color-coded according to whether the contributing protein atoms are polar (red) or nonpolar (brown). The light green cartoon representation shows one of the two β sheets enclosing the nonpolar calyx. The figure was rendered with PyMOL (www.pymol.org) using a 1.4 Å probe to define the (external and internal) molecular surfaces of the protein and the same protein vdW radii as used for the cavity volume calculations.

Fig. 2.

Water ¹⁷O MRD profiles from aqueous solutions of β-lactoglobulin with (holo) or without (apo) bound palmitate at 27°C. The data symbols refer to apo BLG at pH 7.4 (open squares, sample A1) and pH 6.2 (open diamonds, sample A2); holo BLG at pH 7.4 (filled triangles, sample H1; inverted filled triangles, sample H2); and protein-free buffer (open circles). Data from different BLG solutions have been normalized to the protein concentration of sample A1 (0.79 mM, N_W = 69300), using the scaling R₁-R₁⁰ ∝ 1/N_W implicit in Eq. 1. The curves were obtained by fitting the model to the two sets of apo or holo data as described in the text. The parameter values resulting from the fits are given in Table 1.

Fig. 3.

Water ²H MRD profiles from aqueous solutions of β-lactoglobulin with (holo) or without (apo) bound palmitate at 27°C. Symbols and curves as in Fig. 2. The ²H and ¹⁷O data were measured on the same samples.