Disordered water within a hydrophobic protein cavity visualized by x-ray crystallography

Abstract

Water in the hydrophobic cavity of human interleukin 1␤, which was detected by NMR spectroscopy but was invisible by high resolution x-ray crystallography, has been mapped quantitatively by measurement and phasing of all of the low resolution x-ray diffraction data from a single crystal. Phases for the low resolution data were refined by iterative density modification of an initial f lat solvent model outside the envelope of the atomic model. The refinement was restrained by the condition that the map of the difference between the electron density distribution in the full unit cell and that of the atomic model be f lat within the envelope of the well ordered protein structure. Care was taken to avoid overfitting the diffraction data by maintaining phases for the high resolution data from the atomic model and by a resolution-dependent damping of the structure factor differences between data and model. The cavity region in the protein could accommodate up to four water molecules. The refined solvent difference map indicates that there are about two water molecules in the cavity region. This map is compatible with an atomic model of the water distribution refined by using XPLOR. About 70% of the time, there appears to be a water dimer in the central hydrophobic cavity, which is connected to the outside by two constricted channels occupied by single water molecules Ϸ40% of the time on one side and Ϸ10% on the other.Analysis of NMR data from solutions of human interleukin-1␤ (hIL-1␤) has indicated that the hydrophobic cavity in the center of the protein contains positionally disordered water (1). However, this water has escaped detection by four independent high resolution (Ϸ2 Å) crystallographic studies (2-5). On the basis that no water molecules were observed crystallographically in this or similar hydrophobic protein cavities, Matthews et al. (6) have questioned the interpretation of the observed nuclear Overhauser enhancement cross peaks attributed to water molecules that are within Ϸ5 Å of protons of the cavity-forming aliphatic residues.Because the existence of positionally disordered water in hydrophobic protein cavities has been controversial (and because such mobile water can be crucially involved in protein dynamics), we have critically assessed the information available from x-ray crystallography regarding the distribution of disordered solvent in an hIL-1␤ crystal. Refinement of the phases for all of the low resolution data, from 55-to 4.5-Å spacing, restrained by the information about the well ordered protein structure from the high resolution (2.3 Å) data, demonstrates that crystallographically elusive solvent, which is detectable by NMR, can be visualized quantitatively in a difference electron density map. The solvent density distribution is mapped from the difference between the average structure of the whole crystal, calculated from the completely phased data, and that of the atomic model of the ordered protein structure, refined from the high resolution data.Atomic coordinates, occupancies...

Cooperative water filling of a nonpolar protein cavity observed by high-pressure crystallography and simulation

“…Despite the general success of hydrophobic models (1-3), attempts to determine the interior ''hydrophobicity'' of proteins have often given conflicting results (2,(4)(5)(6)(7)(8)(9)(10). Some proteins seem to require water at least transiently in nonpolar cavities for their function (11,12).…”

Consequences of Water between Two Hydrophobic Surfaces on Adhesion and Wetting

“…X-ray crystallography has observed the presence of water within protein cavities of varying hydrophobicity which can affect the strength of protein−ligand binding. 21,22 Simulations have also shown that water can be sequestered between hydrophobic plates with a relatively small centralized hydrophilic patch. 23 Ambiguity also remains as to how dry contact is established underwater.…”

Gating of proton and water transfer in the respiratory enzyme cytochrome c oxidase

“…This important finding implies that the product water indeed takes a very specific route out of the heme a 3 ͞Cu B site, which is next to a hydrophobic cavity that is predicted to at least transiently hold several water molecules (2-4, 17, 18), although they have not been observed in the crystal structures. Hydrophobic cavities in proteins may indeed contain water that is sufficiently mobile to escape detection by x-ray crystallography but may be found by NMR spectroscopy (19). However, there is no obvious route in the static x-ray structures of cytochrome c oxidase by which the product water would be specifically transferred from the heme a 3 ͞Cu B center to the Mg(Mn) site, although a transfer pathway further out from the latter site has been described (20).…”