UO₂²⁺ uptake by proteins: Understanding the binding features of the super uranyl binding protein and design of a protein with higher affinity

The capture of uranyl, UO₂²⁺, by a recently engineered protein (Zhou et al. Nat. Chem. 2014, 6, 236) with high selectivity and femtomolar sensitivity has been examined by a combination of density functional theory, molecular dynamics, and free-energy simulations. It was found that UO₂²⁺ is coordinated to five carboxylate oxygen atoms from four amino acid residues of the super uranyl binding protein (SUP). A network of hydrogen bonds between the amino acid residues coordinated to UO₂²⁺ and residues in its second coordination sphere also affects the protein's uranyl binding affinity. Free-energy simulations show how UO₂²⁺ capture is governed by the nature of the amino acid residues in the binding site, the integrity and strength of the second-sphere hydrogen bond network, and the number of water molecules in the first coordination sphere. Alteration of any of these three factors through mutations generally results in a reduction of the binding free energy of UO₂²⁺ to the aqueous protein as well as of the difference between the binding free energies of UO₂²⁺ and other ions (Ca²⁺, Cu²⁺, Mg²⁺, and Zn²⁺), a proxy for the protein's selectivity over these ions. The results of our free-energy simulations confirmed the previously reported experimental results and allowed us to discover a mutant of SUP, specifically the GLU64ASP mutant, that not only binds UO₂²⁺ more strongly than SUP but that is also more selective for UO₂²⁺ over other ions. The predictions from the computations were confirmed experimentally.