Cryo-EM structure of the Agrobacterium tumefaciens T-pilus reveals the importance of positive charges in the lumen

Abstract

Agrobacterium tumefaciens is a natural genetic engineer that transfers DNA into plants and this is the most frequently applied process for the generation of genetically modified plants. DNA transfer is mediated by a type IV secretion system localized in the cell envelope and extracellular T-pili. We here report the cryo-electron microscopic structures of the T-pilus at 3.2[A] resolution and that of the related plasmid pKM101-determined N-pilus at 3[A] resolution. Both pili contain a main pilus protein (VirB2 in A. tumefaciens and TraM in pKM101) and phospholipids arranged in a 5-start helical assembly. They contain positively charged amino acids in the pilus lumen and the lipids are positively charged in the T-pilus (phosphatidylcholine) conferring overall positive charge to the lumen. Mutagenesis of the lumen-exposed Arg91 residue in VirB2 resulted in protein destabilization and loss of pilus formation. Our results reveal that different phospholipids can be incorporated into type IV secretion system pili and that the charge of the lumen is of functional importance.