CATION-BINDING AND CONFORMATION OF HUMAN CALMODULIN-LIKE PROTEIN

The Ca2+-binding parameters of recombinant human calmodulin-like protein (CLP), a protein specifically expressed in mammary epithelial cells, were studied by flow dialysis in the absence and presence of 2, 10, and 30 mM MgCl2. In general, the four intrinsic binding constants (K'Ca) are about 8-fold lower than in animal and plant calmodulins. In the absence of Mg2+ the K'Ca values of the four binding steps equal 4.0 X 10(3), 3.3 X 10(4), 1.0 X 10(4), and 6.0 x 10(3) M-1, respectively. They allow us to distinguish two pairs of sites: a higher affinity pair with strong positive cooperativity and a lower affinity pair composed of non-interacting sites with different affinities. Mg2+ antagonizes Ca2+ binding by decreasing only Ca2+-binding steps 2 and 3, so that at high Mg2+ concentrations the positive cooperativity in the high-affinity pair has been lost and that the four K'Ca values are very similar with a mean K'Ca of 4 x 10(3) M-1. Direct Mg2+ binding studies by equilibrium gel filtration indicate that 4-5 Mg2+ bind to CLP with a mean K'Mg of 250 M-1. Conformational changes in the unique Tyr138 microenvironment, monitored by fluorimetry and near-UV difference spectrophotometry, indicate that in metal-free CLP this Tyr is shielded from the polar solvent and strongly quenched by a specific chemical group; Ca2+ binding induces a shift of Tyr to a more polar environment and removal of the quenching group, but without full exposure to the solvent. Qualitatively this behavior is reminiscent of that of calmodulins which possess one Tyr in a position identical with that of the single Tyr of CLP. Mg2+ binding has the same effect as Ca2+, but on a smaller scale. The Tyr-related conformational changes occur upon binding of only two Ca2+, suggesting that the C-terminal domain harbors the high-affinity, strongly interacting pair of sites. The Phe-related conformational changes point to the existence of long-range interactions between the two lobes of CLP. We also monitored a conformational probe in the N-terminal domain, i.e., Cys26, by its reactivity toward 5,5'-dithiobis(2-nitrobenzoic acid). In metal-free CLP the pseudo-first-order rate constant (k(SH)) is 8- and 20-fold higher than in the Ca2+- and Mg2+-loaded protein, respectively. The profile of k(SH) during a titration of CLP with Ca2+ is biphasic with an increase from 3.9 to 6.7 min-1 when two Ca2+ are bound, followed by a decrease to 0.5 min-1 when the third Ca2+ binds to CLP. Our data support the model that binding of Ca2+ to the C-terminal domain is directly responsible for Tyr-related conformational changes in this domain, for a modification of the long-range interacting forces between the C- and N-terminal domains, and finally, for the enhancement of the reactivity of the thiol in the N-terminal domain. Subsequent binding of one Ca2+ to the N-terminal domain, presumably to site I, leads to a rearrangement of the Cys environment, which strongly reduces its reactivity. In many aspects the interaction of CLP with Ca2+ and the ensuing conformational changes are reminiscent of those of CaM, especially of plant CaM. However, in CLP these conformational changes have a higher amplitude than in CaM, which may allow the former to act on unique targets.