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The specificity of polygalacturonase-inhibiting protein (PGIP): A single amino acid substitution in the solvent-exposed β-strand/β-turn region of the leucine-rich repeats (LRRs) confers a new recognition capability

TitleThe specificity of polygalacturonase-inhibiting protein (PGIP): A single amino acid substitution in the solvent-exposed β-strand/β-turn region of the leucine-rich repeats (LRRs) confers a new recognition capability
Publication TypeArticolo su Rivista peer-reviewed
Year of Publication1999
AuthorsLeckie, F., Mattei B., Capodicasa Cristina, Hemmings A., Nuss L., Aracri B., De Lorenzo G., and Cervone F.
JournalEMBO Journal
Pagination2352 - 2363
Date Published1999
ISBN Number02614189 (ISSN)
Keywordsamino acid substitution, article, Aspergillus niger, Embryophyta, enzyme inhibitor, enzyme specificity, Fusarium, Fusarium moniliforme, Gibberella fujikuroi, glutamine, higher plant, leucine, Leucine-rich repeat proteins, ligand binding, lysine, Molecular recognition, Multigene Family, Nicotiana benthamiana, Phaseolus vulgaris, polygalacturonase, Polygalacturonase-inhibiting protein (PGIP), priority journal, Site directed mutagenesis, solvent, surface plasmon resonance, vegetable protein

Two members of the pgip gene family (pgip-1 and pgip-2) of Phaseolus vulgaris L. were expressed separately in Nicotiana benthamiana and the ligand specificity of their products was analysed by surface plasmon resonance (SPR). Polygalacturonase-inhibiting protein-1 (PGIP-1) was unable to interact with PG from Fusarium moniliforme and interacted with PG from Aspergillus niger; PGIP-2 interacted with both PGs. Only eight amino acid variations distinguish the two proteins: five of them are confined within the β-sheet/β-turn structure and two of them are contiguous to this region. By site-directed mutagenesis, each of the variant amino acids of PGIP-2 was replaced with the corresponding amino acid of PGIP-1, in a loss-of-function approach. The mutated PGIP-2s were expressed individually in N. benthamiana, purified and subjected to SPR analysis. Each single mutation caused a decrease in affinity for PG from F. moniliforme; residue Q253 made a major contribution, and its replacement with a lysine led to a dramatic reduction in the binding energy of the complex. Conversely, in a gain-of-function approach, amino acid K253 of PGIP-1 was mutated into the corresponding amino acid of PGIP-2, a glutamine. With this single mutation, PGIP-1 acquired the ability to interact with F. moniliforme PG.


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