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• W2155293003 abstract "Erwinia carotovora are phytopathogenic Gram-negative bacteria of agronomic interest as these bacteria are responsible for fruit soft rot and use insects as dissemination vectors. The Erwinia carotovora carotovora strain 15 (Ecc15) is capable of persisting in the Drosophila gut by the sole action of one protein, Erwinia virulence factor (Evf). However, the precise function of Evf is elusive, and its sequence does not provide any indication as to its biochemical function. We have solved the 2.0-Å crystal structure of Evf and found a protein with a complex topology and a novel fold. The structure of Evf confirms that Evf is unlike any virulence factors known to date. Most remarkably, we identified palmitoic acid covalently bound to the totally conserved Cys209, which provides important clues as to the function of Evf. Mutation of the palmitoic binding cysteine leads to a loss of virulence, proving that palmitoylation is at the heart of Evf infectivity and may be a membrane anchoring signal. Fluorescence studies of the sole tryptophan residue (Trp94) demonstrated that Evf was indeed able to bind to model membranes containing negatively charged phospholipids and to promote their aggregation. Erwinia carotovora are phytopathogenic Gram-negative bacteria of agronomic interest as these bacteria are responsible for fruit soft rot and use insects as dissemination vectors. The Erwinia carotovora carotovora strain 15 (Ecc15) is capable of persisting in the Drosophila gut by the sole action of one protein, Erwinia virulence factor (Evf). However, the precise function of Evf is elusive, and its sequence does not provide any indication as to its biochemical function. We have solved the 2.0-Å crystal structure of Evf and found a protein with a complex topology and a novel fold. The structure of Evf confirms that Evf is unlike any virulence factors known to date. Most remarkably, we identified palmitoic acid covalently bound to the totally conserved Cys209, which provides important clues as to the function of Evf. Mutation of the palmitoic binding cysteine leads to a loss of virulence, proving that palmitoylation is at the heart of Evf infectivity and may be a membrane anchoring signal. Fluorescence studies of the sole tryptophan residue (Trp94) demonstrated that Evf was indeed able to bind to model membranes containing negatively charged phospholipids and to promote their aggregation. Flies have been recurrently thought to be involved in the transmission of animal and plant diseases; they live on decaying medium enriched in micro-organisms, and they constitute ideal vectors for microbial dissemination through food contamination because of their close association with animals or plant communities. Although the potential hazard of flies on humans is generally accepted, the interactions between bacteria and potential fly vectors are poorly documented. Unlike the situation observed for plague bacteria and fleas (1Hinnebusch B.J. Rudolph A.E. Cherepanov P. Dixon J.E. Schwan T.G. Forsberg A. Science.. 2002; 296: 733-735Google Scholar) or for many protozoan parasites infecting humans and mosquitoes (2Beerntsen B.T. James A.A. Christensen B.M. Microbiol. Mol. Biol. Rev.. 2000; 64: 115-137Google Scholar), in which the insect is an obligate host, little is known about the mechanisms that allow bacterial persistence within flies (3Vallet-Gely I. Lemaitre B. Boccard F. Nat. Rev. Microbiol.. 2008; 6: 302-313Google Scholar). The identification of one gene, the Erwinia virulence factor (Evf) 3The abbreviations used are: Evf, Erwinia virulence factor; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; ESI-MS, electrospray ionization-mass spectrometry; LC-MS/MS, liquid chromatographytandem mass spectrometry; PC, phosphatidylcholine; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; DTT, dithiothreitol; SUV, small unilamellar vesicle; Bicine, N,N-bis(2-hydroxyethyl)glycine; r.m.s.d., root mean square deviation. that promotes bacterial persistence in the gut of Drosophila larvae, has recently revealed the existence of specific strategies that may enhance survival of bacteria within insects and their dissemination in their environment. The fruit fly Drosophila melanogaster lives in decaying fruits and has been occasionally implicated in the transmission of plant phytopathogens such as the enteric bacteria Erwinia carotovora (4Molina J.J. Harisson M.D. Brewer J.W. Am. Pot. J.. 1974; 51: 245-250Google Scholar, 5Kloepper J.W. Brewer J.W. Harrison M.D. Am. Pot. J.. 1981; 58: 165-175Google Scholar). E. carotovora species induce the soft rotting of many fruits and potatoes through the production of a battery of pectinolytic enzymes (6Barras F. van Gijsegem F. Chatterjee A.K. Annu. Rev. Phytopathol.. 1994; 32: 201-234Google Scholar). A strain of E. carotovora carotovora, Ecc15, was identified that, contrary to most bacterial species, can persist in the gut of Drosophila larvae after natural ingestion (7Basset A. Khush R.S. Braun A. Gardan L. Boccard F. Hoffmann J.A. Lemaitre B. Proc. Natl. Acad. Sci. U. S. A.. 2000; 97: 3376-3381Google Scholar). This infection relies on a very specific bacterium host interaction because only a very limited number of strains is capable of inducing this response. A genetic screen set up to search for infective genes revealed that infectivity is mediated by a single gene, Erwinia virulence factor (Evf) (8Basset A. Tzou P. Lemaitre B. Boccard F. EMBO Rep.. 2003; 4: 205-209Google Scholar). The expression of Evf is regulated by Hor, a member of the family of transcriptional regulators of the SlyA type that are usually involved in the regulation of virulence genes (9Ellison D.W. Miller V.L. Curr. Opin. Microbiol.. 2006; 9: 153-159Google Scholar). Evf is only present in a subset of Erwinia strains that have infectious properties and was characterized as a gene that promotes survival and dissemination (10Acosta Muniz C. Jaillard D. Lemaitre B. Boccard F. Cell. Microbiol.. 2007; 9: 106-119Google Scholar). Strikingly, transfer of Evf into noninfectious, and normally nonpersistent, Gram-negative bacteria enables them to persist in the Drosophila gut and to elicit a systemic immune response. The biochemical function of the Evf protein and the exact mechanism by which it promotes bacterial persistence in the gut remain unknown. However, it was shown that it does not function using previously well known mechanisms of bacterial survival. It does not act by directly counteracting the host immune response; it is not a toxin, and it does not provide protection against antibacterial peptides or against reactive oxygen species. The presence of Evf leads to the accumulation of bacteria in the anterior midgut and dramatically affects gut physiology (10Acosta Muniz C. Jaillard D. Lemaitre B. Boccard F. Cell. Microbiol.. 2007; 9: 106-119Google Scholar). Evf therefore reveals a new aspect of bacterial strategies for colonizing an insect gut niche. Interestingly, Ecc15 accumulated in the infected gut in a nonrandom arrangement reminiscent of biofilms, which might allow the bacteria to interfere with gut peristalsis and therefore prevent elimination by passive transfer through the gut. Biofilm formation in the gut preventing normal blood feeding is also seen in the infection of the flea vector by Yersinia pestis (11Hinnebusch B.J. Perry R.D. Schwan T.G. Science.. 1996; 273: 367-370Google Scholar). Evf has few orthologues in other bacteria and no identified functional sequence signatures. Interestingly, the closest homologue is a gene from Photorhabdus luminescens, a bacterium that exists in symbiosis in the guts of nematodes (12Sicard M. Hering S. Schulte R. Gaudriault S. Schulenburg H. Environ. Microbiol.. 2007; 9: 12-25Google Scholar) suggesting that Evf-like proteins are also virulence factors that promote specific interaction with the host gut allowing persistence. To better understand the function of the Evf protein at the molecular level, we have determined its crystal structure. We show that Evf is an α/β protein with a complex topology and a novel fold. Most remarkably, the crystal structure and mass spectrometry data clearly demonstrate that a palmitoic acid is covalently linked to the 209 cysteine residue via a thioester linkage. To assess whether the association of Evf with palmitoic acid is necessary for infection, mutations of this cysteine residue into alanine or serine were tested for infectivity. All mutants lacking the palmitoic acid-bound residue did not give rise to an immune reaction. We also showed that Evf interacted with model membranes and was able to promote their aggregation. We postulate that the palmitoyl moiety might play a role in these membrane-associated processes. Cloning, Expression, Purification, and Labeling—Evf was amplified by PCR using the plasmid pOM1-evf as a template (7Basset A. Khush R.S. Braun A. Gardan L. Boccard F. Hoffmann J.A. Lemaitre B. Proc. Natl. Acad. Sci. U. S. A.. 2000; 97: 3376-3381Google Scholar). An additional sequence coding for a 6-histidine tag was introduced at the 3′ end of the gene during amplification. The PCR product was then cloned into a derivative of the pET9 vector. Expression was done at 37 °C during 3 h using the transformed Escherichia coli BL21-Gold(DE3) strain (Novagen) and 2× YT medium (Bio 101, Inc., Vista, CA). When the cell culture reached an A600 nm of 1, protein expression was induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside (Sigma), and the cells were grown for a further 4 h. Cells were harvested by centrifugation, resuspended in 40 ml of 20 mm Tris-HCl, pH 7.5, 200 mm NaCl, 5 mm β-mercaptoethanol, and stored overnight at –20 °C. Cell lysis was completed by sonication. The His-tagged protein was purified on a nickel-nitrilotriacetic acid column (Qiagen), eluted with imidazole, and loaded on to a Superdex™ 200 column (Amersham Biosciences), equilibrated against 20 mm Tris-HCl, pH 7.5, 200 mm NaCl, 10 mm β-mercaptoethanol. Selenomethionine-labeled protein was prepared as described and purified as the native protein (13Quevillon-Cheruel S. Collinet B. Tresaugues L. Minard P. Henckes G. Aufrere R. Blondeau K. Zhou C.Z. Liger D. Bettache N. Poupon A. Aboulfath I. Leulliot N. Janin J. van Tilbeurgh H. Methods Mol. Biol.. 2007; 363: 21-37Google Scholar). The homogeneity and selenomethionine labeling of the proteins were checked by SDS-PAGE mass spectrometry. The covalent palmitoylation of Cys209, as suggested by analysis of the crystal structure, was verified by mass spectrometry. All spectra were acquired in positive-ion mode on a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a 337-nm nitrogen laser. Determinations of the molecular masses of Evf proteins were performed in linear mode (accelerating voltage, 25 kV; grid voltage, 93%; guide wire, 0.3%; delay, 600 ns) with external calibration using sinapinic acid as matrix. Crystallization, Data Collection, Structure Solution, and Refinement—Crystals of both the native and selenomethionine-labeled protein were grown from a 1:1 μl mixture of protein (10 mg/ml) with 25% PEG4000, 50 mm Bicine, pH 8, 0.1 mg/ml trypsin. The addition of trypsin resulted in in situ limited proteolysis and was necessary for crystal formation. X-ray diffraction data from a crystal of the selenomethionine-substituted protein were collected on beamline ID23-1 (European Synchrotron Radiation Facility) at a single wavelength of 0.9798 Å, corresponding to the peak position of the selenium K-edge to optimize the anomalous scattering signal strength. The crystals belong to space group P212121 with two Evf copies per asymmetric unit, corresponding to 40% solvent content. Data processing was carried out with the programs MOSFLM (14Leslie A.G.W. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography, No. 26. Daresbury Laboratory, Warrington, UK1992Google Scholar) and SCALA (15Collaborative Computational Project Number Acta Crystallogr.Sect. D Biol. Crystallogr. 1994; 50 (4): 760-763Crossref PubMed Scopus (19797) Google Scholar). The cell parameters and data collection statistics are reported in Table 1. A total of 720 frames of 1° rotation were collected on a single crystal with an attenuated beam, with no evidence of overall radiation damage. Because of the high redundancy, the two selenium atoms were readily localized using the Hyss module of PHENIX (16Grosse-Kunstleve R.W. Adams P.D. Acta Crystallogr. Sect. D Biol. Crystallogr.. 2003; 59: 1966-1973Google Scholar). Phasing was performed using Sharp (17Bricogne G. Vonrhein C. Flensburg C. Schiltz M. Paciorek W. Acta Crystallogr. Sect. D Biol. Crystallogr.. 2003; 59: 2023-2030Google Scholar). A high resolution dataset was recorded on the same crystal without any attenuation. The program SOLOMON (18Abrahams J.P. De Graaff R.A. Curr. Opin. Struct. Biol.. 1998; 8: 601-605Google Scholar) was used for density modification using the phases from Sharp, and the resulting phases were transferred into the high resolution dataset. A combination of Arp/warp (19Perrakis A. Nat. Struct. Biol.. 1999; 6: 458-463Google Scholar) and Resolve (20Terwilliger T. J. Synchrotron Radiat.. 2004; 11: 49-52Google Scholar) was able to build 90% of the structure. Further rebuilding and refinement of the model were performed with O (21Kleywegt G.J. Jones T.A. Acta Crystallogr. Sect. D Biol. Crystallogr.. 1996; 52: 829-832Google Scholar) and refined with REFMAC (22Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr.. 1997; 53: 240-255Google Scholar).TABLE 1Data collection and refinement statisticsProteinEvf selenomethionineSpace groupP212121Unit cell parameters a, b, c69.5 Å, 86.7 Å, 91.4 ÅResolution80.0-2.0 ÅTotal no. of reflections1,056,433Total no. of unique reflections38,113Multiplicity27.7RmergeaRmerge = ΣhΣi|Ihi — 〈Ih〉|/ΣhΣiIhi, where Ihi is the ith observation of the reflection h, and 〈Ih〉 is the mean intensity of reflection h.9.6I/σ(I)25.9Overall completeness (%)100R/Rfree (%)20.1/24.9R.m.s.d. bonds0.011 ÅR.m.s.d. angles1.181°Ramachandran plotDisallowed0.4Most favored5.5Allowed94.1a Rmerge = ΣhΣi|Ihi — 〈Ih〉|/ΣhΣiIhi, where Ihi is the ith observation of the reflection h, and 〈Ih〉 is the mean intensity of reflection h. Open table in a new tab Sample Preparation and Mass Spectrometry Measurements—For ESI-MS measurements, 20 μl of purified Evf protein (4.5 μg/μl) was desalted with size exclusion chromatography columns (Micro Bio-Spin 6 chromatography columns, Bio-Rad), following the manufacturer's recommendations, and eluted in water. Ten μl of desalted Evf protein was mixed with the same volume of acetonitrile (100%) and DTT (10 mm). The reaction was left to proceed for 20 min at 56 °C and then 1% of formic acid was added. Desalted protein samples were loaded into nanoelectrospray needles (Proxeon Biosystems A/S, catalogue number ES 388). Positive ion ESI mass spectra were acquired using a QTOF Premier mass spectrometer (Waters). The instrument was mass-calibrated by spraying a solution of sodium iodide (2 μg/μl). The ion source parameters were as follows: capillary voltage 1.4 kV, cone voltage 40 V, ion source block temperature 80 °C, nebulizer gas flow 25 liters/h. The mass spectra were analyzed using the MassLynx software (Waters) and deconvoluted using the MaxEnt 1 algorithm. For nanoLC-MS/MS analyses, 1 μl of purified Evf protein (4.5 μg/μl) was diluted in 10 μl of 20% acetonitrile, 80% 50 mm NH4HCO3 and enzymatically digested with bovine trypsin or Pseudomonas fragi Asp-N (both purchased from Roche Applied Science) with an enzyme/protein ratio of 1/45 (w/w). The digestion was left to proceed for 4 h at 37 °C. When reduction/alkylation reactions were performed, DTT and iodoacetamide were added after protein dilution at a final concentration of 5 and 18 mm, respectively. DTT was first added for 20 min, and then iodoacetamide was added and left to react for 30 min in the dark at room temperature. Peptide mixtures were Speed-Vac-treated for 10 min and then analyzed with the QTOF Premier mass spectrometer coupled to the Acquity nano-UPLC equipped with a trapping column (Symmetry C18, 180 μm × 20 mm, 5-μm particle size) and an analytical column (BEH130 C18, 75 μm × 100 mm, 1.7-μm particle size) (Waters). The aqueous solvent (buffer A) was 0.1% formic acid in water, and the organic phase (buffer B) was 0.1% formic acid in acetonitrile. A 2–40% B gradient was set for 25 min. Mass spectrometer parameters were as described previously for ESI-MS measurements except for capillary voltage (3 kV) and collision energy (ramping from 15 to 40 eV). For exact mass measurements, Glu-fibrinopeptide reference (m/z = 785.8426) was continuously supplied during nanoLC-MS/MS experiments using the lockspray device. Peptide mass measurements were finally corrected by ProteinLynx Global Server, (Waters) during data processing. Peak lists were generated by ProteinLynx Global Server, and processed data were submitted to searching using the following parameters: data bank His-tagged Evf protein; peptide tolerance 15 ppm; fragment tolerance 0.1 Da; digest reagent trypsin or Asp-N; variable modifications oxidation (methionine) and palmitoylation (cysteine, Serine); fixed modifications carbamido-methylation (cysteine). For MALDI-MS analyses, peptide extracts (0.5 μl) were mixed with an equal volume of 2,5-dihydroxybenzoic acid (10 mg/ml) or α-cyano-4-hydroxycinnamic acid (10 mg/ml) matrix compounds (Sigma). MALDI-MS measurements were carried out on a Voyager-DE STR MALDI-TOF mass spectrometer (Applied Biosystems) in the positive ion reflectron mode. Peak lists were generated by the Data Explorer software (Applied Biosystems), and processed data were submitted to the Find-Mod and Findpep software using the already described parameters except for tolerance at 50 ppm. Steady-state Fluorescence Measurements—Fluorescence emission spectra were recorded with a home-modified SLM spectrofluorimeter with slit width of 4 nm for excitation and 8 nm for emission. Excitation was vertically polarized, and the emission was detected through a polarizer oriented at the magic angle 55°. Measurements were performed in 100-μl microcuvettes. Time-resolved Fluorescence Measurements—Fluorescence intensity and anisotropy decays were obtained from the polarized Ivv(t) and Ivh(t) components measured by the time-correlated single-photon counting technique. A light-emitting diode (PLS 295, serial number PLS-8-2-237 from Picoquant, Berlin-Adlershof, Germany) (maximal emission at 298 nm) operating at 10 MHz was used as excitation source. A short pass Asahi Spectra UV filter ZUS300 was used in front of the LED. A Hamamatsu fast photomultiplier (model R3235-01) was used for detection. Emission wavelength was selected through a Omega 320AELP cutoff filter and a Oriel bandpass filter UG5-005FG09-50S. Fluorescence intensity decay analyses were performed with the maximum entropy method (23Brochon J.C. Methods Enzymol.. 1994; 240: 262-311Google Scholar), using a multiexponential model, Σi αi exp(–t/τi), as described previously. A classical anisotropy model, A(t) = Σi βi exp(–t/θi), in which any rotational correlation time (θ) is coupled with each lifetime (τ), was used to resolve polarized fluorescence decays (24Vincent M. Gallay J. Eur. Biophys. J.. 1991; 20: 183-191Google Scholar). The amplitude of the internal rotation, ωmax, was calculated according to the wobbling-in-cone model (25Kinosita Jr., K. Kawato S. Ikegami A. Biophys. J.. 1977; 20: 289-305Google Scholar). Preparation of Sonicated Phospholipid Vesicles—The phospholipid suspensions were prepared by sonication using egg PC and POPS (both from Avanti Polar Lipids, Alabaster, AL) dissolved in chloroform. The organic solution was evaporated to dryness in a glass tube under a stream of nitrogen. Remaining traces of organic solvent were further removed by submitting the sample to high vacuum during several hours. Hydration of the sample was achieved with either 20 mm Hepes buffer, pH 7.2, or 100 mm cacodylate buffer, pH 5. After vortexing, the multilamellar vesicles formed were sonicated at room temperature with the micro-tip of a Branson-B12 sonifier during 5 min with half-duty cycles. Drosophila Strain and Natural Bacterial Infection—A transgenic strain carrying a diptericin-lacZ was used to monitor the immune response of larvae. Diptericin-lacZ is a P transgene containing a fusion between 2.2 kb of upstream sequence of the antibacterial peptide gene diptericin and the β-galactosidase gene (26Tzou P. Ohresser S. Ferrandon D. Capovilla M. Reichhart J.M. Lemaitre B. Hoffmann J.A. Imler J.L. Immunity.. 2000; 13: 737-748Google Scholar). Approximately 200 third-instar larvae were placed in a 2-ml tube containing 200 μl of concentrated bacteria pellet (A600 = 200) from an overnight culture and 400 μl of crushed banana. The larvae, bacteria, and banana were thoroughly mixed in the microcentrifuge tube; the tube was closed with a foam plug, incubated at room temperature for 30 min, and the mixtures then transferred to a standard cornmeal fly medium and incubated at 29 °C. LacZ titration was performed as described (27Romeo Y. Lemaitre B. Methods Mol. Biol.. 2008; 415: 379-402Google Scholar). Bacterial Strains—The strains and plasmids used in this study are listed in Table 2. Bacteria were cultured in Lennox medium with the appropriate antibiotics (100 μg/ml rifampicin; 100 μg/ml spectinomycin). The rifampicin-resistant Ecc15 and Ecc15 derivatives were grown at 29 °C. E. coli strains were grown at 37 °C.TABLE 2Bacterial strains and plasmidsStrains and plasmidsDescriptionSource or Ref.Bacterial strainsE. carotovora 15 (Ecc15)Wild type8Basset A. Tzou P. Lemaitre B. Boccard F. EMBO Rep.. 2003; 4: 205-209Google ScholarEcc15 evfEvf:Tn10 (KanR)8Basset A. Tzou P. Lemaitre B. Boccard F. EMBO Rep.. 2003; 4: 205-209Google ScholarE. coli K12 MG1655Wild typeLab collectionPlasmidspOM1Cloning vector pSC101 derivative (SpcR)49Espeli O. Moulin L. Boccard F. J. Mol. Biol.. 2001; 314: 375-386Google ScholarpOM1-evfpOM1 expressing evf8Basset A. Tzou P. Lemaitre B. Boccard F. EMBO Rep.. 2003; 4: 205-209Google ScholarpOM1-evf (C209S)pOM1 expressing evf (C209S)This studypOM1-evf (C209A)pOM1 expressing evf (C209A)This study Open table in a new tab Constructions of Evf Derivatives EvfC209S and EvfC209A—The KpnI-HindIII fragment of plasmid pOM1-evf was replaced by KpnI-HindIII PCR fragments generated by using as template pOM1-evf and as primers one oligonucleotide overlapping the KpnI site and converting Cys209 into Ser209 (oligonucleotide S1, 5′-ATCGACGGTACCCATTTCAAGCAGGAGCAAAAGAAGATCGCTACCTCTTCTGCTTCATATCAAGAAGTAA-3′) or into Ala209 (oligonucleotide A1, 5′-ATCGACGGTACCCATTTCAAGCAGGAGCAAAAGAAGATCGCTACCTCTGCTGCTTCATATCAAGAAGTAA-3′) and one oligonucleotide overlapping the HindIII site. Structure Determination and Quality—The full-length Evf protein failed to crystallize. To help crystallization, we have screened crystallization conditions of the Evf protein in presence of trypsin protease. This may enable unstructured regions to be proteolyzed, thereby providing a truncated construct more suitable for crystal formation. This is an often used strategy for obtaining or improving crystals, but usually the truncated construct is further purified to remove the protease. We opted for a different strategy that consists of directly adding the protease to the crystallization drops. In our strategy, the nature and concentration of the protease are used as variable parameters in the crystallization screen. The idea is to provoke proteolysis directly under physicochemical conditions where crystals of the truncated product can nucleate. This strategy has been applied successfully in a number of projects in our laboratory (28Leulliot N. Godin K.S. Hoareau-Aveilla C. Quevillon-Cheruel S. Varani G. Henry Y. van Tilbeurgh H. J. Mol. Biol.. 2007; 371: 1338-1353Google Scholar, 29Leulliot N. Chaillet M. Durand D. Ulryck N. Blondeau K. van Tilbeurgh H. Structure (Lond.).. 2008; 16: 52-61Google Scholar). Once a crystal hit was found, all the subsequent optimizations were performed with the protease present in the crystallization drop. The effect of the protease on crystallogenesis is discussed further. The structure of Evf was solved by single anomalous dispersion using selenomethionine-labeled protein. The presence of only two methionines (excluding the N-terminal methionine) for a total of 270 residues made the detection of the selenium sites challenging. Fortunately, the weakness of the anomalous signal was compensated by the high resistance of the crystals in the x-ray beam, which enabled a highly redundant dataset to be collected. The final model contains residues 8–19 and 24–277 for one chain and residues 10–19 and 27–277 for the other. The two copies of Evf have identical structures (r.m.s.d. 0.38 Å). Refinement statistics are gathered in Table 1 and show good stereochemistry for the final model. Evf Structure Reveals a Novel Fold—The Evf protein adopts a globular fold with approximate dimensions 60 × 50 × 25 Å. The Evf protein contains two 310 helices (H1 and H2), six α-helices (α1 to α6), and 10 β-stands (β1 to β10) with a fairly complex topology (Fig. 1A, generated using Tops (30Westhead D.R. Slidel T.W. Flores T.P. Thornton J.M. Protein Sci.. 1999; 8: 897-904Google Scholar)). A search in the EBI-SSM server found no proteins with a similar fold in the Protein Data Bank. The Evf protein structure is therefore the first representative of a novel protein fold family. The Evf fold can be described as a central mixed β-sheet sandwiched between two α-helical layers (Fig. 1, B and C). The six-stranded β-sheet contains four antiparallel strands (β6β7β8β3/β4) which is completed after β6 by strand β2 and β1 from the N terminus of the protein and β9 from the C terminus as follows: β2 follows β6 and the β1 and β9 strands both form the last “strand” of the β-sheet, respectively, anti-parallel and parallel to β2. The β-sheet is highly curved and forms a half-barrel that cradles one of the α-helices (α4), a structural trait reminiscent of the fold seen in proteins of the hot-dog and related families (31Leulliot N. Quevillon-Cheruel S. Sorel I. Graille M. Meyer P. Liger D. Blondeau K. Janin J. van Tilbeurgh H. J. Biol. Chem.. 2004; 279: 23447-23452Google Scholar, 32Liger D. Quevillon-Cheruel S. Sorel I. Bremang M. Blondeau K. Aboulfath I. Janin J. van Tilbeurgh H. Leulliot N. Proteins.. 2005; 60: 778-786Google Scholar). The helical domain packing on the exterior convex side of the β-sheet is composed of the α1 at the N terminus, the α2, H1 and α3 helices (inserted between β2 and β3), and the α6 and α7 helices (inserted between β8 and β9). The α2 and α3 helices pack on the β-sheet roughly parallel to the β-strands, whereas α1 and α7 pack on the α2 and α3 helices at an ∼20° angle. Although this helical cap seems to form an independent structural domain, it contains elements from the N and C terminus of the protein. The second α-helical layer on the concave side of the β-sheet is composed of the α4 and α5 helices (inserted between β4 and β6) and the H2 310 helix (inserted between β7 and β8). The α4 helix runs inside the half-β-barrel, whereas α5 and H2 are perpendicular to the barrel axis and form a “lid” over the side of the barrel. In Situ Proteolysis during Crystallogenesis—As the protein only crystallized in the presence of trypsin, we set out to identify the crystallized Evf fragment. Mass spectrometry and Edman sequencing of dissolved crystals proved that Evf was cleaved at Arg23. Surprisingly, the electron density maps clearly showed that the peptide between residues 8 and 19 was present and well defined in the crystal structure forming helix α1 and flanking residues. The seven N-terminal residues up to the beginning of helix α1 and the three residues after α1 (Pro20–Gly22) in the α1-β1 loop were not visible in the final electron density map and were therefore not modeled. Trypsin digestion led to cleavage of the polypeptide chain at Arg23, and the residues from the N terminus and from the cut α1-β1 loop are probably disordered. It therefore appears that digestion of the protein by trypsin did not help crystallization by providing a shorter construct but simply by cutting inside the α1-β1 loop. Contrary to the residues 30–36 before β1, which pack against the side of the protein and have B-factors comparable with the rest of the protein residues, residues 23–29 protrude more into the solvent and have higher B-factors suggesting that residues 20–29 form a mobile loop. In our structure, the distance between Gly19 and Arg23 is compatible with the modeling of the three missing residues, but the cut most probably relaxes the structure of the loop away from a conformation that hampers crystallization. Inspection of the crystal packing indeed shows that this region is involved in intermolecular contacts, which could have been hindered with the intact protein. Addition of the protease therefore acts as a surface engineering step rather that removing unstructured regions. Evf Is S-Palmitoylated on Cys209—During refinement, clear residual electron density was detected indicating the presence of a long chain ligand covalently linked to Cys209 (Fig. 2). The hydrophobic nature of the surrounding residues and the form of the electron density suggested that a fatty acid was bound. To gain insight into the chemical nature of the fatty acid, direct ESI-MS measurements of the desalted Evf protein with and without DTT treatment were performed. Note that DTT-induced chemical reduction was left to proceed under denaturing conditions (see “Materials and Methods”). The ESI-MS deconvoluted mass spectra are shown in Fig. 3A. Without DTT treatment, the Evf measured mass was 32,469.8 Da, whereas t" @default.
• W2155293003 created "2016-06-24" @default.
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• W2155293003 date "2009-02-01" @default.
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• W2155293003 title "Evf, a Virulence Factor Produced by the Drosophila Pathogen Erwinia carotovora, Is an S-Palmitoylated Protein with a New Fold That Binds to Lipid Vesicles" @default.
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