De Novo Design of an Allosteric Metalloprotein Assembly with Strained Disulfide Bonds

Abstract

A major goal in metalloprotein design is to build protein scaffolds from scratch that allow precise control over metal coordination. A particular challenge in this regard is the construction of allosteric systems in which metal coordination equilibria are coupled to other chemical events that take place elsewhere in the protein scaffold. We previously developed a metal-templated self-assembly strategy (MeTIR) to build supramolecular protein complexes with tailorable interfaces from monomeric building blocks. Here, using this strategy, we have incorporated multiple disulfide bonds into the interfaces of a Zn-templated cytochrome cb₅₆₂ assembly in order to create mechanical strain on the quaternary structural level. Structural and biophysical analyses indicate that this strain leads to an allosteric system in which Zn²⁺ binding and dissociation are remotely coupled to the formation and breakage of a disulfide bond over a distance of >14 Å. The breakage of this strained bond upon Zn²⁺ dissociation occurs in the absence of any reductants, apparently through a hydrolytic mechanism that generates a sulfenic acid/thiol pair.

Figure 1

(a) Scheme showing structural rearrangements in the tetramer Zn-^C81/C96R1₄ upon Zn²⁺ removal (PDB IDs: 4JE9 and 4JEA). The hydrophobic residues in the i1 interface are shown as cyan sticks. A38-A38 residue pairs are indicated. (b) Close-up view of A38-A38 residue pairs. Dashed lines denote α-C separations.

Figure 2

(a) Non-reducing SDS-PAGE of ^C38/C81/C96R1₄ in the absence and presence of DTT. (b) Sedimentation velocity profile of ^C38/C81/C96R1₄ in the presence of excess EDTA or 1 equivalent of Zn²⁺. (c) Zn-binding isotherm of ^C38/C81/C96R1 determined using Fura-2 as a competing ligand. Vertical lines indicate equivalents of Zn²⁺ added in excess of Fura-2.

Figure 3

(a) X-ray crystal structures of Zn-^C38/C81/C96R1₄ and the metal-free ^C38/C81/C96R1₄. The broken C38-C38 disulfide bond is highlighted with a blue box. Disulfide bonds, Zn-binding sites, and the engineered hydrophobic mutations in i1 (cyan) are shown as sticks. (b) Scheme highlighting rearrangements of interfacial residues in Zn-^C38/C81/C96R1₄ upon Zn²⁺ removal. (c) Close-up view of the broken C38-C38 disulfide bond, forming a cysteine (CYS) and cysteine sulfenic acid (CSO). A water molecule H-bonded to CYS 38 is shown as a pink sphere. (d) Hydrolytic dissociation of a disulfide bond into a sulfenic acid-thiol pair. (e) Close-up view of the W66-W66 π-stacking interaction. All 2F_o-F_c electron density maps are contoured at 1σ (yellow) and 2.5 σ (black).