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 × 10³, 3.3 × 10⁴, 1.0 × 10⁴, and 6.0 × 10³ 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′_caof 4 × 10³ M⁻¹. Direct Mg²⁺ binding studies by equilibrium gel filtration indicate that 4–5 Mg²⁺ bind to CLP with a mean K′_ca 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 o† 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.