Background: The binding of ligands to clusters of complement-type repeat (CR)-domains in proteins of the low-density lipoprotein receptor (LDLR) family is dependent on Ca2+ ions. One reason for this cation requirement was identified from the crystal structure data for a CR-domain from the prototypic LDLR, which showed the burial of a Ca2+ ion as a necessity for correct folding and stabilization of this protein module. Additional Ca2+ binding data to other CR-domains from both LDLR and the LDLR-related protein (LRP) have suggested the presence of a conserved Ca2+ cage within CR-domains from this family of receptors that function in endocytosis and signalling.
Results: We have previously described the binding of several ligands to a fragment comprising the fifth and the sixth CR-domain (CR56) from LRP, as well as qualitatively described the binding of Ca2+ ions to this CR-domain pair. In the present study we have applied the rate dialysis method to measure the affinity for Ca2+, and show that CR56 binds 2 Ca2+ ions with an average affinity of KD = 10.6 microM, and there is no indication of additional Ca2+ binding sites within this receptor fragment.
Conclusions: Both CR-domains of CR56 bind a single Ca2+ ion with an affinity of 10.6 microM within the range of affinities demonstrated for several other CR-domains.