Comparison of the Ca2+-binding properties of human recombinant calretinin-22k and calretinin.
Calretinin-22k (CR-22k) is a splice product of calretinin (CR) found specifically in cancer cells, and possesses four EF-hands and a differently processed C-terminal end. The Ca2+-binding properties of recombinant human calretinin CR-22k were investigated by flow dialysis and spectroscopic methods and compared with those of CR. CR possesses four Ca2+-binding sites with positive cooperativity (nH = 1.3) and a [Ca2+]0.5 of 1.5 microM, plus one low affinity site with an intrinsic dissociation constant (K'D) of 0.5 mM. CR-22k contains three Ca2+-binding sites with nH of 1.3 and [Ca2+]0.5 of 1.2 microM, plus a low affinity site with K'D of 1 mM. All the sites seem to be of the Ca2+-specific type. Limited proteolysis and thiol reactivity suggest that that the C terminus of full-length CR, but not of CR-22k, is in close proximity of site I leading to mutual shielding. Circular dichroism (CD) spectra predict that the content of alpha-helix in CR and CR-22k is similar and that Ca2+ binding leads to very small changes in the CD spectra of both proteins. The optical properties are very similar for CR-22k and CR, even though CR-22k possesses one additional Trp at the C-terminal end, and revealed that the Trp residues are organized into a hydrophobic core in the metal-free proteins and become even better shielded from the aqueous environment upon binding of Ca2+. The fluorescence of the hydrophobic probe 2-p-toluidinylnaphtalene-6-sulfonate is markedly enhanced by the two proteins already in the absence of Ca2+ and is further increased by binding of Ca2+. The trypsinolysis patterns of CR and CR-22k are markedly dependent on the presence or absence of Ca2+. Together, our data suggest the presence of an allosteric conformational unit encompassing sites I-III for CR-22k and I-IV for CR, with a very similar conformation and conformational changes for both proteins. In the allosteric unit of CR, site IV is fully active, whereas in CR-22k this site has a 80-fold decreased affinity, due to the decreased amphiphilic properties of the C-terminal helix of this site. Some very specific Ca2+-dependent conformational changes suggest that both CR and CR-22k belong to the "sensor"-type family of Ca2+-binding proteins.
Schwaller
B
B
Institute of Histology and General Embryology, University of Fribourg, Pérolles, CH-1705 Fribourg, Switzerland.
Durussel
I
I
Jermann
D
D
Herrmann
B
B
Cox
J A
JA
D064032:Calbindin 2; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C576764:CALB2 protein, human; D064032:Calbindin 2; D002147:Calmodulin; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D008274:Magnesium; D002118:Calcium