Structure, binding interface and hydrophobic transitions of Ca ²⁺-loaded calbindin-D_28K

Calbindin-D_28K is a Ca²⁺-binding protein, performing roles as both a calcium buffer and calcium sensor. The NMR solution structure of Ca²⁺-loaded calbindin-D_28K reveals a single, globular fold consisting of six distinct EF-hand subdomains, which coordinate Ca ²⁺ in loops on EF1, EF3, EF4 and EF5. Target peptides from Ran-binding protein M and myo-inositol monophosphatase, along with a new target from procaspase-3, are shown to interact with the protein on a surface comprised of α5 (EF3), α8 (EF4) and the EF2-EF3 and EF4-EF5 loops. Fluorescence experiments reveal that calbindin-D_28K adopts discrete hydrophobic states as it binds Ca²⁺. The structure, binding interface and hydrophobic characteristics of Ca²⁺-loaded calbindin-D _28K provide the first detailed insights into how this essential protein may function. This structure is one of the largest high-resolution NMR structures and the largest monomeric EF-hand protein to be solved to date.