Evolution of Metal Selectivity in Templated Protein Interfaces

Abstract

Selective binding by metalloproteins to their cognate metal ions is essential to cellular survival. How proteins originally acquired the ability to selectively bind metals and evolved a diverse array of metal-centered functions despite the availability of only a few metal-coordinating functionalities remains an open question. Using a rational design approach (Metal-Templated Interface Redesign), we describe the transformation of a monomeric electron transfer protein, cytochrome cb 562 , into a tetrameric assembly ( C96 RIDC-1) that stably and selectively binds Zn 2+ , and displays a metal-dependent conformational change reminiscent of a signaling protein. A thorough analysis of the metal binding properties of C96 RIDC-1 4 reveals that it can also stably harbor other divalent metals with affinities that rival (Ni 2+ ) or even exceed (Cu 2+ ) those of Zn 2+ on a per site basis. Nevertheless, this analysis suggests that our templating strategy also introduces an increased bias towards binding a higher number of Zn 2+ ions (4 high affinity sites) versus Cu 2+ or Ni 2+ (2 high affinity sites), ultimately leading to the exclusive selectivity of C96 RIDC-1 4 for Zn 2 over those ions. More generally, our results indicate that an initial metal-driven nucleation event followed by the formation of a stable protein architecture around the metal provides a straightforward path for generating structural and functional diversity.

An approach to crystallizing proteins by metal‐mediated synthetic symmetrization

“…Such coordination geometries are commonly found in proteins 33,54 and have been engineered previously. [37][38][39][40][41][42] On the basis of the crystal structures of T4L and MBP in each protein, we chose three pairs of solvent-accessible residues that are located close to the ends of helices (Fig. 1).…”

“…37 The crystal structure of this complex reveals a trimer of Atx1 molecules mediated through the bound tetrathiomolybdate molecule. Tezcan and coworkers [38][39][40][41][42] have studied metaldirected protein self-assembly on the model protein, cytochrome b 562 , focusing primarily on the evolution of metal coordination in protein folds and complexes. Their work has shown that by introducing histidine mutations on the alpha helical surface of cytochrome b 562 , the protein can oligomerize to form dimers to tetramers, all of which are mediated through metal binding.…”

In Vitro and Cellular Self-Assembly of a Zn-Binding Protein Cryptand via Templated Disulfide Bonds

“…The Zn(II) affinity of C81/C96 RIDC1 4 was determined by a competition titration using the fluorescent metal indicator Fura-2 (Figure S6 of the SI), whose Zn(II) complex has a dissociation constant ( K d,Zn-Fura ) of 5.7 nM. 49 These titrations revealed that C81/C96 RIDC1 4 bound four Zn(II) ions as expected, and the isotherm was well fit by a 2 + 2 Zn equilibrium model (i.e., two pairs of independent sites), with K d1,2Zn = 2.6 ± 0.3 nM and K d2,2Zn = 25 ± 4 nM (Figure 4b). ICP-OES experiments were carried out in parallel to examine the ability of C81/C96 RIDC1 4 to bind various divalent metal ions.…”

“…The change in excitation intensity at 335 nm was plotted versus Zn(II) concentration, and the resulting curve was fit alternatively to 1 × 4 Zn, 2 × 2 Zn, or 4 × 1 Zn binding models using Dynafit 78 as described previously. 49 A similar protocol was used in the case of A74/C81/C96 RIDC1 4 (13.5 μ M) in competition titration experiments with Mag-Fura-2 (Life Technologies) (8.7 μ M, excitation range of 300–400 nm, emission detection at 505 nm). The change in excitation intensity at 370 nm was plotted versus Zn(II) concentration, and the resulting curve was fit alternatively to 1 × 4 Zn, 2 × 2 Zn, or 4 × 1 Zn binding models by Dynafit.…”

The Outer-Coordination Sphere: Incorporating Amino Acids and Peptides as Ligands for Homogeneous Catalysts to Mimic Enzyme Function

“…Also in the area of protein redesign is the work of Tezcan and coworkers (215)(216)(217)(218)(219). In this series of investigations, they begin with a well-structured electron transfer protein, cytochrome cb562, and utilized metals to direct protein assembly.…”