Interactions between Hofmeister anions and the binding pocket of a protein

Abstract

This paper uses the binding pocket of human carbonic anhydrase II (HCAII, EC 4.2.1.1) as a tool to examine the properties of Hofmeister anions that determine (i) where, and how strongly, they associate with concavities on the surfaces of proteins and (ii) how, upon binding, they alter the structure of water within those concavities. Results from X-ray crystallography and isothermal titration calorimetry show that most anions associate with the binding pocket of HCAII by forming inner-sphere ion pairs with the Zn(2+) cofactor. In these ion pairs, the free energy of anion-Zn(2+) association is inversely proportional to the free energetic cost of anion dehydration; this relationship is consistent with the mechanism of ion pair formation suggested by the "law of matching water affinities". Iodide and bromide anions also associate with a hydrophobic declivity in the wall of the binding pocket. Molecular dynamics simulations suggest that anions, upon associating with Zn(2+), trigger rearrangements of water that extend up to 8 Å away from their surfaces. These findings expand the range of interactions previously thought to occur between ions and proteins by suggesting that (i) weakly hydrated anions can bind complementarily shaped hydrophobic declivities, and that (ii) ion-induced rearrangements of water within protein concavities can (in contrast with similar rearrangements in bulk water) extend well beyond the first hydration shells of the ions that trigger them. This study paints a picture of Hofmeister anions as a set of structurally varied ligands that differ in size, shape, and affinity for water and, thus, in their ability to bind to—and to alter the charge and hydration structure of—polar, nonpolar, and topographically complex concavities on the surfaces of proteins.

Figure 1.

The model system. (A) The Hofmeister series: anions ranked according to their propensity to precipitate proteins from aqueous solution. In this study, we examined the following anions: SO₄²⁻, HPO₄²⁻, CH₃COO^X, HCO₃⁻, Cl⁻, Br⁻, NO₃⁻, I⁻, ClO₄⁻, and SCN⁻. (B) The association of anions with the Zn²⁺ cofactor involves two states: an initial state (left) with the anion and protein in aqueous solution, and a final state (right) with the anion–protein complex in aqueous solution. Thermodynamic parameters measured with ITC (ΔJ°_bind, where J = H, TS, or G) represent a difference between the initial and final states.

Figure 2.

Thermodynamics of anion binding. (A) A plot showing thermodynamic parameters for the association of anions and HCAII (298.15 K, pH = 7.6, 10 mM sodium phsophate buffer; the process depicted in Figure 1B). H/S compensation, revealed by the plot, often arises from rearrangements in the organization of waters that solvate interacting species. (B) A comparison of free energies of hydration (ΔG°_hydration) with free energies of binding (ΔG°_bind,anion). Values of ΔG°_bind,anion decrease linearly with ΔG°_hydration (R² = 0.83), suggesting that anions with a lower free energetic cost of dehydration bind more tightly to the Zn²⁺ cofactor. Values of ΔG°_hydration are taken from Marcus. Error bars represent standard error (n = 23 for the association of HCAII and BTA in the absence of anions, and n ≥ 7 for the association of HCAII and BTA in the presence of each anion; see SI Methods).

Figure 3.

Structural basis of anion binding. (A) X-ray crystal structure of the active site of HCAII complexed with SCN⁻ (PDB entry 4YGK). Both ClO₄⁻ and SCN⁻ displace H₂O-338 (the “so-called” deep water, displayed in SI Figure S3) and shift the position of H₂O-263 (the catalytically important Zn²⁺-bound water). (B) X-ray crystal structure of the active site of HCAII complexed with iodide (PDB entry 4YGN) sites are further elaborated in Appendix 3 of the SI). Iodide sites are numbered in order of their proximity of the Zn²⁺-bound cofactor. I-1 and I-2 denote alternative binding sites for the Zn²⁺-bound iodide (an inner-sphere ion pair and an outer-sphere ion pair, respectively). I-3 denotes a binding site at the border of the hydrophobic and hydrophilic surfaces. I-4 denotes a binding site in the hydrophobic wall. Colors represent amino acids as follows: cyan (within 5 Å of I-3), light purple (within 5 Å of I-4), green (within 5 Å of both I-3 and I-4). (C) A detail of the I-3 binding site. Carbon atoms within 5 Å of the iodide are colored cyan. (D) A detail of the I-4 binding site. Carbon atoms within 5 Å of the iodide are colored light purple. In both (C) and (D), the iodide anions in the I-3 and I-4 positions, respectively, and the Zn²⁺ cofactor are shown as spheres that indicate their solvent-accessible surface area (i.e., the ion/water contact surface); the surface of the protein is also represented in this way.

Figure 4.

Results from WaterMap calculations. (A) A plot showing the contribution of anion-induced rearrangements of water inside the binding pocket of HCAII to the thermodynamics of anion–Zn²⁺ association. Values of ΔJ°_{WM, anion} represent the total difference of thermodynamic properties (enthalpies, entropies, and free energies) of waters in anion-bound and anion-free binding pockets (ΔJ°_WM,anion = ΔJ°_WM,HCA-anion −ΔJ°_WM,HCA, where J = H, TS, or G). (B) A schematic defining regions for calculating ΔH°_WM,anion(d) and −TΔS°_WM,anion(d), the enthalpy and entropy, respectively, associated with rearrangements of water (resulting from anion–Zn²⁺ association) occurring beyond d Å from the surface of the Zn²⁺-bound anion (i.e., waters located between a distance of d Å from the Zn²⁺-bound anion and the edge of the binding pocket). Calculations are based on crystal structures of anion-HCAII complexes. (C) A plot showing values of ΔH°_WM,anion(d) and −TΔS°_WM,anion(d) for the binding of SCN⁻ to Zn²⁺. This plot suggests that SCN⁻ triggers rearrangements of water that extend up to 8 Å from its surface.

Figure 5.

Rearrangements of water in the I-4 binding pocket. (A-B) WaterMap results for the I-4 binding pocket shown in Figure 3D: (top) without iodide bound and (bottom) with iodide bound. Waters are colored according to (A) their enthalpies (ΔH°_WM) and (B) their entropies (−TΔS°_WM), relative to bulk water. Results suggest that the binding of iodide to the I-4 binding pocket causes displacement of two enthalpically and entropically unstable (relative to bulk water) molecules of water (circled and labeled with their corresponding thermodynamic quantities). In all images, the surfaces of the protein (gray) and iodide anion (purple) represent the protein/water and ion/ water contact surfaces, respectively.