CATION-BINDING AND CONFORMATIONAL-CHANGES IN VILIP AND NCS-1, 2 NEURON-SPECIFIC CALCIUM-BINDING PROTEINS

VILIP and NCS-1, neural-specific, 22-kDa Ca2+-binding proteins possess ing four EF-hands, were expressed in Escherichia coli to study their d ivalent cation properties. Flow dialysis (Ca2+ binding) and equilibriu m gel filtration (Mg2+ binding) revealed that both recombinant protein s possess only two active metal-binding sites, which can accommodate e ither Ca2+ or Mg2+. VILIP binds cations without cooperativity with int rinsic affinity constants K'(Ca) of 1.0 x 10(6) M(-1) and K'(Mg) of 4. 8 x 10(3) M(-1). Mg2+ antagonizes Ca2+ binding by shifting the isother ms to higher free Ca2+ concentrations without changing their shape. Th e competition equation yields a K'(Mg, comp) value of 180 M(-1) for bo th sites. NCS-1 binds two Mg2+ without cooperativity with K'(Mg) of 8. 3 x 10(4) M(-1) and two Ca2+ with very strong positive cooperativity ( n(H) = 1.96). In the absence of Mg2+ the K'(Cal) and K'(Ca2) values ar e 8.9 x 10(4) and 1.4 x 10(8) M(-1), respectively, which represent an allosteric increase of 1600-fold. Mg2+ shifts the Ca2+-binding isother ms to higher Ca2+ concentrations, yielding a K'(Mg, comp) value of 800 M(-1) for both sites. Thus VILIP and NCS-1 show three remarkable diff erences in the Ca2+/Mg2+ binding parameters: 1) VILIP binds Ca2+ with much lower affinity than NCS-1; 2) VILIP binds Ca2+ in a noncooperativ e way, whereas NCS-1 shows maximal positive cooperativity; 3) in VILIP the Mg2+/Ca2+ antagonism is much weaker than in NCS-1. Conformational changes monitored by Trp fluorescence indicate that the metal-free fo rms already are highly structured. Ca2+ binding promotes a 20-30% incr ease of fluorescence in both proteins, but whereas the Mg2+ form of VI LIP has the same fluorescence properties as the metal-free form, Mg2+- saturated NCS-1 has those of the Ca2+ form. Near UV difference spectra confirmed that in VILIP the Mg2+ form is very similar to the metal-fr ee form; in NCS-1 it is different, especially in the Tyr region. NCS-1 possesses one unique Cys-38 in EF-hand site I. Its reactivity (k(SH)) toward 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) is the same for th e Ca2+- and Mg2+-loaded protein, but k(SH) is 4-fold higher in metal-f ree NCS-1. VILIP possesses two additional thiols, one of which is inac cessible to DTNB in the native protein. The reactivity of the two acce ssible thiols is identical in the metal-free and Mg2+ forms and 5-fold higher than in the Ca2+ form. In NCS-1 the accessibility of the hydro phobic matrix to 2-p-toluidinylnaphthalene-6-sulfonate is increased by Ca2+ binding but decreased by Mg2+ binding. In VILIP this accessibili ty is highest for the metal-free and Mg2+ forms; Ca2+ binding leads to a 2-fold decrease in fluorescence enhancement. Thus, although these t wo proteins display 57% sequence identity, not only their Ca2+/Mg2+ bi nding properties but also their Ca2+/Mg2+-induced conformational chang es are very different, suggesting that the two proteins play different roles in the cell.