Current Protein and Peptide Science, 2004, 5, 341-349 341 Squash Inhibitors: From Structural Motifs to Macrocyclic Knottins Laurent Chiche1,*, Annie Heitz1, Jean-Christophe Gelly1, Jérôme Gracy1, Pham T.T Chau2, Phan T Ha2, Jean-Franỗois Hernandez3 and Dung Le-Nguyen4 Centre de Biochimie Structurale, CNRS UMR5048, INSERM UMR554, Université Montpellier I, Faculté de Pharmacie, 15, Avenue Flahault, 34093 Montpellier-France; 2Center for Biotechnology, Vietnam National University, 90, Nguyen Trai Street, Hanoï, Vietnam; 3Laboratoire des Aminoacides, Peptides et Protéines CNRS UMR5810 Universités Montpellier I & II Faculté de Pharmacie, 15, avenue Flahault, 34093 Montpellier-France; 4INSERM U376, CHU Arnaud-de-Villeneuve, 371, rue du doyen Giraud, 34295 Montpellier-France Abstract: In this article, we will first introduce the squash inhibitors, a well established family of highly potent canonical serine proteinase inhibitors isolated from Cucurbitaceae The squash inhibitors were among the first discovered proteins with the typical knottin fold shared by numerous peptides extracted from plants, animals and fungi Knottins contain three knotted disulfide bridges, two of them arranged as a Cystine-Stabilized Beta-sheet motif In contrast to cyclotides for which no natural linear homolog is known, most squash inhibitors are linear However, Momordica cochinchinensis Trypsin Inhibitor-I and -II (MCoTI-I and -II), 34-residue squash inhibitors isolated from seeds of a common Cucurbitaceae from Vietnam, were recently shown to be macrocyclic In these circular squash inhibitors, a short peptide linker connects residues that correspond to the N- and C-termini in homologous linear squash inhibitors In this review we present the isolation, characterization, chemical synthesis, and activity of these macrocyclic knottins The solution structure of MCoTI-II will be compared with topologically similar cyclotides, homologous linear squash inhibitors and other knottins, and potential applications of such scaffolds will be briefly discussed Keywords: Macrocyclic proteins, knottins, inhibitor cystine knots, structural motifs, squash inhibitors, disulfide bridges, drug design, serine proteinases INTRODUCTION Nature has many secrets that remain to be discovered, and the discovery of macrocyclic proteins has revealed one such new area that is set to become an important field in structural biology Several examples of macrocyclic proteins have appeared in the last few years strongly suggesting that many are still to come in the near future The cyclotide family reviewed in the preceding articles in this issue [1-3] is particularly remarkable in that it comprises a large number of proteins, all being cyclic Although the stability afforded by the circular feature is clear, it is unclear, however, if linear counterparts exist in nature and will be discovered in the future In this field there is still much to be explained In this article we focus on a protein family that was discovered more than twenty years ago and, until recently, and in contrast to cyclotides, comprised only linear compounds, the squash inhibitors The recent unexpected discovery of circular squash inhibitors from Momordica cochinchinensis, MCoTI-I and -II [4], supports the idea that macrocyclization of proteins may not be uncommon in nature and provides interesting perspectives for structural *Address correspondence to this author at the Centre de Biochimie Structurale, CNRS UMR5048, INSERM UMR554, Université Montpellier I, Faculté de Pharmacie, 15, Avenue Flahault, 34093 Montpellier, France; Tel: (33)[0]-4670-43432; Fax: (33)[0]-4675-29623; E-mail: chiche@ cbs.cnrs.fr 1389-2037/04 $45.00+.00 proteomics, since end-to-end cyclization, like many other post-translational modifications, cannot be simply deduced from genomic sequences We will first present the main historical and structural highlights of the squash inhibitors This will be extended to the intriguing structural class of proteins known as knottins that share a similar scaffold Our recent efforts to organize and standardize knottin data for improved analyses and comparisons will be briefly discussed Then starting from this background, we will describe the macrocyclic squash inhibitors from seeds of Momordica cochinchinensis, including their discovery, isolation, sequence and structure They will be compared to cyclotides, to linear homologs and to structurally similar linear knottins Chemical synthesis and possible applications of this scaffold will be discussed THE FAMILY OF SQUASH INHIBITORS OF SERINE PROTEINASES The so-called 'canonical' inhibitors of serine proteinases interact with their target enzyme in a substrate-like mechanism via a binding loop with characteristic conformation [5, 6] Among these, the squash inhibitors of serine proteinases are small (27-34 residues) disulfide-rich proteins discovered in the late 1970s in seeds of Winter squash [7] So far, all known homologs originate from the Cucurbitaceae plant family [8] Squash inhibitors were for © 2004 Bentham Science Publishers Ltd 342 Current Protein and Peptide Science, 2004, Vol 5, No Chiche et al some time the smallest known natural serine protease inhibitors until the recent discovery of SFTI-1 a 14-residue long circular peptide, which is discussed in detail by Korsinczky et al [9] in this issue Association constants with various serine proteinases may be as high as 10-12 M-1 making these inhibitors among the most potent ones [8] As with other plant protease inhibitors, squash inhibitors are presumed to participate in defense mechanisms by conferring resistance to pests, pathogens or insects [10] Squash inhibitors contain six cysteines involved in three disulfide bridges with I-IV, II-V, III-VI connectivity The first three-dimensional (3D) structure determinations revealed a very specific knotted scaffold achieved when one disulfide bridge (III-VI) crosses the macrocycle formed by the two other disulfides and the interconnecting peptide backbone [11-13] This remarkably stable knotted topology, was previously observed in only one compound, the carboxypeptidase A inhibitor from potato PCI [14] Since these pioneering observations, nearly one hundred proteins have been explicitly shown through structural studies to share this specific knotted scaffold THE KNOTTIN FOLD AND THE CSB MOTIF Small disulfide-rich proteins sharing the disulfide connectivity and topology of the squash inhibitors are now known as knottins [15] or Inhibitor Cystine Knots [16] However, despite similar overall topologies, it soon became apparent that the I-IV disulfide bridge is not structurally conserved between different knottin families and that only two disulfides (II-V and III-VI) were highly conserved [17] This observation was later supported by folding experiments on the squash inhibitor EETI-II in which it was shown that two disulfides are necessary and sufficient to stabilize most of the native structure [18-20] The synthesis and biophysical study of the truncated EETI-II peptide Min-23, comprising only cysteines II, III, V and VI, confirmed that the elementary two-disulfide motif we called the Cystine Stabilized Beta-sheet (CSB) motif is an autonomous folding unit and is the elementary structural motif in knottins [22] Interestingly enough, although this motif has been shown to display high stability (Tm ~ 100°C), it has never been observed in nature alone, without supplementary disulfide bridges The CSB motif is not only found in knottins, but also in numerous other small disulfide rich folds (e.g EGF-like motifs or scorpion toxins), and is actually the most widespread disulfide motif (data not shown) It may thus be hypothesized either that an ancestral CSB-based protein once existed but was lost during evolution, or that the different CSB-based folds appeared independently as a result of convergent evolution Relationships between the CSB motif and knottins are summarized in (Fig 1) As new knottins are discovered, it is becoming more and more apparent that nature has used this stable scaffold in very different contexts to achieve various biological roles At present more than 12 protein families, with virtually no sequence identity, share the knottin fold Due to its small size, its well-defined structure, and its high stability, this scaffold is thought to be an appealing structural template in drug design developments Therefore, to facilitate analyses Fig (1) From elementary CSB motif to macrocyclic knottins The figure indicates structural relationships between knottins and non-knottin CSB-based proteins The images of the CSB motif, the linear and circular squash inhibitors, and the cyclotides were prepared with the MOLMOL [21] and POV-Ray (http://www.povray.org/) programs using coordinates of Min-23, EETI-II, MCoTI-II and kalata B1 The (a)b.c(d)e[f] nomenclature is explained in Fig (2) No cyclic knottins have yet been discovered for which c=0 Note that relationships in this figure not necessarily imply evolutionary relationships (see discussion) and comparisons, we have recently proposed a simple knottin nomenclature based on loop lengths between cysteines, and a unique knottin numbering based on cysteine connectivity and structural conservation of the CSB motif [24] These are illustrated in (Fig 2) and used throughout the rest of the paper Moreover, a KNOTTIN database gathering information on knottins has been set up [24] and can be freely accessed on the Internet (http://knottin.cbs.cnrs.fr or http:knottin com) Database searches by keyword, sequence, nomenclature, or geometrical pattern can be carried out and various displays are proposed Renumbered sequences and structures, as well as structurally fitted PDB files are available All these tools greatly facilitate knottin analyses, and particularly sequence and structure comparisons, as shown in the succeeding sections A sequence alignment between representative knottin sequences is shown in (Fig 3A) Squash Inhibitors Current Protein and Peptide Science, 2004, Vol 5, No 343 Fig (2) Knottin numbering and nomenclature An automatically drawn two-dimensional (2D) Collier de Perles representation [23, 24] of MCoTI-II is shown The line between residues 43 and 58 is a result of the 2D representation and does not indicate chain break The cysteines involved in the knot are displayed with a black or grey background Roman and Arabic numbers indicate the order in the sequence and the new unique numbering of cysteines involved in the knot, respectively Cysteine IV (grey background) does not have a fixed number Letters a-f refer to successive loops between cysteines of the knot, and to the number of amino acids therein The latter values are used to establish the nomenclature {e.g MCoTI-II: (6)5.3(1)5[8]} Numbers between round brackets refer to peptide segments involved in the disulfide macrocycle whereas the number between square brackets refers to the C-to-N linker in cyclic squash inhibitors or cyclotides Fig (3) A Sequence alignment between knottins One representative sequence is shown for most knottin families except conotoxins (GVIA and gm9a) and squash inhibitors (CPTI-II, EETI-II and MCoTI-I, -II and -III) The two-disulfide peptide Min-23 corresponding to the CSB motif is shown at the bottom Disulfide bridges of cysteines I-IV and of the CSB motif are shown on top as thin and thick lines, respectively Additional disulfide bridges are shown as thin boxes and lines Numbering is according to [24] and Fig (2) The X letter in the PCI sequence stands for the ambiguous Glu/Gln residue The "