Crystallographic identification of Ca2+ and Sr2+ coordination sites in synaptotagmin I C2B domain

Yuan Cheng, Sonia M. Sequeira, Lucy Malinina, Valentina Tereshko, Thomas H. Söllner, Dinshaw J. Patel

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Abstract

Synaptotagmin I has two tandem Ca2+-binding C2 domains, which are essential for fast synchronous synaptic transmission in the central nervous system. We have solved four crystal structures of the C 2B domain, one of them in the cation-free form at 1.50 Å resolution, two in the Ca2+-bound form at 1.04 Å (two bound Ca2+ ions) and 1.65 Å (three bound Ca2+ ions) resolution and one in the Sr2+-bound form at 1.18 Å (one bound Sr2+ ion) resolution. The side chains of four highly conserved aspartic acids (D303, D309, D363, and D365) and two main chain oxygens (M302:O and Y364:O), together with water molecules, are in direct contact with two bound Ca2+ ions (sites 1 and 2). At higher Ca2+ concentrations, the side chain of N333 rotates and cooperates with D309 to generate a third Ca2+ coordination site (site 3). Divalent cation binding sites 1 and 2 in the C2B domain were previously identified from NMR NOE patterns and titration studies, supplemented by site-directed mutation analysis. One difference between the crystal and NMR studies involves D371, which is not involved in coordination with any of the identified Ca2+ sites in the crystal structures, while it is coordinated to Ca2+ in site 2 in the NMR structure. In the presence of Sr2+, which is also capable of triggering exocytosis, but with lower efficiency, only one cation binding site (site 1) was occupied in the crystallographic structure.

Original languageEnglish (US)
Pages (from-to)2665-2672
Number of pages8
JournalProtein Science
Volume13
Issue number10
DOIs
StatePublished - Oct 1 2004

Keywords

  • CB domain
  • Calcium binding
  • Strontium binding
  • Synaptotagmin I
  • X-ray crystallography

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    Cheng, Y., Sequeira, S. M., Malinina, L., Tereshko, V., Söllner, T. H., & Patel, D. J. (2004). Crystallographic identification of Ca2+ and Sr2+ coordination sites in synaptotagmin I C2B domain. Protein Science, 13(10), 2665-2672. https://doi.org/10.1110/ps.04832604