Cell wall proteins from Neurospora crassa were isolated and evaluated to demonstrate their metal ability to bind Ca 2+ /Sr 2+ by loading the solubilized protein fraction on to immobilized metal affinity chromatography (IMAC) column pre-equilibrated with Ca 2+ /Sr 2+ . The sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis IMAC eluent, revealed ∼18 proteins with a similarity in the proteome pattern of Ca 2+ /Sr 2+ fractions.Diethyl aminoethyl chromatography showed five proteins in common in binding to Ca 2+ and Sr 2+ , were subjected to N-terminal sequencing. The sequence analysis was studied for the determination of metal-binding site prediction by CHED software indicating that all five were found to have a high affinity toward Ca 2+ . From these five, two were randomly selected and denoted as CWP-A (possess five Ca binding sites of six metal-binding sites) and CWP-B (possess six binding sites of eight metal-binding sites). They were selected for further characterization studies to determine their Ca 2+ bound Sr 2+ binding properties. Surprisingly, these proteins were able to bind Sr 2+ ions (29 μmol) with equal affinity as to Ca 2+ ions (42 μmol) by means of direct binding, and/or by displacing calcium as observed in metal-dependent proteolytic protection, fluorescence-based metal exchange assays, and molecular simulation studies. From the results, we demonstrate for the first time, that there is a stoichiometry between Ca 2+ (an essential macro elemental metal ion) and Sr 2+ ions (a nonessential element for which no reported metabolic activity is reported) for the metal-binding sites on cell wall proteins.