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. 2020 Dec 1;59(49):21940-21944.
doi: 10.1002/anie.202009226. Epub 2020 Sep 28.

Metal-Templated Design of Chemically Switchable Protein Assemblies with High-Affinity Coordination Sites

Albert Kakkis  1 , Derek Gagnon  1 , Julian Esselborn  1 , R David Britt  2 , F Akif Tezcan  1
Affiliations

Metal-Templated Design of Chemically Switchable Protein Assemblies with High-Affinity Coordination Sites

Albert Kakkis et al. Angew Chem Int Ed Engl. .

Abstract

To mimic a hypothetical pathway for protein evolution, we previously tailored a monomeric protein (cyt cb562 ) for metal-mediated self-assembly, followed by re-design of the resulting oligomers for enhanced stability and metal-based functions. We show that a single hydrophobic mutation on the cyt cb562 surface drastically alters the outcome of metal-directed oligomerization to yield a new trimeric architecture, (TriCyt1)3. This nascent trimer was redesigned into second and third-generation variants (TriCyt2)3 and (TriCyt3)3 with increased structural stability and preorganization for metal coordination. The three TriCyt variants combined furnish a unique platform to 1) provide tunable coupling between protein quaternary structure and metal coordination, 2) enable the construction of metal/pH-switchable protein oligomerization motifs, and 3) generate a robust metal coordination site that can coordinate all mid-to-late first-row transition-metal ions with high affinity.

Keywords: bioinorganic chemistry; metalloproteins; protein design; protein structures; supramolecular chemistry.

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Figures

Figure 1.
Figure 1.
A) Structural model of TriCyt1 monomer. B) Crystal structure of the NiII:(TriCyt1)3 trimer (PDB ID: 6WZA), which is essentially identical to that of CuII:(TriCyt1)3 (Figure S2, PDB ID: 6X8X). The upper panels show the side- and top-views of the trimer and the bottom panels depict the primary and secondary coordination environments that comprise the “core motif”. The 2Fo–Fc maps (grey mesh) are contoured at 1σ. C) Sedimentation velocity (SV) profiles of TriCyt1 (30 μM monomer) in the absence and presence of 10 μM MnCl2, NiCl2, and CuCl2 (see Figure S4 for a complete set).
Figure 2.
Figure 2.
A) SV profiles of TriCyt2 (30 μM monomer) in the absence and presence of 10 μM MnCl2. B) Structural overlay of FeII:(TriCyt2)3 (grey, PDB ID: 6WZ0) and NiII:(TriCyt1)3 (cyan, PDB ID: 6WZA). C) Hydrophobic packing (left) and H-bonding (right) interactions at the core interface of FeII:(TriCyt2)3. The 2Fo–Fc maps (grey mesh) are contoured at 1σ.
Figure 3.
Figure 3.
A) SV profiles of TriCyt3 in the absence and presence of MnCl2. B) Crystal structure of CoII:(TriCyt3)3 (PDB ID: 6WZ2), highlighting engineered H-bonding/electrostatic interactions in peripheral interfaces. C) His6-MnII coordination environment in MnII:(TriCyt3)3 (PDB ID: 6WZ1). The 2Fo–Fc (grey) and MnII-anomalous difference (purple) maps are contoured at 1σ and 5σ, respectively.
Figure 4.
Figure 4.
A) X-band EPR spectrum of MnII:(TriCyt3)3. B) MnII-binding isotherm for competitive binding titration of TriCyt3 in the presence of Mag-Fura-2. C) Dissociation constants for MII:(TriCyt3)3 complexes determined by competition titrations (see also Figures S14 and S15, and Tables S7 and S8). Metal was added in 2.5 μM increments from 2.5 mM metal chloride stock solutions. The standard errors shown are the standard errors of the fits as calculated by the data fitting program DynaFit (see Supporting Materials and Methods section of the SI).
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