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Hydration is crucial for a function and a ligand recognition of a protein. The hydration shell constructed on an antifreeze protein (AFP) contains many organized waters, through which AFP is thought to bind to specific ice crystal planes. For a Ca²⁺-dependent species of AFP, however, it has not been clarified how 1 mol of Ca²⁺-binding is related with the hydration and the ice-binding ability. Here we determined the X-ray crystal structure of a Ca²⁺-dependent AFP (jsAFP) from Japanese smelt, <i>Hypomesus nipponensis</i>, in both Ca²⁺-bound and -free states. Their overall structures were closely similar (Root mean square deviation (RMSD) of C&#945; = 0.31 &#197;), while they exhibited a significant difference around their Ca²⁺-binding site. Firstly, the side-chains of four of the five Ca²⁺-binding residues (Q92, D94 E99, D113, and D114) were oriented to be suitable for ice binding only in the Ca²⁺-bound state. Second, a Ca²⁺-binding loop consisting of a segment D94&#8722;E99 becomes less flexible by the Ca²⁺-binding. Third, the Ca²⁺-binding induces a generation of ice-like clathrate waters around the Ca²⁺-binding site, which show a perfect position-match to the waters constructing the first prism plane of a single ice crystal. These results suggest that generation of ice-like clathrate waters induced by Ca²⁺-binding enables the ice-binding of this protein.