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 role 3 . 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 series 5 (Mn 2+  < Fe 2+  < Co 2+  < Ni 2+  < Cu 2+  > Zn 2+ ) with few exceptions 6,7 . Accordingly, metalloproteins overwhelmingly bind Cu 2+ and Zn 2+ 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 Cu 2+ , 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 Cu 2+ 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.