Submitted to: Physiological and Molecular Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/20/2014
Publication Date: 11/28/2014
Citation: Price, N.P., Momany, F.A., Schnupf, U., Naumann, T.A. 2014. Structure and disulfide bonding pattern of the hevein-like peptide domains from plant class IV chitinases. Physiological and Molecular Plant Pathology. 89:25-30.
Interpretive Summary: Proteins that are secreted by cells often have disulfide bond crosslinks that hold different parts of the protein together. This makes the proteins more stable to heat and the environment. This paper describes a new method for measuring the number of disulfide crosslinks that are present in a protein. We applied the method to two peptides, somatostatin and insulin. We also studied the crosslinks in the N-terminal peptides from Arabidopsis and corn chitinases, which play an important role in defense against Fusarium pathogens. The work provided further understanding of the plant-fungal pathogenic interactions, and is of relevance to plant pathologists and crop breeders.
Technical Abstract: Corn (Zea mays) and Arabidopsis (Arabidopsis thaliana) produce GH family 19 plant class IV chitinases. These chitinases contain two domains: a small N-terminal hevein region, and a C-terminal chitinase. Numerous structures of GH19 chitinase domains have been reported, including the chitinase domain of corn ChitA. Structural information on the N-terminal domains, however, is lacking. Fusarium pathogens secrete fungalysin proteases that cleave some class IV chitinases at a well-defined Gly-Cys site between the two domains. To study the structure of the peptide domain we used the fungalysin protease Fv-cmp as a tool to release the hevein domain from plant class IV chitinases, allowing their direct study. MALDI-TOF MS analysis of fungalysin-released peptides from plant class IV chitinases from corn and Arabidopsis allowed visualization of multiple isotopomers, resulting in accurate mass determination. When treated with DTT, peptide ions increased in mass by six mass units, suggesting breakage of three disulfide bonds. When reduced peptides were S-alkylated, peptides were converted to a series of evenly spaced ions with various states of alkylation, confirming the presence of six reduced cysteines. This chemical data was complemented by use of molecular modeling to determine the fold of the peptide and location of disulfide bonds. The chemical data and molecular model combine to create a structural model of a hevein domain from a GH19 chitinase.