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• W2009804131 abstract "Shigella flexneri causes a self-limiting gastroenteritis in humans, characterized by severe localized inflammation and ulceration of the colonic mucosa. Shigellosis most often targets young children in underdeveloped countries. Invasion plasmid antigen C (IpaC) has been identified as the primary effector protein for Shigella invasion of epithelial cells. Although an initial model of IpaC function has been developed, no detailed structural information is available that could assist in a better understanding of the molecular basis for its interactions with the host cytoskeleton and phospholipid membrane. We have therefore initiated structural studies of IpaC, IpaC I′, (residues 101–363 deleted), and IpaC ΔH (residues 63–170 deleted). The secondary and tertiary structure of the protein was examined as a function of temperature, employing circular dichroism and high resolution derivative absorbance techniques. ANS (8-anilino-1-napthalene sulfonic acid) was used to probe the exposure of the hydrophobic surfaces under different conditions. The interaction of IpaC and these mutants with a liposome model (liposomes with entrapped fluorescein) was also examined. Domain III (residues 261–363) was studied using linker-scanning mutagenesis. It was shown that domain III contains periodic, sequence-dependent activity, suggesting helical structure in this section of the protein. In addition to these structural studies, investigation into the actin nucleation properties of IpaC was conducted, and actin nucleation by IpaC and some of the mutants was exhibited. Structure-function relationships of IpaC are discussed. Shigella flexneri causes a self-limiting gastroenteritis in humans, characterized by severe localized inflammation and ulceration of the colonic mucosa. Shigellosis most often targets young children in underdeveloped countries. Invasion plasmid antigen C (IpaC) has been identified as the primary effector protein for Shigella invasion of epithelial cells. Although an initial model of IpaC function has been developed, no detailed structural information is available that could assist in a better understanding of the molecular basis for its interactions with the host cytoskeleton and phospholipid membrane. We have therefore initiated structural studies of IpaC, IpaC I′, (residues 101–363 deleted), and IpaC ΔH (residues 63–170 deleted). The secondary and tertiary structure of the protein was examined as a function of temperature, employing circular dichroism and high resolution derivative absorbance techniques. ANS (8-anilino-1-napthalene sulfonic acid) was used to probe the exposure of the hydrophobic surfaces under different conditions. The interaction of IpaC and these mutants with a liposome model (liposomes with entrapped fluorescein) was also examined. Domain III (residues 261–363) was studied using linker-scanning mutagenesis. It was shown that domain III contains periodic, sequence-dependent activity, suggesting helical structure in this section of the protein. In addition to these structural studies, investigation into the actin nucleation properties of IpaC was conducted, and actin nucleation by IpaC and some of the mutants was exhibited. Structure-function relationships of IpaC are discussed. invasion plasmid antigens invasion plasmid antigen C invasion plasmid antigen D 8-anilino-1-napthalene sulfonic acid type III secretion system minimum Eagle's medium Salmonellainvasion protein C Shigella flexneri causes a self-limiting gastroenteritis called shigellosis, which is characterized by severe localized inflammation and ulceration of the colonic mucosa (1Hale T.L. Microbiol. Rev. 1991; 55: 206-224Google Scholar). An estimated 360,000 Shigella cases occur in the United States each year, although only 18,000 are reported (2Centers for Disease Control and Prevention (2000)Shigellosis, www.cdc.gov/ncidod/dbmd/diseaseinfo/shigellosis_g.htmGoogle Scholar). In developing countries, the disease is present in most villages. It most commonly strikes young children and is responsible for an estimated 600,000 deaths/year worldwide (all Shigella spp. combined) (1Hale T.L. Microbiol. Rev. 1991; 55: 206-224Google Scholar). Shigellosis onset involves bacterial invasion of intestinal epithelial cells by a process called “pathogen-induced phagocytosis” (2Centers for Disease Control and Prevention (2000)Shigellosis, www.cdc.gov/ncidod/dbmd/diseaseinfo/shigellosis_g.htmGoogle Scholar, 3Hueck C. Microbiol. Mol. Biol. 1998; 62: 379-433Google Scholar) and requires the expression of the ipa operon. The invasion plasmid antigens (or Ipa1proteins) are effector proteins that are exported by a dedicated type III secretion system (TTSS) at the host-pathogen interface. There they directly interact with the host cell to promote actin cytoskeleton rearrangements at the site of bacterial contact (2Centers for Disease Control and Prevention (2000)Shigellosis, www.cdc.gov/ncidod/dbmd/diseaseinfo/shigellosis_g.htmGoogle Scholar). These cytoskeletal changes give rise to filopodia, which mature into membrane ruffles that coalesce to trap the pathogen within a membrane-bound vacuole (2Centers for Disease Control and Prevention (2000)Shigellosis, www.cdc.gov/ncidod/dbmd/diseaseinfo/shigellosis_g.htmGoogle Scholar). The vacuole is then quickly lysed to provide the bacterium with access to the host cytoplasm, where it proliferates and is able to directly invade neighboring cells (4Sansonetti P.J. Ryter A. Clerc P. Maurelli A.T. Mounier J. Infect. Immun. 1986; 51: 461-469Google Scholar). IpaC has been identified as the primary effector protein forShigella invasion of epithelial cells (5Marquart M.E. Picking W.L. Picking W.D. Infect. Immun. 1996; 64: 4182-4187Google Scholar, 6Menard R. Prevost M.C. Gounon P. Sansonetti P. Dehio C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1254-1258Google Scholar, 7Tran Van Nhieu G. Caron E. Hall A. Sansonetti P.J. EMBO J. 1999; 18: 3249-3262Google Scholar, 8Davis R. Marquart M.E. Lucius D. Picking W.D. Biochim. Biophys. Acta. 1998; 1429: 45-56Google Scholar). Effector-related functions described for purified IpaC include: (a) enhanced invasion of cultured cells by S. flexneri (5Marquart M.E. Picking W.L. Picking W.D. Infect. Immun. 1996; 64: 4182-4187Google Scholar, 8Davis R. Marquart M.E. Lucius D. Picking W.D. Biochim. Biophys. Acta. 1998; 1429: 45-56Google Scholar, 9Tran N. Serfis A.B. Osiecki J.C. Picking W.L. Coye L. Davis R. Picking W.D. Infect. Immun. 2000; 68: 3710-3715Google Scholar); (b) induced uptake of virulence plasmid-cured S. flexneri (5Marquart M.E. Picking W.L. Picking W.D. Infect. Immun. 1996; 64: 4182-4187Google Scholar); (c) interaction with phospholipid membranes (9Tran N. Serfis A.B. Osiecki J.C. Picking W.L. Coye L. Davis R. Picking W.D. Infect. Immun. 2000; 68: 3710-3715Google Scholar, 10De Geyter C. Wattiez R. Sansonetti P. Falmagne P. Ruysschaert J.M. Parsot C. Cabiaux V. Eur. J. Biochem. 2000; 267: 5769-5776Google Scholar, 11De Geyter C. Vogt B. Benjelloun-Touimi Z. Sansonetti P.J. Ruysschaert J.M. Parsot C. Cabiaux V. FEBS Lett. 1997; 400: 149-154Google Scholar); and (d) triggering of cytoskeletal changes in cultured cells (7Tran Van Nhieu G. Caron E. Hall A. Sansonetti P.J. EMBO J. 1999; 18: 3249-3262Google Scholar, 9Tran N. Serfis A.B. Osiecki J.C. Picking W.L. Coye L. Davis R. Picking W.D. Infect. Immun. 2000; 68: 3710-3715Google Scholar, 12Kuwae A. Yoshida S. Tamano K. Mimuro H. Suzuki T. Sasakawa C. J. Biol. Chem. 2001; 276: 32230-32239Google Scholar). Additional activities associated with IpaC include oligomerization in solution (8Davis R. Marquart M.E. Lucius D. Picking W.D. Biochim. Biophys. Acta. 1998; 1429: 45-56Google Scholar), reconstitution into complexes with IpaB in vitro (8Davis R. Marquart M.E. Lucius D. Picking W.D. Biochim. Biophys. Acta. 1998; 1429: 45-56Google Scholar),in vivo formation of complexes containing IpaB that promote the uptake of latex beads by cultured cells (6Menard R. Prevost M.C. Gounon P. Sansonetti P. Dehio C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1254-1258Google Scholar), and reconstitution with IpaB and IpaD to form a complex that may allow entry of noninvasive Escherichia coli into cultured cells (13Terajima J. Moriishi E. Kurata T. Watanabe H. Microb. Pathog. 1999; 27: 223-230Google Scholar). It has been demonstrated that IpaC possesses a distinct functional organization (Scheme I). The immediate N terminus of IpaC is required for secretion, whereas a region near the N terminus is responsible for association with IpaB, which is probably needed for proper presentation of IpaC to the target cell membrane. The central hydrophobic region of IpaC directs IpaC penetration of phospholipid membranes and contributes to interactions with IpaB (14Picking W.L. Coye L. Osiecki J.C. Barnoski Serfis A. Schaper E. Picking W.D. Mol. Microbiol. 2001; 39: 100-111Google Scholar). 2A. Harrington and W. Picking, manuscript in preparation. The ability of IpaC to interact with phospholipid membranes does not require secretion by the TTSS of S. flexneri (Ref. 9Tran N. Serfis A.B. Osiecki J.C. Picking W.L. Coye L. Davis R. Picking W.D. Infect. Immun. 2000; 68: 3710-3715Google Scholar and this work); however, active insertion of IpaC into target cell membranes by the TTSS greatly increases the efficiency of this process (15Blocker A. Gounon P. Larquet E. Niebuhr K. Cabiaux V. Parsot C. Sansonetti P. J. Cell Biol. 1999; 147: 683-693Google Scholar). The C terminus of IpaC mediates IpaC-IpaC interactions and probably possesses IpaC effector function (7Tran Van Nhieu G. Caron E. Hall A. Sansonetti P.J. EMBO J. 1999; 18: 3249-3262Google Scholar, 14Picking W.L. Coye L. Osiecki J.C. Barnoski Serfis A. Schaper E. Picking W.D. Mol. Microbiol. 2001; 39: 100-111Google Scholar). This information, however, only provides a very general picture of IpaC structure and function and its role in the S. flexneri infection process. Unfortunately, there exists no detailed structural information concerning IpaC that could provide a molecular basis for its interactions with the host cytoskeleton and phospholipid membranes. In this work, we have initiated studies into the structure of IpaC by employing a number of biophysical approaches. Truncated versions of IpaC, specifically IpaC I′ (residues 101–363 deleted) and IpaC ΔH (residues 63–170 deleted), were overexpressed in E. coliand purified, permitting structural analysis and a more comprehensive investigation into the role of the individual domains described above. The interaction of these mutants and full-length IpaC with a liposome model was also examined. In addition to these structural studies, an investigation into the actin nucleation properties of IpaC and these deletion constructs was conducted. Finally, domain III (residues 261–363), which has previously resisted attempts at purification after recombinant expression, was studied using an alternate technique, linker-scanning mutagenesis. From this, it is shown that domain III contains a sequence periodic structure-activity relationship, which suggests that the activity may be dependent on helical structure in this region. Interpretation of IpaC structure with regard to the functional properties of the wild-type protein is then discussed. DOPC (dioleoylphosphatidylcholine) and DOPG (dioleoylphosphatidylglycerol) were purchased from Avanti. ANS (8-anilino-1-naphthalene sulfonic acid) was obtained from Across Organics, and fluorescein (5,6-carboxyfluorescein, high purity) was obtained from Molecular Probes. Dialysis materials were provided by Spectra. All other chemicals were of reagent grade and obtained from Sigma and Fisher. Plasmids used to prepare recombinant IpaC and IpaC ΔH and sipC have been described previously (5Marquart M.E. Picking W.L. Picking W.D. Infect. Immun. 1996; 64: 4182-4187Google Scholar, 16Osiecki J.C. Barker J. Picking W.L. Serfis A.B. Berring E. Shah S. Harrington A. Picking W.D. Mol. Microbiol. 2001; 42: 469-481Google Scholar, 17Picking W.L. Mertz J.A. Marquart M.E. Picking W.D. Protein Expression Purif. 1996; 8: 401-408Google Scholar). pWPI′ was designed to encode an IpaC peptide called region I′, in which residues 101–363 are deleted. This plasmid was generated as pWPC15 except that the 3′ primer was designed to place a stop codon at amino acid 101. All new plasmids were transformed into E. coli BL21(DE3) for high level protein expression. Recombinant proteins were purified via the N-terminal His6 tag by nickel chelation chromatography under denaturing conditions as described in detail previously (5Marquart M.E. Picking W.L. Picking W.D. Infect. Immun. 1996; 64: 4182-4187Google Scholar, 14Picking W.L. Coye L. Osiecki J.C. Barnoski Serfis A. Schaper E. Picking W.D. Mol. Microbiol. 2001; 39: 100-111Google Scholar,17Picking W.L. Mertz J.A. Marquart M.E. Picking W.D. Protein Expression Purif. 1996; 8: 401-408Google Scholar). Purified proteins were step-dialyzed against 10 mmNaPO4, pH 7.2, 150 mm NaCl containing 0.5 mm dithiothreitol to remove the urea. Purified proteins in buffer were stored at −70 °C for long term storage (IpaC only) or 4 °C for short term use (<2 weeks). All protein samples were centrifuged at 13,400 × g for 5 min at 4 °C on the day of use to remove aggregates formed during storage. Tween 20 (0.1%) was added to the IpaC solutions to further stabilize them during freezing. Unfortunately, neither centrifugation nor filtration was effective at completely removing all aggregated material (observed as a small amount of optical density above 300 nm in absorbance spectra). Concentrations were therefore obtained employing derivative absorbance spectroscopy, using N-acetyl-l-tyrosine-ethyl ester derivative minima at 275 and 283 nm to establish a standard curve since IpaC possesses no tryptophan residues. Biophysical studies were conducted in 10 mm NaPO4, 150 mm NaCl, pH 7.2, containing 1 mm dithiothreitol. CD spectra were recorded with a Jasco J-720 spectrophotometer (Tokyo, Japan) equipped with a Peltier temperature controller. Far UV spectra (between 195 and 260 nm) were collected using a 1-mm path length cuvette sealed with a Teflon stopper. A resolution of 0.1 nm and a scanning speed of 20 nm/min with a 2-s response time were employed. Spectra presented are an average of six consecutive spectra. Spectra were recorded at 5 °C intervals employing a thermostated cuvette holder. An incubation time of 3 min at each temperature interval (sufficient for equilibrium to be obtained) and a temperature ramp rate of 20 °C/h were employed. Protein concentrations of 8.3, 26.3, and 11.2 μm were employed for IpaC, IpaC I′, and IpaC ΔH, respectively. Noise reduction and data analysis were performed using Standard Analysis and Temperature/Wavelength Analysis programs (Jasco) and MicroCal Origin™ 6.0 software. Secondary structure content was estimated using the CONTIN (18Provencher S. Glockner J. Biochemistry. 1981; 20: 33-37Google Scholar), SELCON (19Sreerama N. Woody R. Anal. Biochem. 1990; 209: 32-44Google Scholar), and CDSSTR (20Manavalan P. Johnson W.C., Jr. Anal. Biochem. 1987; 167: 76-85Google Scholar) analysis programs provided with the CDPro software suite (21Sreerama, N. CDPro: A Software Package for Analyzing Protein Spectra, lamar.colostate.edu/∼sreeram/CDPro/index.htmlGoogle Scholar). High-resolution absorbance spectra were collected on a Hewlett-Packard 8453 UV-Visible spectrophotometer (Agilent, Palo Alto, CA) fitted with a Peltier temperature controller. Temperature perturbation studies were conducted at protein concentrations of 12.7, 25.3, and 16.9 μm for IpaC, IpaC I′, and IpaC ΔH, respectively. Spectra were collected for 25 s at 2.5 °C intervals with a 3-min equilibration time before collection of each spectrum. Spectral analysis was conducted using UV-Visible Chemstation software (Agilent) and Microcal Origin™ 6.0. Second derivative spectra were calculated employing a nine-point data filter and fifth degree Savitzky-Golay polynomial and subsequently fitted to a cubic function with 99 interpolated points/raw data point, permitting 0.01-nm resolution (22Mach H. Middaugh C.R. Anal. Biochem. 1994; 222: 323-331Google Scholar). Peak positions were then determined from the interpolated curves. Optical density data were simultaneously monitored at 350 nm. Aliquots of DOPC and DOPG in chloroform were dried under nitrogen and vacuum to create thin films of either 100% DOPC or 50:50 [DOPC]:[DOPG]. Films were hydrated in a solution containing 100 mm5,6-carboxyfluorescein at pH 7.0 in water for 10 min and then sonicated in a bath sonicator for 30 min prior to extrusion through a 100-nm pore size membrane 10 times at 45 °C. A final size of ∼150 nm was determined with a Brookhaven ZetaPALS dynamic light scattering instrument (Holtsville, NY) equipped with a 25 mW 626-nm laser. Excess dye was separated from the bulk liposomes by size exclusion chromatography, employing a Sephadex G-25 column coupled to an AKTA FLPC (Amersham Biosciences). The run buffer was 10 mm NaPO4, 150 mm NaCl, pH 7.4. Peak fractions were collected and pooled, and lipid content was determined by a total phosphorous assay, as described (23Avanti Polar Lipids, Determination of Total Phosphorous, www.avantilipids.com/DeterminationOfTotalPhosphorus.html.Google Scholar). Solutions were stored at 4 °C and protected from light. Time-based release studies were conducted with a PTI QuantaMaster spectrophotometer with a thermostated cuvette holder. Samples were excited at 492 nm, and the emission signal was monitored at 517 nm for 10 min. Excitation slits were set at 1 nm, and emission was set at 2 nm. Data points were collected at 0.2-s intervals. Data were collected as follows: buffer was incubated for 3 min at the desired temperature prior to adding liposome solutions. Baseline fluorescence was monitored with liposomes alone for 10 min to determine residual release of fluorescein from the liposomes. Maximum fluorescein release, as defined by release in the presence of 0.1% Triton, was determined by adding Triton to the liposome solution 3 min after the start of signal collection. Protein-induced fluorescein release was examined by monitoring the fluorescein signal at 512 nm after addition of protein to the liposome solution after 3 min of incubation. Both protein and Triton solutions were added during continuous signal collection through a syringe port in the sample compartment. The protein concentration employed was 1 μm, and the total lipid concentration was 100 μm. Release was monitored at 10, 20, 30, 35, 40, 45, 50, and 60 °C. Samples were measured in triplicate. The extent of release was calculated as a percentage of Triton-induced release at 600 s after background correction. Analysis was conducted using Felix (PTI) and MicroCal Origin™ software. The fluorescence of pyrene-labeled G-actin monomers increases following assembly into pyrene F-actin (24Hayward R.D. Koronakis V. EMBO J. 1999; 18: 4926-4934Google Scholar). For monitoring IpaC-mediated actin nucleationin vitro, pyrene G-actin was incubated at 4 °C in G-actin buffer (5 mm Tris-HCl, pH 8.0, 0.1 mm ATP, 0.2 mm CaCl2). Test protein (IpaC or an IpaC mutant) was then added to the sample, and pyrene fluorescence was monitored at 23 °C. A Spex FluoroMax instrument (Jobin Yvon Horiba, Edison, NJ) was used to measure pyrene fluorescence using a time-based acquisition mode with an excitation wavelength of 330 and an emission wavelength of 385 nm. After 15 min, 50× actin polymerization buffer (100 mm MgCl2, 50 mm ATP, 2.5m KCl) was added, and the change in pyrene fluorescence was monitored as a function of time. Negative controls either contained no added protein or contained IpaD, which has no actin-nucleating activity. SipC from Salmonella typhimurium, which has been shown to nucleate actin in vitro, was used as a positive control (24Hayward R.D. Koronakis V. EMBO J. 1999; 18: 4926-4934Google Scholar). Linker-scanning mutagenesis was used to introduce consecutive site-specific mutations into a predicted coiled-coil segment near the C terminus of IpaC. Either NheI cleavage sites encoding Ala-Ser pairs or XhoI sites encoding Leu-Glu pairs were generated throughout the length of the putative coiled-coil region (amino acids 309–344). Primers with a 5′NheI or XhoI site and the appropriate neighboringipaC sequences running in either direction were used for inverse PCR. The product was digested with NheI orXhoI, respectively, and then ligated. The resulting plasmids were electroporated into the S. flexneri IpaC mutant strain SF621, and the ability to restore invasion and contact hemolysis functions was determined as described below. S. flexneri invasion of Henle 407 cells was monitored using a standard gentamycin protection assay as described (16Osiecki J.C. Barker J. Picking W.L. Serfis A.B. Berring E. Shah S. Harrington A. Picking W.D. Mol. Microbiol. 2001; 42: 469-481Google Scholar). Semiconfluent monolayers of Henle 407 cells were seeded into 24-well plates and grown overnight. SF621 harboring the desired plasmid was grown in trypticase soy broth containing 100 μg/ml ampicillin and 50 μg/ml kanamycin to an A 600 of 0.4–0.6. The bacteria were diluted with serum-free MEM containing 0.45% glucose (MEM-glc), centrifuged onto the surface of semiconfluent Henle 407 monolayers, and incubated with the cells for 30 min at 37 °C. Free bacteria were removed by aspiration, and the cells were washed with MEM containing 5% calf serum and 50 μg/ml gentamycin. The cells were incubated in the final gentamycin wash for 2 h (to kill adherent, noninternalized bacteria) and rinsed with MEM-glc. The cells were lysed by overlaying them with 250 μl of 0.5% agarose in water. The agarose was then overlaid with 0.5% agar containing 2× LB medium. After overnight incubation at 37 °C, internalized bacteria formed subsurface colonies that were quantified using a ChemiImager 4400 system (Alpha Innotech Corp., San Leandro, CA). All errors are reported as standard error (S.E., n = 3) unless otherwise indicated. To better understand the function of IpaC in theS. flexneri invasion process, a series of structural studies were conducted to characterize the secondary and tertiary structure of full-length IpaC, IpaC I′, and ΔH mutants and their response to temperature. All three proteins are efficiently secreted by the S. flexneri SF621 TTSS and are able to interact with IpaB, indicating that biological functions associated with the N terminus remain intact (14Picking W.L. Coye L. Osiecki J.C. Barnoski Serfis A. Schaper E. Picking W.D. Mol. Microbiol. 2001; 39: 100-111Google Scholar). The CD spectrum of IpaC at 20 °C exhibits minima at 222 and 204 nm, suggesting the presence of some helical structure (Fig. 1 A). Secondary structure estimates indicate that IpaC contains a mixture of α-helical and β-sheet structure with significant turn and random structure also present (Table I). In contrast, IpaC I′ and IpaC ΔH appear to be less structured, as indicated by a decrease in CD intensity above 210 nm. The spectrum of IpaC I′ exhibits an increase in negative intensity near 200 nm, suggesting increased random structure. Solution conditions prevented collection of data below 200 nm, preventing secondary structure estimation for both mutants. IpaC undergoes a significant change in secondary structure at fairly moderate temperatures (Table I and Fig. 1). This transition suggests a loss of helix content (Table I) and has a midpoint of 43.3 ± 0.3 °C (n = 3). In contrast, IpaC I′ and IpaC ΔH display evidence of only weak transitions between 10 and 40 °C. Although limited aggregation of some samples was observed above 30 °C, no red shifts or strong decreases in intensity indicative of absorption flattening were observed, indicating that the spectral changes are not an artifact of aggregation-dependent phenomena.Table ISecondary structure estimates for IpaCTemperatureα-Helixβ-SheetTurnRandom%%%%20 °C10 (2)1-aErrors are reported as S.E., n = 3.34 (1)22 (2)32 (2)40 °C7 (1)35 (2)23 (2)35 (1)60 °C4 (0)38 (2)23 (2)33 (0)1-a Errors are reported as S.E., n = 3. Open table in a new tab Because IpaC lacks tryptophan residues, derivative absorbance spectroscopy was employed to monitor tertiary structure changes. This method is especially useful in aggregating systems since it is not sensitive to broad spectral components such as light scattering (25Mach H. Middaugh C.R. BioTechniques. 1993; 15: 240-242Google Scholar). The spectra display two phenylalanine (Fig. 2, A and B, 253 and 260 nm) and three tyrosine (Fig. 2, C–E, 268, 276, and 285 nm) minima (22Mach H. Middaugh C.R. Anal. Biochem. 1994; 222: 323-331Google Scholar). Most plots of peak position versustemperature show similar linear increases with temperature, which is an intrinsic property of the aromatic amino acids. 3L. A. Kueltzo and C. R. Middaugh, manuscript in preparation. However, deviations from these linear plots consistent with structural alterations are observed in many cases. The IpaC Phe minimum at 253 nm is shifted to longer wavelengths relative to the two mutants and shows no temperature dependence at lower temperatures. Upon reaching the temperature at which the protein begins aggregating, the noise in the data begins to increase. The second Phe minimum shows a difference in minimum position for each of the proteins with the IpaC peak once again at the longest wavelength. An expanded version of the IpaC I′ data (Fig. 2 F) shows that IpaC I′ actually undergoes a small but very reproducible transition between 30 and 55 °C. The 267 nm IpaC I′ tyrosine minimum is observed at longer wavelengths, disappearing from the derivative spectrum at temperatures above 62 °C. Two strong transitions are observed in the plot of the 276-nm tyrosine minimum of IpaC with no similar transitions seen for either mutant. The 284-nm IpaC I′ and IpaC ΔH tyrosine minima again manifest no evidence of conformational change, whereas the IpaC absorption band shows evidence of the two transitions and is again present at a longer wavelength (Fig. 2 E). Trends in protein-associative behavior can be seen by monitoring the turbidity (OD) at 350 nm as the temperature is increased (Fig. 3). Full-length IpaC shows a large change in turbidity starting near 30 °C. Transitions are observed between 30 and 70 °C and above 70 °C. Although the changes are small, a distinct transition is observed starting at 55 °C for IpaC I′, whereas IpaC ΔH shows a small but steady increase in OD starting at 20 °C (Fig. 3, bottom panels). Both results are consistent with the formation of soluble aggregates. The effect of IpaC, IpaC I′, and IpaC ΔH upon fluorescein-containing liposomes was examined as a function of the temperature and lipid composition. Fluorescein can be sequestered in liposomes at concentrations that produce self-quenching (26Mach H. Middaugh C.R. Biochemistry. 1995; 34: 9913-9920Google Scholar). Any interaction of a protein with such loaded liposomes that is sufficient to significantly perturb the bilayer should cause a leakage of the fluorescein, leading to its dilution and an increase in the fluorescence intensity of the fluorescein dye. Using this approach, two types of vesicles were employed: 100% DOPC, producing a neutral surface charge, and 50:50 [DOPC]:[DOPG] to produce an overall negatively charged surface. The extent of dye release at 600 s at different temperatures is shown in Fig. 4. No release is observed for IpaC I′ and IpaC ΔH. IpaC shows little release at low temperatures with DOPC liposomes, but a dramatic increase is observed at 30 °C and higher. Release at lower temperatures is greater with DOPC:DOPG liposomes. The rate of release is similar to that of the Triton control (results not illustrated). The liposomes remained above the phase transitions of the component lipids (−20 and −18 °C for DOPC and DOPG, respectively (27Silvius J.R. Jost P.C. Griffith O.H. Lipid-Protein Interactions. 2nd Ed. John Wiley & Sons, Inc., New York1982: 515Google Scholar)) under all conditions examined. The region between amino acids 309 and 344 of IpaC is predicted to possess a coiled-coil trimerization domain based on primary structure analysis (28Pallen M.J. Dougan G. Frankel G. Mol. Microbiol. 1997; 25: 423-425Google Scholar). Attempts to purify a recombinant form of this region have been unsuccessful, so a structural analysis of this region was conducted using linker-scanning mutagenesis. Initially, XhoI linkers were used to substitute Leu-Glu amino acid pairs for existing amino acids in this region of IpaC. Sequence periodicity in the generation of inactivating mutations with respect to restoring invasiveness toS. flexneri SF621 was consistent with the presence of a coiled α-helix in this region (Fig. 5 A). Although linker-scanning mutations near the C-terminal end of the putative coiled-coil show periodicity in reducing invasiveness, they do not eliminate IpaC activity completely. In contrast, when NheI linkers (encoding Ala-Ser) were introduced into the same region, substitution for the Leu335-Ile336 pair did completely eliminate IpaC invasion function, as did substitution for Leu339-Leu340 (Fig. 5 B). This difference in the effect of amino acid substitutions at this location could be explained by the fact that the Leu-Glu led to the replacement of a nonpolar pair of amino acids by a nonpolar/polar pair. On the other hand, NheI scanning results in the replacement of nonpolar residues by a polar pair. In total, these data are consistent with the presence of an α-helix within this portion of IpaC. Because a single amino acid change at Ile336 and Leu340 in the putative IpaC coiled region only partially reduced the ability of IpaC to restore invasiveness, other single amino acid changes were introduced, using Pro as the substituted amino acid because of its incompatibility with α-helix formation. When Pro was used to replace Ser3" @default.
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• W2009804131 date "2003-01-01" @default.
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• W2009804131 title "Structure-Function Analysis of Invasion Plasmid Antigen C (IpaC) from Shigella flexneri" @default.
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