Characterization and 2D NMR study of the stable [9–21, 15–27] 2 disulfide intermediate in the folding of the 3 disulfide trypsin inhibitor EETI II

The Ecballium elaterium trypsin inhibitor II (EETI-II) contains 28 amino acids and three disulfides forming a cystine knot. Reduced EETI-II refolds spontaneously and quantitatively in vitro and regains its native structure. Due to its high propensity to form a reverse turn, the GPNG sequence of segment 22-25 comprising a ␤-turn in native EETI-II is a possible candidate for a folding initiation site. We generated a molecular repertoire of EETI-II variants with variegated 22-25 tetrapeptide sequences and presented these proteins on the outer membrane of Escherichia coli cells via fusion to the Iga ␤ autotransporter. Functional trypsin-binding variants were selected by combination of magnetic and fluorescence-activated cell sorting. At least 1-5% of all possible tetrapeptide sequences were compatible with formation of the correct three disulfides. Occurrence of amino acid residues in functional variants is positively correlated with their propensity to be generally found in ␤-turns. The folding pathway of two selected variants, EETI-␤ NEDE and EETI-␤ TNNK , was found to be indistinguishable from EETI-II and occurs through formation of a stable 2-disulfide intermediate. Substantial amounts of misfolded byproducts, however, were obtained upon refolding of these variants corroborating the importance of the wild type EETI-II GPNG sequence to direct quantitative formation of the cystine knot architecture.Numerous small proteins, typically not longer than 40 residues in length, share a common structural motif consisting of a cystine knot and a small triple-stranded ␤-sheet. Members of the "knottin" family (1) of small proteins have a common architecture, but diverse biological activities and negligible amino acid sequence identity. Examples are (i) -conotoxin MVIIa, a 26-residue polypeptide found in the venom of the cone snail Conus magus, which acts as a neurotoxin by its high affinity binding to voltage-gated Ca 2ϩ channels (2); (ii) potato carboxypeptidase inhibitor (PCI), 1 a 39-amino acid peptide (3); and (iii) EETI-II from the squirting cucumber Ecballium elaterium, a member of the squash family of protease inhibitors (4). These proteins are mainly stabilized by three intramolecular disulfide bonds, where the first cysteine residue in the polypeptide chain is connected with the fourth, the second with the fifth, and the third with the sixth (Fig. 1, top panel). The cystine knot formed by these three disulfide bonds is defined by a ring formed by the first and the second disulfide bond in the peptide sequence and the intervening polypeptide backbone, through which the third disulfide bond passes (Fig.

mentioning

confidence: 98%

mentioning

confidence: 99%

See 1 more Smart Citation

Sequence Requirements of the GPNG β-Turn of the Ecballium elaterium Trypsin Inhibitor II Explored by Combinatorial Library Screening

Wentzel¹,

Christmann²,

Krätzner³

et al. 1999

“…3b). Although the N-terminal disulfide bridge of the inhibitory cystine knot motif does not generally contribute to the hydrophobic core of inhibitory cystine knot toxins and is not essential for formation of the basic inhibitory cystine knot fold (6,29), a complete cystine knot motif has been found in all four atracotoxin structures reported to date (this study and Refs. 6, 9, and 11).…”

Section: Isolation Of a Novel Insecticidal Toxin-mentioning

confidence: 77%

Discovery and Structure of a Potent and Highly Specific Blocker of Insect Calcium Channels

Wang

Connor

Wilson

et al. 2001

We have isolated a novel family of insect-selective neurotoxins that appear to be the most potent blockers of insect voltage-gated calcium channels reported to date. These toxins display exceptional phylogenetic specificity, with at least a 10,000-fold preference for insect versus vertebrate calcium channels. The structure of one of the toxins reveals a highly structured, disulfide-rich core and a structurally disordered C-terminal extension that is essential for channel blocking activity. Weak structural/functional homology with -agatoxin-IVA/B, the prototypic inhibitor of vertebrate P-type calcium channels, suggests that these two toxin families might share a similar mechanism of action despite their vastly different phylogenetic specificities.

“…A few residues are labeled for comparison. cystine knots and other three-and four-disulfide proteins, including, for example, the trypsin inhibitor EETI-II, which also shares high sequence and structural similarity with MCoTI-II (16,33). Both EETI-II and MCoTI-II have 11 residues embedded in the backbone ring that form in the core of the cystine knot, whereas kalata B1 has only eight residues because of a shorter loop 1, as illustrated in Fig.…”

Section: Discussionmentioning

confidence: 99%

Knots in Rings

Cemazar¹,

Daly²,

Häggblad

et al. 2006

The aim of this work was to elucidate the oxidative folding mechanism of the macrocyclic cystine knot protein MCoTI-II. We aimed to investigate how the six-cysteine residues distributed on the circular backbone of the reduced unfolded peptide recognize their correct partner and join up to form a complex cystine-knotted topology. To answer this question, we studied the oxidative folding of the naturally occurring peptide using a range of spectroscopic methods. Although it is widely accepted that the folding of proteins is governed exclusively by their amino acid sequence (1), the prediction of the threedimensional structure of a biologically active protein from its primary sequence remains an unsolved challenge. The importance of protein folding is highlighted by the causes of debilitating diseases such as Alzheimer disease, cystic fibrosis, and Creutzfeld-Jakob disease (2, 3), which are attributed to the loss of biological functions of specific proteins due to their inability to either fold or remain correctly folded. An understanding of protein folding should provide a greater opportunity to devise novel approaches for the treatment of these and other protein misfolding diseases.In proteins containing cysteine residues, the oxidative formation of native disulfide bonds is an integral part of the folding process. In such proteins, conformational folding is coupled with disulfide formation in the process of oxidative folding. Because disulfide bonds play key roles in the stabilization of three-dimensional structures in vivo but their formation in a cellular environment is poorly understood, the study of oxidative folding in vitro offers a valuable tool to understand the complex pathways by which native disulfide formation takes place. In particular, the study of oxidative folding is facilitated by the ability to isolate discrete (disulfide-bonded) intermediates, something that cannot be achieved in studies of conformational folding of non-disulfide-containing proteins.Model studies of oxidative folding of cysteine-rich proteins, such as bovine pancreatic trypsin inhibitor (4, 5) and ribonuclease A (6, 7) have established a basis for understanding this process. Most oxidative folding studies reported to date have involved the isolation of stable intermediates by liquid chromatographic purification of acid-quenched folding reactions followed by structural elucidation and disulfide connectivity analysis. The oxidative folding pathways analyzed so far can be classified in terms of the number and types (i.e. native or nonnative) of disulfide bonds present in the intermediate species. Interestingly, different combinations of intermediates have been found for different proteins, and it is not clear how the primary amino acid sequence or a particular three-dimensional fold relates to a particular type of oxidative folding pathway. Thus there is a need for detailed studies on specific protein classes, and in this paper, we report on a class where the final disulfide network is particularly interesting in that it forms a "...