The Legionella pneumophila GTPase activating protein LepB accelerates Rab1 deactivation by a non-canonical hydrolytic mechanism

Abstract

GTPase activating proteins (GAPs) from pathogenic bacteria and eukaryotic host organisms deactivate Rab GTPases by supplying catalytic arginine and glutamine fingers in trans and utilizing the cis-glutamine in the DXXGQ motif of the GTPase for binding rather than catalysis. Here, we report the transition state mimetic structure of the Legionella pneumophila GAP LepB in complex with Rab1 and describe a comprehensive structure-based mutational analysis of potential catalytic and recognition determinants. The results demonstrate that LepB does not simply mimic other GAPs but instead deploys an expected arginine finger in conjunction with a novel glutamic acid finger, which forms a salt bridge with an indispensible switch II arginine that effectively locks the cis-glutamine in the DXXGQ motif of Rab1 in a catalytically competent though unprecedented transition state configuration. Surprisingly, a heretofore universal transition state interaction with the cis-glutamine is supplanted by an elaborate polar network involving critical P-loop and switch I serines. LepB further employs an unusual tandem domain architecture to clamp a switch I tyrosine in an open conformation that facilitates access of the arginine finger to the hydrolytic site. Intriguingly, the critical P-loop serine corresponds to an oncogenic substitution in Ras and replaces a conserved glycine essential for the canonical transition state stereochemistry. In addition to expanding GTP hydrolytic paradigms, these observations reveal the unconventional dual finger and non-canonical catalytic network mechanisms of Rab GAPs as necessary alternative solutions to a major impediment imposed by substitution of the conserved P-loop glycine.

Keywords: GAP; GTPase; GTPase Activating Protein; Host-Pathogen Interactions; Legionella pneumophila; LepB; Rab; Rab Proteins; Rab1; X-ray Crystallography.

FIGURE 1.

Identification and characterization of the LepB GAP core. A, the region corresponding to the LepB GAP core identified by limited proteolysis with Glu-C is depicted above the predictions of consensus secondary structure (47), heptad repeats (hr) with Coils (48), and transmembrane (TM) helices with TMHMM (49). B, shown are time courses for Rab1 GTP hydrolysis at the indicated concentrations of the LepB GAP core. C, shown is stopped flow analysis of the kinetics for Rab1 GTP hydrolysis as a function of the concentration of the LepB GAP core. D, shown is a profile of the catalytic efficiency (k_cat/K_m) of the LepB GAP core for GST-fusions of 30 Rab GTPases. E, shown is gel filtration chromatography after incubation of the LepB GAP core with Rab1-GDP, Rab1-GDP-AlF₃, or Rab1-GppNHp in a 1:2 stoichiometric ratio. On the right is an SDS-polyacrylamide gel of the fractions corresponding to the first peak (elution volume 8.5–10.5 ml; 0.5-ml fractions). a.u., arbitrary units.

FIGURE 2.

Hydrolytic activities of Rab1A and Rab3A in the presence and absence of LepB. A, stopped flow experiments with excitation at 297 nm and detection through a 320-nm long pass filter are shown. Note that the small transient signal for Rab3A (right panel) requires Mg²⁺ and is also observed for the GppNHp-loaded protein (data not shown), indicating that it cannot be due to GTP hydrolysis. B, shown are microplate experiments with excitation at 297 nm and detection at 340 nm. C, shown are microplate experiments with phosphate release detected using the MDCC-phosphate-binding protein.

FIGURE 3.

Stereoviews of electron density contoured at 1.0 σ from a σA-weighted 2wF_o − DF_c map calculated with MAD phases improved by solvent flipping and NCS averaging. A, shown are chains A and B. B, shown are chains C and D. C, shown are chains E and F. The final refined model is also shown. D, shown is the extended solvent channel in the vicinity of chains E and F.

FIGURE 4.

Structural basis for Rab1 recognition by LepB. A, shown is the overall view of the LepB catalytic core in complex with Rab1-GDP and AlF₃. CTD, C-terminal domain; NTD, N-terminal domain. B, shown is the non-polar interface between LepB and the switch/interswitch regions of Rab1. LepB is rendered as spheres beneath a semi-transparent surface with carbon, nitrogen, and oxygen atoms colored light slate, blue, and red, respectively. Rab1 is rendered as tubes and colored as in panel A. C, shown is are polar interactions between LepB and Rab1, defined using a 3.4 Å distance cutoff with appropriate stereochemistry. D, shown is the location of AMPylated (Tyr-80) and phosphocholinated (Ser-79) residues.

FIGURE 5.

Non-canonical structural mechanism for acceleration of GTP hydrolysis. A, shown is the view of the GTP hydrolytic site in the LepB-Rab1 complex with canonical and non-canonical polar interactions, colored as indicated. B, shown is the view of the GTP hydrolytic site in the VirA-Rab1 complex (PDB ID 4FMB). C, shown is catalytic efficiency of mutations involving LepB and/or Rab1 residues in the non-canonical polar network (left), LepB residues in the polar/non-polar interface with the switch and interswitch regions (middle), and Rab1 residues in the non-canonical polar network with respect to the TBC domain of TBC1D20 (right). Bars are colored according to the location of the mutated residues using the color scheme in Fig. 2A. a.u., arbitrary units.

FIGURE 6.

Role of the tandem domain architecture and clamp interaction. A, shown are polar and non-polar interactions between Tyr-40 in the switch I region of Rab1 and the interface of the N-terminal and C-terminal domains of the LepB GAP core. CTD, C-terminal domain; NTD, N-terminal domain. B, shown is a comparison of Rab1 from the LepB complex with AMPylated Rab1-GTP (PDB ID 3NKV) after superposition. C, shown are polar interactions involving the Ser-20, Tyr-40, and Ser-42 in the closed conformation of AMPylated Rab1-GTP. D, shown is a comparison of Tyr-40 conformations in AMPylated Rab1-GTP alone and in the complexes with LepB and VirA (PDB ID 4FMB) after superposition of the Rab1 molecules. E, shown is catalytic efficiency of LepB and/or Rab1 mutations involving residues or domains implicated in the clamp interaction with Tyr-40. F, shown are rate constants for intrinsic GTP hydrolysis by Rab1 mutants.

FIGURE 7.

Comparison of hydrolytic site configurations in GAP-GTPase transition state mimetic complexes. Note the steric clash of the P-loop serine with the cis-Gln and arginine finger in the RasGAP complex with Ras G12S. The arginine and glutamine fingers in VirA and Gyp1 avoid conflict with the P-loop serine by inserting from compatible orientations. LepB avoids conflict by inserting the arginine finger from a compatible orientation and supplying a trans-glutamate to redirect the cis-glutamine into a non-canonical polar network involving Ser-20, Ser-42, and Arg-72.

FIGURE 8.

Annotated alignment of the P-loop and switch regions of Rab GTPases. Conserved residues in GTPase motifs are highlighted in black, residues in the non-canonical polar network are in red, and the switch I tyrosine is in blue.