Indirect role of Ca2+ in the assembly of extracellular matrix proteins.

We have investigated through molecular dynamics the binding properties at the interface between the C-type lectin subdomain (CLD) of aggrecan and the fibronectin type III domains ((4-5)FnIII) of tenasin, in particular the mechanistically unknown, but essential role of Ca(2+) in the binding. The binding between the CLD and (4-5)FnIII is critical in cross-linking aggrecan, hyaluronan, and tenascin to form extended protein networks found in tissues such as cartilage. None of the structurally resolved Ca(2+) ions in the complex of the CLD and (4-5)FnIII is directly bridging the two proteins. However, one of the Ca(2+) ions (Ca2) is found to play the role of maintaining the structure of the L4 loop at the CLD binding surface, thus facilitating a high affinity binding between the CLD and (4-5)FnIII. Removal of this Ca(2+) ion causes a drastic structural change in the L4 loop, which presumably hinders the binding of the CLD to the (4-5)FnIII. The other bound Ca(2+) ions (Ca1 and Ca3) have no significant effect on the structure of the CLD binding surface, and thus are not expected to affect the binding. Our results might also suggest that the role of Ca2 in maintaining the structure of the L4 loop is most important during the binding process. Once the complex is formed, the dependence of the complex on the structuring role of Ca2 is reduced. In response to tensile force, the CLD and (4-5)FnIII separate by breaking first the electrostatic interactions at the interface, followed by the hydrophobic ones. The sequence of the unbinding events and the maximum force required to separate the two proteins are independent of the presence of the Ca(2+) ions, underlying the indirect role of Ca(2+) in the binding.