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) (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 organismsto 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 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.

Automated summary

Metalloproteins rely on selective metal coordination for their functions, with natural proteins predominantly binding Cu2+ and Zn2+ ions. An artificial dimeric protein was designed to favor lower-Irving-Williams transition metals over Cu2+, showcasing the importance of flexibility in protein design and offering insights for constructing selective metal sequestration agents.