GDP-bound and Nucleotide-free Intermediates of the Guanine Nucleotide Exchange in the Rab5·Vps9 System

Abstract

Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7⅐VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg 2؉ and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP ␤-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP ␤-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.Small GTPases work as a molecular switch, which is turned off by its intrinsic GTPase activity hydrolyzing GTP to GDP. To turn the switch on, the bound GDP should be removed to introduce a GTP. Normally, this exchange reaction is much slower than the rate of intrinsic GTPase activity because nucleotide binds to small GTPase tightly with the Mg 2ϩ ion. GEF 5 enhances the nucleotide exchange of its cognate GTPase by destabilizing the Mg 2ϩ ion and GDP binding to the GTPase. Recognition of the GDP-bound GTPase by GEF is a critical but transient step prior to the formation of a nucleotide-free GTPase⅐GEF binary complex (1, 2). Because the nucleotide-free complex is stable in vitro, most GTPase⅐GEF complexes have been crystallized and structurally analyzed as the nucleotidefree form in the past, with a few exceptions containing nucleotides. One of them is a structure of eFF1A⅐eEF1B␣ in complex with different di-and triphosphate nucleotides, in which ␤-and ␥-phosphate electron densities of the nucleotides were ambiguous and only GMP could have been modeled (3). Another example is the Arf1⅐Sec7 complex, in which introducing either an abortive inhibitor or a mutation on the GEF allowed for GDP binding into the Arf1⅐Sec7 complex (4, 5). The complex structure between ROP4⅐GDP, a member of the plant Rho family, and its GEF PRONE8 indicated that GDP bound to the ROP4⅐PRONE8 complex loosely (6). A complex between Cdc42 and the DHR2 GEF domain of DOCK9 GEF has been crystallized in three different nucleotide forms: nucleotide-free, GDPbound, and GTP/Mg 2ϩ -bound. These structures demonstrate that the nucleotide sensor found in the ␣10 helix of the DHR2 domain is responsible for the exclusion of GDP/Mg 2ϩ and the introduction of GTP/Mg 2ϩ (7). It should be noted that none of the nucleotides were directly recognized by the GEFs in the complex structures mentioned above. These exceptions have encouraged us to investigate the transient nucleotide recognition in the GEF-catalyzed reaction.Rab small GTPases regulate vesicular transport in eukaryotes (8, 9), including ...

ENDOSOMAL RAB EFFECTOR WITH PX-DOMAIN, an Interacting Partner of RAB5 GTPases, Regulates Membrane Trafficking to Protein Storage Vacuoles in Arabidopsis

“…Among these subgroups, the RAB5 group (also called RABF) consists of three members, which are further classified into two subtypes: plant-unique ARA6 (also known as RABF1) and the canonical RAB5 group (ARA7 and RAB HOMOLOG1 [RHA1], also known as RABF2b and RABF2a, respectively) (Ueda et al, 2001Ebine and Ueda, 2009). Despite the differences in their primary structures, these RAB5 members are activated by the same GEF, VACUOLAR PROTEIN SORTING 9a (VPS9a), whose loss of function results in embryonic lethality (Goh et al, 2007;Uejima et al, 2010Uejima et al, , 2013. The double mutant of canonical RAB5, ara7 rha1, also exhibits gametophytic lethality (Dhonukshe, 2009).…”

A disappearing act performed by magnesium: the nucleotide exchange mechanism of Ran GTPase by quantum mechanics/molecular mechanics studies

“…Another difference in the reported guanine nucleotide exchange mechanism of Ran and other Ras-like GTPases is the lack of intrusion into the magnesium-binding site by residues from the GEF. In some GTPases, residues of the GEF have been reported to block the metal-binding site in the GDPbound-GTPase-GEF complexes [14,16,39,40]. However, this is not the case in the Ran-RCC1 complex interaction.…”

“…The fact that the Switch 1 motif adopts the 'open' conformation prior to GEF interaction indicates that Ran employs a nucleotide exchange mechanism dissimilar to that of RhoA. Ran also appears to adopt a different nucleotide exchange mechanism than that represented in the Cdc42-DOCK9 and ARA7-VPS9a complexes [16,39], which may be caused by the different GEF proteins. In addition, it is postulated that the Cdc42-DOCK9 requires a specific α10 motif which functions to block the magnesiumbinding site and the ARA7-VPS9a interaction requests utilization of an 'aspartate finger' in forcing the removal of the ion.…”

“…That is, the residues 32-37 in the Switch 1 loop (comprising residues 29-46 Ran ) were unresolved and missing in the crystal structure as well as residues 233-238 of RCC1. A common conception in the Ras-like GTPase nucleotide exchange mechanism is the stabilization of the nucleotide-free intermediate state by the interaction of the P-loop lysine residue (K23 Ran ) and either the aspartate at the beginning of Switch 2 or the glutamate at the end of the Switch 2 motif [19,23,39]. In the Ran-RCC1 crystal structure, K23 Ran forms a salt bridge interaction with D65 Ran on the Switch 2.…”

“…Authors suggested that opening of the Switch 1 loop aids exchange and also identified the α10 insert which orients into the nucleotide-binding site, utilizing a valine residue to block the metal-binding site which in turn stabilizes the nucleotide-free complex. Further nucleotide exchange activity in the ARA7-VPS9a was reported, suggesting that removal of the magnesium ion from the nucleotide-binding site of the GEF-bound GTPase is effected by a so-called 'aspartate finger' which inserts into the nucleotide-binding site, causing removal of the ion and formation of an interaction with the P-loop lysine [17]. This destabilizes the interaction of GDP with ARA7 GTPase.…”