Structural basis for selective AMPylation of Rac-subfamily GTPases by Bartonella effector protein 1 (Bep1)

Abstract

Small GTPases of the Ras-homology (Rho) family are conserved molecular switches that control fundamental cellular activities in eukaryotic cells. As such, they are targeted by numerous bacterial toxins and effector proteins, which have been intensively investigated regarding their biochemical activities and discrete target spectra; however, the molecular mechanism of target selectivity has remained largely elusive. Here we report a bacterial effector protein that selectively targets members of the Rac subfamily in the Rho family of small GTPases but none in the closely related Cdc42 or RhoA subfamilies. This exquisite target selectivity of the FIC domain AMP-transferase Bep1 from Bartonella rochalimae is based on electrostatic interactions with a subfamily-specific pair of residues in the nucleotide-binding G4 motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 enable modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural understanding of target selectivity toward Rac-subfamily GTPases and provides a highly selective tool for their functional analysis.

Evolutionary Diversification of Host-Targeted Bartonella Effectors Proteins Derived from a Conserved FicTA Toxin-Antitoxin Module

“…All members of branch 1 are predicted to catalyze AMP transfer (indicated in green in the cladogram). This has been verified for Bro _Bep1, which AMPylates the switch 1 loop of small GTPases [ 16 ]. Most members of branch A are putative AMP transferases as well.…”

“…[ 1 , 44 ]) and shown for Bhe_BepC further down, the ATP substrate binds to a crevice formed by helices α1, α2, α4, and α6 with the α-phosphate moiety interacting with the N-terminus of α5. Suspended above the bound ATP substrate, there is an irregular β-hairpin (flap, orange) that has been shown to interact with target proteins (e.g., the switch-1 loop of small GTPases [ 14 , 16 ]) through β-sheet augmentation. Another β-hairpin (Bep element, shown in green) found exclusively in BepFICs precedes the flap.…”

“…As the basis for a detailed structure–function relationship study, Bep constructs were subjected to crystallization and resulted in eight new crystal structures of representative FICBeps with resolutions ranging between 3 and 1.6 Å ( Table S3 ). Figure 5 shows a side-by-side view and a superposition of these crystal structures and the recently determined Bro_Bep1 structure [ 16 ]. The comparison shows that the conformation and relative position/orientation of N-ext, the Bep element (green), and the FIC signature loop (red) are invariant.…”

“…α inh is found N-terminally or C-terminally at the FIC core in class II and III Fic proteins, respectively, or is part of a small interacting protein known as antitoxin in the case of class I Fic proteins [ 1 ]. Additionally, FIC domains generally contain a β-hairpin between helix α2 and helix α3 that is referred to as the “flap” and mediates docking of a target segment in extended conformation via β-strand augmentation [ 5 , 14 , 15 , 16 ]. This sequence-independent interaction ensures the productive insertion of the modifiable hydroxyl residue of the target into the FIC active site.…”

“…Although novel types of effectors without FIC domains arose in all three linages, a prominent fraction of prototypic Beps in L1 (12 out of 21), L3 (9 out of 12), and L4 (5 out of 10) contain FIC domains that are generally assumed to engage with target proteins in the host, although the molecular activity and biological role of these BepFIC domains have only begun to be revealed [ 21 ]. So far, it has been shown that Bep1 AMPylates Rac-subfamily GTPases [ 16 ], Bep2 AMPylates the host intermediate filament protein vimentin [ 25 ], and BepA causes the AMPylation of two unknown host proteins of ca. 40–50 kDa size [ 6 ].…”