The role of the conserved switch II glutamate in guanine nucleotide exchange factor-mediated nucleotide exchange of GTP-binding proteins

Guanine nucleotide exchange factors (GEFs) regulate the activity of small G proteins by catalysing the intrinsically slow exchange of GDP for GTP. The mechanism involves the formation of trimeric G protein-nucleotide-GEF complexes, followed by the release of nucleotide to form stable binary G protein-GEF complexes. A number of structural studies of G protein-GEF complexes have shown large structural changes induced in the nucleotide binding site. Together with a recent structure of a trimeric complex, these studies have suggested not only some common principles but also large differences in detail in the GEF-mediated exchange reaction. Several structures suggested that a glutamic acid residue in switch II, which is part of the DxxGQE motif and highly conserved in Ras-like G proteins, might have a decisive mechanistic role in GEF-mediated nucleotide exchange reactions. Here we show that mutation of the switch II glutamate to Ala severely impairs GEF-catalysed nucleotide exchange in most, but not all, Ras family G proteins, explaining its high sequence conservation. The residue determines the initial approach of GEF to the nucleotide-loaded G protein and does not appreciably affect the formation of a binary nucleotide-free complex. Its major effect thus appears to be the removal of the P-loop lysine from its interaction with the nucleotide.     

Intermediates in the Guanine Nucleotide Exchange Reaction of Rab8 Protein Catalyzed by Guanine Nucleotide Exchange Factors Rabin8 and GRAB

Small G-proteins of the Ras superfamily control the temporal and spatial coordination of intracellular signaling networks by acting as molecular on/off switches. Guanine nucleotide exchange factors (GEFs) regulate the activation of these G-proteins through catalytic replacement of GDP by GTP. During nucleotide exchange, three distinct substrate·enzyme complexes occur: a ternary complex with GDP at the start of the reaction (G-protein·GEF·GDP), an intermediary nucleotide-free binary complex (G-protein·GEF), and a ternary GTP complex after productive G-protein activation (G-protein·GEF·GTP). Here, we show structural snapshots of the full nucleotide exchange reaction sequence together with the G-protein substrates and products using Rabin8/GRAB (GEF) and Rab8 (G-protein) as a model system. Together with a thorough enzymatic characterization, our data provide a detailed view into the mechanism of Rabin8/GRAB-mediated nucleotide exchange.     

Kinetics of Interaction between ADP-ribosylation Factor-1 (Arf1) and the Sec7 Domain of Arno Guanine Nucleotide Exchange Factor,Modulation by Allosteric Factors,and the Uncompetitive Inhibitor Brefeldin A

The GDP/GTP nucleotide exchange of Arf1 is catalyzed by nucleotide exchange factors (GEF), such as Arno, which act through their catalytic Sec7 domain. This exchange is a complex mechanism that undergoes conformational changes and intermediate complex species involving several allosteric partners such as nucleotides, Mg²⁺, and Sec7 domains. Using a surface plasmon resonance approach, we characterized the kinetic binding parameters for various intermediate complexes. We first confirmed that both GDP and GTP counteract equivalently to the free-nucleotide binary Arf1-Arno complex stability and revealed that Mg²⁺ potentiates by a factor of 2 the allosteric effect of GDP. Then we explored the uncompetitive inhibitory mechanism of brefeldin A (BFA) that conducts to an abortive pentameric Arf1-Mg²⁺-GDP-BFA-Sec7 complex. With BFA, the association rate of the abortive complex is drastically reduced by a factor of 42, and by contrast, the 15-fold decrease of the dissociation rate concurs to stabilize the pentameric complex. These specific kinetic signatures have allowed distinguishing the level and nature as well as the fate in real time of formed complexes according to experimental conditions. Thus, we showed that in the presence of GDP, the BFA-resistant Sec7 domain of Arno can also associate to form a pentameric complex, which suggests that the uncompetitive inhibition by BFA and the nucleotide allosteric effect combine to stabilize such abortive complex.     

Mechanism of nucleotide release from Rho by the GDP dissociation stimulator protein

Guanine nucleotide dissociation stimulator (GDS) promotes the release of tightly bound GDP from various Ras superfamily proteins, including RhoA, Rac1, K-Ras, Rap1A, and Rap1B. It displays no significant sequence homology to other known exchange factors for small G-proteins. Studies are reported here of the mechanism of GDS-mediated nucleotide release from RhoA using a combination of equilibrium and stopped-flow kinetic measurements, employing fluorescent N-methylanthraniloyl (mant) derivatives of GDP and 2'-deoxyGDP. It is proposed that GDS operates by an associative displacement mechanism where stimulated nucleotide release from the Rho.mantGDP complex occurs via a transiently populated ternary complex (Rho.GDS.mantGDP). In kinetic experiments where excess GDS was mixed with the Rho.mantGDP complex, stimulated mantGDP dissociation rates of 1 s(-)(1) were measured during a single turnover, representing a 5000-fold enhancement over the intrinsic rate of mantGDP dissociation from Rho. The stable, nucleotide-free binary complex Rho.GDS was isolated. When the Rho.GDS complex was mixed with excess mantGDP, a biphasic increase in fluorescence occurred, the observed rate constants of which both reached saturating values at high mantGDP concentrations. This is compelling evidence that an isomerization of the Rho.GDS.mantGDP ternary complex is an important feature of the mechanism of nucleotide release.     

On the mode of inhibition of eukaryotic protein synthesis by ADP-ribosylation of elongation factor 2

The exchange of free guanine nucleotides with guanine nucleotides bound to elongation factor 2 (EF-2) and to the EF-2-ribosome complex, and the effect of ADP-ribosylation of the EF-2 thereon, were investigated by nitrocellulose filter assay. Under the experimental conditions, stoichiometric amounts of guanine nucleotides were bound, in particular, to ternary complexes of EF-2 with biphasic kinetics. The exchange kinetics were similarly biphasic in all cases. Ribosomes appeared to have variable effects on the exchange kinetics, depending on the type of nucleotide bound. Thus, in their presence, the rate and magnitude of the fast exchange of nucleotides revealed increasing values in the order GTP (GXP) > GTP gamma S > GDP. ADP-ribosylation had no inhibitory effect on the binding of guanine nucleotides to EF-2 or to the EF-2-ribosome complex but reduced significantly the fast exchange of GTP (GXP) and GTP gamma S bound to the EF-2-ribosome complex. The effect of ADP-ribosylation on the fast exchange of GDP in binary and ternary complexes was less pronounced. The mechanism of inhibition of protein synthesis by ADP-ribosylation of EF-2 is discussed in view of these data.     

Structure of Galpha(i1) bound to a GDP-selective peptide provides insight into guanine nucleotide exchange

Heterotrimeric G proteins are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive, GDP bound state and an active, GTP bound state. Under basal conditions, G proteins exist in the inactive, GDP bound state; thus, nucleotide exchange is crucial to the onset of signaling. Despite our understanding of G protein signaling pathways, the mechanism of nucleotide exchange remains elusive. We employed phage display technology to identify nucleotide state-dependent Galpha binding peptides. Herein, we report a GDP-selective Galpha binding peptide, KB-752, that enhances spontaneous nucleotide exchange of Galpha(i) subunits. Structural determination of the Galpha(i1)/peptide complex reveals unique changes in the Galpha switch regions predicted to enhance nucleotide exchange by creating a GDP dissociation route. Our results cast light onto a potential mechanism by which Galpha subunits adopt a conformation suitable for nucleotide exchange.     

Magnesium fluoride-dependent binding of small G proteins to their GTPase-activating proteins.

GTPase-activating proteins (GAPs) enhance the intrinsic GTPase activity of small G proteins, such as Ras and Rho, by contributing a catalytic arginine to the active site. An intramolecular arginine plays a similar role in heterotrimeric G proteins. Aluminum fluoride activates the GDP form of heterotrimeric G proteins, and enhances binding of the GDP form of small G proteins to their GAPs. The resultant complexes have been interpreted as analogues of the transition state of the hydrolytic reaction. Here, equilibrium binding has been measured using scintillation proximity assays to provide quantitative information on the fluoride-mediated interaction of Ras and Rho proteins with their respective GAPs, neurofibromin (NF1) and RhoGAP. High-affinity fluoride-mediated complex formation between Rho.GDP and RhoGAP occurred in the absence of aluminum; however, under these conditions, magnesium was required. Additionally, the novel observation was made of magnesium-dependent, fluoride-mediated binding of Ras.GDP to NF1 in the absence of aluminum. Aluminum was required for complex formation when the concentration of magnesium was low. Thus, either aluminum fluoride or magnesium fluoride can mediate the high-affinity binding of Rho. GDP or Ras.GDP to GAPs. It has been reported that magnesium fluoride can activate heterotrimeric G proteins. Thus, magnesium-dependent fluoride effects might be a general phenomenon with G proteins. Moreover, these data suggest that some protein.nucleotide complexes previously reported to contain aluminum fluoride may in fact contain magnesium fluoride.     

Structural Evidence for a Sequential Release Mechanism for Activation of Heterotrimeric G Proteins

Heptahelical G-protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors couple to heterotrimeric G proteins to relay extracellular signals to intracellular signaling networks, but the molecular mechanism underlying guanosine 5′-diphosphate (GDP) release by the G protein α-subunit is not well understood. Amino acid substitutions in the conserved α5 helix of G_i, which extends from the C-terminal region to the nucleotide-binding pocket, cause dramatic increases in basal (receptor-independent) GDP release rates. For example, mutant Gα_i1-T329A shows an 18-fold increase in basal GDP release rate and, when expressed in culture, it causes a significant decrease in forskolin-stimulated cAMP accumulation. The crystal structure of Gα_i1-T329A·GDP shows substantial conformational rearrangement of the switch I region and additional striking alterations of side chains lining the catalytic pocket that disrupt the Mg⁺² coordination sphere and dislodge bound Mg⁺². We propose a “sequential release” mechanism whereby a transient conformational change in the α5 helix alters switch I to induce GDP release. Interestingly, this mechanistic model for heterotrimeric G protein activation is similar to that suggested for the activation of the plant small G protein Rop4 by RopGEF8.     

The Nucleotide‐Dependent Interactome of Rice Heterotrimeric G‐Protein α ‐Subunit

The rice heterotrimeric G‐protein complex, a guanine‐nucleotide‐dependent on‐off switch, mediates vital cellular processes and responses to biotic and abiotic stress. Exchange of bound GDP (resting state) for GTP (active state) is spontaneous in plants including rice and thus there is no need for promoting guanine nucleotide exchange in vivo as a mechanism for regulating the active state of signaling as it is well known for animal G signaling. As such, a master regulator controlling the G‐protein activation state is unknown in plants. Therefore, an ab initio approach is taken to discover candidate regulators. The rice Gα subunit (RGA1) is used as bait to screen for nucleotide‐dependent protein partners. A total of 264 proteins are identified by tandem mass spectrometry of which 32 were specific to the GDP‐bound inactive state and 22 specific to the transition state. Approximately, 10% are validated as previously identified G‐protein interactors.     

Structural mimicry of DH domains by Arfaptin suggests a model for the recognition of Rac-GDP by its guanine nucleotide exchange factors.

Small G proteins cycle between an inactive form bound to GDP, and an active form bound to GTP. The two forms have different conformations and interact specifically with different partners, hence, the ability of G proteins to function as molecular switches. This view has been challenged by recent structural and biochemical studies of the Arfaptin/Por protein, which interacts equally well with the GDP- and GTP-bound forms of the G protein Rac. Here it is shown that the dimeric helical domain of Arfaptin superimposes with a monomeric helical domain from the Dbl homology domain of Tiam, a guanine nucleotide exchange factor (GEF) for Rac, in their respective complexes with Rac. This unexpected structural mimicry suggests that the Rac-GDP-Arfaptin complex resembles the low-affinity Rac-GDP-GEF complex that initiates the exchange reaction. This provides a model for the exchange mechanism where DH domains first dock onto Rac-GDP at the switch 2 before they undergo domain closure to catalyze GDP dissociation.     

Conformational states of the small G protein Arf-1 in complex with the guanine nucleotide exchange factor ARNO-Sec7

Arf1 is a small G protein involved in vesicular trafficking, and although it is only distantly related to Ras, it adopts a similar three-dimensional structure. In the present work, we study Arf1 bound to GDP and GTP and its interactions with one of its guanosine nucleotide exchange factors, ARNO-Sec7. The (31)P NMR spectra of Arf1.GDP.Mg(2+) and Arf1.GTP.Mg(2+) share the general features typical for all small G proteins studied so far. Especially, the beta-phosphate resonances of the bound nucleotide are shifted strongly downfield compared with the resonance positions of the free magnesium complexes of GDP and GTP. However, no evidence for an equilibrium between two conformational states of Arf1.GDP.Mg(2+) or Arf1.GTP.Mg(2+) could be observed as it was described earlier for Ras and Ran. Glu(156) of ARNO-Sec7 has been suggested to play as "glutamic acid finger" an important role in the nucleotide exchange mechanism. In the millimolar concentration range used in the NMR experiments, wild type ARNO-Sec7 and ARNO-Sec7(E156D) do weakly interact with Arf1.GDP.Mg(2+) but do not form a strong complex with magnesium-free Arf1.GDP. Only wild type ARNO-Sec7 competes weakly with GDP on Arf1.GDP.Mg(2+) and leads to a release of GDP when added to the solution. The catalytically inactive mutants ARNO-Sec7(E156A) and ARNO-Sec7(E156K) induce a release of magnesium from Arf1.GDP.Mg(2+) but do not promote GDP release. In addition, ARNO-Sec7 does not interact or only very weakly interacts with the GTP-bound form of Arf1, opposite to the observation made earlier for Ran, where the nucleotide exchange factor RCC1 forms a complex with Ran.GTP.Mg(2+) and is able to displace the bound GTP.     

Kinetic studies on the role of elongation factors 1 beta and 1 gamma in protein synthesis

An equilibrium isotope exchange technique was used to measure in an Artemia system the catalytic influence of elongation factor (EF) 1 beta gamma on the dissociation of GDP from the complex of elongation factor 1 alpha.[3H] GDP in the presence of an excess of free GDP. The kinetic data demonstrate that, in analogy to procaryotes, dissociation of GDP occurs via the formation of a transient ternary complex of EF-1 alpha.GDP.EF-1 beta gamma. The rate constants for the dissociation of GDP from EF-1 alpha.GDP and from the ternary complex EF-1 alpha.GDP.EF-1 beta gamma were found to be 0.7 x 10(-3) and greater than or equal to 0.7 s-1, respectively. The equilibrium association constants of GDP to EF-1 alpha.EF-1 beta gamma and of EF-1 beta gamma to EF-1 alpha.GDP were found to be 2.3 x 10(5) and 4.2 x 10(5) M-1, respectively. Judged from the known elongation rate in vivo and kinetic constants of nucleotide exchange, it was estimated that the recycling of EF-1 alpha may be a rate-controlling step in eucaryotic translation. As a model for GTP exchange, the formation of the ternary EF-1 alpha.guanylyl (beta gamma-methylene)diphosphonate.EF-1 beta gamma complex was also studied. It was observed that both an increase of the level of aminoacyl-tRNA and of temperature favored the dissociation of this complex, thereby enabling EF-1 beta gamma to recycle as a catalyst. This behavior would explain the frequent occurrence of a heavy form of elongation factor 1 in extracts of the eucaryotic cell.     

The interaction between the archaeal elongation factor 1alpha and its nucleotide exchange factor 1beta.

In Sulfolobus solfataricus the binding of the exchange factor 1beta (SsEF-1beta) to SsEF-1alpha-GDP displaces the nucleotide and the SsEF-1alpha-SsEF-1beta complex is formed. The complex itself is stable, but it dissociates upon the addition of GDP or Gpp(NH)p but not ATP. Since the rate of the formation of the SsEF-1alpha-SsEF-1beta complex is significatively slower than the rate of the nucleotide exchange catalyzed by SsEF-1beta it can be inferred that in vivo the GDP/GTP exchange reaction proceeds via an SsEF-1alpha-SsEF-1beta interaction without involving the formation of a stable binary complex as an intermediate.     

Mechanism of domain closure of Sec7 domains and role in BFA sensitivity

Activation of small G proteins of the Arf family is initiated by guanine nucleotide exchange factors whose catalytic Sec7 domain stimulates the dissociation of the tightly bound GDP nucleotide. The exchange reaction involves distinct sequential steps that can be trapped by the noncompetitive inhibitor brefeldin A, by mutation of an invariant catalytic glutamate, or by removal of guanine nucleotides. Arf-GDP retains most characteristics of its GDP-bound form at the initial low-affinity Arf-GDP-Sec7 step. It then undergoes large conformational changes toward its GTP-bound form at the next step, and eventually dissociates GDP to form a nucleotide-free high-affinity Arf-Sec7 complex at the last step. Thus, Arf proteins evolve through different conformations that must be accommodated by Sec7 domains in the course of the reaction. Here the contribution of the flexibility of Sec7 domains to the exchange reaction was investigated with the crystal structure of the unbound Sec7 domain of yeast Gea2. Comparison with Gea2 in complex with nucleotide-free Arf1 Delta 17 [Goldberg, J. (1998) Cell 95, 237-248] reveals that Arf induces closure of the two subdomains that form the sides of its active site. Several residues that determine sensitivity to brefeldin A are involved in interdomain and local movements, pointing to the importance of the flexibility of Sec7 domains for the inhibition mechanism. Altogether, this suggests a model for the initial steps of the exchange reaction where Arf docks onto the C-terminal domain of the Sec7 domain before closure of the N-terminal domain positions the catalytic glutamate to complete the reaction.     

Nucleotide binding and filament assembly of recombinant yeast septin complexes

Septins are filament-forming GTPases involved in cytokinesis and cortical organization. In the yeast Saccharomyces cerevisiae, the septins encoded by CDC3, CDC10, CDC11, and CDC12 form a high-molecular-weight complex, localized at the cytoplasmic face of the plasma membrane in the mother-bud neck. While septin function at the cellular level is fairly well understood, progress on structure-function analysis of these proteins has been slow and limited by the lack of large amounts of pure complex. While monomeric septins form apparently non-native aggregates, stable recombinant complexes of two, three, or four yeast septins can be produced by co-expression from bi-cistronic vectors in E. coli. The septin polypeptides show various degrees of saturation with guanine nucleotides in different complexes. The binary core Cdc3p-Cdc12p complex contains no bound nucleotide. While ternary complexes are partially saturated and can bind extraneously added nucleotide with micromolar affinity, only the complete four-component septin complex is fully coordinated with tightly bound GDP/GTP after chromatographic purification. We show here that the nucleotide-binding sites of the septins show drastic changes on formation of higher oligomers. Although the binary core Cdc3p-Cdc12p complex does not form filaments, the ternary and quaternary complexes form bundles of paired filaments. In the case of ternary complexes, filament formation is stimulated by guanine nucleotide, but is not dependent on the presence or absence of the gamma-phosphate.     

The nucleotide exchange factor Ric-8A is a chaperone for the conformationally dynamic nucleotide-free state of Gαi1

Heterotrimeric G protein α subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of Gα, catalyzed by guanine nucleotide exchange factors (GEFs). In addition to transmembrane G protein-coupled receptors (GPCRs), which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class Gα subunits. Ric-8A binds to Gα?GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free Gαi1 is stable, but dissociates upon binding of GTP to Gαi1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from Gαi1, experiments were conducted to characterize the physical state of nucleotide-free Gαi1 (hereafter referred to as Gαi1[ ]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free Gαi1 is more accessible to trypsinolysis than Gαi1?GDP, but less so than Gαi1[ ] alone. The TROSY-HSQC spectrum of [(15)N]Gαi1[ ] bound to Ric-8A shows considerable loss of peak intensity relative to that of [(15)N]Gαi1?GDP. Hydrogen-deuterium exchange in Gαi1[ ] bound to Ric-8A is 1.5-fold more extensive than in Gαi1?GDP. Differential scanning calorimetry shows that both Ric-8A and Gαi1?GDP undergo cooperative, irreversible unfolding transitions at 47° and 52°, respectively, while nucleotide-free Gαi1 shows a broad, weak transition near 35°. The unfolding transition for Ric-8A:Gαi1[ ] is complex, with a broad transition that peaks at 50°, suggesting that both Ric-8A and Gαi1[ ] are stabilized within the complex, relative to their respective free states. The C-terminus of Gαi1 is shown to be a critical binding element for Ric-8A, as is also the case for GPCRs, suggesting that the two types of GEF might promote nucleotide exchange by similar mechanisms, by acting as chaperones for the unstable and dynamic nucleotide-free state of Gα.     

The effect of Mg2+ and guanine nucleotide exchange factor on the binding of guanine nucleotides to eukaryotic initiation factor 2

A major site of regulation of polypeptide chain initiation is the binding of Met-tRNA to 40 S ribosomal subunits which is mediated by eukaryotic initiation factor 2 (eIF-2). The formation of ternary complex, eIF-2.GTP.Met-tRNA, is potently inhibited by GDP. Measurement of the parameters for guanine nucleotide binding to eIF-2 is critical to understanding the control of protein synthesis by fluctuations in cellular energy levels. We have compared the dissociation constants (Kd) of eIF-2.GDP and eIF-2.GTP and find that GDP has a 400-fold higher affinity for GDP than GTP. The Kd for GDP is almost an order of magnitude less than has been reported previously. The difference between the Kd values for the two nucleotides is the result of a faster rate constant for GTP release, the rate constants for binding being approximately equal. This combination of rate constants and low levels of contaminating GDP in preparations of GTP can explain the apparently unstable nature of eIF-2.GTP observed by others. Mg2+ stabilizes binary complexes slowing the rates of release of nucleotide from both eIF-2.GDP and eIF-2.GTP. The competition between GTP and GDP for binding to eIF-2.guanine nucleotide exchange factor complex has been measured. A 10-fold higher GTP concentration than GDP is required to reduce [32P] GDP binding to eIF-2.guanine nucleotide exchange factor complex by 50%. The relevance of this competition to the regulation of protein synthesis by energy levels is discussed.     

eIF-2B and the exchange of guanine nucleotides bound to eIF-2

Available data for the formation of the ternary complex eIF-2 X GTP X methionyl-tRNAi involved in eukaryotic initiation and of the inhibition of ternary complex formation by GDP have been examined with a view to determining the mechanism by which eIF-2B facilitates nucleotide exchange. Two mechanisms have been considered--first a displacement reaction in which eIF-2B displaces GDP and GTP in a manner analogous to a "ping-pong" enzyme mechanism, and secondly the possibility that binding of eIF-2B to eIF-2 nucleotide complexes enhances the rate of nucleotide exchange without itself inducing nucleotide displacement. Comparison has been made between the properties of eIF-2 and eIF-2B and of the bacterial elongation factors Tu and Ts. It seems most probable that, as previously suggested by others for Ts, eIF-2B effectively catalyses an exchange reaction through a "ping-pong" type mechanism. Possible explanations of data suggesting otherwise are put forward. Both eIF-2 and bacterial Tu are complex allosteric proteins subject to a variety of influences which in the case of eIF-2 include phosphorylation of the alpha subunit. This phosphorylation appears to change the equilibria in the reaction mechanism such that the transferred entity (eIF-2) becomes firmly bound to the catalyst (eIF-2B). Minimum rate constants for the formation of eIF-2 X eIF-2B from eIF-2 X GDP and eIF-2 X GTP and reverse reactions are derived. These values suggest that the initiation factors are likely to have to operate in a restricted environment if rates of protein synthesis seen in vivo are to be sustained.     

Kinetic mechanism of elongation factor Ts-catalyzed nucleotide exchange in elongation factor Tu.

The interaction of Escherichia coli elongation factor Tu (EF-Tu) with elongation factor Ts (EF-Ts) and guanine nucleotides was studied by the stopped-flow technique, monitoring the fluorescence of tryptophan 184 in EF-Tu or of the mant group attached to the guanine nucleotide. Rate constants of all association and dissociation reactions among EF-Tu, EF-Ts, GDP, and GTP were determined. EF-Ts enhances the dissociation of GDP and GTP from EF-Tu by factors of 6 x 10(4) and 3 x 10(3), respectively. The loss of Mg(2+) alone, without EF-Ts, accounts for a 150-300-fold acceleration of GDP dissociation from EF-Tu.GDP, suggesting that the disruption of the Mg(2+) binding site alone does not explain the EF-Ts effect. Dissociation of EF-Ts from the ternary complexes with EF-Tu and GDP/GTP is 10(3)-10(4) times faster than from the binary complex EF-Tu.EF-Ts, indicating different structures and/or interactions of the factors in the binary and ternary complexes. Rate constants of EF-Ts binding to EF-Tu in the free or nucleotide-bound form or of GDP/GTP binding to the EF-Tu.EF-Ts complex range from 0.6 x 10(7) to 6 x 10(7) M(-1) s(-1). At in vivo concentrations of nucleotides and factors, the overall exchange rate, as calculated from the elemental rate constants, is 30 s(-1), which is compatible with the rate of protein synthesis in the cell.     

The isolation and characterization from rabbit reticulocytes of two forms of eukaryotic initiation factor 2 having different beta-polypeptides

We have isolated from the high salt wash of rabbit reticulocyte ribosomes two forms of the polypeptide chain initiation factor 2 (eIF-2) which differ with respect to their beta-subunit, GDP content, and sensitivity to Mg2+ in ternary (eIF-2 X GTP X Met-tRNAf) and binary (eIF-2 X GDP) complex formation. The form of eIF-2 eluting first from a cation exchange (Mono S, Pharmacia) column has a beta-subunit of lower molecular weight (eIF-2(beta L] and a more acidic pI value than the form eluting at a higher salt concentration (eIF-2(beta H]. These two forms of eIF-2 beta-polypeptides are also detected in reticulocyte lysates when the proteins are resolved by two-dimensional isoelectric focusing-dodecyl sulfate polyacrylamide gel electrophoresis followed by immunoblotting. The peptide mapping of the isolated beta-subunits after limited proteolysis by papain, pancreatic protease, alpha-chymotrypsin, or Staphylococcus aureus V8 protease further demonstrates that the two forms of beta-subunits are not the product of a non-specific proteolytic action that occurred during the purification procedure, but rather reflects the existence in vivo of both forms of eIF-2. The GDP content of eIF-2(beta L) and eIF-2(beta H) is approximately 0.85 and 0.22 mol of GDP/mol of eIF-2, respectively. The KD for GDP of eIF-2(beta L) was lower (2.2 X 10(-9) M) than that of eIF-2(beta H) (6.0 X 10(-8) M). In the presence of 1 mM Mg2+, the activities of eIF-2(beta L) and eIF-2(beta H) in forming a binary and a ternary complex are inhibited 90 and 25%, respectively. The extent of Mg2+ inhibition and its reversal by the guanine nucleotide exchange factor is directly proportional to the amount of GDP bound to eIF-2. No inhibition by Mg2+ is observed when eIF-2-bound GDP is removed by alkaline phosphatase. In the presence of the guanine nucleotide exchange factor, both forms of eIF-2 are equally active in ternary complex formation, and the complex formed is quantitatively transferred to 40 S ribosomal subunits.