THERMODYNAMIC AND MOLECULAR-PROPERTIES OF THE INTERACTION BETWEEN AMPHIOXUS CALCIUM VECTOR PROTEIN AND ITS 26-KDA TARGET

Calcium vector protein (CaVP) of amphioxus shares some common structur al features with Ca2+-dependent activators such as troponin C and calm odulin, and is associated in vivo with a 26 kDa (CaVPT), a multidomain protein with one IQ- and two IglI-motifs. Isolated CaVP binds two Ca2 + ions with very different intrinsic affinity constants: K'(Ca1) = 4.9 x 10(6) M(-1) and K'(Ca2) = 7.3 x 10(3) M(-1), respectively. In the c omplex with CaVPT, CaVP also binds two Ca2+, but with strong positive cooperativity (n(H) = 1.9) and with distinctly higher affinity: K'(Ca1 ) = 2.4 x 10(5) M(-1) and K'(Ca2) = 1.0 X 10(8) M(-1). Since neither i n the isolated CaVP nor in the complex Ca2+ binding is influenced by 2 mM MgCl2, both sites can be considered as Ca2+-specific. In the absen ce of Ca2+, the complex is stable under physiogical conditions, but th e interaction is governed by the principle of linked functions and Ca2 + binding to CaVP reinforces the affinity between CaVP and CaVPT 70-fo ld. Both proteins interact with the hydrophobic probe 2 p-toluidinylna phthalene-6-sulfonate (TNS), but CaVPT enhances the fluoresence 45-fol d, CaVP.Ca-2 and metal-free CaVP only 10- and 5-fold, respectively. Co mplex formation between CaVPT and CaVP leads to a 3-fold reduction of the fluorescence enhancement, suggesting that a strong solvent-shielde d hydrophobic core is formed. CaVP contains two highly reactional thio ls (k(SH) > 0.3 s(-1)) for 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) ; CaVPT contains three thiols, two of them also with k(SH) > 0.3 s(-1) in the native state. The buried thiol could be identified as Cys132. In the CaVP-CaVPT complex Cys132 remains inaccessible, but the reactiv ities of the other four thiols are dramatically reduced with k(SH) = 7 .7 x 10(-3) s(-1) for the two thiols of CaVP and 5.8 x 10(-4) s(-1) fo r those of CaVPT. Interestingly, Ca2+ binding does not change the conf ormation of the complex, as probed with TNS and DTNB. In conclusion, t he complex is strengthened by Ca2+ binding, but not strictly Ca2+-depe ndent; whereas isolated CaVPT and CaVP show the characteristics of hig hly dynamic, interactive proteins, complex formation leads to a rigid structure with a very stable hydrophobic core, likely yielding a nonin teractive end product.