UC San Diego

Proteins are one of the main building blocks of life. Among their numerous functions are roles as biocatalysts for essential chemical reactions, signaling agents that coordinate communication down to the sub-cellular level, and as modules for constructing cellular superstructures. In many cases, these functions rely on the ability of proteins to form stable and specific associative interactions with other proteins, as well as with metal ions. Drawing inspiration from nature, we deploy these functional features as design elements that aid in the construction of artificial metalloprotein assemblies. Using an iterative design approach, we have engineered the monomeric protein cytochrome cb562 by application orthogonal strategies: Metal Designed Protein Self-Assembly (MDPSA), Metal-Templated Interface Redesign (MeTIR), and the installation of intermolecular disulfide bond crosslinks. This synergistic design approach afforded a self-assembling protein variant, C38/C81/C96R1, which bears metal chelating groups, a designed dimerization interface (termed i1), and surface-exposed cysteine residues. In the presence of ZnII this engineered variant self-assembled into a tetramer, Zn-C38/C81/C96R14. Removal of ZnII from the assembled tetramer resulted in hydrolysis of a single C38-C38 disulfide bond, leaving the five remaining crosslinks intact. Thus, C38/C81/C96R14 is an allosteric protein, capable of remotely coupling ZnII binding at is central binding sites to the breakage of a peripheral disulfide bond. In this work, we discuss the design of the C38/C81/C96R14 protein as well as the demonstration of this allosteric behavior. We further carry out biochemical and biophysical characterization of this protein and related variants to determine the structural and energetic basis of this Zn-disulfide allostery. The C38-C38 disulfide bonds are embedded in the i1interface which, crucially, forms malleable protein-protein contacts. The flanking disulfide crosslinks of C38/C81/C96R14 serve as structural conduits for coupling the pair of i1 interfaces, which is critical to develop the requisite driving force to effect disulfide bond hydrolysis. In this designed system, it is the underlying malleability of the structure that gives rise to the allosteric behavior. We consider this as a successful application of synergistic design to give a coordinated protein function, and anticipate that adoption of similar design paradigms will greatly benefit ongoing protein engineering efforts in the community at large.