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• W2010920218 abstract "The adhesive phenotype of Candida albicans contributes to its ability to colonize the host and cause disease. Als proteins are one of the most widely studied C. albicans virulence attributes; deletion of ALS3 produces the greatest reduction in adhesive function. Although adhesive activity is thought to reside within the N-terminal domain of Als proteins (NT-Als), the molecular mechanism of adhesion remains unclear. We designed mutations in NT-Als3 that test the contribution of the peptide-binding cavity (PBC) to C. albicans adhesion and assessed the adhesive properties of other NT-Als3 features in the absence of a functional PBC. Structural analysis of purified loss-of-PBC-function mutant proteins showed that the mutations did not alter the overall structure or surface properties of NT-Als3. The mutations were incorporated into full-length ALS3 and integrated into the ALS3 locus of a deletion mutant, under control of the native ALS3 promoter. The PBC mutant phenotype was evaluated in assays using monolayers of human pharyngeal epithelial and umbilical vein endothelial cells, and freshly collected human buccal epithelial cells in suspension. Loss of PBC function resulted in an adhesion phenotype that was indistinguishable from the Δals3/Δals3 strain. The adhesive contribution of the Als3 amyloid-forming-region (AFR) was also tested using these methods. C. albicans strains producing cell surface Als3 in which the amyloidogenic potential was destroyed showed little contribution of the AFR to adhesion, instead suggesting an aggregative function for the AFR. Collectively, these results demonstrate the essential and principal role of the PBC in Als3 adhesion. The adhesive phenotype of Candida albicans contributes to its ability to colonize the host and cause disease. Als proteins are one of the most widely studied C. albicans virulence attributes; deletion of ALS3 produces the greatest reduction in adhesive function. Although adhesive activity is thought to reside within the N-terminal domain of Als proteins (NT-Als), the molecular mechanism of adhesion remains unclear. We designed mutations in NT-Als3 that test the contribution of the peptide-binding cavity (PBC) to C. albicans adhesion and assessed the adhesive properties of other NT-Als3 features in the absence of a functional PBC. Structural analysis of purified loss-of-PBC-function mutant proteins showed that the mutations did not alter the overall structure or surface properties of NT-Als3. The mutations were incorporated into full-length ALS3 and integrated into the ALS3 locus of a deletion mutant, under control of the native ALS3 promoter. The PBC mutant phenotype was evaluated in assays using monolayers of human pharyngeal epithelial and umbilical vein endothelial cells, and freshly collected human buccal epithelial cells in suspension. Loss of PBC function resulted in an adhesion phenotype that was indistinguishable from the Δals3/Δals3 strain. The adhesive contribution of the Als3 amyloid-forming-region (AFR) was also tested using these methods. C. albicans strains producing cell surface Als3 in which the amyloidogenic potential was destroyed showed little contribution of the AFR to adhesion, instead suggesting an aggregative function for the AFR. Collectively, these results demonstrate the essential and principal role of the PBC in Als3 adhesion. Candida albicans is the most common cause of opportunistic mycoses worldwide (1Pfaller M.A. Diekema D.J. Epidemiology of invasive candidiasis: a persistent public health problem.Clin. Microbiol. Rev. 2007; 20: 133-163Crossref PubMed Scopus (3082) Google Scholar). Among these conditions are oropharyngeal candidiasis that afflicts many HIV-infected patients, vaginal thrush, and denture stomatitis (2Gendreau L. Loewy Z.G. Epidemiology and etiology of denture stomatitis.J. Prosthodont. 2011; 20: 251-260Crossref PubMed Scopus (415) Google Scholar, 3Sobel J.D. Vulvovaginal candidosis.Lancet. 2007; 369: 1961-1971Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar, 4Thompson 3rd, G.R. Patel P.K. Kirkpatrick W.R. Westbrook S.D. Berg D. Erlandsen J. Redding S.W. Patterson T.F. Oropharyngeal candidiasis in the era of antiretroviral therapy.Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 2010; 109: 488-495Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Candida spp. are among the top four pathogens causing nosocomial bloodstream infections (5Wisplinghoff H. Bischoff T. Tallent S.M. Seifert H. Wenzel R.P. Edmond M.B. Nosocomial bloodstream infections in United States hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study.Clin. Infect. Dis. 2004; 39: 309-317Crossref PubMed Scopus (3402) Google Scholar), which carry mortality rates ranging between 30 and 40% (1Pfaller M.A. Diekema D.J. Epidemiology of invasive candidiasis: a persistent public health problem.Clin. Microbiol. Rev. 2007; 20: 133-163Crossref PubMed Scopus (3082) Google Scholar, 6Tortorano A.M. Peman J. Bernhardt H. Klingspor L. Kibbler C.C. Faure O. Biraghi E. Canton E. Zimmermann K. Seaton S. Grillot R. Epidemiology of candidaemia in Europe: results of 28-month European Confederation of Medical Mycology (ECMM) hospital-based surveillance study.Eur. J. Clin. Microbiol. Infect. Dis. 2004; 23: 317-322Crossref PubMed Scopus (425) Google Scholar). Cell surface glycoproteins in the C. albicans Als family (Als1–Als7 and Als9) are most commonly associated with adhesion of the fungus to host cells, as well as more complex interactions such as invasion and biofilm formation, for which adhesion is a prerequisite (7Chandra J. Kuhn D.M. Mukherjee P.K. Hoyer L.L. McCormick T. Ghannoum M.A. Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance.J. Bacteriol. 2001; 183: 5385-5394Crossref PubMed Scopus (1270) Google Scholar, 8Phan Q.T. Myers C.L. Fu Y. Sheppard D.C. Yeaman M.R. Welch W.H. Ibrahim A.S. Edwards Jr., J.E. Filler S.G. Als3 is a Candida albicans invasin that binds to cadherins and induces endocytosis by host cells.PLoS Biol. 2007; 5: e64Crossref PubMed Scopus (415) Google Scholar, 9Silverman R.J. Nobbs A.H. Vickerman M.M. Barbour M.E. Jenkinson H.F. Interaction of Candida albicans cell wall Als3 protein with Streptococcus gordonii SspB adhesin promotes development of mixed-species communities.Infect. Immun. 2010; 78: 4644-4652Crossref PubMed Scopus (168) Google Scholar). Deletion of ALS genes from C. albicans or expression of ALS genes in Saccharomyces cerevisiae leads to reduction or gain of adhesive function, respectively (10Hoyer L.L. Green C.B. Oh S.H. Zhao X. Discovering the secrets of the Candida albicans agglutinin-like sequence (ALS) gene family: a sticky pursuit.Med. Mycol. 2008; 46: 1-15Crossref PubMed Scopus (242) Google Scholar, 11Zhao X. Oh S.H. Cheng G. Green C.B. Nuessen J.A. Yeater K. Leng R.P. Brown A.J. Hoyer L.L. ALS3 and ALS8 represent a single locus that encodes a Candida albicans adhesin: functional comparisons between Als3p and Als1p.Microbiology. 2004; 150: 2415-2428Crossref PubMed Scopus (192) Google Scholar). The N-terminal domain of Als proteins (NT-Als) 4The abbreviations used are:NT-AlsN-terminal domain of Als proteinsAFRamyloid-forming regionBECbuccal epithelial cellFaDuhuman pharyngeal epithelial cellFg-γfibrinogen-γgkgatekeeperITCisothermal titration calorimetryPBCpeptide-binding cavityr.m.s.d.root mean square deviationsNT-Als3shorter version of NT-Als3 that excludes the C-terminal AFR. was implicated in adhesive function (12Loza L. Fu Y. Ibrahim A.S. Sheppard D.C. Filler S.G. Edwards Jr., J.E. Functional analysis of the Candida albicans ALS1 gene product.Yeast. 2004; 21: 473-482Crossref PubMed Scopus (61) Google Scholar, 13Zhao X. Daniels K.J. Oh S.H. Green C.B. Yeater K.M. Soll D.R. Hoyer L.L. Candida albicans Als3p is required for wild-type biofilm formation on silicone elastomer surfaces.Microbiology. 2006; 152: 2287-2299Crossref PubMed Scopus (136) Google Scholar), prompting work to solve its structure (14Salgado P.S. Yan R. Taylor J.D. Burchell L. Jones R. Hoyer L.L. Matthews S.J. Simpson P.J. Cota E. Structural basis for the broad specificity to host-cell ligands by the pathogenic fungus Candida albicans.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 15775-15779Crossref PubMed Scopus (66) Google Scholar). X-ray crystallographic analysis of NT-Als9-2 (encoded by an ALS9 allele) showed that it is characterized by two immunoglobulin-like domains and possesses a peptide-binding cavity (PBC) that can bury up to 6 residues from flexible C termini of polypeptides (14Salgado P.S. Yan R. Taylor J.D. Burchell L. Jones R. Hoyer L.L. Matthews S.J. Simpson P.J. Cota E. Structural basis for the broad specificity to host-cell ligands by the pathogenic fungus Candida albicans.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 15775-15779Crossref PubMed Scopus (66) Google Scholar). An invariant Lys (Lys-59) at the end of the PBC recognizes the carboxyl group at the C terminus of a peptide ligand, allowing the remaining peptide backbone to associate in parallel orientation with β-strand G2. In the structure of different NT-Als·ligand complexes, several water molecules anchor polypeptides with different C-terminal sequences in the PBC. These structural observations suggest that the PBC is responsible for Als adhesive interactions and may account for the ability of Als proteins, particularly Als3, to bind to the numerous ligands documented in the literature (e.g. 2Gendreau L. Loewy Z.G. Epidemiology and etiology of denture stomatitis.J. Prosthodont. 2011; 20: 251-260Crossref PubMed Scopus (415) Google Scholar, 3Sobel J.D. Vulvovaginal candidosis.Lancet. 2007; 369: 1961-1971Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar, 4Thompson 3rd, G.R. Patel P.K. Kirkpatrick W.R. Westbrook S.D. Berg D. Erlandsen J. Redding S.W. Patterson T.F. Oropharyngeal candidiasis in the era of antiretroviral therapy.Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 2010; 109: 488-495Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 12Loza L. Fu Y. Ibrahim A.S. Sheppard D.C. Filler S.G. Edwards Jr., J.E. Functional analysis of the Candida albicans ALS1 gene product.Yeast. 2004; 21: 473-482Crossref PubMed Scopus (61) Google Scholar, 13Zhao X. Daniels K.J. Oh S.H. Green C.B. Yeater K.M. Soll D.R. Hoyer L.L. Candida albicans Als3p is required for wild-type biofilm formation on silicone elastomer surfaces.Microbiology. 2006; 152: 2287-2299Crossref PubMed Scopus (136) Google Scholar, 14Salgado P.S. Yan R. Taylor J.D. Burchell L. Jones R. Hoyer L.L. Matthews S.J. Simpson P.J. Cota E. Structural basis for the broad specificity to host-cell ligands by the pathogenic fungus Candida albicans.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 15775-15779Crossref PubMed Scopus (66) Google Scholar, 15Otoo H.N. Lee K.G. Qiu W. Lipke P.N. Candida albicans Als adhesins have conserved amyloid-forming sequences.Eukaryot. Cell. 2008; 7: 776-782Crossref PubMed Scopus (96) Google Scholar, 16Lipke P.N. Garcia M.C. Alsteens D. Ramsook C.B. Klotz S.A. Dufrêne Y.F. Strengthening relationships: amyloids create adhesion nanodomains in yeasts.Trends Microbiol. 2012; 20: 59-65Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). N-terminal domain of Als proteins amyloid-forming region buccal epithelial cell human pharyngeal epithelial cell fibrinogen-γ gatekeeper isothermal titration calorimetry peptide-binding cavity root mean square deviation shorter version of NT-Als3 that excludes the C-terminal AFR. Here, we describe use of the NT-Als9-2 structure to guide a mutagenesis strategy aimed at disrupting PBC function in Als3. Als3 was selected for this analysis because deleting ALS3 results in a larger decrease in C. albicans adhesion than for any other gene in the family (10Hoyer L.L. Green C.B. Oh S.H. Zhao X. Discovering the secrets of the Candida albicans agglutinin-like sequence (ALS) gene family: a sticky pursuit.Med. Mycol. 2008; 46: 1-15Crossref PubMed Scopus (242) Google Scholar). We also targeted the NT-Als C terminus, which contains a conserved amyloid-forming region (AFR) (15Otoo H.N. Lee K.G. Qiu W. Lipke P.N. Candida albicans Als adhesins have conserved amyloid-forming sequences.Eukaryot. Cell. 2008; 7: 776-782Crossref PubMed Scopus (96) Google Scholar) for which adhesive properties have been proposed in the literature (16Lipke P.N. Garcia M.C. Alsteens D. Ramsook C.B. Klotz S.A. Dufrêne Y.F. Strengthening relationships: amyloids create adhesion nanodomains in yeasts.Trends Microbiol. 2012; 20: 59-65Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). We used biophysical techniques to solve the NT-Als3 structure and assessed the structural impact of the site-directed mutations. Finally, we produced the mutant Als3 proteins on the C. albicans surface, under control of the native ALS3 promoter, and tested the strains for their adhesive phenotype in assays using cultured or freshly collected human cells. The complementary information provided from the study of purified proteins using biophysical techniques and phenotypic analysis of mutant proteins displayed in native conditions on the C. albicans surface demonstrates the essential contribution of the PBC to Als3 adhesive function. DNA encoding the N-terminal domain of Als3 (NT-Als3, amino acids 1–315 of the mature protein, KTIT—IVIVA, GenBank Accession No. AY223552), was subcloned in plasmid pET32-Xa-LIC by ligation-independent cloning (Novagen). From this construct, a shorter version of NT-Als3 that excludes the C-terminal AFR (sNT-Als3), was generated by replacing the codon corresponding to Asn-303 with a stop codon. Other mutations that probe the function of the PBC and the AFR in these constructs are summarized in Fig. 1. Mutagenesis was performed with the QuikChange II Kit (Agilent Technologies). Oligonucleotides were designed according to the manufacturer's instructions. Expression and purification of unlabeled and U-15N-labeled NT-Als3 constructs/mutants were performed as described for NT-Als1 (17Yan R. Simpson P.J. Matthews S.J. Cota E. Backbone 1H, 15N, 13C and Ile, Leu, Val methyl chemical shift assignments for the 33.5 kDa N-terminal domain of Candida albicans Als1.Biomol. NMR Assign. 2010; 4: 187-190Crossref PubMed Scopus (8) Google Scholar). DNAs were transformed for protein expression in the Origami Escherichia coli strain (New England Biolabs) and grown at 37 °C in LB agar plates with 50 μg/ml carbenicillin, 15 μg/ml kanamycin, and 12.5 μg/ml tetracycline. Cultures grown in this medium were clarified by centrifugation at 2000 × g and the cell pellets resuspended in 500 ml of LB or M9 minimal medium with 0.07% w/v 15N ammonium chloride (Spectra Gases) plus carbenicillin and kanamycin for expression of uniformly labeled 15N proteins. Cell cultures at an A600 0.5–0.7 were induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside and left to grow at 18 °C overnight. Cells were then harvested by centrifugation, resuspended in ice-cold 50 mm Tris, pH 8, 300 mm NaCl plus Complete protease inhibitor (Roche Applied Science), and lysed by French press. The lysate was centrifuged at 30,000 × g for 20 min to obtain a cleared supernatant. Soluble thioredoxin-His6-Als3 fusions were affinity-purified using nickel-nitrilotriacetic acid resin (Qiagen) in 50 mm Tris, pH 8.0, 300 mm NaCl, 10 mm imidazole, and eluted with 50 mm Tris, pH 8, 300 mm NaCl, 250 mm imidazole. The N-terminal thioredoxin and His6 tags were removed from Als3 proteins by Factor Xa (Novagen) and a second nickel affinity purification. Als3 proteins were further purified by size exclusion chromatography on a HiLoad 16/60 Superdex 75 column (GE Healthcare) in 50 mm Tris, pH 8.0, and 300 mm NaCl.FIGURE 2NMR spectra (1H-15N TROSY-HSQC) showing the effect of mutations in the PBC and AFR on NT-Als3. A, the NMR spectrum of purified NT-Als3 showed considerably reduced solubility. Two potential mechanisms explain this effect: oligomerization caused by insertion of the free C terminus (CT) of the protein as a ligand into the binding pocket of an adjacent NT-Als3 molecule (“self-complementation,” left) (14Salgado P.S. Yan R. Taylor J.D. Burchell L. Jones R. Hoyer L.L. Matthews S.J. Simpson P.J. Cota E. Structural basis for the broad specificity to host-cell ligands by the pathogenic fungus Candida albicans.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 15775-15779Crossref PubMed Scopus (66) Google Scholar) and aggregation mediated by exposed AFR sequences (30Garcia M.C. Lee J.T. Ramsook C.B. Alsteens D. Dufrêne Y.F. Lipke P.N. A role for amyloid in cell aggregation and biofilm formation.PloS One. 2011; 6: e17632Crossref PubMed Scopus (88) Google Scholar). Note that self-complementation is suppressed in full-length Als proteins on the C. albicans surface, where the C terminus is linked to the cell wall via a glycosylphosphatidylinositol anchor remnant (10Hoyer L.L. Green C.B. Oh S.H. Zhao X. Discovering the secrets of the Candida albicans agglutinin-like sequence (ALS) gene family: a sticky pursuit.Med. Mycol. 2008; 46: 1-15Crossref PubMed Scopus (242) Google Scholar). B, deletion of the AFR resulted in sNT-Als3, a construct free of oligomerization and aggregation effects. C, mutations in the PBC (K59M, A116V, and Y301F to generate NT-Als3-pbc) improved protein solubility compared with A, presumably due to elimination of self-complementation and decreased aggregation because the AFR was associated with the rest of the NT-Als3 folded structure (Fig. 3). D, solubility of the protein in C was further improved by removing residual AFR activity via mutations I311S/I313S (NT-Als3-pbc-afr). These results demonstrate at the molecular level the role of the PBC and the AFR in aggregation among purified NT-Als proteins.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 1Als3 constructs and mutants for structural analysis. Top, as a reference, the arrangement of domains in full-length Als3 is shown to scale: the signal sequence (pink) is followed by the N-terminal region, containing the amyloid-forming region (red and blue, respectively), the threonine-rich region (light gray), the central repeats (light green), and the C-terminal region (dark gray). Bottom, an expanded N-terminal region is used to show the location of Als3 mutants and the truncation at the C terminus (CT) to remove the AFR in sNT-Als3 and sNT-Als3-gk. The numbering of amino acids is consistent with the alignment of mature Als proteins described previously (14Salgado P.S. Yan R. Taylor J.D. Burchell L. Jones R. Hoyer L.L. Matthews S.J. Simpson P.J. Cota E. Structural basis for the broad specificity to host-cell ligands by the pathogenic fungus Candida albicans.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 15775-15779Crossref PubMed Scopus (66) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Samples for NMR experiments were dialyzed extensively against 50 mm sodium phosphate buffer, pH 8.0, 50 mm NaCl and concentrated to 200 μm protein. 10% D2O was added to lock the deuterium signal. Proton and 15N TROSY-HSQC spectra were recorded at 308 K on a Bruker 800 MHz Avance II spectrometer equipped with a TXI Cryoprobe (Cross Faculty NMR Centre, Imperial College London). ITC experiments were carried out in a VP-ITC MicroCalorimeter (Microcal). Binding isotherms were obtained upon titration of 2.8 mm hepta-Thr (Biomatik) into solutions of 100 μm sNT-Als3 or sNT-Als3-gk. Titrations were conducted in 50 mm sodium phosphate, pH 6.0, and 150 mm NaCl at 303 K. Prior to ITC analysis, protein samples in 3 m urea were dialyzed twice against the buffer above to remove proteins/peptides bound to sNT-Als3 and sNT-Als3-gk during protein purification. sNT-Als3 (10 mg/ml) in 20 mm Tris-HCl, pH 8.0, 100 mm NaCl was incubated with 100-fold molar excess hepta-Thr (sNT-Als3/hepta-Thr) crystallized at 293 K by sitting drop vapor diffusion against 20% v/v PEG 400, 30% w/v PEG 4000, 100 mm sodium citrate, 50 mm ammonium acetate, pH 5.6. NT-Als3-pbc (10 mg/ml) in 20 mm Tris-HCl, pH 8, 100 mm NaCl was crystallized at 293 K by sitting drop vapor diffusion against 25% w/v PEG 4000 and 30% v/v ethylene glycol. sNT-Als3/hepta-Thr crystals were briefly washed in paraffin oil before flash freezing in liquid N2. Crystals of NT-Als3-pbc were flash frozen straight in liquid N2. Data for sNT-Als3/hepta-Thr were collected at 100 K on station I04-1 of the Diamond Light Source, UK. Data were processed using XDS (18Kabsch W. XDS.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 125-132Crossref PubMed Scopus (11225) Google Scholar) and then scaled with SCALA (19Evans P.R. An introduction to data reduction: space-group determination, scaling and intensity statistics.Acta Crystallogr. D Biol. Crystallogr. 2011; 67: 282-292Crossref PubMed Scopus (994) Google Scholar), within XIA2 (20Winter G. Lobley C.M. Prince S.M. Decision making in xia2.Acta Crystallogr. D Biol. Crystallogr. 2013; 69: 1260-1273Crossref PubMed Scopus (381) Google Scholar). Initial phases were calculated with molecular replacement with NT-Als9-2 (Protein Data Bank ID code 2Y7N) (14Salgado P.S. Yan R. Taylor J.D. Burchell L. Jones R. Hoyer L.L. Matthews S.J. Simpson P.J. Cota E. Structural basis for the broad specificity to host-cell ligands by the pathogenic fungus Candida albicans.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 15775-15779Crossref PubMed Scopus (66) Google Scholar) as the search model using MOLREP (21Vagin A. Teplyakov A. Molecular replacement with MOLREP.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 22-25Crossref PubMed Scopus (2701) Google Scholar). Model building was carried out in COOT (22Emsley P. Cowtan K. COOT: model-building tools for molecular graphics.Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23224) Google Scholar), and refinement with TLS groups (23Painter J. Merritt E.A. TLSMD web server for the generation of multi-group TLS models.J. Appl. Crystallogr. 2006; 39: 109-111Crossref Scopus (647) Google Scholar) was done in REFMAC (24Murshudov G.N. Vagin A.A. Dodson E.J. Refinement of macromolecular structures by the maximum-likelihood method.Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13853) Google Scholar), where 5% of the reflections were omitted for cross-validation. The final model contains 1 molecule of hepta-Thr in the asymmetric unit, and all residues could be built except for the C-terminal Arg-302 (sNT-Als3) and the side chains of Lys-1, Lys-75, Lys-106, Lys-132, Lys-148 (sNT-Als3), and Thr-1 (hepta-Thr). Data for NT-Als3-pbc were collected at 100 K on station I24 of the Diamond Light Source, UK. Data were processed using XDS (18Kabsch W. XDS.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 125-132Crossref PubMed Scopus (11225) Google Scholar) and then scaled with SCALA (19Evans P.R. An introduction to data reduction: space-group determination, scaling and intensity statistics.Acta Crystallogr. D Biol. Crystallogr. 2011; 67: 282-292Crossref PubMed Scopus (994) Google Scholar), within XIA2 (20Winter G. Lobley C.M. Prince S.M. Decision making in xia2.Acta Crystallogr. D Biol. Crystallogr. 2013; 69: 1260-1273Crossref PubMed Scopus (381) Google Scholar). The presence of twinning and significant pseudotranslational symmetry (u = 0.01, v = 0.23, w = −0.5) was detected using XTRIAGE (25Zwart P.H. Grosse-Kunstleve R.W. Adams P.D. Xtriage and Fest: automatic assessment of x-ray data and substructure structure factor estimation.CCP4 Newsl. 2005; 43 (Contribution 7)Google Scholar). Initial phases were calculated with molecular replacement with NT-Als9-2 as the search model using MOLREP (21Vagin A. Teplyakov A. Molecular replacement with MOLREP.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 22-25Crossref PubMed Scopus (2701) Google Scholar). Model building was carried out in COOT (22Emsley P. Cowtan K. COOT: model-building tools for molecular graphics.Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23224) Google Scholar) and refinement with TLS groups (23Painter J. Merritt E.A. TLSMD web server for the generation of multi-group TLS models.J. Appl. Crystallogr. 2006; 39: 109-111Crossref Scopus (647) Google Scholar), NCS, and twin refinement (a = 0.49) was done in REFMAC (24Murshudov G.N. Vagin A.A. Dodson E.J. Refinement of macromolecular structures by the maximum-likelihood method.Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13853) Google Scholar), where 5% of the reflections were omitted for cross-validation. The final model contains 4 molecules of NT-Als3-pbc in the asymmetric unit, and all residues could be built except for the C-terminal Thr-313 (all chains) and the side chains of Lys-1, Asn-53, Lys-75, Lys-106, Glu-129, Lys-132, Lys-148, Lys-149, Tyr-203, Lys-219, Glu-231, Glu-278, Arg-288, Phe-298 (chain A); Lys-1, Lys-106, Glu-129, Lys-132, Asp-145, Lys-148, Lys-149, Lys-164, Lys-178, Gln-207, Lys-219, Glu-231, Tyr-285 (chain B); Lys-1, Lys-75, Lys-106, Lys-132, Lys-148, Lys-164, Lys-178, Gln-187, Met-197, Lys-237, Lys-249, Asn-250, Tyr-285 (chain C); and Lys-1, Lys-75, Lys-106, Lys-132, Lys-148, Arg-158, Lys-164, Arg-294 (chain D). Processing and refinement statistics of the final models are in Table 1.TABLE 1Crystallographic data and refinement statisticssNT-Als3/hepta-ThrNT-Als3-pbcCrystal parameters Space groupP21P21 Cell dimensionsa = 49.4, b = 58.2, c = 57.2, β = 114.5a = 112.6, b = 67.1, c = 112.7, β = 103.5Data collection BeamlineDLS I04–1DLS I24 Wavelength (̊)0.91730.9790 Resolution (̊)1.40–29.09 (1.40–1.48)3.00–48.33 (3.00–3.08) Unique observations53890 (7562)32262 (2312) RsymaRsym = Σ|I−<I>|/ΣI where I is the integrated intensity of a given reflection and <I> is the mean intensity of multiple corresponding symmetry-related reflections.0.054 (0.179)0.097 (0.398) <I>/σI17.8 (7.7)9.6 (2.7) Completeness (%)93.1 (89.9)97.5 (95.5) Redundancy6.6 (6.8)2.8 (2.8)Refinement Rwork/Rfree bRwork = Σ‖Fo| − |Fc‖/ΣFo where Fo and Fc are the observed and calculated structure factors, respectively. Rfree = Rwork calculated using ∼5% random data excluded from the refinement. (%)13.6/17.722.9/24.2 Number of protein residues6051252 r.m.s.d. stereochemistrycr.m.s.d. stereochemistry is the deviation from ideal values. Bond lengths (̊)0.0190.009 Bond angles (°)1.7621.439 Ramachandran analysisdRamachandran analysis was carried out using MOLPROBITY (40). Residues in outlier regions00 Residues in favored regions97.0%93.7% Residues in allowed regions100%100%a([a-z])% Rsym = Σ|I−<I>|/ΣI where I is the integrated intensity of a given reflection and <I> is the mean intensity of multiple corresponding symmetry-related reflections.b([a-z])% Rwork = Σ‖Fo| − |Fc‖/ΣFo where Fo and Fc are the observed and calculated structure factors, respectively. Rfree = Rwork calculated using ∼5% random data excluded from the refinement.c r.m.s.d. stereochemistry is the deviation from ideal values.d Ramachandran analysis was carried out using MOLPROBITY (40Davis I.W. Murray L.W. Richardson J.S. Richardson D.C. MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes.Nucleic Acids Res. 2004; 32: W615-W619Crossref PubMed Scopus (814) Google Scholar). Open table in a new tab C. albicans strains that produce mutant Als3 proteins were made using constructs in a modified version of the plasmid pUL (11Zhao X. Oh S.H. Cheng G. Green C.B. Nuessen J.A. Yeater K. Leng R.P. Brown A.J. Hoyer L.L. ALS3 and ALS8 represent a single locus that encodes a Candida albicans adhesin: functional comparisons between Als3p and Als1p.Microbiology. 2004; 150: 2415-2428Crossref PubMed Scopus (192) Google Scholar). All PCR amplifications used SC5314 genomic DNA as template and proofreading polymerase. Primers ALS3dnF and ALS3dnR (Table 2) were used to amplify a 358-bp fragment downstream of the ALS3 coding region. The resulting PCR product was digested with SstII-NgoMIV and cloned into identically digested pUL, resulting in plasmid 1765. Primers were synthesized to add a polylinker to plasmid 1765, expanding its 5′ end multiple cloning site to include HindIII-PstI-BamHI-NcoI-XhoI-SphI. The resulting plasmid was named 2303. Primers 3upPstF and 3upBamR were used to amplify a fragment that includes sequences from upstream of the ALS3 coding region as well as the first 141 nucleotides of ALS3. This fragment was digested with PstI-BamHI and cloned into identically digested plasmid 2303, producing plasmid 2329. Another portion of the ALS3 5′ domain was amplified using primers 3cdBamF and 3cdNcoR. The product was digested with BamHI-NcoI and cloned into plasmid 2329, yielding plasmid 2370. The remainder of the ALS3 coding region was amplified using primers 3cdNcoF and 3dnXhoR, digested with NcoI-XhoI, and cloned into plasmid 2370. The resulting plasmid (2386) included the entire ALS3 coding region, with flanking sequences to direct integration into the ALS3 locus, and the URA3 selectable marker. The ALS3 coding region included BamHI and NcoI sites within the 5′ domain to provide easy swapping of cassettes encoding mutant ALS3 sequences. Gene fragments encoding the desired mutations were synthesized by Genewiz Inc., digested with BamHI-NcoI, and cloned into plasmid 2386 to replace the wild-type sequence. The DNA sequence of each resulting construct was verified, and the plasmid was digested with PstI-SstI t" @default.
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• W2010920218 date "2014-06-01" @default.
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• W2010920218 title "The Peptide-binding Cavity Is Essential for Als3-mediated Adhesion of Candida albicans to Human Cells" @default.
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• W2010920218 doi "https://doi.org/10.1074/jbc.m114.547877" @default.
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