Metal templated design of protein interfaces

Abstract

Metal coordination is a key structural and functional component of a large fraction of proteins. Given this dual role we considered the possibility that metal coordination may have played a templating role in the early evolution of protein folds and complexes. We describe here a rational design approach, Metal Templated Interface Redesign (MeTIR), that mimics the time course of a hypothetical evolutionary pathway for the formation of stable protein assemblies through an initial metal coordination event. Using a folded monomeric protein, cytochrome cb(562), as a building block we show that its non-self-associating surface can be made self-associating through a minimal number of mutations that enable Zn coordination. The protein interfaces in the resulting Zn-directed, D(2)-symmetrical tetramer are subsequently redesigned, yielding unique protein architectures that self-assemble in the presence or absence of metals. Aside from its evolutionary implications, MeTIR provides a route to engineer de novo protein interfaces and metal coordination environments that can be tuned through the extensive noncovalent bonding interactions in these interfaces.

Fig. 1.

(A) Cartoon outline for MeTIR and the species involved. Red arrows represent engineering or hypothetical evolutionary steps. Red and blue spheres represent metal ions 1 and 2 with different preferential coordination geometries. 1. Protein/peptide with a non-self-associating surface; 2. 1 modified with metal-coordinating groups; 3. initial metal1-templated protein/peptide complex with noncomplementary interfaces; 4. metal1-templated protein/peptide complex with optimized, complementary interfaces; 5. protein/peptide with a self-associating surface; 6. metal-independent protein/peptide complex biased towards metal1 binding; 7. protein/peptide complex with distorted metal2 coordination. (B) The structure of MBPC-1, corresponding to species 2, showing the two i, i + 4 diHis motifs on helix3.

Fig. 2.

Three pairs of interfaces (i1, i2, i3) formed within the D₂-symmetrical Zn₄:MBPC-1₄ tetramer; the Zn coordination environment in each interface is listed below.

Fig. 3.

Side chain conformations in the core regions of interfaces i1 and i2 in (A) Zn₄:MBPC-1₄, (B) the Rosetta-calculated model, and (C) Zn₄:RIDC-2₄. Highlighted are six positions in each interface that were subjected to redesign, as well as those involved in Zn coordination. Water molecules observed in the Zn₄:RIDC-2₄ structure are shown as small red spheres.

Fig. 4.

(A) From left to right: sedimentation coefficient distributions for various concentrations of MBPC-1, RIDC-1 and RIDC-2 in the presence of equimolar Zn(II). (B) Backbone superposition of Zn₄:MBPC-1₄ (green), Zn₄:RIDC-1₄ (blue), and Zn₄:RIDC-2₄. (C) Tetrahedral Zn-coordination environment in Zn₄:RIDC-2₄, with the corresponding F_o-F_c omit electron density map (3.2σ).

Fig. 5.

(A) Sedimentation coefficient distributions for various concentrations of RIDC-1 in the presence of 5 mM EDTA, showing the population of the dimeric species at increasing concentrations. (B) Representative sedimentation equilibrium profile for RIDC-1 (see Fig. S4 for the complete set of measurements). Shown here are the scans for 20 μM RIDC-1 in 20 mM Tris (pH 7) and 5 mM EDTA collected at 20,000, 25,000, and 30,000 rpm, which are best fit with a monomer-dimer equilibrium model. (C) Side and top views of the RIDC-1₂ crystal structure, whereby the interfacial residues are shown as sticks. (D) Close-up views of the two (nearly) symmetrical interaction zones in the dimer interface detailing the hydrophobic and H-bonding contacts.

Fig. 6.

(A) The influence of Zn-templated interfacial mutations in i1 on the conformations of Cu-mediated dimeric assemblies. (B) Backbone superposition of Cu₂:RIDC-1₂ (gray) and a dimeric half of Zn₄:RIDC-1₄ (orange) that contains i1. Interfacial residues of Cu₂:RIDC-1₂ are shown as sticks; see Fig. S3 for detailed comparison of Cu₂:RIDC-1₂ and Zn₄:RIDC-1₄ interfaces. (C) Cu coordination environment in Cu₂:RIDC-1₂ (Site 1), highlighting the open coordination sites occupied by two water molecules. The Glu81 side chain from a crystallographic symmetry-related dimer that forms H-bonds to the coordinated water molecules is shown in light gray. The F_o-F_c omit electron density map is contoured at 3σ.