Crystallographic study of hydration of an internal cavity in engineered proteins with buried polar or ionizable groups

Abstract

Although internal water molecules are essential for the structure and function of many proteins, the structural and physical factors that govern internal hydration are poorly understood. We have examined the molecular determinants of internal hydration systematically, by solving the crystal structures of variants of staphylococcal nuclease with Gln-66, Asn-66, and Tyr-66 at cryo (100 K) and room (298 K) temperatures, and comparing them with existing cryo and room temperature structures of variants with Glu-66, Asp-66, Lys-66, Glu-92 or Lys-92 obtained under conditions of pH where the internal ionizable groups are in the neutral state. At cryogenic temperatures the polar moieties of all these internal side chains are hydrated except in the cases of Lys-66 and Lys-92. At room temperature the internal water molecules were observed only in variants with Glu-66 and Tyr-66; water molecules in the other variants are probably present but they are disordered and therefore undetectable crystallographically. Each internal water molecule establishes between 3 and 5 hydrogen bonds with the protein or with other internal water molecules. The strength of interactions between internal polar side chains and water molecules seems to decrease from carboxylic acids to amides to amines. Low temperature, low cavity volume, and the presence of oxygen atoms in the cavity increase the positional stability of internal water molecules. This set of structures and the physical insight they contribute into internal hydration will be useful for the development and benchmarking of computational methods for artificial hydration of pockets, cavities, and active sites in proteins.

FIGURE 1

(A) Stereo view of electron density maps of the Tyr-66 protein at room temperature after molecular replacement, contoured over the final refined coordinates. The Tyr side chain and water molecules 1 and 2 are unambiguously shown in the 1.25 σ 2F_o–F_c (blue) and 3.0 σ F_o–F_c (orange) electron density. (B) Ribbon representation of the final structure of the variant with Tyr-66 at room temperature with water molecules 1 and 2 displayed.

FIGURE 2

(A) Stereo view of composite representation of all the internal water molecules observed in variants of SNase with Glu, Gln, Asp, Asn, or Tyr at positions 66 or 92. Water molecules are represented as labeled blue spheres. Glu-66 is displayed, with carboxylic oxygens in red, and Ile-92 is shown in yellow. (B) Composite representation of the potential hydrogen bonding interactions of the internal water molecules as observed for waters in sites 1, 2, 3, 4, and 7 in the structure with I92E (yellow), for waters in sites 5 and 6 in the structure with I92D (orange), and for water in site 8 for the structure with V66D (green). All distances are expressed in Å.

FIGURE 3

(A) Superposition of side chains and water molecules near Glu-66 at 100 K (blue) and 298 K (cyan) and near Gln-66 at 100 K (orange) and 296 K (yellow). (B) Superposition of side chains and water molecules near Asp-66 at 100 K (blue) and 298 K (cyan) and near Asn-66 at 100 K (orange) and 296 K (yellow). (C) Superposition of side chains and water molecules near Glu-66 at 100 K (blue) and near Tyr-66 at 100 K (orange) and 296 K (yellow). Water molecules at sites 1, 2, 3, 7, and 8 are labeled.