Overcoming universal restrictions on metal selectivity by protein design

Selective metal coordination is central to the functions of metalloproteins:^1,2 each metalloprotein must pair with its cognate metallocofactor to fulfil its biological role³. However, achieving metal selectivity solely through a three-dimensional protein structure is a great challenge, because there is a limited set of metal-coordinating amino acid functionalities and proteins are inherently flexible, which impedes steric selection of metals^3,4. Metal-binding affinities of natural proteins are primarily dictated by the electronic properties of metal ions and follow the Irving-Williams series⁵ (Mn²⁺ < Fe²⁺ < Co²⁺ < Ni²⁺ < Cu²⁺ > Zn²⁺) with few exceptions^6,7. Accordingly, metalloproteins overwhelmingly bind Cu²⁺ and Zn²⁺ in isolation, regardless of the nature of their active sites and their cognate metal ions^1,3,8. This led organisms to evolve complex homeostatic machinery and non-equilibrium strategies to achieve correct metal speciation^1,3,8-10. Here we report an artificial dimeric protein, (AB)₂, that thermodynamically overcomes the Irving-Williams restrictions in vitro and in cells, favouring the binding of lower-Irving-Williams transition metals over Cu²⁺, the most dominant ion in the Irving-Williams series. Counter to the convention in molecular design of achieving specificity through structural preorganization, (AB)₂ was deliberately designed to be flexible. This flexibility enabled (AB)₂ to adopt mutually exclusive, metal-dependent conformational states, which led to the discovery of structurally coupled coordination sites that disfavour Cu²⁺ ions by enforcing an unfavourable coordination geometry. Aside from highlighting flexibility as a valuable element in protein design, our results illustrate design principles for constructing selective metal sequestration agents.