Structural insights into the function of a unique tandem GTPase EngA in bacterial ribosome assembly

Many ribosome-interacting GTPases, with proposed functions in ribosome biogenesis, are also implicated in the cellular regulatory coupling between ribosome assembly process and various growth control pathways. EngA is an essential GTPase in bacteria, and intriguingly, it contains two consecutive GTPase domains (GD), being one-of-a-kind among all known GTPases. EngA is required for the 50S subunit maturation. However, its molecular role remains elusive. Here, we present the structure of EngA bound to the 50S subunit. Our data show that EngA binds to the peptidyl transferase center (PTC) and induces dramatic conformational changes on the 50S subunit, which virtually returns the 50S subunit to a state similar to that of the late-stage 50S assembly intermediates. Very interestingly, our data show that the two GDs exhibit a pseudo-two-fold symmetry in the 50S-bound conformation. Our results indicate that EngA recognizes certain forms of the 50S assembly intermediates, and likely facilitates the conformational maturation of the PTC of the 23S rRNA in a direct manner. Furthermore, in a broad context, our data also suggest that EngA might be a sensor of the cellular GTP/GDP ratio, endowed with multiple conformational states, in response to fluctuations in cellular nucleotide pool, to facilitate and regulate ribosome assembly.     

Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu

Elongation factor Tu from Thermus thermophilus was treated successively with periodate-oxidized GDP or GTP and cyanoborohydride. Covalently modified cyanogen bromide or trypsin fragments of the protein were isolated, and the position of their modification was determined. Lysine residues 52 and 137 were heavily labeled, lysine-137 being considerably more reactive in the GTP form as compared to the GDP form of the protein. These residues are in the proximity of the GDP/GTP binding site. Lys-325 was also labeled, but to a lower extent. The part of the EF-Tu containing residue 52 is missing in crystallized EF-Tu.GDP from Escherichia coli [Jurnak, F. (1985) Science (Washington, D.C.) 230, 32-36]. These results place the part of T. thermophilus EF-Tu corresponding to the missing fragment in E. coli EF-Tu in the vicinity of the nucleotide binding site and allow its role in the interaction with aminoacyl-tRNA and elongation factor Ts to be evaluated. Cross-linking of EF-Tu.GDP by irradiation at 257 nm showed that a sequence of 10 amino acids residues which is found in the Thermus thermophilus elongation factor Tu but not in other homologous bacterial proteins is located in the vicinity of the GDP/GTP binding site.     

The T-cell receptor zeta chain contains a GTP/GDP binding site.

In a search for nucleotide binding proteins associated with the T-cell receptor (TCR)-CD3 complex, a novel labeling technique involving introduction of [alpha-32P]GTP or [alpha-32P]ATP into permeabilized cells followed by in situ periodate oxidation was developed. To test the method we first demonstrated that p21ras and other classical GTP binding proteins could be labeled in a GTP-specific manner. In human T lymphocytes the TCR zeta chain was found to be specifically labeled by GTPoxi but not by ATPoxi or CTPoxi. Labeling kinetics and competition experiments demonstrated that zeta had a capacity to bind GTP and GDP but not GMP or ATP. Proteolytic cleavage experiments identified lysine 128 as the GTP crosslinking site. This result was confirmed by studies using oligonucleotide-directed mutagenesis. Lysine residues 128, 135 and 149 were each replaced by arginine and glycine 134 by valine and mutated proteins were expressed in CHO cells. Labeling of mutants K128R and G134V was abrogated whereas mutant proteins K135R and K148R could still be specifically crosslinked to GTP. We conclude that Lys128 and Gly134 are part of a GTP/GDP binding site suggesting that zeta is a unique GTP/GDP binding structure.     

Vav, a GDP/GTP nucleotide exchange factor, interacts with GDIs, proteins that inhibit GDP/GTP dissociation

Vav functions as a specific GDP/GTP nucleotide exchange factor which is regulated by tyrosine phosphorylation in the hematopoietic system. Loss of the amino-terminus sequences of Vav was sufficient to control its transforming potential and its function in T cells. We report here the identification of the hematopoietic GDP dissociation inhibitor protein, Ly-GDI, as a protein that interacts with the amino-terminus of Vav. Further analysis confirmed that Vav and Ly-GDI interact both in in vitro and in in vivo assays. This association is maximal only when the amino region of Vav is intact and requires an intact carboxy-terminus of Ly-GDI. The interaction between Vav and Ly-GDI is not dependent on the tyrosine phosphorylation status of Vav. In addition, Rho-GDI, the highly homologous protein to Ly-GDI, associates with Vav as well. The contribution of the interaction between Vav and GDIs, proteins that are involved in the GDP/GTP exchange processes, to the biological function of Vav is further discussed.     

Identification of the mitochondrial GTP/GDP transporter in Saccharomyces cerevisiae

The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 16 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates, and cofactors across the inner membrane. Here the identification and functional characterization of the mitochondrial GTP/GDP carrier (Ggc1p) is described. The ggc1 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. It transported GTP and GDP and, to a lesser extent, the corresponding deoxynucleotides and the structurally related ITP and IDP by a counter-exchange mechanism. Transport was saturable with an apparent K(m) of 1 microm for GTP and 5 microm for GDP. It was strongly inhibited by pyridoxal 5'-phosphate, bathophenanthroline, tannic acid, and bromcresol purple but little affected by the inhibitors of the ADP/ATP carrier carboxyatractyloside and bongkrekate. Furthermore, in contrast to the ADP/ATP carrier, the Ggc1p-mediated GTP/GDP heteroexchange is H(+)-compensated and thus electroneutral. Cells lacking the ggc1 gene had reduced levels of GTP and increased levels of GDP in their mitochondria. Furthermore, the knock-out of ggc1 results in lack of growth on nonfermentable carbon sources and complete loss of mitochondrial DNA. The physiological role of Ggc1p in S. cerevisiae is probably to transport GTP into mitochondria, where it is required for important processes such as nucleic acid and protein synthesis, in exchange for intramitochondrially generated GDP.     

Structured mRNAs regulate translation initiation by binding to the platform of the ribosome

Gene expression can be regulated at the level of initiation of protein biosynthesis via structural elements present at the 5' untranslated region of mRNAs. These folded mRNA segments may bind to the ribosome, thus blocking translation until the mRNA unfolds. Here, we report a series of cryo-electron microscopy snapshots of ribosomal complexes directly visualizing either the mRNA structure blocked by repressor protein S15 or the unfolded, active mRNA. In the stalled state, the folded mRNA prevents the start codon from reaching the peptidyl-tRNA (P) site inside the ribosome. Upon repressor release, the mRNA unfolds and moves into the mRNA channel allowing translation initiation. A comparative structure and sequence analysis suggests the existence of a universal stand-by site on the ribosome (the 30S platform) dedicated for binding regulatory 5' mRNA elements. Different types of mRNA structures may be accommodated during translation preinitiation and regulate gene expression by transiently stalling the ribosome.     

The structural GDP/GTP cycle of human Arf6

The small GTP-binding protein Arf6 coordinates membrane traffic at the plasma membrane with aspects of cytoskeleton organization. This function does not overlap with that of other members of the ADP-ribosylation factor (Arf) family, although their switch regions, which are their major sites of interaction with regulators and effectors, have virtually identical sequences. Here we report the crystal structure of full-length, non-myristoylated human Arf6 bound to GTPγS. Unlike their GDP-bound forms, the active forms of Arf6 and Arf1 are very similar. Thus, the switch regions are discriminatory elements between Arf isoforms in their inactive but not in their active forms, a property that may generalize to other families of small G proteins. This suggests that GTP-bound Arfs may establish specific interactions outside the switch regions and/or be recognized in their cellular context rather than as isolated proteins. The structure also allows further insight into the lack of spontaneous GTPase activity of Arf proteins.     

GTP/GDP binding stabilizes bacterial cell division protein FtsZ against degradation by FtsH protease in vitro

Factors contributing to the stability of bacterial cell division protein FtsZ remain unknown. In order to identify FtsZ-stabilizing factor(s), we exploited FtsH protease-based in vitro FtsZ degradation assay system. Whole cell lysate from an ftsH-null strain of Escherichia coli inhibited degradation of FtsZ by FtsH in vitro. However, activated charcoal-treated lysate did not inhibit degradation. The loss of ability of the activated charcoal-treated lysate to inhibit degradation of FtsZ was restored when it was replenished with GTP, but not when replenished with other NTPs or dNTPs. The lysate did not protect either FtsZ deletion mutants, which do not bind GTP, or FtsH substrates, sigma(32) and cI-108 proteins, against FtsH. GDP and GTPgammaS also stabilized FtsZ against FtsH. Neither GTP nor GDP inhibited proteolytic activity of FtsH per se. These observations demonstrate that binding of GTP/GDP ligands is responsible for the proteolytic stability of FtsZ against FtsH.     

Constitutive GDP/GTP exchange and secretion-dependent GTP hydrolysis activity for Rab27 in platelets

We have previously demonstrated that Rab27 regulates dense granule secretion in platelets. Here, we analyzed the activation status of Rab27 using the thin layer chromatography method analyzing nucleotides bound to immunoprecipitated Rab27 and the pull-down method quantifying Rab27 bound to the GTP-Rab27-binding domain (synaptotagmin-like protein (Slp)-homology domain) of its specific effector, Slac2-b. We found that Rab27 was predominantly present in the GTP-bound form in unstimulated platelets due to constitutive GDP/GTP exchange activity. The GTP-bound Rab27 level drastically decreased due to enhanced GTP hydrolysis activity upon granule secretion. In permeabilized platelets, increase of Ca(2+) concentration induced dense granule secretion with concomitant decrease of GTP-Rab27, whereas in non-hydrolyzable GTP analogue GppNHp (beta-gamma-imidoguanosine 5'-triphosphate)-loaded permeabilized platelets, the GTP (GppNHp)-Rab27 level did not decrease upon the Ca(2+)-induced secretion. These data suggested that GTP hydrolysis of Rab27 was not necessary for inducing the secretion. Taken together, Rab27 is maintained in the active status in unstimulated platelets, which could function to keep dense granules in a preparative status for secretion.     

NMR studies of the conformational change in human N-p21ras produced by replacement of bound GDP with the GTP analog GTP gamma S.

1H-Detected 15N-edited NMR in solution was used to study the conformational differences between the GDP- and GTP gamma S-bound forms of human N-p21ras. The amide protons of 15N-labeled glycine and isoleucine were observed. Resonances were assigned to residues of particular interest, glycines-60 and -75 and isoleucines-21 and -36, by incorporating various 13C-labeled amino acids in addition to [15N]glycine and [15N]iosleucine and by replacing Mg2+ by Co2+. When GTP gamma S replaced GDP in the active site of p21ras, only 5 of the 14 glycine amide resonances show major shifts, indicating that the conformational effects are fairly localized. Responsive glycines-10, -12, -13, and -15 are in the active site. Gly-75, located at the far end of a conformationally-active loop and helix, also responds to substitution of GTP gamma S for GDP, while Gly-77 does not, supporting a role for Gly-75 as a swivel point for the conformational change. The amide proton resonances of isoleucines-36 and -21 and a third unidentified isoleucine also undergo major shifts upon replacement of GDP by GTP gamma S. Thus, the effector-binding loop containing Ile-36 is confirmed to be involved in the conformational change, and the alpha-helix containing Ile-21 is also shown to be affected.     

Probing the binding states of GDP to Cdc42 using urea interaction

The inactive state of the small G protein Cdc42, the Cdc42.GDP.Mg(2+) ternary complex, was investigated using fluorescence, Mn(2+) substituted electron paramagnetic resonance, and (31)P nuclear magnetic resonance spectroscopy at various urea concentrations. The urea interaction with the protein was used to probe the binding state of GDP.Mg(2+) to Cdc42. Two binding states of the Cdc42.GDP.Mg(2+) ternary complex with different binding stability were observed. The two binding states were characterized by two sets of (31)P resonance of GDP phosphate groups, namely P(alpha) and P(beta), P('alpha), and P('beta). The high populated binding state I (P(alpha) and P(beta)) was more stable and less sensitive to the urea interaction. Yet the population of binding state II (P('alpha) and P('beta)) was lower, and the binding of GDP.Mg(2+) to Cdc42 in this state was more sensitive to the urea interaction. The release of GDP.Mg(2+) from the ternary complex in binding state II was faster than in state I.     

Crystal structure of the ribosome recycling factor bound to the ribosome

In bacteria, disassembly of the ribosome at the end of translation is facilitated by an essential protein factor termed ribosome recycling factor (RRF), which works in concert with elongation factor G. Here we describe the crystal structure of the Thermus thermophilus RRF bound to a 70S ribosomal complex containing a stop codon in the A site, a transfer RNA anticodon stem-loop in the P site and tRNA(fMet) in the E site. The work demonstrates that structures of translation factors bound to 70S ribosomes can be determined at reasonably high resolution. Contrary to earlier reports, we did not observe any RRF-induced changes in bridges connecting the two subunits. This suggests that such changes are not a direct requirement for or consequence of RRF binding but possibly arise from the subsequent stabilization of a hybrid state of the ribosome.     

F-actin-dependent translocation of the Rap1 GDP/GTP exchange factor RasGRP2

RasGRPs constitute a new group of diacylglycerol-dependent GDP/GTP exchange factors that activate Ras subfamily GTPases. Despite a common structure, Ras-GRPs diverge in their GTPase specificity, subcellular distribution, and downstream biological effects. The more divergent family member is RasGRP2, a Rap1-specific exchange factor with low affinity toward diacylglycerol. The regulation of RasGRP2 during signal transduction has remained elusive up to now. In this report, we show that the subcellular localization of Ras-GRP2 is highly dependent on actin dynamics. Thus, the induction of F-actin by cytoskeletal regulators such as Vav, Vav2, Dbl, and Rac1 leads to the shift of RasGRP2 from the cytosol to membrane ruffles and its co-localization with F-actin. Treatment of cells with cytoskeletal disrupting drugs abolishes this effect, leading to an abnormal localization of RasGRP2 in cytoplasmic clusters of actin. The use of Rac1 effector mutants indicates that the RasGRP2 translocation is linked exclusively to actin polymerization and is independent of other pathways such as p21-activated kinase JNK, or superoxide production. Biochemical experiments demonstrate that the translocation of RasGRP2 to membrane ruffles is mediated by the direct association of this protein with F-actin, a property contained within its 150 first amino acids. Finally, we show that the RasGRP2/F-actin interaction promotes the regionalized activation of Rap1 in juxtamembrane areas of the cell. These results reveal a novel function of the actin cytoskeleton in mediating the spatial activation of Ras subfamily GTPases through the selective recruitment of GDP/GTP exchange factors.     

Crystal structures of a type-1 ribosome inactivating protein from Momordica balsamina in the bound and unbound states

The ribosome inactivating proteins (RIPs) of type 1 are plant toxins that eliminate adenine base selectively from the single stranded loop of rRNA. We report six crystal structures, type 1 RIP from Momordica balsamina (A), three in complexed states with ribose (B), guanine (C) and adenine (D) and two structures of MbRIP-1 when crystallized with adenosine triphosphate (ATP) (E) and 2′-deoxyadenosine triphosphate (2′-dATP) (F). These were determined at 1.67 Å, 1.60 Å, 2.20 Å, 1.70 Å, 2.07 Å and 1.90 Å resolutions respectively. The structures contained, (A) unbound protein molecule, (B) one protein molecule and one ribose sugar, (C) one protein molecule and one guanine base, (D) one protein molecule and one adenine base, (E) one protein molecule and one ATP-product adenine molecule and (F) one protein molecule and one 2′-dATP-product adenine molecule. Three distinct conformations of the side chain of Tyr70 were observed with (i) χ¹ = − 66°and χ² = 165° in structures (A) and (B); (ii) χ¹ = − 95° and χ² = 70° in structures (C), (D) and (E); and (iii) χ¹ = − 163° and χ² = 87° in structure (F). The conformation of Tyr70 in (F) corresponds to the structure of a conformational intermediate. This is the first structure which demonstrates that the slow conversion of DNA substrates by RIPs can be trapped during crystallization.     

Stereochemistry of the guanyl nucleotide binding site of transducin probed by phosphorothioate analogues of GTP and GDP

The stereochemistry of the guanyl nucleotide binding site of transducin from bovine retinal rod outer segments was probed with phosphorothioate analogues of GTP and GDP. Transducin has markedly different affinities for the five thio analogues of GTP, as measured by their effectiveness in inhibiting GTPase activity, competing with GTP for entry into transducin, and displacing GDP bound to transducin. The order of binding affinities is GTP gamma S = (Sp)-GTP alpha S greater than (Rp)-GTP alpha S greater than (Sp)-GTP beta S much greater than (Rp)-GTP beta S. The affinity of transducin for GTP gamma S is greater than 10(4) higher than that for (Rp)-GTP beta S. These five analogues have the same relative potencies in eliciting the release of transducin from the membrane and in activating the phosphodiesterase. Transducin hydrolyzes (Sp)-GTP alpha S with a l/e time of 55 s, compared with 28 s for GTP. In contrast, (Rp)-GTP alpha S, like GTP gamma S, is not hydrolyzed on the time scale of several hours. The order of effectiveness of thio analogues of GDP in displacing bound GDP is (Sp)-GDP alpha S greater than GDP greater than (Rp)-GDP alpha S greater than GDP beta S. The affinity of transducin for (Sp)-GDP alpha S is about 10-fold higher than that for GDP beta S. Mg2+ is required for the binding of GTP and GDP to transducin. Cd2+ does not lead to a reversal of stereospecificity at either the alpha- or beta-phosphorus atom of GTP. These results lead to the following conclusions: The pro-R oxygen atom at the alpha-phosphorus of GTP does not bind Mg2+ but instead interacts with the protein. The pro-S oxygen at the alpha-phosphorus does not appear to be involved in a critical interaction with transducin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a gephyrin-binding motif in the GDP/GTP exchange factor collybistin.

The brain-specific GDP/GTP exchange factor collybistin interacts with the receptor-anchoring protein gephyrin and activates the Rho-like GTPase Cdc42, which is known to regulate actin cytoskeleton dynamics. Alternative splicing creates two collybistin variants, I and II. In coexpression experiments, collybistin II has been shown to induce the formation of submembraneous gephyrin aggregates which cluster with hetero-oligomeric glycine receptors (GlyRs). Here we identified residues critical for interaction with gephyrin in the linker region between the SH3 and the DH domains of collybistin. Respective collybistin deletion mutants failed to bind gephyrin upon coexpression in heterologous cells, in GST pull-down assays and in the yeast two-hybrid system. Site-directed mutagenesis revealed polar amino acid residues as essential determinants of gephyrin binding. Furthermore, in vitro gephyrin bound simultaneously to both collybistin and the GlyR beta-subunit binding motif. Our data are consistent with collybistin-gephyrin interactions occuring during inhibitory postsynaptic membrane formation.     

X-Ray structures of the universal translation initiation factor IF2/eIF5B: conformational changes on GDP and GTP binding

X-ray structures of the universal translation initiation factor IF2/eIF5B have been determined in three states: free enzyme, inactive IF2/eIF5B.GDP, and active IF2/eIF5B.GTP. The "chalice-shaped" enzyme is a GTPase that facilitates ribosomal subunit joining and Met-tRNA(i) binding to ribosomes in all three kingdoms of life. The conserved core of IF2/eIF5B consists of an N-terminal G domain (I) plus an EF-Tu-type beta barrel (II), followed by a novel alpha/beta/alpha-sandwich (III) connected via an alpha helix to a second EF-Tu-type beta barrel (IV). Structural comparisons reveal a molecular lever, which amplifies a modest conformational change in the Switch 2 region of the G domain induced by Mg(2+)/GTP binding over a distance of 90 A from the G domain active center to domain IV. Mechanisms of GTPase function and ribosome binding are discussed.     

The influence of bound GDP on the kinetics of guanine nucleotide binding to G proteins

Purified guanine nucleotide-binding regulatory proteins, as either the oligomers or the isolated nucleotide-binding alpha subunits, display anomalous kinetics of nucleotide binding. This is due to the presence of tightly bound GDP in these preparations. The dissociation of bound GDP is the rate-limiting step for nucleotide binding. GDP can be removed by chromatography in the presence of 1 M (NH4)2SO4 and 20% glycerol, which yields preparations of G proteins that contain less than 0.1 mol of GDP/mol of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S)-binding site. When the GDP is removed, the binding of GTP gamma S displays kinetics consistent with a bimolecular reaction.     

Global conformational rearrangements during the activation of the GDP/GTP exchange factor Vav3

Activation of Rho/Rac GTPases during cell signaling requires the participation of GDP/GTP exchange factors of the Dbl family. Although the structure of the catalytic core of Dbl proteins has been established recently, the molecular changes that the full-length proteins experience during normal or oncogenic conditions of stimulation are still unknown. Here, we have used single-particle electron microscopy to solve the structures of the inactive (unphosphorylated), active (phosphorylated), and constitutively active (N-terminally deleted) versions of the exchange factor Vav3. Comparison of these forms has revealed the interdomain interactions maintaining the inactive Vav3 state and the dynamic changes that the overall Vav3 structure undergoes upon tyrosine phosphorylation. We have also found that the conformations of phosphorylated Vav3 and N-terminally deleted Vav3 are distinct, indicating that the acquisition of constitutive activity by exchange factors is structurally more complex than the mere elimination of inhibitory interactions between structural domains.