Calcium-modulated S100 protein-phospholipid interactions. An NMR study of calbindin D_9k and DPC

The cellular functions of several S100 proteins involve specific interactions with phospholipids and the cell membrane. The interactions between calbindin D_9k (S100D) and the detergent dodecyl phosphocholine (DPC) were studied using NMR spectroscopy. In the absence of Ca²⁺, the protein associates with DPC micelles. The micelle-associated state has intact helical secondary structures but no apparent tertiary fold. At neutral pH, Ca²⁺-loaded calbindin D_9k does not associate with DPC micelles. However, a specific interaction is observed with individual DPC molecules at a site close to the linker between the two EF-hands. Binding to this site occurs only when Ca²⁺ is bound to the protein. A reduction in pH in the absence of Ca²⁺ increases the stability of the micelle-associated state. This along with the corresponding reduction in Ca ²⁺ affinity causes a transition to the micelle-associated state also in the presence of Ca²⁺ when the pH is lowered. Site-specific analysis of the data indicates that calbindin D_9k has a core of three tightly packed helices (A, B, and D), with a dynamic fourth helix (C) more loosely associated. Evidence is presented that the Ca²⁺-binding characteristics of the two EF-hands are distinctly different in a micelle environment. The role of calbindin D_9k in the cell is discussed, along with the broader implications for the function of the S100 protein family.