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.