Cation- and Peptide-Binding Properties of Human Calmodulin-like Skin Protein

• Published in: Biochemistry (Easton) Vol. 41; no. 17; pp. 5439 - 5448
• Main Authors: Durussel, Isabelle, Méhul, Bruno, Bernard, Dominique, Schmidt, Rainer, Cox, Jos A
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 30.04.2002
• Subjects: Binding Sites; Calcium - metabolism; Calcium-Binding Proteins - chemistry; Calcium-Binding Proteins - metabolism; Calmodulin - metabolism; Cations, Divalent - metabolism; Circular Dichroism; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes - chemistry; Humans; Magnesium - metabolism; Melitten - chemistry; Melitten - metabolism; Naphthalenesulfonates - chemistry; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Skin - enzymology; Skin - metabolism; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Transglutaminases - chemistry; Transglutaminases - metabolism; Tryptophan - chemistry; Tyrosine - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi016062z

Human CLSP, a new Ca2+-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 Ca2+ ions at two pairs of sites with [Ca2+]0.5 values of 1.2 and 150 μM, respectively. Mg2+ at millimolar concentrations strongly decreases the affinity for Ca2+ of the two high-affinity sites, but has no effect on the low-affinity sites. The protein can also bind two Mg2+ ([Mg2+]0.5 = 57 μM) at the sites of high Ca2+ affinity. Thus, as fast skeletal muscle troponin C (TnC), CLSP possesses two high-affinity Ca2+−Mg2+ mixed sites and two low-affinity Ca2+-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 Ca2+−Mg2+ mixed sites reside in the N-terminal half. The binding of cations modifies the intrinsic fluorescence of the two Tyr residues. Upon Ca2+ binding, hydrophobicity is exposed at the protein surface that can be monitored with a fluorescent probe. The Ca2+-dependency of the two conformational changes is biphasic in the absence of Mg2+, but monophasic in the presence of 2 mM Mg2+, both corresponding closely to direct binding of Ca2+ to CLSP. In the presence of Ca2+, 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 Ca2+ 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.