Structural basis for the acetylation mechanism of the Legionella effector VipF

Abstract

The pathogen Legionella pneumophila, which is the causative agent of Legionnaires' disease, secrets hundreds of effectors into host cells via its Dot/Icm secretion system to subvert host-cell pathways during pathogenesis. VipF, a conserved core effector among Legionella species, is a putative acetyltransferase, but its structure and catalytic mechanism remain unknown. Here, three crystal structures of VipF in complex with its cofactor acetyl-CoA and/or a substrate are reported. The two GNAT-like domains of VipF are connected as two wings by two β-strands to form a U-shape. Both domains bind acetyl-CoA or CoA, but only in the C-terminal domain does the molecule extend to the bottom of the U-shaped groove as required for an active transferase reaction; the molecule in the N-terminal domain folds back on itself. Interestingly, when chloramphenicol, a putative substrate, binds in the pocket of the central U-shaped groove adjacent to the N-terminal domain, VipF remains in an open conformation. Moreover, mutations in the central U-shaped groove, including Glu129 and Asp251, largely impaired the acetyltransferase activity of VipF, suggesting a unique enzymatic mechanism for the Legionella effector VipF.

An acetyltransferase effector conserved across Legionella species targets the eukaryotic eIF3 complex to modulate protein translation

“…1F ). These results are in line with our structural analysis, indicating that only the C-terminal domain of the VipF effectors is responsible for observed in vitro acetyltransferase activity, which was also shown in a previous report of Lpg0103 ( 23 ).…”

“…Among tested substrates, we observed that Lha0223 activity was highest against chloramphenicol, in line with the general activity reported for L. pneumophila VipF/Lpg0103 ( Fig. 1B ) ( 22 , 23 ). However, Lha0223 also demonstrated strong activity against poly-lysine substrate suggesting that it can be active against peptide or protein targets.…”

“…The phylogenetic reconstruction revealed that VipF homologs in Legionella species shared between 35% and 83% of primary sequence identity, suggesting a significant diversification across this effector family. Along with previous data ( 23 ), our analysis of the primary sequences of VipF orthologs confirmed the presence of conserved motifs corresponding to two GNAT domains typically found in a small subfamily, called tandem GNAT enzymes (Fig. S1A).…”

“…1A ). Recently, the structure of Lpg0103/VipF in complex with CoA and chloramphenicol was reported ( 23 ). The defined structure of Lha0223 acetyl-CoA complex superimposes with Lpg0103 complex structure with RMSD of 1.08 Å over 264 α-carbons, thus suggesting that VipF orthologs, despite having significant variation in primary sequence, share a conserved overall structure (Fig.…”

“…A previous report suggested that both GNAT domains of Lpg0103 are active against chloramphenicol substrate. However, a more recent report established that VipF follows the general trend of tandem GNATs with only the C-terminal domain retaining the catalytic activity ( 22 , 23 ). Despite these insights into the biochemical activity of VipF, the significance of its acetyltransferase activity function for Legionella ’s invasion of the host cell has never been elucidated.…”