Ionic interaction of positive amino acid residues of fungal hydrophobin RolA with acidic amino acid residues of cutinase CutL1

Abstract

Summary: Hydrophobins are amphipathic proteins secreted by filamentous fungi. When the industrial fungus Aspergillus oryzae is grown in a liquid medium containing the polyester polybutylene succinate co-adipate (PBSA), it produces RolA, a hydrophobin, and CutL1, a PBSA-degrading cutinase. Secreted RolA attaches to the surface of the PBSA particles and recruits CutL1, which then condenses on the particles and stimulates the hydrolysis of PBSA. Here, we identified amino acid residues that are required for the RolA-CutL1 interaction by using site-directed mutagenesis. We quantitatively analyzed kinetic profiles of the interactions between RolA variants and CutL1 variants by using a quartz crystal microbalance (QCM). The QCM analyses revealed that Asp142, Asp171 and Glu31, located on the hydrophilic molecular surface of CutL1, and His32 and Lys34, located in the N-terminus of RolA, play crucial roles in the RolA-CutL1 interaction via ionic interactions. RolA immobilized on a QCM electrode strongly interacted with CutL1 (KD=6.5nM); however, RolA with CutL1 variants, or RolA variants with CutL1, showed markedly larger KD values, particularly in the interaction between the double variant RolA-H32S/K34S and the triple variant CutL1-E31S/D142S/D171S (KD=78.0nM). We discuss a molecular prototype model of hydrophobin-based enzyme recruitment at the solid-water interface. The Aspergillus oryzae hydrophobin RolA attaches to the PBSA surface, then recruits and condenses cutinase CutL1 on the surface. Here, we identified amino acid residues required for the RolA-CutL1 interaction. The QCM analysis revealed that negatively charged residues (Asp142, Asp171, and Glu31) of CutL1, and positively charged residues (His32 and Lys34) of RolA play crucial roles in the RolA-CutL1 interaction via ionic interactions. We discuss a molecular prototype model of hydrophobin-based enzyme recruitment at the solid-water interface.