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Type: Artigo de periódico
Title: Characterization of the C-terminal half of human juvenile myoclonic epilepsy protein EFHC1: Dimer formation blocks Ca2+ and Mg2+ binding to its functional EF-hand
Author: Murai, MJ
Sassonia, RC
Zamboni, AH
Conte, FF
Martins-De-Souza, D
Aparicio, R
de Oliveira, MG
Lopes-Cendes, I
Abstract: Human EFHC1 is a member of the EF-hand superfamily of Ca2+-binding proteins with three DM10 domains of unclear function. Point mutations in the EFHC1 gene are related to juvenile myoclonic epilepsy, a fairly common idiopathic generalized epilepsy. Here, we report the first structural and thermodynamic analyses of the EFHC1C-terminus (residues 403-640; named EFHC1C), comprising the last DM10 domain and the EF-hand motif. Circular dichroism spectroscopy revealed that the secondary structure of EFHC1C is composed by 34% of alpha helices and 17% of beta -strands. Size exclusion chromatography and mass spectrometry showed that under oxidizing condition EFHC1C dimerizes through the formation of disulfide bond. Tandem mass spectrometry (MS/MS) analysis of peptides generated by trypsin digestion suggests that the Cys575 is involved in intermolecular S-S bond. In addition, DTNB assay showed that each reduced EFHC1C molecule has one accessible free thiol. Isothermal titration calorimetry (ITC) showed that while the interaction between Ca2+ and EFHC1C is enthalpically driven (Delta H= -58.6 to -67 kj/mol and T Delta S = -22.5 to -31 kj/mol) the interaction between Mg2+ and EFHC1 C involves an entropic gain,and is similar to 5 times less enthalpically favorable (Delta H= -11.7 to -14kj/mol and T Delta S=21.9 to 19kj/mol) than for Ca2+ binding. It was also found that under reducing condition Ca2+ or Mg2+ ions bind to EFH1C in a1/1 molar ratio, while under oxidizing condition this ratio is reduced, showing that EFH1C dimerization block Ca2+ and Mg2+ binding.
Subject: EFHC1
DM10 domain
EF-hand motif
divalent ion binding
isothermal titration calorimetry
Country: EUA
Editor: Elsevier Science Inc
Citation: Archives Of Biochemistry And Biophysics. Elsevier Science Inc, v. 477, n. 1, n. 131, n. 138, 2008.
Rights: fechado
Identifier DOI: 10.1016/
Date Issue: 2008
Appears in Collections:Unicamp - Artigos e Outros Documentos

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