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J. Biol. Chem., Vol. 283, Issue 40, 27110-27120, October 3, 2008

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The NMR Structures of the Major Intermediates of the Two-domain Tick Carboxypeptidase Inhibitor Reveal Symmetry in Its Folding and Unfolding Pathways^*

Joan L. Arolas¹², David Pantoja-Uceda¹², Salvador Ventura, Francisco J. Blanco^¶3, and Francesc X. Aviles⁴

From the Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain, the Departamento de Espectroscopia y Estructura Molecular, Instituto de Química-Física Rocasolano, Consejo Superior de Investigaciones Científicas, Serrano 119, 28006 Madrid, Spain, and the ^¶Structural Biology Unit, CIC bioGUNE, Parque Tecnológico de Bizkaia, Edificío 800, 48160 Derio, Spain

There is a lack of experimental structural information about folding intermediates of multidomain proteins. Tick carboxypeptidase inhibitor (TCI) is a small, disulfide-rich protein consisting of two domains that fold and unfold autonomously through the formation of two major intermediates, IIIa and IIIb. Each intermediate contains three native disulfide bonds in one domain and six free cysteines in the other domain. Here we have determined the NMR structures of these two intermediates trapped and isolated at acidic pH in which they are stable and compared their structures with that of the native protein analyzed under the same conditions. Both IIIa and IIIb were found to contain a folded region that corresponds to the N- and C-terminal domains of TCI, respectively, with structures very similar to the corresponding regions of the native protein. The remainder of the polypeptide chains of the intermediates was shown to be unfolded in a random coil conformation. Solvent exchange measurements further indicated that the two protein domains are not completely independent, but affect each other in terms of dynamics and stability, in agreement with reported inhibitory activity data. The derived results provide structural evidence for symmetric TCI folding and unfolding mechanisms that converge in IIIa and IIIb and reveal the structural basis that accounts for the strong and simultaneous accumulation of both intermediates. Altogether, this work has important implications for a better understanding of the folding mechanisms of multidomain, disulfide-rich proteins.

Received for publication, May 26, 2008 , and in revised form, July 14, 2008.

The atomic coordinates and structure factors (codes 2K2X (N), 2K2Y (IIIa), and 2K2Z (IIIb)) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The NMR chemical shifts have been deposited in the BioMagResBank under accession numbers 15729 (N), 15730 (IIIa), and 15731 (IIIb).

^* This work was supported in part by the Ministerio de Educación y Ciencia, Spain, Grants BIO2007-68046 (to F. X. A.) and GEN2003-20642-C09-05 (to F. J. B. and F. X. A.) and by EU-Grant 3D repertoire, Contract LSHG-CT-2005-512028 (to F. J. B.). 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 on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1–S4 and Figs. S1–S2.

¹ Both authors contributed equally to this work.

² Supported by Juan de la Cierva research contracts awarded by the Ministerio de Educación y Ciencia, Spain.

³ Supported by Ikerbasque (Basque Foundation for Science, Spain).To whom correspondence may be addressed. Tel.: 34-946572521; Fax: 34-946572502; E-mail: fblanco{at}cicbiogune.es.

⁴ To whom correspondence may be addressed. Tel.: 34-935811315; Fax: 34-935812011; E-mail: francescxavier.aviles{at}uab.es.

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