CA2+-Dependent antifreeze proteins: Modulation of conformation and activity by divalent metal ions

The antifreeze proteins (AFPs) are structurally diverse molecules that share an ability to bind to ice crystals and inhibit their growth. The type II fish AFPs of Atlantic herring and smelt are homologous with the carbohydrate-recognition domains of Ca²⁺-dependent (C-type) lectins and they also require Ca-4+ for their activity. To investigate the role of metal ions in the structure and function of type II AFPs, the binding of Ca-4+ and other divalent cations to herring AFP was investigated. Binding studies using 45ca2+ demonstrated that the AFP has a single Ca²⁺-binding site with a Kd of 8.9 uM. Proteolysis protection studies and measurement of antifreeze activity revealed a conformational change from a proteasesensitive and inactive apoAFP to a protease-resistant active AFP upon Ca²⁺ binding. This transition was also detected using fluorescence spectroscopy. Other divalent metal ions including Mn2+, Ba-4+, and Zn-4+ bind at the Ca²⁺ binding site and induce a similar change. Antifreeze activity appeared normal when Ca-4+ or Mn-4+ were bound. In contrast, activity was much lower in the presence of other metal ions. Examination of ice crystals in the presence of Ba-4 + revealed a distinct morphology. These studies demonstrate that herring AFP binds Ca-4+ and, consequently, adopts a conformation that is essential for its ice-binding activity. (Supported by MRC, Canada).