Solution NMR structure of a designed metalloprotein and complementary molecular dynamics refinement

Abstract

We report the solution NMR structure of a designed dimetal-binding protein, di-Zn(II) DFsc, along with a secondary refinement step employing molecular dynamics techniques. Calculation of the initial NMR structural ensemble by standard methods led to distortions in the metal-ligand geometries at the active site. Unrestrained molecular dynamics using a nonbonded force field for the metal shell, followed by quantum mechanical/molecular mechanical dynamics of DFsc, were used to relax local frustrations at the dimetal site that were apparent in the initial NMR structure and provide a more realistic description of the structure. The MD model is consistent with NMR restraints, and in good agreement with the structural and functional properties expected for DF proteins. This work demonstrates that NMR structures of metalloproteins can be further refined using classical and first-principles molecular dynamics methods in the presence of explicit solvent to provide otherwise unavailable insight into the geometry of the metal center.

Figure 1. Di-Zn(II) DFsc NMR Structure

(A) α carbon trace of the ensemble of di-Zn(II) DFsc NMR structures. (B) Sausage representation of the 36 NMR structures of di-Zn(II) DFsc. The color and tube size are based on the B factor, where the color scale is from dark blue to red, with red corresponding to the highest B factor and dark blue to the lowest B factor. (C) Target designed model (orange) of DFsc overlaid with the lowest-energy NMR structure (gray). (D) Amino acid sequence of DFsc; blue-highlighted residues consist of the primary coordination sphere. All figures were generated with PyMOL (DeLano, 2002) unless otherwise noted.

Figure 2. Superposition of di-Zn(II) DFsc Models and Structure

The di-Zn(II) DFsc target design model (orange) was superimposed versus (A) di-Zn(II) DFsc calculated NMR structure (gray), rmsd 1.2 Å and (B) average MD model of di-Zn(II) DFsc (green), rmsd 0.8 Å. The superpositions demonstrate the large movement of helix 2 in the di-Zn(II) DFsc calculated NMR structure relative to the targeted design and MD model. Helices 1, 3, and 4 are included in the superpositions: residues 6–16, 69–82, and 99–112; the loops are not included.

Figure 3. Surface Representation of di-Zn(II) DFsc Displaying the Accessibility to the Active Site

(A) Target design model of di-Zn(II) DFsc. (B) Calculated NMR structure of di-Zn(II) DFsc, where the metal site is inaccessible owing to the shift in helix 2. (C) MD model of di-Zn(II) DFsc. All the atoms of the protein are colored gray and the metal ions and primary ligands are shown in magenta. Hydrogen atoms are not depicted in this figure.

Figure 4. B Factors of the MD Model of di-Zn(II)

(A) MD model of di-Zn(II) DFsc. The color scale is from blue to red, where red corresponds to the highest B factor and blue to the lowest B factor. (B) Plot of MD-based B factor/Ų versus residue number.

Figure 5. Di-Zn(II) Cluster in the Average MD Model

Dashed lines indicate hydrogen bonding. The Zn(II)-Zn(II) distance is 4.2 Å. Compared to the calculated NMR structure, E11 undergoes a carboxylate shift to monodentate and two water molecules solvate the dimetal site. Hydrogen bonds between the water molecules and ligands are shown as red dashed lines.