The specificity of polygalacturonase-inhibiting protein (PGIP): a single amino acid substitution in the solvent-exposed beta-strand/beta-turn region of the leucine-rich repeats (LRRs) confers a new recognition capability.
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 beta-sheet/beta-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.
Leckie
F
F
Dipartimento di Biologia Vegetale, Università di Roma 'La Sapienza', Piazzale Aldo Moro 5, 00185, Roma, Italy.
Mattei
B
B
Capodicasa
C
C
Hemmings
A
A
Nuss
L
L
Aracri
B
B
De Lorenzo
G
G
Cervone
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D007887:Fabaceae / Q000235:genetics; D005670:Fusarium / Q000201:enzymology; D015723:Gene Library; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D010946:Plants, Medicinal; D010947:Plants, Toxic; D011096:Polygalacturonase / Q000037:antagonists & inhibitors*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D020449:Repetitive Sequences, Amino Acid; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000235:genetics
D018076:DNA, Complementary; C081479:PGIP protein, plant; D010940:Plant Proteins; D011994:Recombinant Proteins; D011096:Polygalacturonase