Folding units in calcium vector protein of amphioxus: Structural and functional properties of its amino‐ and carboxy‐terminal halves

Muscle of amphioxus contains large amounts of a four EF‐hand Ca 2+ ‐binding protein, CaVP, and its target, CaVPT. To study the domain structure of CaVP and assess the structurally important determinants for its interaction with CaVPT, we expressed CaVP and its amino (N‐CaVP) and carboxy‐terminal halves (C‐CaVP). The interactive properties of recombinant and wild‐type CaVP are very similar, despite three post‐translational modifications in the wild‐type protein. N‐CaVP does not bind Ca 2+ , shows a well‐formed hydrophobic core, and melts at 44°C. C‐CaVP binds two Ca 2+ with intrinsic dissociation constants of 0.22 and 140 μM (i.e., very similar to the entire CaVP). The metal‐free domain in CaVP and C‐CaVP shows no distinct melting transition, whereas its 1Ca 2+ and 2Ca 2+ forms melt in the 111°–123°C range, suggesting that C‐CaVP and the carboxy‐ domain of CaVP are natively unfolded in the metal‐free state and progressively gain structure upon binding of 1Ca 2+ and 2Ca 2+ . Thermal denaturation studies provide evidence for interdomain interaction: the apo, 1Ca 2+ and 2Ca 2+ states of the carboxy‐domain destabilize to different degrees the amino‐domain. Only C‐CaVP forms a Ca 2+ ‐dependent 1:1 complex with CaVPT. Our results suggest that the carboxy‐terminal domain of CaVP interacts with CaVPT and that the amino‐terminal lobe modulates this interaction.