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 d ) 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.