Cystine Knot Mutations Affect the Folding of the Glycoprotein Hormone α-Subunit

110

The extracellular AVR4 elicitor of the pathogenic fungus Cladosporium fulvum induces defense responses in the tomato genotype Cf-4. Here, the four disulfide bonds of AVR4 were identified as Cys-11-41, Cys-21-27, Cys-35-80, and Cys-57-72 by partial reduction with Tris-(2-carboxyethyl)-phosphine hydrochloride, subsequent cyanylation, and base-catalyzed chain cleavage. The resulting peptide fragments were analyzed by mass spectrometry. Sequence homology and the disulfide bond pattern revealed that AVR4 contains an invertebrate (inv) chitin-binding domain (ChBD). Binding of AVR4 to chitin was confirmed experimentally. The three disulfide bonds encompassing the inv ChBD motif are also required for protein stability of AVR4. Independent disruption of each of the three conserved disulfide bonds in AVR4 resulted in a protease-sensitive protein, whereas the fourth disulfide bond appeared not to be required for protein stability. Most strains of C. fulvum virulent on Cf-4 tomato contain Cys to Tyr substitutions in AVR4 involving two (Cys-11-41, Cys-35-80) of the three disulfide bonds present in the inv ChBD motif. These natural Cys to Tyr mutant AVR4 proteins did retain their chitin binding ability and when bound to chitin were less sensitive to proteases. Thus, the widely applied tomato Cf-4 resistance gene is circumvented by C. fulvum by amino acid substitutions affecting two disulfide bonds in AVR4 resulting in the absence of the corresponding AVR4 isoforms in apoplastic fluid. However, these natural isoforms of AVR4 appear to have retained their intrinsic function, i.e. binding to chitin present in the cell wall of C. fulvum, most likely to protect it against the deleterious effects of plant chitinases.Gene-for-gene-based disease resistance in plants commonly requires two complementary genes, an avirulence (Avr) 1 gene in the pathogen and a matching resistance gene in the host (1, 2). The Cf resistance genes of tomato mediate specific recognition of extracellular elicitor proteins encoded by Avr genes of the pathogenic fungus Cladosporium fulvum (3). The Avrs of C. fulvum and their matching Cf genes have become valuable instruments to investigate signal transduction pathways leading to plant disease resistance (4 -7, 9).2 To obtain sustainable resistance, the Cf resistance genes were introgressed from wild Lycopersicon species into commercial tomato cultivars. However, because of selection pressure new strains of C. fulvum emerged that had overcome the introgressed resistance traits by modification of the Avr genes (10). Although some Avr genes in these virulent C. fulvum strains were found to be absent (11), others contained point mutations (12) or transposon insertions (13). The natural strains of C. fulvum carrying these mutated Avr genes did not exhibit significantly reduced virulence under laboratory conditions (11-14), suggesting that AVR proteins are not essential for virulence or that the modified isoforms of AVRs still contribute to virulence of C. fulvum. The genetic variation is so far strictly limited to t...

Section: Figsupporting

confidence: 80%

Natural Disulfide Bond-disrupted Mutants of AVR4 of the Tomato Pathogen Cladosporium fulvum Are Sensitive to Proteolysis, Circumvent Cf-4-mediated Resistance, but Retain Their Chitin Binding Ability

Burg

Westerink

Françoijs

et al. 2003

110

“…Previous studies have revealed the importance of the disulfide bridges for the folding of glycoprotein hormone ␣-subunit in vivo (30). Here we showed that in vitro, it is possible to generate cystine-deficient mutants of the cystine knot growth factor VEGF and to subsequently investigate their crystal structures.…”

Section: Function Of the Cystine Knot In Vegfmentioning

confidence: 90%

“…Unfortunately, comparisons with other growth factors cannot be drawn because to the best of our knowledge, no thermodynamic characterization of a cystine knot growth factor has been reported to date. Data on the function of the individual disulfide bridges in these growth factors are restricted to observations on impaired expression in mammalian cells (30), as well as to measurements of residual biological activity in mutants in which single cysteines have been removed (31,32).…”

Section: Enthalpic and Entropic Contributions To ⌬Gmentioning

confidence: 99%

The Cystine Knot Promotes Folding and Not Thermodynamic Stability in Vascular Endothelial Growth Factor

Muller

Heiring

Misselwitz

et al. 2002

Cystine knots consist of three intertwined disulfide bridges and are considered major determinants of protein stability in proteins in which they occur. We questioned this function and observed that removal of individual disulfide bridges in human vascular endothelial growth factor (VEGF) does not reduce its thermodynamic stability but reduces its unexpected high thermal stability of 108°C by up to 40°C. In wild-type VEGF (⌬G u . A detailed crystallographic analysis of the mutant structures suggests a role of the cystine knot motif in protein folding rather than in the stabilization of the folded state. When assuming that the sequential order of the disulfide bridge formation is conserved between VEGF and glycoprotein ␣-subunit, the crystal structure of the mutant C61A-C104A, which deviates by a root mean square deviation of more than 2.2 Å from wild-type VEGF, identifies a true folding intermediate of VEGF.The cystine knot structural motif consists of three highly intertwined disulfide bridges and occurs in the small inhibitor cystine knot proteins, the cyclotides and the growth factor cystine knot proteins. The first two classes of proteins consist of small polypeptides ranging from 25 to 30 amino acids with functions as diverse as protease inhibition (squash family of inhibitors), ion channel blocking (toxins), and antimicrobial activity (cyclotides) (1-3). The third class consists of the growth factor cystine knot proteins, with members such as transforming growth factor-␤, ␣-nerve growth factor, platelet-derived growth factor, vascular endothelial growth factor (VEGF), 1 bone morphogenic proteins, IL-17s, and many others. These physiologically important growth factors share a common monomer structure but differ in their mode of dimerization (3), thus introducing molecular diversity into the structural mechanism by which these growth factors activate their cognitive receptors (4). Whole genome analyses hint that the cystine knot motif might be very common in extracellular signaling molecules (5).The molecular function of the cystine knot motif is poorly understood. In this motif, two disulfide bridges connect two neighboring chain segments and form a ring structure; a third disulfide bridge penetrates this ring segment and cross-links two additional chain segments. By implication, the cystine knot motif interlocks four separate chain segments. Considerations on the small size of the inhibitor cystine knot proteins and the lack of extended hydrophobic core regions in both the inhibitor and growth factor cystine knot proteins lead to the assumption that this structural motif is a major determinant for the thermodynamic stability of these proteins. Thus, amide exchange experiments estimated the thermodynamic stability of the toxin -MVIIA to be about 4 kcal mol Ϫ1 (6). This equals the stabilization often introduced by single disulfide bridges of 3-5 kcal mol Ϫ1 (7, 8), and therefore it is plausible that removal of single cystines impairs the stability of these proteins.We challenged this assumption and studied t...

“…Oxidative folding has now been studied for representatives of all three subfamilies of cystine knots, namely the inhibitory cystine knots (12,14,15), the growth factor knots (36,37), and the cyclic cystine knot (20), and the present study is the first report on the oxidative folding of a representative of the trypsin inhibitor subfamily of CCK. Some studies have reported that the cystine knot is either necessary for folding or has a role in directing the folding (36,37), but that it is not required for the overall thermodynamic stability of the protein (37).…”

Section: Discussionmentioning

confidence: 99%

“…Some studies have reported that the cystine knot is either necessary for folding or has a role in directing the folding (36,37), but that it is not required for the overall thermodynamic stability of the protein (37). Several reports have also emphasized that the cystine knot is a structural feature that governs the exceptional thermal (as opposed to thermodynamic) stability of proteins containing it (22,37).…”

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 "...