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• W2070407927 abstract "FeoB is a prokaryotic membrane protein responsible for the import of ferrous iron (Fe2+). A defining feature of FeoB is that it includes an N-terminal 30-kDa soluble domain with GTPase activity, which is required for iron transport. However, the low intrinsic GTP hydrolysis rate of this domain appears to be too slow for FeoB either to function as a channel or to possess an active Fe2+ membrane transport mechanism. Here, we present crystal structures of the soluble domain of FeoB from Streptococcus thermophilus in complex with GDP and with the GTP analogue derivative 2′-(or -3′)-O-(N-methylanthraniloyl)-β,γ-imidoguanosine 5′-triphosphate (mant-GMPPNP). Unlike recent structures of the G protein domain, the mant-GMPPNP-bound structure shows clearly resolved, active conformations of the critical Switch motifs. Importantly, biochemical analyses demonstrate that the GTPase activity of FeoB is activated by K+, which leads to a 20-fold acceleration in its hydrolysis rate. Analysis of the structure identified a conserved asparagine residue likely to be involved in K+ coordination, and mutation of this residue abolished K+-dependent activation. We suggest that this, together with a second asparagine residue that we show is critical for the structure of the Switch I loop, allows the prediction of K+-dependent activation in G proteins. In addition, the accelerated hydrolysis rate opens up the possibility that FeoB might indeed function as an active transporter. FeoB is a prokaryotic membrane protein responsible for the import of ferrous iron (Fe2+). A defining feature of FeoB is that it includes an N-terminal 30-kDa soluble domain with GTPase activity, which is required for iron transport. However, the low intrinsic GTP hydrolysis rate of this domain appears to be too slow for FeoB either to function as a channel or to possess an active Fe2+ membrane transport mechanism. Here, we present crystal structures of the soluble domain of FeoB from Streptococcus thermophilus in complex with GDP and with the GTP analogue derivative 2′-(or -3′)-O-(N-methylanthraniloyl)-β,γ-imidoguanosine 5′-triphosphate (mant-GMPPNP). Unlike recent structures of the G protein domain, the mant-GMPPNP-bound structure shows clearly resolved, active conformations of the critical Switch motifs. Importantly, biochemical analyses demonstrate that the GTPase activity of FeoB is activated by K+, which leads to a 20-fold acceleration in its hydrolysis rate. Analysis of the structure identified a conserved asparagine residue likely to be involved in K+ coordination, and mutation of this residue abolished K+-dependent activation. We suggest that this, together with a second asparagine residue that we show is critical for the structure of the Switch I loop, allows the prediction of K+-dependent activation in G proteins. In addition, the accelerated hydrolysis rate opens up the possibility that FeoB might indeed function as an active transporter. Small regulatory GTPase proteins (G proteins) are key binary switches in cellular processes such as differentiation, proliferation, and cell motility (1Cachero T.G. Morielli A.D. Peralta E.G. Cell. 1998; 93: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 2Finlin B.S. Correll R.N. Pang C. Crump S.M. Satin J. Andres D.A. J. Biol. Chem. 2006; 281: 23557-23566Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). As such, their aberrant functions result in a number of pathophysiological disorders, such as asthma and cancer (3Schaafsma D. Roscioni S.S. Meurs H. Schmidt M. Cell Signal. 2008; 20: 1705-1714Crossref PubMed Scopus (30) Google Scholar, 4Karnoub A.E. Weinberg R.A. Nat. Rev. Mol. Cell Biol. 2008; 9: 517-531Crossref PubMed Scopus (1128) Google Scholar). G proteins cycle between a GDP-bound inactive state and a GTP-bound active state. When bound to GTP, they interact with effector proteins through active conformations of certain polypeptide segments designated Switch I and Switch II (5Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1384) Google Scholar). The active GTP-bound conformations of the Switch segments are stabilized by their interactions with the nucleotide γ-phosphate and a Mg2+ ion at the nucleotide-binding site. Hydrolysis of GTP to GDP releases the γ-phosphate and the Mg2+ ion, leading to the relaxation of the Switch I and II segments to their inactive conformations (5Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1384) Google Scholar). The prokaryotic G protein-coupled ferrous iron (Fe2+) transporter B (FeoB) is unique in that its cytoplasmic G protein domain is directly tethered to a polytopic membrane domain (6Marlovits T.C. Haase W. Herrmann C. Aller S.G. Unger V.M. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16243-16248Crossref PubMed Scopus (118) Google Scholar). The G protein domain belongs to the TrmE-Era-EngA-YihA-Septin-like (TEES) 4The abbreviations used are: TEESTrmE-Era-EngA-YihA-Septin-likeGAPGTPase-activating proteinmant-GMPPNP2′-(or -3′)-O-(N-methylanthraniloyl)-β,γ-imidoguanosine 5′-triphosphate. superfamily of bacterial GTPases, which are recognized by sequence conservation between the G1 and G2 motifs. (see Fig. 1A) (7Leipe D.D. Wolf Y.I. Koonin E.V. Aravind L. J. Mol. Biol. 2002; 317: 41-72Crossref PubMed Scopus (856) Google Scholar). GTP binding to the G protein domain (FeoGP) initiates the transport of Fe2+ across the membrane, which is halted by the hydrolysis of GTP to GDP. Previous studies have shown that FeoGP has slow GTPase activity (Escherichia coli kcat = 0.0015 s−1, equivalent to the hydrolysis of one GTP molecule in 11 min) (6Marlovits T.C. Haase W. Herrmann C. Aller S.G. Unger V.M. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16243-16248Crossref PubMed Scopus (118) Google Scholar, 8Eng E.T. Jalilian A.R. Spasov K.A. Unger V.M. J. Mol. Biol. 2008; 375: 1086-1097Crossref PubMed Scopus (35) Google Scholar, 9Guilfoyle A. Maher M.J. Rapp M. Clarke R. Harrop S. Jormakka M. EMBO J. 2009; 28: 2677-2685Crossref PubMed Scopus (38) Google Scholar). A slow intrinsic GTPase rate is not uncommon for G proteins, because their activity can be accelerated by GTPase-activating proteins (GAPs), which can stimulate GTP hydrolysis by several orders of magnitude (10Bos J.L. Rehmann H. Wittinghofer A. Cell. 2007; 129: 865-877Abstract Full Text Full Text PDF PubMed Scopus (1315) Google Scholar). One mode of activation by many GAPs is through the insertion of an arginine residue (arginine finger) into the active site of the G protein, which neutralizes the developing negative charge on the nucleotide during hydrolysis (11Rittinger K. Walker P.A. Eccleston J.F. Nurmahomed K. Owen D. Laue E. Gamblin S.J. Smerdon S.J. Nature. 1997; 388: 693-697Crossref PubMed Scopus (223) Google Scholar, 12Scheffzek K. Ahmadian M.R. Kabsch W. Wiesmüller L. Lautwein A. Schmitz F. Wittinghofer A. Science. 1997; 277: 333-338Crossref PubMed Scopus (1202) Google Scholar). Such GAPs are an essential aspect of the function and regulation of G protein-coupled processes, yet despite extensive efforts, no such GAP or other activating factor has been identified for FeoB. The slow intrinsic hydrolysis rate of FeoB has thus remained somewhat puzzling, being too slow to support an active Fe2+ transport mechanism (8Eng E.T. Jalilian A.R. Spasov K.A. Unger V.M. J. Mol. Biol. 2008; 375: 1086-1097Crossref PubMed Scopus (35) Google Scholar). Yet should it rather function as a G protein-coupled channel, the channel would remain in an “open” state for an unfeasible period of time after GTP binding. TrmE-Era-EngA-YihA-Septin-like GTPase-activating protein 2′-(or -3′)-O-(N-methylanthraniloyl)-β,γ-imidoguanosine 5′-triphosphate. To gain further insight into the mechanism of FeoB, we and other groups have recently structurally characterized the soluble G protein domain from four different organisms: E. coli, Methanococcus jannaschii, Thermotoga maritima, and Legionella pneumophilia (9Guilfoyle A. Maher M.J. Rapp M. Clarke R. Harrop S. Jormakka M. EMBO J. 2009; 28: 2677-2685Crossref PubMed Scopus (38) Google Scholar, 13Hattori M. Jin Y. Nishimasu H. Tanaka Y. Mochizuki M. Uchiumi T. Ishitani R. Ito K. Nureki O. Structure. 2009; 17: 1345-1355Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 14Köster S. Wehner M. Herrmann C. Kühlbrandt W. Yildiz O. J. Mol. Biol. 2009; 392: 405-419Crossref PubMed Scopus (24) Google Scholar, 15Petermann N. Hansen G. Schmidt C.L. Hilgenfeld R. FEBS Lett. 2010; 584: 733-738Crossref PubMed Scopus (25) Google Scholar). The structures revealed a canonical G protein similar to Ras, followed by a helical domain with a proposed effector (9Guilfoyle A. Maher M.J. Rapp M. Clarke R. Harrop S. Jormakka M. EMBO J. 2009; 28: 2677-2685Crossref PubMed Scopus (38) Google Scholar) or GDP dissociation inhibitor role (8Eng E.T. Jalilian A.R. Spasov K.A. Unger V.M. J. Mol. Biol. 2008; 375: 1086-1097Crossref PubMed Scopus (35) Google Scholar, 13Hattori M. Jin Y. Nishimasu H. Tanaka Y. Mochizuki M. Uchiumi T. Ishitani R. Ito K. Nureki O. Structure. 2009; 17: 1345-1355Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). However, in all FeoB structures of the G protein complexed with GTP analogues, the crucial Switch I loop was either disordered (9Guilfoyle A. Maher M.J. Rapp M. Clarke R. Harrop S. Jormakka M. EMBO J. 2009; 28: 2677-2685Crossref PubMed Scopus (38) Google Scholar, 14Köster S. Wehner M. Herrmann C. Kühlbrandt W. Yildiz O. J. Mol. Biol. 2009; 392: 405-419Crossref PubMed Scopus (24) Google Scholar) or situated far from the nucleotide-binding site (13Hattori M. Jin Y. Nishimasu H. Tanaka Y. Mochizuki M. Uchiumi T. Ishitani R. Ito K. Nureki O. Structure. 2009; 17: 1345-1355Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). This indicated that the Switch I region was not essential for nucleotide or Mg2+ binding, consistent with mutational analysis of conserved residues in Switch I of E. coli FeoB, which did not significantly affect its GTPase activity (8Eng E.T. Jalilian A.R. Spasov K.A. Unger V.M. J. Mol. Biol. 2008; 375: 1086-1097Crossref PubMed Scopus (35) Google Scholar). Conversely, studies upon full-length FeoB harboring the same mutations in Switch I were unable to restore Fe2+ uptake in a feoB-deficient background (6Marlovits T.C. Haase W. Herrmann C. Aller S.G. Unger V.M. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16243-16248Crossref PubMed Scopus (118) Google Scholar). This apparent disparity has left the role played by the Switch I region in FeoB open. To probe the function of Switch I in FeoB and to extend the current structural information, we have determined the structure of the 30-kDa soluble domain of FeoB from Streptococcus thermophilus (NFeoBSt, residues 1–270) complexed with GDP and with the nonhydrolyzable GTP analogue (5′[β,γ-imido]triposphate) derivative, mant-GMPPNP. The latter structure reveals an ordered Switch I motif in the canonical, active conformation, in which the loop hydrogen bonds with the nucleotide γ-phosphate and coordinates the Mg2+ ion. Hydrolysis measurements conducted in the presence of K+ show FeoB to be a potassium-activated G protein, with the cation increasing the rate of GTP hydrolysis by up to 20-fold. Furthermore, from structural comparison and mutational analysis, we propose that two conserved asparagine residues (Asn11 and Asn19) facilitate the activation by potassium and that these residues confer the general characteristic of potassium-dependent activation in the TEES superfamily of G proteins. DNA encoding residues 1–270 from FeoB was amplified from S. thermophilus genomic DNA (strain LMG 18311) and cloned into a pGEX-4T-1 glutathione S-transferase fusion vector (GE Healthcare). NFeoBSt was expressed and purified from E. coli BL21 (DE3) cells as described for E. coli NFeoB (9Guilfoyle A. Maher M.J. Rapp M. Clarke R. Harrop S. Jormakka M. EMBO J. 2009; 28: 2677-2685Crossref PubMed Scopus (38) Google Scholar). Briefly, NFeoBSt was purified by glutathione S-transferase affinity chromatography in 20 mm Tris, pH 8.0, 100 mm NaCl. Thrombin cleavage to remove the glutathione S-transferase tag was performed for 24 h at 25 °C. NFeoBSt was then applied to a Superdex 75 size exclusion column (GE Healthcare Life Sciences), and the eluted protein concentrated to ∼12 mg/ml as determined by the BCA assay method. The concentrated protein was stored at −80 °C until further use. The plasmid pGEX-4T1/NFeoBSt was used to generate an expression vector with a single amino acid mutation (N11A) by using a Stratagene QuikChange site-directed mutagenesis kit. The mutant was expressed and purified as for the wild-type protein, with the addition of 5% glycerol in all purification buffers. For crystallization of mant-GMPPNP-bound NFeoBSt, protein (7 mg/ml in 20 mm Tris, pH 8.0, 10 mm MgCl2, 100 mm NaCl) was incubated overnight at 4 °C with a 0.5 mm mant-GMPPNP. GDP-bound NFeoBSt was crystallized from a solution containing protein (3 mg/ml in 20 mm Tris, pH 8.0, 2 mm MgCl2, 100 mm KCl), which was incubated for 2 h at 4 °C with 0.2 mm GDP, 2 mm NaF, and 0.2 mm AlCl3. Crystallization conditions were screened at room temperature using the hanging drop vapor diffusion method with Qiagen JCSG+ and PACT screens. All crystallization drops were set up in 96-well plates (Greiner Bio-One) using a Mosquito nanolitre liquid handling robot (Molecular Dimensions) and contained equal volumes of reservoir solution (150 nl) and protein solution. For mant-GMPPNP-bound NFeoBSt, a large crystal plate appeared after 1 week in PACT screen condition D5 (0.1 m MMT buffer, pH 8.0, 25% (w/v) polyethylene glycol 1500), which had the approximate dimensions 350 × 100 × 20 μm. GDP-bound NFeoBSt crystallized after 3 days in the JCSG+ condition H8 (0.2 m NaCl, 0.1 m bis-Tris, pH 5.5, 25% (w/v) polyethylene glycol 3350). The crystal had the approximate dimensions 100 × 100 × 10 μm. Single crystals were flash frozen directly into a cold N2 gas stream (100 K) using 20% (v/v) glycerol in reservoir solution as cryoprotectant. Diffraction data for each crystal were recorded on a Mar345 image plate detector using copper Kα X-rays from either a Rigaku RU-200 (mant-GMPPNP-bound NFeoBSt), or a Rigaku MicroMax-007 HF (GDP-bound NFeoBSt) rotating anode generator. The data were processed and scaled using HKL2000 (16Otwinowski Z. Minor W. Charles W. Carter J. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38570) Google Scholar). The structure of NFeoBSt complexed with mant-GMPPNP was solved by molecular replacement using the structure of the G domain of E. coli NFeoB as a search model (Protein Data Bank accession code 3HYR, residues 1–168). The waters were removed prior to molecular replacement, and all of the selenomethionine residues were mutated to methionine. CHAINSAW (17Stein N. J. Appl. Cryst. 2008; 41: 641-643Crossref Scopus (451) Google Scholar) from the CCP4 program suite (18Collaborative Computational Project, Number 4 Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19768) Google Scholar) was used to truncate nonconserved side chains to the gamma atom. Molecular replacement was performed using the program PHASER (19McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. J. Appl. Cryst. 2007; 40: 658-674Crossref PubMed Scopus (14552) Google Scholar), using diffraction data to 2.5 Å. The G domain was unambiguously positioned with a single unique solution, which had a log likelihood gain of 84 and a Z-score of 7.4. Molecular replacement was then attempted using the helical domain of E. coli FeoB (residues 171–260); however, no clear solution could be found. Refinement thus commenced with the G domain only, which represented ∼60% of the NFeoBSt structure. Refinement of all structures was carried out using REFMAC5.5 (20Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13869) Google Scholar) with TLS (21Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. D Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1651) Google Scholar), and all model building was performed in COOT (22Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23377) Google Scholar). No σ cut-off was employed during refinement. After several rounds of refinement of mant-GMPPNP-bound NFeoBSt, the electron density was of sufficient quality to manually position the C-terminal helix of the helical domain (residues 242–259), and iterative rounds of manual building and refinement produced a model of 199 residues. Model phases were then input into ARP/warp (23Cohen S.X. Morris R.J. Fernandez F.J. Ben Jelloul M. Kakaris M. Parthasarathy V. Lamzin V.S. Kleywegt G.J. Perrakis A. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2222-2229Crossref PubMed Scopus (152) Google Scholar), which built a further 59 residues. The final model contains protein residues 2–161 and 167–265. The structure of GDP-bound NFeoBSt was solved by molecular replacement using PHASER, with the coordinates of mant-GMPPNP-bound NFeoBSt as a search model. Molecular replacement identified a single solution with a log likelihood gain of 174 and a Z-score of 16.5. The final model consists of protein residues 1–258. A Malachite Green phosphate assay kit (BioAssay Systems) was used to measure the GTPase activity of wild-type and mutant NFeoBSt in the presence of various monovalent cations. NFeoBSt (0.3 μm) was incubated with 250 μm GTP and 5 mm MgCl2 at 37 °C in 20 mm Tris, pH 8, and 100 or 200 mm salt (either LiCl, NaCl, KCl, NH4Cl, RbCl, or CsCl). After the addition of protein, aliquots were removed at frequent intervals for up to 3 h and mixed in a 1:4 ratio with the Malachite Green reagent according to the manufacturer's instructions. For each aliquot, color was allowed to develop for 30 min at room temperature before measuring the absorbance at 620 nm in a 96-well plate (Greiner Bio-One) on a POLARstar Omega microplate reader (BMG LABTECH). The turnover number (kcat), was calculated from the slope of each time course. All of the hydrolysis assays were performed in triplicate. The rate of GTP hydrolysis by wild-type and mutant NFeoBSt in NaCl and KCl were determined by intrinsic tryptophan fluorescence in single-turnover experiments. All of the experiments contained 70 μm NFeoBSt and 30 μm GTP and were conducted on a POLARstar Omega microplate reader (BMG LABTECH) in black flat-bottomed 96-well plates (Greiner Bio-One). A 280 ± 5-nm filter was used for excitation, and fluorescence was measured using a 355 ± 5-nm emission filter. NFeoBSt was preincubated for 5 min at 37 °C in buffer containing 20 mm Tris, pH 8, and either NaCl (100 or 200 mm) or KCl (50–200 mm). This solution was used to set the relative gain of the plate reader, which was adjusted to give a fluorescence signal of 90% of the maximum possible value. GTP was then added to the mixture, and the fluorescence was monitored for 1 min. The GTPase reaction was initiated with the addition of 1 mm MgCl2 using the POLARstar reagent injection apparatus. After the addition of MgCl2, hydrolysis was monitored every 5 s for 4 min or every 20 s for 30 min for reactions performed in KCl and NaCl, respectively. The data were fit to a single-exponential association function using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA), where the rate constant is equal to kcat. Because of the presence of a 60-s lag phase in the NaCl reactions, these data were excluded in the calculation of kcat in NaCl. All of the experiments were performed in triplicate. Intrinsic tryptophan fluorescence was also used to determine the binding affinity of wild-type and mutant NFeoBSt for GTP. Different concentrations of GTP (0.02–0.8 mm) were added to protein (15 μm) in 20 mm Tris, pH 8, with either 100 mm NaCl or 100 mm KCl at 23 °C. The gain of each sample in the POLARstar plate reader was adjusted to give a signal of 90% of the maximum value. The fluorescence was monitored every 10 s for 1 min, after which time nucleotide binding was initiated by the addition of 3 mm MgCl2 using the POLARstar reagent injectors. For wild-type measurements conducted in NaCl and for mutant measurements, the slow rate of nucleotide hydrolysis precluded any hydrolysis-related increase in fluorescence on the time scale of the experiment. Therefore, the fluorescence of the samples after MgCl2 addition was recorded every 10 s for a further minute, and the steady-state fluorescence values (both before and after nucleotide binding) were individually averaged. The relative change in fluorescence for each nucleotide concentration was used to plot a binding isotherm. For wild-type measurements in the presence of KCl, the data were collected every 5 s for 3 min after the addition of MgCl2. At lower nucleotide concentrations, rapid hydrolysis caused a significant increase in fluorescence after MgCl2 addition. Therefore, the data were fit to an exponential association function, and the calculated fluorescence at 0 s was used to determine the change in fluorescence upon nucleotide binding. Binding curves were fit to a rectangular hyperbola using GraphPad Prism 5.0, and all of the experiments were performed in triplicate. The rates of mant-GMPPNP and mant-GTP binding to mutant and wild-type NFeoBSt were measured using a stopped flow apparatus (SF-61; Hi-Tech Scientific Ltd., Salisbury, UK) as previously described (6Marlovits T.C. Haase W. Herrmann C. Aller S.G. Unger V.M. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16243-16248Crossref PubMed Scopus (118) Google Scholar, 9Guilfoyle A. Maher M.J. Rapp M. Clarke R. Harrop S. Jormakka M. EMBO J. 2009; 28: 2677-2685Crossref PubMed Scopus (38) Google Scholar). Briefly, protein (10 μm in 20 mm Tris, pH 8.0, 100 mm MgCl2, and either 100 mm NaCl or KCl) was rapidly mixed with 500 μm mant-GMPPNP or mant-GTP (in 20 mm Tris, pH 8.0, and 100 mm of the corresponding salt). To measure the rate of mant-GDP release, protein (10 μm in 20 mm Tris, pH 8.0, and either 100 mm NaCl or KCl) was incubated for 10 min with 0.5 μm mant-GDP and then rapidly mixed with 500 μm GTP in the corresponding buffer. All of the measurements were conducted at 25 °C. The mant fluorophore was excited at 355 nm using a mercury lamp (Osram, Berlin, Germany), and the fluorescence was collected at wavelengths of ≥400 nm using a GG400 glass cut-off filter (Schott, Mainz, Germany). All of the experiments were performed 7–10 times, and the data were averaged before calculating the final kobs or koff values. NFeoBSt (residues 1–270) co-crystallized with the nonhydrolyzable GTP analogue mant-GMPPNP in space group C2221, with one molecule in the asymmetric unit. The crystal diffracted to 1.9 Å, and refinement converged with residuals Rwork = 0.197 and Rfree = 0.241. The final model contains residues 2–161 and 167–265, a mant-GMPPNP molecule, a Mg2+ ion, and a glycerol molecule. The mant group from the nucleotide is disordered in the structure and makes no contacts with the protein (supplemental Fig. S1). The GDP-bound form of NFeoBSt crystallized in space group P41212, with one molecule in the asymmetric unit. The structure was determined and refined to 2.9-Å resolution with Rwork = 0.239 and Rfree = 0.298. Coordinates and structure factors have been deposited under Protein Data Bank codes 3LX5 and 3LX8 for the mant-GMPPNP- and GDP-bound forms, respectively. The data collection and refinement statistics are summarized in Table 1.TABLE 1Crystallographic data processing and refinement statisticsmant-GMPPNP-NFeoBStGDP-NFeoBStData processingWavelength (Å)1.54121.5412Space groupC2221P41212Unit cell dimensionsa (Å)49.142.8b (Å)82.242.8c (Å)148.8282.1Resolution (Å)50-1.90 (1.93-1.90)50-2.90 (2.95-2.90)Total reflections135,00342,655Unique reflections23,847 (1026)6,494 (209)Completeness (%)97.8 (85.4)97.9 (71.1)〈I/σ(I)〉14.2 (3.5)11.2 (2.0)Redundancy5.7 (4.4)6.6 (2.6)RmergeaRmerge = ΣhklΣj |Ij(hkl) − 〈I(hkl)〉|/ΣhklΣj Ij(hkl).0.118 (0.336)0.133 (0.414)RefinementNo. molecules/asymmetric unit11No. protein atoms2,0941,934No. water molecules1120Total no. atoms2,2451,962Resolution (Å)23.6-1.90 (1.95-1.90)50-2.90 (2.97-2.90)Unique reflections23,665 (1503)6,427 (374)Completeness (%)98.0 (87.3)98.3 (84.0)Root mean square deviationBonds (Å)0.0140.010Angles (°)1.4791.494Wilson B factor (Å2)24.966.9〈B factor〉 (Å2)Protein27.954.8Ligand/ion26.778.9Solvent21n/aRamachandran plotbAs calculated by MolProbity (43).Favored (%)98.295.4Additionally allowed (%)1.84.6RworkcRwork = Σhkl|Fobs − Fcalc|/Σhkl |Fobs|.0.197 (0.254)0.239 (0.348)RfreedCalculated using 5 and 10% of the diffraction data that was excluded during refinement for mant-GMPPNP- and GDP-bound forms, respectively.0.241 (0.277)0.298 (0.411)Protein Data Bank code3LX53LX8a Rmerge = ΣhklΣj |Ij(hkl) − 〈I(hkl)〉|/ΣhklΣj Ij(hkl).b As calculated by MolProbity (43Davis I.W. Leaver-Fay A. Chen V.B. Block J.N. Kapral G.J. Wang X. Murray L.W. Arendall 3rd, W.B. Snoeyink J. Richardson J.S. Richardson D.C. Nucleic Acids Res. 2007; 35: W375-W383Crossref PubMed Scopus (3030) Google Scholar).c Rwork = Σhkl|Fobs − Fcalc|/Σhkl |Fobs|.d Calculated using 5 and 10% of the diffraction data that was excluded during refinement for mant-GMPPNP- and GDP-bound forms, respectively. Open table in a new tab Both structures of NFeoBSt show the G protein domain (residues 1–170) followed by the helical domain (residues 171–270) (Fig. 1B). The GTPase domain forms the archetypical G protein fold, which consists of a six-stranded β-sheet surrounded by six α-helices. The helical domain is composed of a five-helix bundle (h1–h5), which contacts the G domain via helices h2, h3, and h5. Previous structures of the soluble domain of FeoB have indicated putative biological dimer (13Hattori M. Jin Y. Nishimasu H. Tanaka Y. Mochizuki M. Uchiumi T. Ishitani R. Ito K. Nureki O. Structure. 2009; 17: 1345-1355Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 14Köster S. Wehner M. Herrmann C. Kühlbrandt W. Yildiz O. J. Mol. Biol. 2009; 392: 405-419Crossref PubMed Scopus (24) Google Scholar) and trimer (9Guilfoyle A. Maher M.J. Rapp M. Clarke R. Harrop S. Jormakka M. EMBO J. 2009; 28: 2677-2685Crossref PubMed Scopus (38) Google Scholar) configurations, whereas gel filtration indicates NFeoBSt is monomeric in solution. In the crystal of mant-GMPPNP-bound NFeoBSt, a symmetric dimer (generated by symmetry operation −x, y, −z + 1/2) lies across a crystallographic 2-fold axis. The dimer interface is formed from the Switch I loop, yet its low total buried surface area (409 Å2/protomer) suggests that it is most likely a product of crystal packing (24Jones S. Thornton J.M. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 13-20Crossref PubMed Scopus (2275) Google Scholar). For GDP-bound NFeoBSt, there are likewise no significant contacts within the crystal that are suggestive of any biologically relevant higher order oligomer, with the largest buried interface area being 376 Å2/protomer. GDP-bound NFeoBSt was crystallized from a solution containing the components necessary for formation of the transition-state analogue of GTP (25Bigay J. Deterre P. Pfister C. Chabre M. FEBS Lett. 1985; 191: 181-185Crossref PubMed Scopus (325) Google Scholar). Despite intrinsic tryptophan fluorescence indicating that the transition state was successfully formed in solution (supplemental Fig. S2), the resultant crystals contained only GDP-bound protein. The GDP molecule is contacted by a number of residues at the nucleotide-binding site, including Asn113, Asn116, Ser16, and the backbone amides of Asn11–Gly13. The Switch I loop (residues 24–36) does not interact with the GDP and is instead oriented away from the nucleotide-binding site in its inactive conformation. The loop forms an additional anti-parallel β-strand against the β-sheet core of the protein (Fig. 1B), as has been observed for the GDP-bound forms of NFeoB from other organisms (13Hattori M. Jin Y. Nishimasu H. Tanaka Y. Mochizuki M. Uchiumi T. Ishitani R. Ito K. Nureki O. Structure. 2009; 17: 1345-1355Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 14Köster S. Wehner M. Herrmann C. Kühlbrandt W. Yildiz O. J. Mol. Biol. 2009; 392: 405-419Crossref PubMed Scopus (24) Google Scholar). Following the nomenclature of the Ras-related protein Ran (26Scheffzek K. Klebe C. Fritz-Wolf K. Kabsch W. Wittinghofer A. Nature. 1995; 374: 378-381Crossref PubMed Scopus (174) Google Scholar) and ADP-ribosylation factor 1 (27Amor J.C. Harrison D.H. Kahn R.A. Ringe D. Nature. 1994; 372: 704-708Crossref PubMed Scopus (253) Google Scholar), where the additional β-strand in Switch I has also been observed, we refer to this strand as β2E. The core structures of GDP-NFeoBSt and mant-GMPPNP-NFeoBSt are highly similar, and superposition of the two structures, excluding the Switch regions, gives a root mean square deviation of 1.2 Å in 218 Cα positions. However, three principal regions have undergone structural rearrangement between the two forms: the Switch I motif, the Switch II motif, and the helical domain. The most dramatic of these changes is in Switch I, where the β2E strand has been broken and the loop has been shifted almost 30 Å toward the nucleotide-binding site (Fig. 1B). Now in its GTP-bound orientation, Switch I forms a lid over the nucleotide, the apex of which is comprised of residues 29–33. This lid leaves the nucleotide base and ribose solvent-exposed yet completely buries all three of its phosphate groups (supplemental Fig. S3). There is a distinctive bend in the Switch I backbone near residue 26, which lies at the base of the loop. This bend is caused by two hydrogen bonds between both side chain groups of a conserved asparagine residue, Asn19, and the backbone amide and carbonyl groups of Asn26 from the Switch I" @default.
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