Calbindin D28k exhibits properties characteristic of a Ca2+ sensor.
Calbindin D(28k) is a member of the calmodulin superfamily of Ca(2+)-binding proteins and contains six EF-hands. The protein is generally believed to function as a Ca(2+) buffer, but the studies presented in this work indicate that it may also act as a Ca(2+) sensor. The results show that Mg(2+) binds to the same sites as Ca(2+) with an association constant of approximately 1.4.10(3) m(-1) in 0.15 m KCl. The four high affinity sites in calbindin D(28k) bind Ca(2+) in a non-sequential, parallel manner. In the presence of physiological concentrations of Mg(2+), the Ca(2+) affinity is reduced by a factor of 2, and the cooperativity, which otherwise is modest, increases. Based on the binding constants determined in the presence of physiological salt concentrations, we estimate that at the Ca(2+) concentration in a resting cell calbindin D(28k) is saturated to 40-75% with Mg(2+) but to less than 9% with Ca(2+). In contrast, the protein is expected to be nearly fully saturated with Ca(2+) at the Ca(2+) level of an activated cell. A substantial conformational change is observed upon Ca(2+) binding, but only minor structural changes take place upon Mg(2+) binding. This suggests that calbindin D(28k) undergoes Ca(2+)-induced structural changes upon Ca(2+) activation of a cell. Thus, calbindin D(28k) displays several properties that would be expected for a protein involved in Ca(2+)-induced signal transmission and hence may function not only as a Ca(2+) buffer but also as a Ca(2+) sensor. Digestion patterns resulting from limited proteolysis of the protein suggest that the loop of EF-hand 2, a variant site that does not bind Ca(2+), becomes exposed upon Ca(2+) binding.
Berggård
Tord
T
Department of Biophysical Chemistry, Lund University, SE-221 00 Lund, Sweden. tord.berggard@bpc.lu.se
Miron
Simona
S
Onnerfjord
Patrik
P
Thulin
Eva
E
Akerfeldt
Karin S
KS
Enghild
Jan J
JJ
Akke
Mikael
M
Linse
Sara
S
D000595:Amino Acid Sequence; D000818:Animals; D064092:Calbindin 1; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D013058:Mass Spectrometry; D015233:Models, Statistical; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000737:chemistry*; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000494:pharmacology; D014466:Ultraviolet Rays
C576498:CALB1 protein, human; D064092:Calbindin 1; D064026:Calbindins; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D014357:Trypsin; D008274:Magnesium; D002118:Calcium