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 microM. 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 1.2x10(-7) M in the presence of Ca2+ and 1.5x10(-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.
Rogstam
Annika
A
Department of Biophysical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden.
Linse
Sara
S
Lindqvist
Anders
A
James
Peter
P
Wagner
Ludwig
L
Berggård
Tord
T
D002118:Calcium / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002151:Calorimetry; D002942:Circular Dichroism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D064046:Secretagogins; D015398:Signal Transduction; D013056:Spectrophotometry, Ultraviolet; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism; D014364:Tryptophan / Q000032:analysis
D002135:Calcium-Binding Proteins; C576556:SCGN protein, human; C494208:SNAP25 protein, human; D064046:Secretagogins; D050825:Synaptosomal-Associated Protein 25; D014364:Tryptophan; D002118:Calcium