Disulfide Bond Structure of the AVR9 Elicitor of the Fungal Tomato Pathogen Cladosporium fulvum: Evidence for a Cystine Knot
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- 作者：Henno W. van den Hooven ; Harrold A. van den Burg ; Paul Vossen ; Sjef Boeren ; Pierre J. G. M. de Wit ; and Jacques Vervoort
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：March 27, 2001
- 年：2001
- 卷：40
- 期：12
- 页码：3458 - 3466
- 全文大小：137K
- 年卷期：v.40,no.12(March 27, 2001)
- ISSN：1520-4995
文摘
Disease resistance in plants is commonly activated by the product of an avirulence (Avr) geneof a pathogen after interaction with the product of a matching resistance (R) gene in the host. In susceptibleplants, Avr products might function as virulence or pathogenicity factors. The AVR9 elicitor from thefungus Cladosporium fulvum induces defense responses in tomato plants carrying the Cf-9 resistancegene. This 28-residue 
-sheet AVR9 peptide contains three disulfide bridges, which were identified inthis study as Cys2-Cys16, Cys6-Cys19, and Cys12-Cys26. For this purpose, AVR9 was partially reduced,and the thiol groups of newly formed cysteines were modified to prevent reactions with disulfides. AfterHPLC purification, the partially reduced peptides were sequenced to determine the positions of the modifiedcysteines, which originated from the reduced disulfide bridge(s). All steps involving molecules with freethiol groups were performed at low pH to suppress disulfide scrambling. For that reason, cysteinemodification by N-ethylmaleimide was preferred over modification by iodoacetamide. Upon (partial)reduction of native AVR9, the Cys2-Cys16 bridge opened selectively. The resulting molecule was furtherreduced to two one-bridge intermediates, which were subsequently completely reduced. The (partially)reduced cysteine-modified AVR9 species showed little or no necrosis-inducing activity, demonstratingthe importance of the disulfide bridges for biological activity. Based on peptide length and cysteine spacing,it was previously suggested that AVR9 isa cystine-knotted peptide. Now, we have proven that the bridgingpattern of AVR9 is indeed identical to that of cystine-knotted peptides. Moreover, NMR data obtainedfor AVR9 show that it is structurally closely related to the cystine-knotted carboxypeptidase inhibitor.However, AVR9 does not show any carboxypeptidase inhibiting activity, indicating that the cystine-knotfold is a commonly occurring motif with varying biological functions.
-sheet AVR9 peptide contains three disulfide bridges, which were identified inthis study as Cys2-Cys16, Cys6-Cys19, and Cys12-Cys26. For this purpose, AVR9 was partially reduced,and the thiol groups of newly formed cysteines were modified to prevent reactions with disulfides. AfterHPLC purification, the partially reduced peptides were sequenced to determine the positions of the modifiedcysteines, which originated from the reduced disulfide bridge(s). All steps involving molecules with freethiol groups were performed at low pH to suppress disulfide scrambling. For that reason, cysteinemodification by N-ethylmaleimide was preferred over modification by iodoacetamide. Upon (partial)reduction of native AVR9, the Cys2-Cys16 bridge opened selectively. The resulting molecule was furtherreduced to two one-bridge intermediates, which were subsequently completely reduced. The (partially)reduced cysteine-modified AVR9 species showed little or no necrosis-inducing activity, demonstratingthe importance of the disulfide bridges for biological activity. Based on peptide length and cysteine spacing,it was previously suggested that AVR9 isa cystine-knotted peptide. Now, we have proven that the bridgingpattern of AVR9 is indeed identical to that of cystine-knotted peptides. Moreover, NMR data obtainedfor AVR9 show that it is structurally closely related to the cystine-knotted carboxypeptidase inhibitor.However, AVR9 does not show any carboxypeptidase inhibiting activity, indicating that the cystine-knotfold is a commonly occurring motif with varying biological functions.