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J. Biol. Chem., Vol. 281, Issue 34, 24193-24203, August 25, 2006

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Structural Determinants of Salmon Calcitonin Bioactivity

THE ROLE OF THE LEU-BASED AMPHIPATHIC -HELIX^*

Giuseppina Andreotti¹, Blanca López Méndez¹², Pietro Amodeo, Maria A. Castiglione Morelli, Hiromichi Nakamuta^¶, and Andrea Motta³

From the Istituto di Chimica Biomolecolare del Consiglio Nazionale delle Ricerche, Comprensorio Olivetti, Edificio A, 80078 Pozzuoli (Napoli), Italy, Dipartimento di Chimica, Università della Basilicata, 85100 Potenza, Italy, and the ^¶Laboratory of Pharmacology, Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Hiroshima International University, Hirokoshingai 5-1-1, Kure, Hiroshima 737-0112, Japan

Salmon calcitonin (sCT) forms an amphipathic helix in the region 9–19, with the C-terminal decapeptide interacting with the helix (Amodeo, P., Motta, A., Strazzullo, G., Castiglione Morelli, M. A. (1999) J. Biomol. NMR 13, 161–174). To uncover the structural requirements for the hormone bioactivity, we investigated several sCT analogs. They were designed so as to alter the length of the central helix by removal and/or replacement of flanking residues and by selectively mutating or deleting residues inside the helix. The helix content was assessed by circular dichroism and NMR spectroscopies; the receptor binding affinity in human breast cancer cell line T 47D and the in vivo hypocalcemic activity were also evaluated. In particular, by NMR spectroscopy and molecular dynamics calculations we studied Leu²³,Ala²⁴-sCT in which Pro²³ and Arg²⁴ were replaced by helix inducing residues. Compared with sCT, it assumes a longer amphipathic -helix, with decreased binding affinity and one-fifth of the hypocalcemic activity, therefore supporting the idea of a relationship between a definite helix length and bioactivity. From the analysis of other sCT mutants, we inferred that the correct helix length is located in the 9–19 region and requires long range interactions and the presence of specific regions of residues within the sequence for high binding affinity and hypocalcemic activity. Taken together, the structural and biological data identify well defined structural parameters of the helix for sCT bioactivity.

Received for publication, April 12, 2006 , and in revised form, June 8, 2006.

^* This work was supported in part by Consiglio Nazionale delle Ricerche/Ministero dell'Istruzione, Università e Ricerca Legge 449/97 DM 30/10/2000 (to AM). 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.

The atomic coordinates and structure factors (codes 2GLH and 2GLG) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

This paper is dedicated to the memory of Gianfranco Borin.

¹ Contributed equally to this work.

² Supported in part by European Community Grant ERBFMRXCT960069. Present address: Centro de Investigaciones Biológicas, CSIC, C/Ramiro de Maetzu 9, 28040 Madrid, Spain.

³ To whom correspondence should be addressed: Istituto di Chimica Biomolecolare del CNR, Comprensorio Olivetti, Edificio A, Via Campi Flegrei 34, I-80078 Pozzuoli (Napoli), Italy. Tel.: 39-081-8675-228; Fax: 39-081-8041-770; E-mail: amotta{at}icmib.na.cnr.it.

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