Understanding the structural principles of REE binding in proteins is essential for developing selective protein-based ligands for efficient REE recovery. Lanmodulin (LanM) proteins, particularly Hans-LanM, offer a promising bio-based alternative for selective REE recovery, motivating computational studies to understand and optimize their binding mechanisms. This study integrates computational and experimental approaches to elucidate the binding preferences of Hans-Lanmodulin (Hans-LanM) for REEs, focusing on binding affinity trends, EF-hand selectivity, and structural stability. We evaluate the correlation between experimental binding measurements (apparent Kd) and computational binding energy scores for WT and R100K Hans-LanM. Our results demonstrate consistent binding affinity rankings, with Dy³⁺ exhibiting weaker binding compared to La³⁺ and Nd³⁺. Competitive binding simulations further highlight dynamic metal-specific recognition, where La³⁺ stabilizes the dimeric conformation while Nd³⁺ and Dy³⁺ induce monomerization. The R100K mutation disrupts EF-hand integrity, reducing lanthanide affinity, particularly for La³⁺, underscoring the critical role of residue-specific interactions such as R100–D93 electrostatic coupling. Additionally, we performed PyRosetta-based alanine scanning mutagenesis of EF-hand motifs in Hans-LanM, identifying key residues that influence binding affinity and stability, particularly within the primary interaction sphere. The analysis highlighted the crucial importance of negatively charged and polar residues in facilitating metal binding interactions. Finally, by analyzing conformational dynamics analysis, we identified key residues that undergo structural compaction upon Nd³⁺, Dy³⁺ and La³⁺ binding, highlighting critical residues those targeted mutations could fine-tune metal selectivity while preserving overall structural integrity, thereby providing a rational framework for engineering Hans-LanM variants with enhanced rare earth element specificity.
