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

Authors: Naumann, Todd; DOWLING, NICOLE - University Of Waterloo; Price, Neil; ROSE, DAVID - University Of Waterloo

Submitted to: Biochemical and Biophysical Research Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2024
Publication Date: 3/6/2024
Citation: Naumann, T.A., Dowling, N.V., Price, N.P., Rose, D.R. 2024. In vitro functional analysis and in silico structural modelling of pathogen-secreted polyglycine hydrolases. Biochemical and Biophysical Research Communications. https://doi.org/10.1016/j.bbrc.2024.149746.
DOI: https://doi.org/10.1016/j.bbrc.2024.149746

Interpretive Summary: Fungi can attack corn in the field, causing disease and contaminating the grain with harmful toxins called mycotoxins. While plants have proteins that target fungi and help to defend against disease, fungi have proteins that target these plant defenses. ARS researchers in Peoria, Illinois, worked with scientists in Waterloo, Ontario, Canada, to study how one of the fungal proteins, polyglycine hydrolase, targets a corn defense protein. They combined laboratory experimentation, computational analyses and computer modeling to determine the structure and function of this fungal anti-defense protein. They found that the protein has two distinct parts. One part resembled proteins in bacteria that inactivate antibiotics but the structure and function of the second part was novel. They found that that part of the protein may help guide its delivery in the plant. These discoveries will help breeders produce plants with improved disease resistance and, ultimately, save farmers money and improve safety of food for consumers.

Technical Abstract: Polyglycine hydrolases are fungal effectors composed of an N-domain with unique sequence and structure and a C-domain that resembles beta-lactamases, with serine protease activity. These secreted fungal proteins cleave Gly-Gly bonds within a polyglycine sequence in corn ChitA chitinase. The polyglycine hydrolase N-domain (PND) function is unknown. In this manuscript we provide evidence that the PND does not directly participate in ChitA cleavage. In vitro analysis of site-directed mutants in conserved residues of the PND of polyglycine hydrolase Es-cmp did not specifically impair protease activity. Also, purified Galma1_244932, a fungal protein consisting of a PND without accessory domains, did not bind ChitA. Furthermore, in silico structural models of three ChitA-bound polyglycine hydrolases created by High Ambiguity Driven protein-protein DOCKing (HADDOCK) did not predict significant interactions between the PND and ChitA. Together these results suggest that the PND has another function. To determine what types of PND-containing proteins exist in nature we performed a computational analysis of Foldseek-identified PND-containing proteins. The analysis showed that proteins with PNDs are present throughout biology as either single domain proteins or fused to accessory domains that are diverse but are usually proteases or kinases.