In vitro characterization of the homogalacturonan-binding domain of the wall-associated kinase WAK1 using site-directed mutagenesis.
Wall-associated kinase 1--WAK1 is a transmembrane protein containing a cytoplasmic Ser/Thr kinase domain and an extracellular domain in contact with the pectin fraction of the plant cell wall in Arabidopsis thaliana (L.) HEYNH. In a previous paper [Decreux, A., Messiaen, J., 2005. Wall-associated kinase WAK1 interacts with cell wall pectins in a calcium-induced conformation. Plant Cell Physiol. 46, 268-278], we showed that a recombinant peptide expressed in yeast corresponding to amino acids 67-254 of the extracellular domain of WAK1 specifically interacts with commercial non-methylesterified homogalacturonic acid, purified homogalacturonans from Arabidopsis and oligogalacturonides in a calcium-induced conformation. In this report, we used a receptor binding domain sequence-based prediction method to identify four putative binding sites in the extracellular domain of WAK1, in which cationic amino acids were selected for substitution by site-directed mutagenesis. Interaction studies between mutated forms of WAK1 and homogalacturonans allowed us to identify and confirm at least five specific amino acids involved in the interaction with homogalacturonan dimers and multimers. The presence of this homogalacturonan-binding domain within the extracellular domain of WAK1 is discussed in terms of cell wall architecture and signal transduction.
Decreux
Annabelle
A
FUNDP, Unité de Recherche en Biologie Cellulaire et Moléculaire Végétale, 61 rue de Bruxelles, 5000 Namur, Belgium.
Thomas
Annick
A
Spies
Benoît
B
Brasseur
Robert
R
Van Cutsem
Pierre
P
Messiaen
Johan
J
D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010368:Pectins / Q000378:metabolism*; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012964:Sodium / Q000494:pharmacology
D029681:Arabidopsis Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D010368:Pectins; D012964:Sodium; D011494:Protein Kinases; C101093:WAK1 protein, Arabidopsis; D002118:Calcium; C003181:polygalacturonic acid