Catalytic mechanism of a mammalian Rab·RabGAP complex in atomic detail

Abstract

Rab GTPases, key regulators of vesicular transport, hydrolyze GTP very slowly unless assisted by Rab GTPase-activating proteins (RabGAPs). Dysfunction of RabGAPs is involved in many diseases. By combining X-ray structure analysis and time-resolved FTIR spectroscopy we reveal here the detailed molecular reaction mechanism of a complex between human Rab and RabGAP at the highest possible spatiotemporal resolution and in atomic detail. A glutamine residue of Rab proteins (cis-glutamine) that is essential for intrinsic activity is less important in the GAP-activated reaction. During generation of the RabGAP·Rab:GTP complex, there is a rapid conformational change in which the cis-glutamine is replaced by a glutamine from RabGAP (trans-glutamine); this differs from the RasGAP mechanism, where the cis-glutamine is also important for GAP catalysis. However, as in the case of Ras, a trans-arginine is also recruited to complete the active center during this conformational change. In contrast to the RasGAP mechanism, an accumulation of a state in which phosphate is bound is not observed, and bond breakage is the rate-limiting step. The movement of trans-glutamine and trans-arginine into the catalytic site and bond breakage during hydrolysis are monitored in real time. The combination of X-ray structure analysis and time-resolved FTIR spectroscopy provides detailed insight in the catalysis of human Rab GTPases.

Fig. 1.

Structure of Rab1b in complex with TBC1D20. (A) Ribbon representation of Rab1b·GDP·BeF₃⁻·TBC1D20 complex with TBC1D20 shown in gray and Rab1b in green, with switch regions colored as indicated by labeling. (B) Interactions in the active site of the complex with coloring as above and functional groups as indicated by labeling. (C) Overlay of the crystal structures of TBC1D20-Rab1b (white) and Gyp1-Rab33 (cyan, PDB ID 2G77). The electron density map contoured at 1.0 σ of the R105_T side chain is highlighted in black. The indices G and T are used for Gyp1 and TBC1D20 amino acid legends, respectively.

Fig. 2.

Mutational analysis of the active site of the Rab·RabGAP complex. Kinetics of GTP hydrolysis by Rab1b for the intrinsic (393 K), GAP-catalyzed (368 K), and mutant cases (386 K). The data points represent the normalized difference between adjacent GDP (1,100 cm⁻¹) and GTP (1,129 cm⁻¹) bands, which eliminates baseline drifts for the long measurements. Fast kinetics result from 15 independent measurements, slow kinetics of mutant proteins and the intrinsic reaction from two independent measurements. Errors are given as SDs, which were not calculated for the slow measurements due to the low number of experiments needed to achieve good signal-to-noise ratio. Lines represent monoexponential fits.

Fig. 3.

Kinetics of the TBC1D20-catalyzed GTP hydrolysis in Rab1b. Time-dependent absorbance changes of marker bands of the free P_i (1,078 cm⁻¹); overlapping vibrations of the GAP glutamine finger and a tyrosine’s backbone (1,651 cm⁻¹); and the α-phosphate (1,256 cm⁻¹) in ²H₂O at 368 K as assigned later in the text. Fifteen independent measurements were averaged. The continuous lines correspond to a global fit with two exponential functions.

Fig. 4.

Band assignment of the glutamine finger of TBC1D20. Amplitude spectra of the GTPase reaction using unlabeled (cyan) and [5-¹³C]glutamine-labeled (red) TBC1D20 in ²H₂O. In k₁, negative bands belong to the GTP state, positive bands to the intermediate state. In k₂, negative bands belong to the intermediate state, positive bands to the GDP + P_i state. The double difference (labeled − unlabeled) is shown in green. The band at 1,648/1,651 cm⁻¹ can be assigned to a glutamine side-chain and most likely represents the catalytic glutamine finger moving into the active site with k₁ and in the reverse direction with k₂.

Fig. 5.

Summary of the reaction mechanism. (A) Illustration of the observed processes. With k₁, the arginine and glutamine fingers of GAP move into the active site, whereas the intrinsic glutamine of the GTPase moves out to interact with the backbone of GAP. Thus, the catalytically active conformation is formed. With k₂, these conformational changes are reversed, and, simultaneously, bond cleavage occurs and P_i is released. (B) Reaction scheme of the Rab·RabGAP reaction.