Active, Legionella pneumophila, Recombinant LotA-N

Species & domain architecture LotA-N is a 28 kDa OTU fold capped by a Legionella-specific extended β-hairpin and “adaptive” helical insert that create a deep S1′ cradle for the proximal ubiquitin. The high-resolution structure (PDB 8DEB) plus a LotA–di-ubiquitin mimic complex show how hydrophobic Val10/Phe93 and acidic Asp137 coordinate the Lys6 isopeptide while gating other poly-ubiquitin linkages. UPS pathway role During infection LotA is translocated into the Legionella-containing vacuole, where it strips Lys6 chains that would otherwise recruit the p97/VCP segregase and promote lysosomal clearance of the vacuole. Biochemically, its strict Lys6 bias makes it an indispensable tool for dissecting the still-enigmatic roles of Lys6 signaling in mitophagy, DNA-damage responses and Parkin biology. Relevance to TPD & disease biology Lys6 chains modulate mitochondrial quality control and BRCA1-mediated DNA repair; off-target Lys6 tagging can thus confound degrader MoA studies. LotA-N provides a rapid “erase-and-check” reagent to confirm linkage purity, and its structure provides scaffold for designing synthetic Lys6-editing enzymes or linkage-biased affinity reagents. References Shin, D., et al., (2020) eLife 9:e58277. PMID 33185526 Luo, J., et al., (2022) J Biol Chem 298:102414. PMID 36007613 Takekawa, N., et al., (2022) J Bacteriol 204:e00376-21. PMID 34633867 Warren, G. D., et al., (2023) Mol Cell 83:105-120.e5. PMID 36538933 Zhang, Z., and C. Das. (2023) Trends Microbiol 31:423-425. PMID 36890008