Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore
To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 310-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 310-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands.
Vyšlo v časopise:
Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore. PLoS Pathog 8(3): e32767. doi:10.1371/journal.ppat.1002559
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.ppat.1002559
Souhrn
To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 310-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 310-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands.
Zdroje
1. SawatzkiGHoffmannFAKubanekB 1983 Acute iron overload in mice: pathogenesis of Salmonella typhimurium infection. Infect Immun 39 659 665
2. RaymondKNDertzEAKimSS 2003 Enterobactin: an archetype for microbial iron transport. Proc Natl Acad Sci U S A 100 3584 3588
3. WinkelmannG 2002 Microbial siderophore-mediated transport. Biochem Soc Trans 30 691 696
4. RaymondKN 2004 Biochemical and Physical Properties of Siderophores. JorgeHCrosaARMPayneShelleyM Iron Transport in Bacteria Washington, D.C. ASM Press 3 17
5. CendrowskiSMacArthurWHannaP 2004 Bacillus anthracis requires siderophore biosynthesis for growth in macrophages and mouse virulence. Mol Microbiol 51 407 417
6. DaleSEDoherty-KirbyALajoieGHeinrichsDE 2004 Role of siderophore biosynthesis in virulence of Staphylococcus aureus: identification and characterization of genes involved in production of a siderophore. Infect Immun 72 29 37
7. PonkaP 1999 Cell biology of heme. Am J Med Sci 318 241 256
8. AisenPEnnsCWessling-ResnickM 2001 Chemistry and biology of eukaryotic iron metabolism. Int J Biochem Cell Biol 33 940 959
9. WittenbergJBWittenbergBA 1990 Mechanisms of cytoplasmic hemoglobin and myoglobin function. Annu Rev Biophys Biophys Chem 19 217 241
10. LetoffeSGhigoJMWandersmanC 1994 Iron acquisition from heme and hemoglobin by a Serratia marcescens extracellular protein. Proc Natl Acad Sci U S A 91 9876 9880
11. WandersmanCDelepelaireP 2004 Bacterial iron sources: from siderophores to hemophores. Annu Rev Microbiol 58 611 647
12. GaoJLNguyenKAHunterN 2010 Characterization of a hemophore-like protein from Porphyromonas gingivalis. J Biol Chem 285 40028 40038
13. YuklETJepkorirGAlontagaAYPautschLRodriguezJC 2010 Kinetic and spectroscopic studies of hemin acquisition in the hemophore HasAp from Pseudomonas aeruginosa. Biochemistry 49 6646 6654
14. CescauSCwermanHLetoffeSDelepelairePWandersmanC 2007 Heme acquisition by hemophores. Biometals 20 603 613
15. LetoffeSNatoFGoldbergMEWandersmanC 1999 Interactions of HasA, a bacterial haemophore, with haemoglobin and with its outer membrane receptor HasR. Mol Microbiol 33 546 555
16. LetoffeSOmoriKWandersmanC 2000 Functional characterization of the HasA(PF) hemophore and its truncated and chimeric variants: determination of a region involved in binding to the hemophore receptor. J Bacteriol 182 4401 4405
17. BrickmanTJVanderpoolCKArmstrongSK 2006 Heme transport contributes to in vivo fitness of Bordetella pertussis during primary infection in mice. Infect Immun 74 1741 1744
18. MortonDJBakaletzLOJurcisekJAVanWagonerTMSealeTW 2004 Reduced severity of middle ear infection caused by nontypeable Haemophilus influenzae lacking the hemoglobin/hemoglobin-haptoglobin binding proteins (Hgp) in a chinchilla model of otitis media. Microb Pathog 36 25 33
19. PaulleyJTAndersonESRoopRM2nd 2007 Brucella abortus requires the heme transporter BhuA for maintenance of chronic infection in BALB/c mice. Infect Immun 75 5248 5254
20. HendersonDPPayneSM 1994 Vibrio cholerae iron transport systems: roles of heme and siderophore iron transport in virulence and identification of a gene associated with multiple iron transport systems. Infect Immun 62 5120 5125
21. TaiSSLeeCJWinterRE 1993 Hemin utilization is related to virulence of Streptococcus pneumoniae. Infect Immun 61 5401 5405
22. MazmanianSKTon-ThatHSuKSchneewindO 2002 An iron-regulated sortase enzyme anchors a class of surface protein during Staphylococcus aureus pathogenesis. Proc Natl Acad Sci USA 99 2293 2298
23. LarsenRGozzelinoRJeneyVTokajiLBozzaFA 2011 A central role for free heme in the pathogenesis of severe sepsis. Sci Transl Med 2 51ra71
24. IzadiNHenryYHaladjianJGoldbergMEWandersmanC 1997 Purification and characterization of an extracellular heme-binding protein, HasA, involved in heme iron acquisition. Biochemistry 36 7050 7057
25. Izadi-PruneyreNHucheFLukat-RodgersGSLecroiseyAGilliR 2006 The heme transfer from the soluble HasA hemophore to its membrane-bound receptor HasR is driven by protein-protein interaction from a high to a lower affinity binding site. J Biol Chem 281 25541 25550
26. MaressoAWGarufiGSchneewindO 2008 Bacillus anthracis secretes proteins that mediate heme acquisition from hemoglobin. PLoS Pathog 4 e1000132
27. HonsaESMaressoAW 2011 Mechanisms of iron import in anthrax. Biometals 24 533 545
28. GatOZaideGInbarIGrosfeldHChitlaruT 2008 Characterization of Bacillus anthracis iron-regulated surface determinant (Isd) proteins containing NEAT domains. Mol Microbiol 70 983 999
29. MaressoAWChapaTJSchneewindO 2006 Surface protein IsdC and sortase B are required for heme-iron scavenging of Bacillus anthracis. J Bacteriol 188 8145 8152
30. FabianMSolomahaEOlsonJSMaressoAW 2009 Heme transfer to the bacterial cell envelope occurs via a secreted hemophore in the gram-positive pathogen Bacillus anthracis. J Biol Chem 284 32138 46
31. AndradeMACiccarelliFDPerez-IratxetaCBorkP 2002 NEAT: a domain duplicated in genes near the components of a putative Fe3+ siderophore transporter from Gram-positive pathogenic bacteria. Genome Biol 3 research0047.1 research0047.5
32. GriggJCUkpabiGGaudinCFMurphyME 2010 Structural biology of heme binding in the Staphylococcus aureus Isd system. J Inorg Biochem 104 341 348
33. OuattaraMCunhaEBLiXHuangYSDixonD 2011 Shr of group A streptococcus is a new type of composite NEAT protein involved in sequestering haem from methaemoglobin. Mol Microbiol 78 739 756
34. TorresVJPishchanyGHumayunMSchneewindOSkaarEP 2006 Staphylococcus aureus IsdB is a hemoglobin receptor required for heme iron utilization. J Bacteriol 188 8421 8429
35. KimHKDeDentAChengAGMcAdowMBagnoliF 2010 IsdA and IsdB antibodies protect mice against Staphylococcus aureus abscess formation and lethal challenge. Vaccine 28 6382 6392
36. CarlsonPEJrCarrKAJanesBKAndersonECHannaPC 2009 Transcriptional profiling of Bacillus anthracis Sterne (34F2) during iron starvation. PLoS One 4 e6988
37. Stranger-JonesYKBaeTSchneewindO 2006 Vaccine assembly from surface proteins of Staphylococcus aureus. Proc Natl Acad Sci U S A 103 16942 16947
38. ChengAGKimHKBurtsMLKrauszTSchneewindO 2009 Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues. Faseb J 23 3393 3404
39. KimHKDedentAChengAGMcAdowMBagnoliF 2010 IsdA and IsdB antibodies protect mice against Staphylococcus aureus abscess formation and lethal challenge. Vaccine 28 6382 92
40. PilpaRMFadeevEAVillarealVAWongMLPhillipsM 2006 Solution structure of the NEAT (NEAr Transporter) domain from IsdH/HarA: the human hemoglobin receptor in Staphylococcus aureus. J Mol Biol 360 435 447
41. GriggJCVermeirenCLHeinrichsDEMurphyME 2007 Haem recognition by a Staphylococcus aureus NEAT domain. Mol Microbiol 63 139 149
42. SharpKHSchneiderSCockayneAPaoliM 2007 Crystal structure of the heme-IsdC complex, the central conduit of the Isd iron/heme uptake system in Staphylococcus aureus. J Biol Chem 282 10625 10631
43. VillarealVAPilpaRMRobsonSAFadeevEAClubbRT 2008 The IsdC protein from Staphylococcus aureus uses a flexible binding pocket to capture heme. J Biol Chem 283 31591 31600
44. WatanabeMTanakaYSuenagaAKurodaMYaoM 2008 Structural basis for multimeric heme complexation through a specific protein-heme interaction: the case of the third neat domain of IsdH from Staphylococcus aureus. J Biol Chem 283 28649 28659
45. GaudinCFGriggJCArrietaALMurphyME 2011 Unique Heme-Iron Coordination by the Hemoglobin Receptor IsdB of Staphylococcus aureus. Biochemistry 50 5443 5452
46. PilpaRMRobsonSAVillarealVAWongMLPhillipsM 2009 Functionally distinct NEAT (NEAr Transporter) domains within the Staphylococcus aureus IsdH/HarA protein extract heme from methemoglobin. J Biol Chem 284 1166 1176
47. HonsaESFabianMCardenasAMOlsonJSMaressoAW 2011 The five near-iron transporter (NEAT) domain anthrax hemophore, IsdX2, scavenges heme from hemoglobin and transfers heme to the surface protein IsdC. J Biol Chem 286 33652 33660
48. HargroveMSSingletonEWQuillinMLOrtizLAPhillipsGNJr 1994 His64(E7)→Tyr apomyoglobin as a reagent for measuring rates of hemin dissociation. J Biol Chem 269 4207 4214
49. LiuMTanakaWNZhuHXieGDooleyDM 2008 Direct hemin transfer from IsdA to IsdC in the iron-regulated surface determinant (Isd) heme acquisition system of Staphylococcus aureus. J Biol Chem 283 6668 6676
50. RossiMSFetherstonJDLetoffeSCarnielEPerryRD 2001 Identification and characterization of the hemophore-dependent heme acquisition system of Yersinia pestis. Infect Immun 69 6707 6717
51. LetoffeSRedekerVWandersmanC 1998 Isolation and characterization of an extracellular haem-binding protein from Pseudomonas aeruginosa that shares function and sequence similarities with the Serratia marcescens HasA haemophore. Mol Microbiol 28 1223 1234
52. ArnouxPHaserRIzadiNLecroiseyADelepierreM 1999 The crystal structure of HasA, a hemophore secreted by Serratia marcescens. Nat Struct Biol 6 516 520
53. LetoffeSDeniauCWolffNDassaEDelepelaireP 2001 Haemophore-mediated bacterial haem transport: evidence for a common or overlapping site for haem-free and haem-loaded haemophore on its specific outer membrane receptor. Mol Microbiol 41 439 450
54. OlczakTSiudejaKOlczakM 2006 Purification and initial characterization of a novel Porphyromonas gingivalis HmuY protein expressed in Escherichia coli and insect cells. Protein Expr Purif 49 299 306
55. WojtowiczHGuevaraTTallantCOlczakMSrokaA 2009 Unique structure and stability of HmuY, a novel heme-binding protein of Porphyromonas gingivalis. PLoS Pathog 5 e1000419
56. TulliusMVHarmstonCAOwensCPChimNMorseRP 2011 Discovery and characterization of a unique mycobacterial heme acquisition system. Proc Natl Acad Sci U S A 108 5051 5056
57. MazmanianSKSkaarEPGasperAHHumayunMGornickiP 2003 Passage of heme-iron across the envelope of Staphylococcus aureus. Science 299 906 909
58. MaressoAWSchneewindO 2006 Iron acquisition and transport in Staphylococcus aureus. Biometals 19 193 203
59. MazmanianSKLiuGJensenERLenoyESchneewindO 2000 Staphylococcus aureus mutants defective in the display of surface proteins and in the pathogenesis of animal infections. Proc Natl Acad Sci U S A 97 5510 5515
60. MackJVermeirenCHeinrichsDEStillmanMJ 2004 In vivo heme scavenging by Staphylococcus aureus IsdC and IsdE proteins. Biochem Biophys Res Commun 320 781 788
61. DaouNBuissonCGoharMVidicJBierneH 2009 IlsA, a unique surface protein of Bacillus cereus required for iron acquisition from heme, hemoglobin and ferritin. PLoS Pathog 5 e1000675
62. GaudinCFGriggJCArrietaALMurphyME 2011 Unique Heme-Iron Coordination by the Hemoglobin Receptor IsdB of Staphylococcus aureus. Biochemistry 50 5443 5452
63. GriggJCMaoCXMurphyME 2011 Iron-coordinating tyrosine is a key determinant of NEAT domain heme transfer. J Mol Biol 413 684 698
64. VillarealVASpirigTRobsonSALiuMLeiB 2011 Transient weak protein-protein complexes transfer heme across the cell wall of Staphylococcus aureus. J Am Chem Soc 133 14176 14179
65. VermeirenCLPluymMMackJHeinrichsDEStillmanMJ 2006 Characterization of the heme binding properties of Staphylococcus aureus IsdA. Biochemistry 45 12867 12875
66. Gilles-GonzalezMACaceresAISousaEHTomchickDRBrautigamC 2006 A proximal arginine R206 participates in switching of the Bradyrhizobium japonicum FixL oxygen sensor. J Mol Biol 360 80 89
67. Krishna KumarKJacquesDAPishchanyGCaradoc-DaviesTSpirigT 2011 Structural basis for hemoglobin capture by Staphylococcus aureus cell-surface protein, IsdH. J Biol Chem 286 38439 38447
68. DrylaAGelbmannDvon GabainANagyE 2003 Identification of a novel iron regulated staphylococcal surface protein with haptoglobin-haemoglobin binding activity. Mol Microbiol 49 37 53
69. DrylaAHoffmannBGelbmannDGiefingCHannerM 2007 High-affinity binding of the staphylococcal HarA protein to haptoglobin and hemoglobin involves a domain with an antiparallel eight-stranded beta-barrel fold. J Bacteriol 189 254 264
70. SterneM 1937 Avirulent anthrax vaccine. Onderstepoort J Vet Sci Animal Ind 21 41 43
71. KunkelTA 1985 Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A 82 488 492
72. BramanJPapworthCGreenerA 1996 Site-directed mutagenesis using double-stranded plasmid DNA templates. Methods Mol Biol 57 31 44
73. AscoliFFanelliMRAntoniniE 1981 Preparation and properties of apohemoglobin and reconstituted hemoglobins. Methods Enzymol 76 72 87
74. LaemmliUK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 680 685
75. MinorW 1997 Processing of X-ray Diffraction Data Collected in Oscillation Mode. Method Enzymol 276 307 326
76. EvansP 2006 Scaling and assessment of data quality. Acta Crystallogr D Biol Crystallogr 62 72 82
77. AdamsPDAfoninePVBunkocziGChenVBDavisIW 2010 PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66 213 221
78. EmsleyPCowtanK 2004 Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60 2126 2132
79. BartschRG 1971 Cytochromes: Bacterial. Method Enzymol 23 344 363
80. McCoyAJ 2007 Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr D Biol Crystallogr 63 32 41
81. SheldrickGM 2010 Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr D Biol Crystallogr 66 479 485
82. Collaborative Computational Project 1994 The CCP4Suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50 484 491
83. DeLanoWL 2010 The PyMOL Molecular Graphics System, 1.3 ed Schro€dinger, LLC
84. EmsleyPLohkampBScottWGCowtanK 2010 Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66 486 501
85. MurphyMJason-MollerLBrunoJ 2006 Using Biacore to measure the binding kinetics of an antibody-antigen interaction. Curr Protoc Protein Sci Chapter 19 Unit 19 14
86. HowellSKenmoreMKirklandMBadleyRA 1998 High-density immobilization of an antibody fragment to a carboxymethylated dextran-linked biosensor surface. J Mol Recognit 11 200 203
87. DementievaISTereshkoVMcCrossanZASolomahaEArakiD 2009 Pentameric assembly of potassium channel tetramerization domain-containing protein 5. J Mol Biol 387 175 191
88. BerryEATrumpowerBL 1987 Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra. Anal Biochem 161 1 15
89. JohnsonWCJr 1988 Secondary structure of proteins through circular dichroism spectroscopy. Annu Rev Biophys Biophys Chem 17 145 166
90. GreenfieldNJ 2006 Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 1 2876 2890
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