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Arch Biochem Biophys. 2008 Sep 1;477(1):131-8. doi: 10.1016/j.abb.2008.06.008. Epub 2008 Jun 19.

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 information

1
Department of Medical Genetics, Faculty of Medical Sciences, Tessália Vieira de Camargo, 126, CEP 13083-970, University of Campinas, UNICAMP, Campinas, SP, Brazil.

Abstract

Human EFHC1 is a member of the EF-hand superfamily of Ca(2+)-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 Ca(2+) and EFHC1C is enthalpically driven (DeltaH=-58.6 to -67 kJ/mol and TDeltaS=-22.5 to -31 kJ/mol) the interaction between Mg(2+) and EFHC1C involves an entropic gain, and is approximately 5 times less enthalpically favorable (DeltaH=-11.7 to -14 kJ/mol and TDeltaS=21.9 to 19 kJ/mol) than for Ca(2+) binding. It was also found that under reducing condition Ca(2+) or Mg(2+) ions bind to EFHC1C in a 1/1 molar ratio, while under oxidizing condition this ratio is reduced, showing that EFHC1C dimerization blocks Ca(2+) and Mg(2+) binding.

PMID:
18593566
DOI:
10.1016/j.abb.2008.06.008
[Indexed for MEDLINE]

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