In silico characterization of SAP55: insights into a predicted phytoplasmal M41-like metallopeptidase effector with potential eukaryotic host dual lipidation motifs

Abstract Phytoplasmas are cell wall-less, phloem-limited plant pathogenic bacteria that cause devastating agricultural losses globally. Although phytoplasma pathogenicity is driven by secreted effector proteins translocated via the Sec pathway, their identification and functional characterization remain severely hindered by the fastidious nature of these pathogens. Here, we present a comprehensive structural, evolutionary, and functional in silico characterization of SAP55, an uncharacterized candidate effector from the Aster yellows witches’-broom strain. AlphaFold 3 modeling predicted an N-terminal signal peptide with a cleavage-compatible structural architecture that is predicted to interact with phytoplasmal signal peptidase I. Genomic and phylogenetic analyses revealed that SAP55 is linked to potential mobile units and virulence islands, suggesting potential evolutionary mobility across lineages. Structural and sequence-based annotation identified a core domain with similarities to the M41 zinc-dependent metallopeptidase family with a conserved HEXXH motif. Notably, SAP55 is predicted to represent an atypical protease variant; structural comparisons and HSYMDOCK/PDBePISA thermodynamic simulations suggest that it lacks the AAA+ ATPase domain, the central loop, and hexameric subunit affinity, operating instead as a putative monomeric form with an elongated antiparallel β4-strand that may facilitate substrate interaction. Our analyses suggest that the conserved N-terminal methionine may represent a potential stabilization feature under N-end rule principle. Its C-terminal hypervariable region contains a conserved CXCAAL motif and polybasic cluster predicted to be compatible with host geranylgeranyltransferase type I and S-palmitoylation machinery, potentially supporting association with the cytoplasmic leaflet of the plasma membrane By providing a comprehensive computational framework for a candidate membrane-associated candidate effector, this study proposes a working model for SAP55-mediated host manipulation, and establishes a foundation for future experimental investigation.