Characterization of the calcium-binding sites of calcineurin B

Lazaros T. Kakalis, Michael Kennedy, Robert Sikkink, Frank Rusnak, Ian M. Armitage

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Abstract

Calcineurin (CaN) is a calcium- and calmodulin-dependent serine/threonine phosphatase whose inhibition by the immunosuppressant-immunophilin complexes (cyclosporin-cyclophilin and FK506-FKBP) is considered key to the mechanism of immunosuppression. CaN is a heterodimer, consisting of a 59 kDa catalytic subunit (A) and a 19 kDa calcium-binding regulatory subunit (B). The latter is postulated to harbor four calcium binding domains of the EF hand type. The titration of the CaN B apoprotein with the isomorphic Cd2+ was followed by 113Cd NMR and these data support one high-affinity metal binding site and three lower-affinity ones. Flow dialysis data with Ca2+ indicate one high affinity calcium binding site with Kd ∼ 2.4 × 10-8 M and three other sites with Kd ∼ 1.5 × 10-5 M. The chemical shifts of all four 113Cd resonances (-75, -93, -106 and -119 ppm) are in the same range as found in other 113Cd substituted calcium-binding proteins, and are indicative of all-oxygen coordination of pentagonal bipyramidal geometry.

Original languageEnglish (US)
Pages (from-to)55-58
Number of pages4
JournalFEBS Letters
Volume362
Issue number1
DOIs
StatePublished - Mar 27 1995

Bibliographical note

Funding Information:
Acknowledgements." This study was supported by grants from NIH (GM49858 to I.M.A. and GM46865 to F.R.). NMR instrumentation and computational facilities were provided by grants from NIH (RR03475), NSF (DMB8610557), and ACS (RD259). Access to the atomic absorption spectrophotometer at the Clinical Chemistry Laboratory of the Yale-New Haven Hospital is gratefully acknowledged.

Keywords

  • Cadmium-113 NMR
  • Calcineurin
  • Calcium binding
  • EF hand

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