Binding of calcium ions and SNAP-25 to the hexa EF-hand protein secretagogin

Secretagogin is a hexa EF-hand protein, which has been identified as a novel potential tumour marker. In the present study, we show that secretagogin binds four Ca²⁺ ions (log K₁=7.1±0.4, log K₂=4.7±0.6, log K₃=3.6±0.7 and log K₄=4.6±0.6 in physiological salt buffers) with a [Ca²⁺]_0.5 of approx. 25 μM. The tertiary structure of secretagogin changes significantly upon Ca²⁺ binding, but not upon Mg²⁺ binding, and the amount of exposed hydrophobic surface in secretagogin increases upon Ca²⁺ binding, but not upon Mg²⁺ binding. These properties suggest that secretagogin belongs to the ‘sensor’ family of Ca²⁺-binding proteins. However, in contrast with the prototypical Ca²⁺ sensor calmodulin, which interacts with a very large number of proteins, secretagogin is significantly less promiscuous. Only one secretagogin-interacting protein was reproducibly identified from insulinoma cell lysates and from bovine and mouse brain homogenates. This protein was identified as SNAP-25 (25 kDa synaptosome-associated protein), a protein involved in Ca²⁺-induced exocytosis in neurons and in neuroendocrine cells. K_d was determined to be 1.2×10⁻⁷ M in the presence of Ca²⁺ and 1.5×10⁻⁶ M in the absence of Ca²⁺. The comparatively low Ca²⁺ affinity for secretagogin and the fact that it undergoes Ca²⁺-induced conformational changes and interacts with SNAP-25 raise the possibility that secretagogin may link Ca²⁺ signalling to exocytotic processes.