Information Transfer in the Penta-EF-hand Protein Sorcin Does Not Operate via the Canonical Structural/Functional Pairing
A STUDY WITH SITE-SPECIFIC MUTANTS*
- Manuela Mella,
- Gianni Colotti,
- Carlotta Zamparelli,
- Daniela Verzili,
- Andrea Ilari and
- Emilia Chiancone‡
- Consiglio Nazionale delle Ricerche Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi Fanelli, University of Rome La Sapienza, 00185 Rome, Italy
- ‡To whom correspondence should be addressed. Tel.: 39-06-49910761; Fax: 39-06-4440062; E-mail: emilia.chiancone{at}uniroma1.it.
Abstract
Sorcin is a typical penta-EF-hand protein that participates in Ca2+-regulated processes by translocating reversibly from cytosol to membranes, where it interacts with different target proteins in different tissues. Binding of two Ca2+/monomer triggers translocation, although EF1, EF2, and EF3 are potentially able to bind calcium at micromolar concentrations. To identify the functional pair, the conserved bidentate -Z glutamate in these EF-hands was mutated to yield E53Q-, E94A-, and E124A-sorcin, respectively. Limited structural perturbations occur only in E124A-sorcin due to involvement of Glu-124 in a network of interactions that comprise the long D helix connecting EF3 to EF2. The overall affinity for Ca2+ and for two sorcin targets, annexin VII and the ryanodine receptor, follows the order wild-type > E53Q- > E94A- > E124A-sorcin, indicating that disruption of EF3 has the largest functional impact and that disruption of EF2 and EF1 has progressively smaller effects. Based on this experimental evidence, EF3 and EF2, which are not paired in the canonical manner, are the functional EF-hands. Sorcin is proposed to be activated upon Ca2+ binding to EF3 and transmission of the conformational change at Glu-124 via the D helix to EF2 and from there to EF1 via the canonical structural/functional pairing. This mechanism may be applicable to all penta-EF-hand proteins.
Footnotes
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↵1 The abbreviations used are: PEF, penta EF-hand; Ryr, ryanodine receptor; PVDF, polyvinylidene difluoride; SPR, surface plasmon resonance; RU, resonance units.
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This paper is dedicated to the memory of Eraldo Antonini, beloved and unforgettable master, deceased prematurely 20 years ago, on March 19, 1983.
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↵* This work was supported by the Consiglio Nazionale delle Ricerche Target Project Biotechnology (to E. C.), by local grants from the Ministero dell'Istruzione Università e Ricerca (to E. C. and C. Z.), and by a special grant for the Centro di Eccellenza Biologia e Medicina Molecolare (to E. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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- Received December 30, 2002.
- Revision received March 28, 2003.
- The American Society for Biochemistry and Molecular Biology, Inc.
