title: Cysteine 81 is critical for the interaction of S100A4 and myosin-IIA.
abstract: Overexpression of S100A4, a member of the S100 family of Ca(2+)-binding proteins, is associated with a number of human pathologies, including fibrosis, inflammatory disorders, and metastatic disease. The identification of small molecules that disrupt S100A4/target interactions provides a mechanism for inhibiting S100A4-mediated cellular activities and their associated pathologies. Using an anisotropy assay that monitors the Ca(2+)-dependent binding of myosin-IIA to S100A4, NSC 95397 was identified as an inhibitor that disrupts the S100A4/myosin-IIA interaction and inhibits S100A4-mediated depolymerization of myosin-IIA filaments. Mass spectrometry demonstrated that NSC 95397 forms covalent adducts with Cys81 and Cys86, which are located in the canonical target binding cleft. Mutagenesis studies showed that covalent modification of just Cys81 is sufficient to inhibit S100A4 function with respect to myosin-IIA binding and depolymerization. Remarkably, substitution of Cys81 with serine or alanine significantly impaired the ability of S100A4 to promote myosin-IIA filament disassembly. As reversible covalent cysteine modifications have been observed for several S100 proteins, we propose that modification of Cys81 may provide an additional regulatory mechanism for mediating the binding of S100A4 to myosin-IIA.
authors: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors: Natalya G
authors: NG
authors: Dulyaninova
authors: Karen M
authors: KM
authors: Hite
authors: Wendy D
authors: WD
authors: Zencheck
authors: Dominic A
authors: DA
authors: Scudiero
authors: Steven C
authors: SC
authors: Almo
authors: Robert H
authors: RH
authors: Shoemaker
authors: Anne R
authors: AR
authors: Bresnick
mesh_terms: D002853:Chromatography, Liquid; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D003599:Cytoskeleton; D006801:Humans; D009285:Naphthoquinones / Q000494:pharmacology*; D024507:Nonmuscle Myosin Type IIA / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020687:cdc25 Phosphatases / Q000037:antagonists & inhibitors
chemical_list: C451261:2,3-bis(2-hydroxyethylsulfanyl)-(1,4)naphthoquinone; D009285:Naphthoquinones; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C077591:S100A4 protein, human; D020687:cdc25 Phosphatases; D024507:Nonmuscle Myosin Type IIA; D003545:Cysteine