title: Dimerization and protease resistance: new insight into the function of PR-1.
abstract: The group 1 pathogenesis-related (PR-1) proteins have long been considered hallmarks of hypersensitive response/defense pathways in plants, but their biochemical functions are still obscure despite resolution of the NMR/X-ray structures of several PR-1-like proteins, including P14a (the prototype PR-1). We report here the characterization of two basic PR-1 proteins (PR-1-1 and PR-1-5) recently identified from hexaploid wheat (Triticum aestivum). Both proteins were expressed in Pichia pastoris as a single major species of ∼15 kDa. Sequence identity of the expressed PR-1 proteins was verified by MALDI-TOF/TOF analysis. Accumulation of the native PR-1-5 protein in pathogen-challenged wheat was confirmed by protein gel blot analysis. Low-temperature SDS-PAGE and yeast two-hybrid assays revealed that PR-1-1 exists primarily as a monomer whereas PR-1-5 forms homodimers. Both PR-1 proteins are resistant to proteases compared to bovine serum albumin, but PR-1-1 shows resistance mainly to subtilisin and protease K (serine proteases) whereas PR-1-5 shows resistance to subtilisin, protease K and papain (a cysteine protease). Site-specific mutations at the five putative active sites in the PR-1 domain all affected dimerization, with the mutations at Glu-72 and Glu-102 (in the PR-1-5 numeration) also diminishing protease resistance. Sequence analysis revealed that the Glu-72 and Glu-102 residues are located in motif-like sequences that are conserved in both PR-1 and the human apoptosis-related caspase proteins. These findings prompt us to examine the function of PR-1 for a role in protease-mediated programmed cell death pathways in plants.
authors.affiliation: USDA-ARS, Cereal Crops Research Unit, Fargo, ND 58102, USA. Shunwen.Lu@ars.usda.gov
authors.forename: Shunwen
authors.initials: S
authors.lastname: Lu
authors.forename: Justin D
authors.initials: JD
authors.lastname: Faris
authors.forename: Robert
authors.initials: R
authors.lastname: Sherwood
authors.forename: Michael C
authors.initials: MC
authors.lastname: Edwards
mesh_terms: D020816:Amino Acid Motifs; D020134:Catalytic Domain; D019281:Dimerization; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014908:Triticum / Q000235:genetics / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; C053376:pathogenesis-related proteins, plant; D010447:Peptide Hydrolases