Cd²⁺ as a Ca²⁺ Surrogate in Protein–Membrane Interactions: Isostructural but Not Isofunctional

Due to its favorable spectroscopic properties, Cd²⁺ is frequently used as a probe of Ca²⁺ sites in proteins. We investigate the ability of Cd²⁺ to act as a structural and functional surrogate of Ca²⁺ in protein–membrane interactions. C2 domain from protein kinase Cα (C2α) was chosen as a paradigm for the Ca²⁺-dependent phosphatidylserine-binding peripheral membrane domains. We identified the Cd²⁺-binding sites of C2α using NMR spectroscopy, determined the 1.6 Å crystal structure of Cd²⁺-bound C2α, and characterized metal-ion-dependent interactions between C2α and phospholipid membranes using fluorescence spectroscopy and ultracentrifugation experiments. We show that Cd²⁺ forms a tight complex with the membrane-binding loops of C2α but is unable to support its membrane-binding function. This is in sharp contrast with Pb²⁺, which is almost as effective as Ca²⁺ in driving the C2α-membrane association process. Our results provide the first direct evidence for the specific role of divalent metal ions in mediating protein–membrane interactions, have important implications for metal substitution studies in proteins, and illustrate the potential diversity of functional responses caused by toxic metal ions.