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.
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.
Murai
Marcelo J
MJ
Department of Medical Genetics, Faculty of Medical Sciences, Tessália Vieira de Camargo, 126, CEP 13083-970, University of Campinas, UNICAMP, Campinas, SP, Brazil.
Sassonia
Rogério C
RC
Zamboni
André H
AH
Conte
Fábio F
FF
Martins-de-Souza
Daniel
D
Aparicio
Ricardo
R
de Oliveira
Marcelo G
MG
Lopes-Cendes
Iscia
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D017931:DNA Primers; D019281:Dimerization; D006801:Humans; D008274:Magnesium / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D020190:Myoclonic Epilepsy, Juvenile / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary
D002135:Calcium-Binding Proteins; D017931:DNA Primers; C489648:EFHC1 protein, human; D008274:Magnesium; D002118:Calcium