Low-pH Molten Globule-Like Form of CIA17, a Chitooligosaccharide-Specific Lectin from the Phloem Exudate of Coccinia indica, Retains Carbohydrate-Binding Ability.

Abstract

pH-induced changes in the conformation, structural dynamics, and carbohydrate-binding activity of Coccinia indica agglutinin (CIA17), a PP2-type lectin, were investigated employing biophysical approaches. The secondary structure of CIA17 remains nearly unaltered over a wide pH range (2.0-8.5), while the tertiary structure of the protein exhibits considerable changes. A decrease in the fluorescence intensity and excited-state lifetime at low pH indicated perturbation in the local conformation (near Trp residues) of CIA17, which was further supported by enhancement in the Trp accessibility toward charged quenchers under acidic conditions. Fluorescence correlation spectroscopic studies indicated that at pH 2.0, CIA17 exists as a monomer over the concentration range of 10-200 nM and forms dimers at higher concentrations (KD ∼ 387 nM) but could not form higher oligomers even at ∼150-fold higher concentrations, unlike under native conditions at pH 7.4. Thermal unfolding of the low pH intermediate involves two distinct steps: dissociation of a dimer to a monomer, followed by the unfolding of the monomer. These results strongly suggest that the acid-induced unfolding pathway of CIA17 involves the formation of a monomeric molten globule-like intermediate, which retains appreciable carbohydrate-binding ability. These observations are of great physiological significance since the PP2 proteins are involved in plant defense responses.