In vitro functional analysis and in silico structural modelling of pathogen-secreted polyglycine hydrolases

Area of Science:

• Fungal biology
• Protease biochemistry
• Structural biology

Background:

• Polyglycine hydrolases are fungal secreted proteins with serine protease activity.
• They cleave Gly-Gly bonds in corn ChitA chitinase via their C-domain.
• The function of the N-domain (PND) remains uncharacterized.

Purpose of the Study:

• To investigate the function of the polyglycine hydrolase N-domain (PND).
• To determine if the PND directly participates in chitinase cleavage.
• To explore the natural occurrence and potential roles of PND-containing proteins.

Main Methods:

• In vitro site-directed mutagenesis of conserved PND residues in Es-cmp polyglycine hydrolase.
• High Ambiguity Driven protein-protein DOCKing (HADDOCK) for in silico modeling of ChitA-bound hydrolases.
• Computational analysis of Foldseek-identified PND-containing proteins.

Main Results:

• Mutagenesis of conserved PND residues did not impair protease activity.
• In silico models showed no significant predicted interactions between PND and ChitA.
• PND-containing proteins are widespread in biology, often fused with proteases or kinases.

Conclusions:

• The PND is unlikely to be directly involved in ChitA cleavage.
• The PND likely possesses a distinct, yet unknown, biological function.
• PNDs are versatile protein domains found across various biological contexts.