Mechanism of the intrinsic arginine finger in heterotrimeric G proteins

Abstract

Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gα i1 , which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine "finger." We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gα i1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gα i1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gα i1 ·RGS4 mechanism. This movement induces a charge shift toward β-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis.GTPase | FTIR spectroscopy | QM/MM calculations | arginine finger | reaction mechanism

Evolution of cation binding in the active sites of P-loop nucleoside triphosphatases in relation to the basic catalytic mechanism

“…It still remains unclear whether there is some universal catalytic mechanism that is common for all P-loop NTPases, see (Wittinghofer, 2006; Kamerlin et al, 2013) for reviews. Recently, Gerwert and colleagues proposed that the Arg finger promotes GTP hydrolysis in small GTPases by rotating the α-phosphate with respect to β- and γ-phosphates towards an eclipsed conformation, which would favor the bond cleavage because of repulsion between the oxygen atoms of all three phosphate groups (Rudack et al, 2012; Mann et al, 2016; Gerwert et al, 2017). Blackburn and colleagues proposed that the insertion of the activating Arg residue leads to the reshuffling of the hydrogen bonded network, which drives the displacement of the attacking water molecule into the reactive position (Jin et al, 2016; Jin et al, 2017a; Jin et al, 2017b).…”

Common mechanisms of catalysis in small and heterotrimeric GTPases and their respective GAPs

“…In the X-ray structural models, the nucleotide is substituted by GTPγS or GDP · AlFx (Coleman et al, 1994;Kaya et al, 2016), and the position of the arginine is artificially altered due to the presence of the γS and AlF 4 − analogs. In contrast to this, QM/MM calculations of Gα i1 with the natural nucleotide GTP show a direct hydrogen bond to the γ-phosphate ( Figure 6A) (Mann et al, 2016). This interaction was further confirmed experimentally by FTIR spectroscopy: A mutation deleting this hydrogen bond leads to a blue-shift of the absorption band of the γ-phosphate only, whereas the α-and β-phosphate bands are unchanged.…”

“…For example, although it is not possible to measure coupling of the arginine finger to GTP in the Ras-GAP system, this was nicely resolved in the Gα i1 system (Mann et al, 2016). Similarly, mutation of Lys16, which is found in the conserved GTPase GxxxxGKS/T motif, was not possible in small GTPases due to the fact that mutated proteins were unstable (Du and Sprang, 2009).…”

Monitoring transient events in infrared spectra using local mode analysis

“…A combination of theoretical quantum mechanics (QM) (or quantum mechanics/molecular mechanics [QM/MM] for larger systems) and experimental approaches in vibrational spectroscopy is well suited to connect atomic structural properties with spectral features. This combination is well‐established and has been demonstrated in many applications …”

“…This combination is well-established and has been demonstrated in many applications. [3][4][5][6][7][8][9][10][11][12] Using time-resolved Fourier transformed infrared (FTIR) difference spectroscopy, it is possible to study the structural transitions and reaction steps in proteins in short time periods. This already enabled a deeper understanding of various reaction mechanisms, such as the light-driven proton pump bacteriorhodopsin 13 or the GTPase mechanism of the Ras protein.…”