Affinity of S100A1 protein for calcium increases dramatically upon glutathionylation.
S100A1 is a typical representative of a group of EF-hand calcium-binding proteins known as the S100 family. The protein is composed of two alpha subunits, each containing two calcium-binding loops (N and C). At physiological pH (7.2) and NaCl concentration (100 mm), we determined the microscopic binding constants of calcium to S100A1 by analysing the Ca(2+)-titration curves of Trp90 fluorescence for both the native protein and its Glu32 --> Gln mutant with an inactive N-loop. Using a chelator method, we also determined the calcium-binding constant for the S100A1 Glu73 --> Gln mutant with an inactive C-loop. The protein binds four calcium ions in a noncooperative way with binding constants of K(1) =4 +/- 2 x 10(3) m(-1) (C-loops) and K(2) approximately 10(2) m(-1) (N-loops). Only when both loops are saturated with calcium does the protein change its global conformation, exposing to the solvent hydrophobic patches, which can be detected by 2-p-toluidinylnaphthalene-6-sulfonic acid - a fluorescent probe of protein-surface hydrophobicity. S-Glutathionylation of the single cysteine residue (85) of the alpha subunits leads to a 10-fold increase in the affinity of the protein C-loops for calcium and an enormous - four orders of magnitude - increase in the calcium-binding constants of its N-loops, owing to a cooperativity effect corresponding to DeltaDeltaG = -6 +/- 1 kcal.mol(-1). A similar effect is observed upon formation of the mixed disulfide with cysteine and 2-mercaptoethanol. The glutathionylated protein binds TRTK-12 peptide in a calcium-dependent manner. S100A1 protein can act, therefore, as a linker between the calcium and redox signalling pathways.
Goch
Grazyna
G
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Poland.
Vdovenko
Sergiusz
S
Kozłowska
Hanna
H
Bierzyñski
Andrzej
A
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051347:CapZ Actin Capping Protein; D002417:Cattle; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D009154:Mutation; D009282:Naphthalenesulfonates / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010084:Oxidation-Reduction; D010446:Peptide Fragments; D011485:Protein Binding; D011487:Protein Conformation; D009418:S100 Proteins; D014364:Tryptophan / Q000378:metabolism
C109928:5-nitro-1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid; D002135:Calcium-Binding Proteins; D051347:CapZ Actin Capping Protein; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D009842:Oligopeptides; D010446:Peptide Fragments; D009418:S100 Proteins; C110294:S100A1 protein; C475482:TRTK-12 peptide; D004533:Egtazic Acid; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D014364:Tryptophan; D005978:Glutathione; D002118:Calcium