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, Th e type II fish AFPs of Atlantic herring and smelt are unique among kno wn AFPs in their requirement of a cofactor for antifreeze activity. Th ese AFPs are homologous with the carbohydrate-recognition domains of C a2+-dependent (C-type) lectins and require Ca2+ for their activity. To investigate the role of metal ions in the structure and function of t ype II AFPs, the binding of Ca2+ and other divalent cations to herring AFP was investigated. Binding studies using Ca-45(2+) demonstrated th at the AFP has a single Ca2+-binding site with a K-d of 9 mu M. Proteo lysis protection studies and measurement of antifreeze activity reveal ed a conformational change from a protease-sensitive and inactive apoA FP to a protease resistant active AFP upon Ca2+ binding. Other divalen t metal ions including Mn2+, Ba2+, and Zn2+ bind at the Ca2+-binding s ite and induce a similar change. A saturatable increase in tryptophan emission intensity at 340 nm also occurred upon Ca2+ addition, Whereas antifreeze activity appeared normal when Ca2+ or Mn2+ were bound, it was much lower in the presence of other metal ions. When Ba2+ was boun d to the AFP, ice crystals showed a distinct difference in morphology. These studies demonstrate that herring AFP specifically binds Ca2+ an d, consequently, adopts a conformation that is essential for its ice b inding activity.