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

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 1999 May 6

PMID 10228150

Citations 67

Authors

F Leckie

B Mattei

C Capodicasa

A Hemmings

L Nuss

B Aracri

G De Lorenzo

F Cervone

Affiliations

Soon will be listed here.

Abstract

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.

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