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J. Biol. Chem., Vol. 281, Issue 33, 23668-23675, August 18, 2006

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The Absolute Structural Requirement for a Proline in the P3'-position of Bowman-Birk Protease Inhibitors Is Surmounted in the Minimized SFTI-1 Scaffold^*

Norelle L. Daly¹, Yi-Kuang Chen, Fiona M. Foley, Paramjit S. Bansal, Rekha Bharathi, Richard J. Clark, Christian P. Sommerhoff, and David J. Craik²

From the Institute for Molecular Bioscience and Australian Research Council Special Research Centre for Functional and Applied Genomics, the University of Queensland, Brisbane, Queensland 4072, Australia and the Department of Clinical Chemistry and Clinical Biochemistry, Ludwig-Maximilians-University, D-80336 Munich, Germany

SFTI-1 is a small cyclic peptide from sunflower seeds that is one of the most potent trypsin inhibitors of any naturally occurring peptide and is related to the Bowman-Birk family of inhibitors (BBIs). BBIs are involved in the defense mechanisms of plants and also have potential as cancer chemopreventive agents. At only 14 amino acids in size, SFTI-1 is thought to be a highly optimized scaffold of the BBI active site region, and thus it is of interest to examine its important structural and functional features. In this study, a suite of 12 alanine mutants of SFTI-1 has been synthesized, and their structures and activities have been determined. SFTI-1 incorporates a binding loop that is clasped together with a disulfide bond and a secondary peptide loop making up the circular backbone. We show here that the secondary loop stabilizes the binding loop to the consequences of sequence variations. In particular, full-length BBIs have a conserved cis-proline that has been shown previously to be required for well defined structure and potent activity, but we show here that the SFTI-1 scaffold can accommodate mutation of this residue and still have a well defined native-like conformation and nanomolar activity in inhibiting trypsin. Among the Ala mutants, the most significant structural perturbation occurred when Asp¹⁴ was mutated, and it appears that this residue is important in stabilizing the trans peptide bond preceding Pro¹³ and is thus a key residue in maintaining the highly constrained structure of SFTI-1. This aspartic acid residue is thought to be involved in the cyclization mechanism associated with excision of SFTI-1 from its 58-amino acid precursor. Overall, this mutational analysis of SFTI-1 clearly defines the optimized nature of the SFTI-1 scaffold and demonstrates the importance of the secondary loop in maintaining the active conformation of the binding loop.

Received for publication, February 14, 2006 , and in revised form, May 31, 2006.

^* This work was supported in part by a grant from the National Health and Medical Research Council (to N. L. D.). 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.

¹ National Health and Medical Research Industry Fellow.

² Australian Research Council Professorial Fellow. To whom correspondence should be addressed. Tel.: 61-7-3346-2019; Fax: 61-7-3346-2101; E-mail: d.craik{at}imb.uq.edu.au.

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