Cation- and peptide-binding properties of human calmodulin-like skin protein.
Human CLSP, a new Ca(2+)-binding protein specifically expressed in differentiated keratinocytes, is a 15.9 kDa, four EF-hand containing protein with 52% sequence identity to calmodulin (CaM). The protein binds four Ca(2+) ions at two pairs of sites with [Ca(2+)](0.5) values of 1.2 and 150 microM, respectively. Mg(2+) at millimolar concentrations strongly decreases the affinity for Ca(2+) of the two high-affinity sites, but has no effect on the low-affinity sites. The protein can also bind two Mg(2+) ([Mg(2+)](0.5) = 57 microM) at the sites of high Ca(2+) affinity. Thus, as fast skeletal muscle troponin C (TnC), CLSP possesses two high-affinity Ca(2+)-Mg(2+) mixed sites and two low-affinity Ca(2+)-specific sites. Studies on the isolated recombinant N- (N-CLSP) and C-terminal half domains of CLSP (C-CLSP) revealed that, in contrast to the case of TNC, the high-affinity Ca(2+)-Mg(2+) mixed sites reside in the N-terminal half. The binding of cations modifies the intrinsic fluorescence of the two Tyr residues. Upon Ca(2+) binding, hydrophobicity is exposed at the protein surface that can be monitored with a fluorescent probe. The Ca(2+)-dependency of the two conformational changes is biphasic in the absence of Mg(2+), but monophasic in the presence of 2 mM Mg(2+), both corresponding closely to direct binding of Ca(2+) to CLSP. In the presence of Ca(2+), human CLSP forms a high-affinity 1:1 complex with melittin, a natural peptide considered to be a model for the interaction of CaM with its targets. In the complex, CLSP binds Ca(2+) with high affinity to all four binding sites. Isolated N- and C-CLSP show only a weak interaction with melittin, which is enhanced when both halves are simultaneously presented to the model peptide.
Durussel
Isabelle
I
Department of Biochemistry, University of Geneva, 1211 Genève, Switzerland.
Méhul
Bruno
B
Bernard
Dominique
D
Schmidt
Rainer
R
Cox
Jos A
JA
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008555:Melitten / Q000737:chemistry / Q000378:metabolism; D009282:Naphthalenesulfonates / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012867:Skin / Q000201:enzymology / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D011503:Transglutaminases / Q000737:chemistry / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
C408467:CALML5 protein, human; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002413:Cations, Divalent; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; D008555:Melitten; D014443:Tyrosine; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D014364:Tryptophan; D011503:Transglutaminases; D008274:Magnesium; D002118:Calcium