PROTEINS: Structure, Function, and Genetics 28:285–288 (1997) Expression, Crystallization and Preliminary X-Ray Diffraction Study of FtsY, the Docking Protein of the Signal Recognition Particle of E. coli Guillermo Montoya,1 Cecilia Svensson,1 Joen Luirink,2 and Irmgard Sinning1* 1European Molecular Biology Laboratory, Structural Biology Programme, Heidelberg, Germany 2Department of Microbiology, Institute of Molecular Biological Sciences, Biocentrum Amsterdam, DeBoelelaan 1087, Amsterdam, The Netherlands ABSTRACT FtsY is the docking protein or SRa homologue in E. coli. It is involved in targeting secretory proteins to the cytoplasmic membrane by interacting with the signal recognition particle, controlled by guanosine 58-triphosphate. Two different constructs have been used in crystallization studies: the fulllength protein and a truncated fragment with a his-tag at the C terminus. Only the second construct resulted in crystals suitable for x-ray diffraction. The crystals belong to the monoclinic space group P21 with cell dimensions a 5 32.20 Å, b 5 79.57 Å, c 5 59.21 Å, and b 5 94.45, and contain one molecule per asymmetric unit. At cryogenic temperatures the crystals diffract to a resolution limit of 2.5 Å by using a rotating anode, and beyond 1.8 Å by using synchrotron radiation. Proteins 28:285–288, 1997. r 1997 Wiley-Liss, Inc. Key words: SRP; SRP receptor; GTPase; expression; crystallization; x-ray diffraction INTRODUCTION Targeting and translocation of proteins into and across membranes in eukaryotes and prokaryotes, is mediated by hydrophobic N-terminal signal sequences. This suggests similarities in their basic mechanisms of targeting and translocation.1 In mammalian cells, targeting of most proteins to the endoplasmic reticulum is mediated by the SRP, which consists of a 7S RNA and six polypeptides of 9, 14, 19, 54, 68, and 72 kDa.2 The SRP binds via its 54-kDa subunit (SRP54) to the signal sequence of the nascent polypeptide chain as soon as it becomes available at the ribosome, thereby arresting elongation.3 The complex formed by the ribosome, the nascent polypeptide chain, and the SRP is targeted to the endoplasmic reticulum by interacting with a docking protein complex (or SRP receptor) formed by two proteins. Both the SRa and the SRb have GTPase activity.4,5 The SRP and the receptor are released after GTP hydrolysis, and the remaining ribosomer 1997 WILEY-LISS, INC. nascent chain complex interacts with a complex of membrane proteins, which catalyzes membrane insertion and translocation of the nascent chain. In E. coli, several factors of the general secretory pathway have been identified using genetic and biochemical approaches.6 Molecular chaperones, such as SecB, function to maintain certain precursor proteins in a translocation competent conformation in the cytosol and to target them to the cytoplasmic membrane where a complex composed of SecA/Y/E/G and SecD/F assists in membrane insertion and translocation. The role of the SRP in E. coli is poorly understood. It is much simpler than the eukaryotic SRP consisting of only one protein—p48, Ffh—and a 4.5 S RNA.7,8 SRP may support cotranslational translocation in a separate secrectory pathway or could form part of the above-mentioned general secretory pathway. The first hypothesis is supported by identification of E. coli FtsY as the homologue of SRa. FtsY displays significant sequence similarity to SRa9,10 and was shown to be essential for efficient secretion of a subset of preproteins.11 Furthermore, the GTPdependent interaction of Ffh and FtsY was demonstrated,12 and it was shown that Ffh and FtsY act as regulatory proteins for one another.13 Earlier, a role in cell division was proposed based on the location of ftsY in an operon together with ftsE and ftsX, in which mutations have been identified that affect cell division.14 FtsY consists of 497 amino acids (54 kDa) and behaves as a monomer in solution—although it displays an unusual electrophoretic behavior. FtsY consists of three domains: an acidic N-terminal domain, an N domain (named in analogy to the N domain in Ffh and SRP54) and a C-terminal G domain, which contains the consensus elements for GTP binding. Together with SRP54, Ffh, the SRa Abbreviations: SRP, signal recognition particle; Ffh, fifty-four homologe; DTT, dithiothreitol; IPTG, isopropyl-thio-bd-galactopyranoside; GDP, guanosine diphosphate; GMP-PNP, guanylylimidodiphosphate; MAD, multiple wavelength anomalous diffraction; Se-Met, selenomethionine; GTP, guanosine triphosphate *Correspondence to: Irmgard Sinning, EMBL, Meyerhofstr. 1, D-69117. Heidelberg, Gemany. Received 20 September 1996; Accepted 11 November 1996 286 G. MONTOYA ET AL. and SRb proteins, FtsY forms a distinct family of GTPases.15 So far, no high-resolution structural information is available for any member of the SRP family of GTPases. In this report we describe the expression and crystallization of the full-length protein and of the NG domain of FtsY. MATERIALS AND METHODS Expression and Purification Expression and purification of recombinant FtsY have been performed in principle as described earlier.11 Some modifications to improve the expression and purification of the protein have been included, for example, the application of a molecular sieve column after the ionic exchange step. Besides the full-length protein, the NG fragment of FtsY containing the residues 197 to 497 and 6 histidine residues at the C terminus was expressed in E. coli, strain BL21(DE3)pLys S (Novagen), by using the pET9 plasmid. Cells were transformed by electroporation, and colonies were selected by double resistance against kanamycin (30 µg/ml) and chloramphenicol (34 µg/ml). Cells were grown in LB medium supplemented with 0.4% glucose plus kanamycin and chloramphenicol. Cells were induced with 0.4 mM IPTG at OD600 5 0.3. After 2 hours the cells were collected by centrifugation and stored at 280°C until use. The cells were disrupted by two passes through a french press in 50 mM Tris-HCl pH 7.4, 300 mM NaCl, 10 mM MgCl2. Unlysed cells were removed by centrifugation and 1 mM GDP was added to the supernatant. The sample was loaded on a nickel column (Chelating Sepharose Fast Flow, Pharmacia) equilibrated with 50 mM Tris-HCl pH 7.4, 300 mM NaCl, and 10 mM MgCl2. The column was first washed with a gradient from 0 to 100 mM imidazol and then a gradient from 100 to 800 mM imidazol was applied. The protein eluted as a single peak around 300 mM imidazol. The sample was concentrated to 8 mg/ml, and then subjected to dialysis against 50 mM TrisHCl pH 8.0, 200 mM (NH4)2SO4, 0.2 mM ZnCl2, 5mM MgCl2, 5 mM DTT for 1 hour. GDP was exchanged by GMP-PNP following the procedure described by John et al.16 The GMP-PNP exchanged sample was then subjected to gel filtration chromatography using a Superose 12 column (Pharmacia) with a mobile phase of 10 mM Tris-HCl pH 7.4, 100 mM NaCl, and 10 mM MgCl2. The protein eluted as a single peak followed by the excess of GMP-PNP. The nucleotide content of the protein before and after exchange was monitored by HPLC analysis.16 Seleno-methionine (Se-Met) containing NG fragment was produced using the E. coli methionine auxotroph strain B834 (DE3). Cells were transformed by electroporation, and colonies were selected by using kanamycin. The procedure was similar to the selection procedure described above. A preculture was grown overnight at 37°C in minimal medium plus methionine (40 mg/ml) and kanamycin. The preculture was inoculated in 1l minimal medium plus 40 mg/ml methionine and kanamycin and incubated at 37°C until it reached 0.8 OD at 600 nm. The cells were collected by centrifugation, and the pellet was dissolved in 1 l minimal medium plus kanamycin but without methionine. The culture was incubated overnight at 30°C. In the morning Se-Met was added (40 mg/ml), and the culture was incubated for 1 hour at 37°C and then induced with 0.4 mM IPTG. After 4 hours, the cells were collected and stored at 280°C until use. The purification and crystallization procedure for the Se-Met-containing protein was the same used for the native protein. The purity of the full-length protein, the NG domain, and the Se-Met-NG domain were checked by SDSPAGE and mass spectrometry, and was higher than 98%. Crystallization The full-length protein and the NG fragment of FtsY were concentrated to 5 and 10 mg/ml, and crystallization experiments were performed by using the hanging drop vapor diffusion method at 20 and 4°C using Linbro 24-well culture plates. The NG domain was crystallized with the his tag present at the C terminus. Crystals were obtained for both protein samples at 4°C. Crystals of the full-length protein were obtained in 100 mM NaCitrate, 50 mM bis-Tris pH 6.5 and 15% isopropanol. The crystals of the NG domain grew in 50 mM bis-Tris pH 6.0, 200 mM Li2SO4,and 18% PEG 8000 at 4°C within 1 week to a maximum size of around 0.3 3 0.4 3 3.0 mm. These crystals were suitable for x-ray diffraction. Data Collection and Reduction Initially, inspection of the crystals of the fulllength protein, the NG domain, as well as heavy metal soaks were done at room temperature on an MAR research image plate on a rotating anode. For data collection, crystals of the NG domain were mounted in loops and flash-frozen with liquid nitrogen to 100 K with an Oxford Cryosystems cryostream. Before freezing the crystals were soaked in a cryobuffer containing 50 mM bis-Tris pH 6.0, 18% PEG 8000, and 10% PEG 400, for 2 minutes. Native data were collected using BL-19 at the European Synchrotron Radiation Facility (ESRF) in Grenoble. Data were collected with a detector-to-crystal distance of 14 cm (180-mm diameter plate, l 5 1.0 Å) in 1-in. frames and with a 2-minute exposure time. All data have been integrated with Denzo and Scalepack. 17 Results and Discussion Using the expression protocol described above, full-length FtsY and the NG domain can routinely be produced in E. coli with yields ranging from 10 to 30 mg/l. The Se-Met containing NG domain is slightly less well expressed and yields about 7 mg/l. The 287 DOCKING PROTEIN FTSY OF E. COLI TABLE I. Data Statistics Total observations/ unique Complete- Rsym† dmin* reflections ness (%) (Å) (%) Crystal NG-FtsY in house NG-FtsY BL19 Se-NG-FtsY BL19 Fig. 1. a: Needle-like crystals of the full-length FtsY protein. b: Monoclinic crystals of the NG-domain of FtsY (with C-terminal his tag). The approximate size is 0.2 3 0.2 3 0.3 mm. incorporation of Se-Met was tested by mass spectrometry showing that all six methionines have been replaced by Se-Met. The exchange of GDP versus GMP-PNP seems to be crucial for obtaining well ordered crystals—even when the nucleotide is no longer present in the protein used for crystallization, as determined by HPLC analysis. Without following the exchange protocol or using cocrystallization, only small bundles of needles were obtained. So far, we have no explanation for this puzzling observation. One possibility is that an impurity is removed by the additional gel filtration step after the nucleotide exchange. However, including this step in the purification without following the exchange protocol did not have the same effect. It seems more likely that the exchange is needed to get the protein in one conformation. Needle-like crystals were obtained for the fulllength protein (Fig. 1a), but the size was not sufficient to allow crystallographic experiments. Further attempts to improve the quality of these crystals were unsuccessful so far. Most likely, growth in the third dimension is hampered by the acidic Nterminal domain. The existence of a stable proteolytic fragment of FtsY containing only the NG domain18 suggested the use of this fragment for crystallographic studies. Monoclinic crystals of the NG domain were obtained, see Figure 1b. The crystals belong to the space group P21 with unit cell dimensions a 5 32.2, b 5 79.57, c 5 59.21, and b angle 5 94.45. Assuming one protein molecule per asymmetric unit, the crystal volume per protein mass (Vm) is 2.3 Å3/Da, corresponding to a solvent content of 37%. This is within the range commonly observed for protein crystals.19 The crystals diffract beyond 3 Å at room temperature, but radiation damage decreased the diffracton limit to 4 Å after 7 to 9 hours. Cryocooling effectively eliminated radiation damage and also increased the resolution to about 2.5 Å. Cryocooling caused changes in the b angle and in the b axis of the crystals. A native dataset at 110 K was collected by using synchrotron radiation at the ESRF in Grenoble 2.5 1.8 52959/10181 190204/24107 97.2 88.2 3.0 6.9 2.7 45999/7468 89.1 9.0 *Minimum Bragg spacing with (I )/s at least 2.5. †R sym 5 S 0 Iobs 2 Iavg 0 /S Iavg . BL-19. Synchrotron radiation increased the averaged resolution to beyond 1.8 Å. For the statistics of the different native data sets see Table I. Heavy metal soaks are in progress—so far without success due to problems caused by nonisomorphism of the soaked crystals with the native ones. Since the NG domain contains six methionine residues it seems possible to use the MAD approach for solving the structure.20 Crystals of the Se-Met-containing NG domain have been obtained following the same protocol as for the ‘‘native’’ protein. The Se-Met crystals are isomorphous with the ‘‘native’’ crystals and diffract to about 2.8 Å on a rotating anode (Table I). 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