Water in the polar and nonpolar cavities of the protein interleukin-1β

Abstract

Water in the protein interior serves important structural and functional roles and is also increasingly recognized as a relevant factor in drug binding. The nonpolar cavity in the protein interleukin-1β has been reported to be filled by water on the basis of some experiments and simulations and to be empty on the basis of others. Here we study the thermodynamics of filling the central nonpolar cavity and the four polar cavities of interleukin-1β by molecular dynamics simulation. We use different water models (TIP3P and SPC/E) and protein force fields (amber94 and amber03) to calculate the semigrand partition functions term by term that quantify the hydration equilibria. We consistently find that water in the central nonpolar cavity is thermodynamically unstable, independent of force field and water model. The apparent reason is the relatively small size of the cavity, with a volume less than ∼80 Å(3). Our results are consistent with the most recent X-ray crystallographic and simulation studies but disagree with an earlier interpretation of nuclear magnetic resonance (NMR) experiments probing protein-water interactions. We show that, at least semiquantitatively, the measured nuclear Overhauser effects indicating the proximity of water to the methyl groups lining the nonpolar cavity can, in all likelihood, be attributed to interactions with buried and surface water molecules near the cavity. The same methods applied to determine the occupancy of the polar cavities show that they are filled by the same number of water molecules observed in crystallography, thereby validating the theoretical and simulation methods used to study the water occupancy in the nonpolar protein cavity.

Figure 1

Structure of IL-1β (PDB code 6I1B24), with nonpolar residues (cyan) lining the central cavity (green). For a spherical probe of radius 1.4 Å, the volume accessible to the probe center is ∼10 ų, and the volume covered by the probe sphere (shown in green) is ∼140 ų. In the most recent crystal structure of IL-1β by Quillin et al. (PDB code 2NVH) the volume covered by a probe sphere of 1.4 Å radius is ∼40 ų. In the other crystal structures,-, the volume varies between ∼45 and ∼80 ų. In the amber03/TIP3P MD simulations with occupancy N=0, starting from the NMR structure 6I1B, the volume fluctuates about a mean of ∼80 ų. The larger cavity volume in the NMR structure 6I1B is partly a reflection of the fact that it was refined before the use of compactness restraints.

Figure 2

Distribution of water insertion and removal energies. (a) Distribution of removal energies p_rem,1(ΔU) (open squares) and insertion energies p_ins,0(ΔU) (circles), and reweighted distribution $p_{\mathit{\text{ins}},0}(\Delta U)exp\left[-\beta (\Delta U+{\mu }_{0,1}^{ex})\right]$ (filled squares), obtained from amber03. (b) Logarithm of the ratio of the normalized distributions of insertion and removal energies for transitions between N=0 and 1 (filled circles), 1 and 2 (filled squares), 2 and 3 (open squares), and 3 and 4 (open circles). Also shown are straight-line fits with fixed slope β, and intercept $\beta {\mu }_{N,N+1}^{ex}$.

Figure 3

Free energy ΔA_n of transferring N water molecules from bulk into the nonpolar cavity of IL-1β, obtained from the logarithm of P(N)/P(0). Results are shown for simulations using the amber03 force field with TIP3P water (circles), amber94 with TIP3P water (squares), and amber94 with SPC/E water (triangles). Lines are guides to the eye.

Figure 4

(a) Energy ΔU_n /N and (b) entropy ΔS_n /Nk_B per molecule of transferring N water molecules from bulk into the nonpolar cavity of IL-1β, estimated under the assumption of negligible protein reorganization energy. Results are shown for simulations using the amber03 force field with TIP3P water (circles), and the amber94 force field with TIP3P (squares) and SPC/E water (triangles).

Figure 5

Free energies ΔA_n of transferring N water molecules from bulk into the polar cavities 1-4 of IL-1β, numbered according to Quillin et al., obtained from simulations using the amber94 force field with TIP3P water. Lines are guides to the eye.

Figure 6

NMR NOE analysis. The effective distance between water protons and the methyl protons of nonpolar residues lining the cavity is shown for the MD simulations with an empty nonpolar cavity (blue) using actual proton distances (open triangles) and methyl carbon-oxygen distances (filled circles). Also shown are results for five different crystal structures (red; curves labeled by the corresponding PDB codes) and the NMR structure (green) with only six water molecules, calculated by using the methyl carbon-oxygen distances. Lines are guides to the eye.

Figure 7

Distribution of removal energies for water in the nonpolar cavity of IL-1β. Results are shown for N=1 to 4 (thick lines; curves peaking from right to left). Thin lines show the corresponding Gaussian distributions. The inset shows the distributions on a logarithmic scale.

Figure 8

Hydrogen bonded tetramer formed by four water molecules in the central nonpolar cavity of IL-1β at 298 K.