The Role of Asparagine as a Gatekeeper Residue in the Selective Binding of Rare Earth Elements by Lanthanide-Binding Peptides.

Lanthanide-binding tag (LBT) peptides selectively complex lanthanide cations (Ln) in their binding pockets and are promising for lanthanide separation. However, designing LBTs that selectively target specific Ln cations remains a challenge due to limited molecular-level understanding and control of interactions within the lanthanide-binding pocket. In this study, we reveal that the N5 asparagine residue acts as a gatekeeper in the binding pocket, resulting in a 100-fold selectivity for smaller Lu over larger La cations. Nuclear magnetic resonance spectroscopy and molecular dynamics simulations show that the N5 residue weakly binds to the larger La cation, permitting HO molecules inside the pocket. For the smaller Lu cations, the N5 residue forms an inter-arm hydrogen bond with the E14 glutamic acid residue, locking the Lu cation in the pocket and preventing HO infiltration. Mutating the N5 asparagine to a D5 aspartic acid prevents such a hydrogen bond, eliminating the gatekeeping mechanism and precipitously reducing selectivity. The resulting binding affinity to Ln cations is non-monotonic but generally increases with cation size. These results suggest a molecular design paradigm: the reduced affinity for larger lanthanides is due to open pocket conformations, while the selectivity of smaller Ln cations over larger ones is due to the gatekeeping hydrogen bond.