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 proteins 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 proteins 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.