Computationally derived structural insights into Rare Earth selectivity in lanmodulin and its variants

Understanding rare earth element (REE) binding to proteins enables the engineering of selective protein-based ligands for precise REE recovery. Lanmodulin (LanM), with notable REE selectivity and picomolar binding affinity, is a promising candidate. This study shows that LanM variants employ distinct inter-residue interactions for REE binding. We detail the thermodynamics and structural aspects of binding events in wild-type (WT) Methylorubrum extorquens LanM and five EF-hand residue variants (4P₂A and 4D₉X, X = N, A, H, M), using protein variant structure prediction, molecular dynamics simulations and binding motif exploration. We demonstrate strong agreement between experimental binding measurements (apparent K ) and in silico binding energy scores of WT, 4 P₂A, and 4D₉X LanMs. We systematically investigate the role of solvent dielectric, sample multiple force fields, and initial protein structure bias on metal ion-binding energetics. In addition, we identify amino acids outside the direct metal binding motif crucial for coordinating the binding events which is corroborated with experimental binding characteristics of 4D₉X variants. Computationally measured binding affinity with contribution from this secondary set of residues show agreement with the experimental K _d values and suggests how some point mutations can induce long-range structural perturbations to regulate metal ion-protein recognition and interactions. Finally, we analyze structural changes arising from alterations in side-chain flexibility of each amino acid on the protein backbone at the instant of metal binding and recognition - which manifests as altered helicity at a specific locus of the protein, a result that is corroborative of the observations from circular dichroism experiments.