Characterizing metal-binding sites in proteins with X-ray crystallography

Abstract

Metals have crucial roles in many physiological, pathological, toxicological, pharmaceutical, and diagnostic processes. Proper handling of metal-containing macromolecule samples for structural studies is not trivial, and failure to handle them properly is often a source of irreproducibility caused by issues such as pH changes, incorporation of unexpected metals, or oxidization/reduction of the metal. This protocol outlines the guidelines and best practices for characterizing metal-binding sites in protein structures and alerts experimenters to potential pitfalls during the preparation and handling of metal-containing protein samples for X-ray crystallography studies. The protocol features strategies for controlling the sample pH and the metal oxidation state, recording X-ray fluorescence (XRF) spectra, and collecting diffraction data sets above and below the corresponding metal absorption edges. This protocol should allow experimenters to gather sufficient evidence to unambiguously determine the identity and location of the metal of interest, as well as to accurately characterize the coordinating ligands in the metal binding environment within the protein. Meticulous handling of metal-containing macromolecule samples as described in this protocol should enhance experimental reproducibility in biomedical sciences, especially in X-ray macromolecular crystallography. For most samples, the protocol can be completed within a period of 7-190 d, most of which (2-180 d) is devoted to growing the crystal. The protocol should be readily understandable to structural biologists, particularly protein crystallographers with an intermediate level of experience.

Figure 1.. Overview of the protocol depicted in a flow chart.

The anticipated timing is denoted on the top of each stage.

Figure 2.. Representative structures of horse serum albumin (ESA)-Zn²⁺ complexes showing the dynamic behavior of His-247.

Both structures were crystallized in 100 mM Tris buffer, and the pH refers to the final pH in the crystallization drop. (a) ESA-Zn²⁺, 2.5 mM Zn²⁺, pH 7.4; PDB ID: 5IIH. (b) ESA-Zn²⁺, 15 mM Zn²⁺, pH 6.5; PDB ID: 5IIX. Residues are shown as sticks, zinc ion in gray, oxygen in red, nitrogen in dark blue, and carbon in green. Coordination bonds are marked with black dashed lines. Gray grid represents 2mF_o – DF_c map (σ – 1.0), orange – anomalous map (σ – 3.0). Data for this figure were taken from Handing K.B., et al.

Figure 3.. The influence of transition metals on the stability of STM1931 protein from S. typhimurium analyzed by thermal shift assay (TSA) experiments.

The STM1931 protein in the presence of 189 mM NaCl was used as a reference. The presence of 0.273 mM CdCl₂ decreased the stability of this protein, while the concentrations of 4.38 mM of ZnCl₂ and 2.35 mM of (NH₄)₂MoO₄ (ammonium molybdate) increased the stability of the protein. The diffraction experiments showed that the C103A mutant of STM1931 protein from S. typhimurium crystallized in the presence of zinc ions (PDB ID: 4K2H).

Figure 4.. Fluorescence spectra, fluorescence scan, and electron density maps of a zinc-containing protein dihydroorotase from Yersinia pestis CO92.

(a) X-ray fluorescence spectrum of the sample excited at the selenium K-edge (12664 eV). The peak around 12600 eV corresponds to the energy of the incident X-ray beam. The peak around 8600 eV corresponds to the Kα emission energy of zinc. The peak at 3300 eV may correspond to the Kα emission energy of potassium. The small peak at 6400 eV may correspond to the Kα emission energy of iron present in the loop pin. (b) Fluorescence spectra of the crystal at excitation energies below (9618 eV – orange) and above (9668 eV – blue) the theoretical value of zinc absorption K-edge (9659 eV). The peaks at 3300 eV and 6400 eV are similar to those in panel a. (c) X-ray fluorescence scan of the sample at zinc K-edge region. The f” plot and the observed absorption edge (9663 eV, or 1.283 Å) correspond to the table values for zinc K-edge. (d) Models and the electron density maps based on the data collected above the zinc absorption edge (left, model in cyan) and below the edge (right, model in yellow). Residues are shown in sticks, zinc ion in gray, oxygen in red, nitrogen in dark blue, carbon in cyan or yellow. Gray grid represents 2mF_o – DF_c map (σ – 1.5), pink - anomalous map (σ – 4.0).

Figure 5.. Use of restraints for a zinc binding site in horse serum albumin (ESA).

(a) 2Fo-Fc map with model and anomalous map, no metal. (b) Fragment of restraint file with LINK field present. (c) Refined model with metal and distances between ligands and metal shown. Residues are shown in sticks, oxygen in red, nitrogen in dark blue, carbon in cyan, water shown as a red sphere, and zinc shown as a gray sphere. Coordination bonds are marked with black dashed lines. Gray grid represents 2mF_o – DF_c map (σ – 1.0), pink - anomalous map (σ – 3.0).

Figure 6.

An example of a validation report for a zinc binding site ESA.