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Biogenesis of [Fe-S] proteins in Escherichia coli

Frederic Barras, LCB, CNRS, Marseille. Biogenesis of [Fe-S] proteins in Escherichia coli. Marburg, 21 april 2004. Biogenesis of [Fe-S] proteins in Escherichia coli. Background The IscS system: other’s story The Suf system: our story The Csd system: the new story. Some [Fe-S] clusters.

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Biogenesis of [Fe-S] proteins in Escherichia coli

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  1. Frederic Barras, LCB, CNRS, Marseille Biogenesis of [Fe-S] proteins in Escherichia coli Marburg, 21 april 2004

  2. Biogenesis of [Fe-S] proteins in Escherichia coli • Background • The IscS system: other’s story • The Suf system: our story • The Csd system: the new story

  3. Some [Fe-S] clusters Kiley and Beinert, 2003

  4. Biological functions of [Fe-S] clusters • Electron transfer • Sulfur transfer • Sensing O2 and derivatives

  5. [Fe-S] cluster containing proteins FNR PJ Kiley

  6. [Fe-S] cluster containing proteins Aconitase/IRE-BP Aconitase [4Fe-4S] cluster IRE-binding site not accessible IRE-BP No [4Fe-4S] cluster IRE-binding site accessible

  7. [Fe-S] cluster containing proteins SoxR [2Fe-2S]2+ [2Fe-2S]2+ -35 -10 [2Fe-2S]3+ [2Fe-2S]3+ -35 -10 soxS mRNA Pomposiello and Demple, 2001

  8. How to make [Fe-S] proteins? 1- Chemist’s answer Apo-protein [Fe-S] protein Fe2+ Na2S DTT

  9. How to make [Fe-S] proteins? 2- Biologist’s answer • Source of Fe and S? • (toxicity) • Assembly of [Fe-S] cluster • (insertion and folding)

  10. Biogenesis of [Fe-S] proteins in Escherichia coli • Background • The IscS system: other’s story • The Suf system: our story • The Csd system: the new story

  11. Study of nitrogen fixation in Azotobacter vinelandii Dr. Dean’s laboratory (1993) Apo-nitrogenase [4Fe-4S] nitrogenase Fe2+ Cysteine DTT NifS

  12. Enzymatic activity of Cysteine desulfurases

  13. A. vinelandii A. Vinelandii (isc) E. coli (isc) iscR nifS iscU iscA hscB hscA fdx R. prowazekii (isc) iscS1 iscS2 iscU iscA1 iscA2 hscB hscA fdx NFS1 ISU1,2 ISA1,2 JAC1 SSQ1 YAH1 ARH1 S. cerevisiae NifS-like gene clusters Frazzon and Dean, 2003 Muhlenhoff and Lill, 2000

  14. Functions of the isc gene products iscR iscS iscU iscA hscB hscA fdx - Cysteine desulfurase [2Fe-2S] Ferredoxin [2Fe-2S] [Fe-S] cluster assembly scafold H2O2 Molecular chaperones

  15. Model ADP HscA ATP B Apo IscU Cysteine [Fe-S] IscS S Alanine Cysteine IscS S IscA [Fe-S] [Fe-S] Fdx Alanine

  16. 3 NifS-like in E. coli iscS csdA (CSD) sufS (csdB) location 53.7’ 37.8’ 63.4’ Cysteine Selenocysteine 0.38 3.1 0.9 6.2 0.02 5.5 I II II AminotransferaseClass V SSGSACTS RXGHHCA + + Structure 3D

  17. Loci containing NifS homologues in E. coli iscR iscS iscU iscA sufA sufB sufC sufD sufS sufE csdA ygdK ygdL

  18. Biogenesis of [Fe-S] proteins in Escherichia coli • Background • The IscS system: other’s story • The Suf system: our story • The Csd system: the new story

  19. Erwinia chrysanthemi Enterobacteria Plant pathogen Search for virulence genes by transposon mutagenesis Beaulieu and van Gijsegem, J Bact, 1990

  20.  ‚ ƒ pin10: suf genes RT-PCR C PCR sufC sufD sufS sufE sufA sufB ‚ ƒ  wt - FeSO4 fur - FeSO4 2000 fur + FeSO4 1500 b-glucuronidase actvity 1000 500 wt + FeSO4 0 0 200 400 600 800 min Fur regulated operon Patzer and Hantke, J Bact 1999, Nachin et al., Mol. Microbiol. 2001

  21. SufD SufB Functional Prediction sufC sufD sufS sufE sufA sufB IscA Cluster [Fe-S] formation NifS-like Cysteine desulfurase Signatures ABC ATPase ? ? ?

  22. Takahashi et al., JBC, 2002

  23. sufA sufB sufC sufD sufS sufE Erwinia chrysanthemi Archaebacteria Eubacteria Aquifex aeolicus Archeoglobus fulgidus Bacillus subtilis Methanococcus jannaschii M. thermoautotrophicum Chlamidiae pneumoniae - Pyrococcus abyssi Chlamidiae trachomatis - Pyrococcus horikoshii Deinococcus radiodurans Escherichia coli Mycobacterium tuberculosis Synechocystis spp. Thermotoga maritima Treponema pallidium Xylella fastidiosa -

  24. ABC transporter ? sufC sufD sufB No TM No TM Walker A and B boxes C region

  25. SufC exhibits ATPase activity 4,5 4 3,5 3 Vm: 4.45 mmole min-1 2,5 M . min-1 2 Km: 0.29 mM m 1,5 1 0,5 0 0 0,5 1 1,5 2 [ATP] (mM)

  26. Act LexA SufC SufB SufD Interactions SufB-SufC SufD-SufC Yeast Two-Hybrid SufC SufB SufD

  27. SufC (chromosomal) Ha-SufB (plasmidic) SufC and SufB are cytoplasmic Mbrs Peri Cyto Total Mbrs Peri Cyto Total SufC (plasmidic) Mbrs Peri Cyto Mbrs Total Peri Cyto Total MsrA MsrA OutF OutF Cel5 Cel5

  28. SufB SufC SufD Suf C: an unorthodox cytoplasmic ABC ATPase ATP ADP Nachin et al., EMBO J. 2003

  29. Fe-S cluster transfer from HoloSufA/IscA to apoBiotin Synthase. X holoSufA holoIscA (Fe2+ and S2-) Mature BioB Ollagnier de Choudens et al., JBC 2003

  30. Fe-S transfer « en bloc » ApoBioB was incubated with 5 mM DTT and a two-fold molar excess of either holoSufA (X) or holoIscA () or a four-fold molar excess of Fe2+ and S2- () and increasing concentrations of bathophenantroline. After 30 minutes incubation at 18°C, biotin synthase activity was measured.

  31. [Fe-S] SufB SufA SufC SufD Apoprotein Protein [Fe-S] ATP ADP Fe-S

  32. Biochemical analysis Units : µmol Ala / min

  33. Flexible loop Cys324 Structural studies Black: IscS from T. maritima White: SufS from E. coli Mihara, H. et al. (2002) J. Biochem. 131, 679-685 IscS from E. coli Cupp-Vickery, JR et al. (2004) J. Mol. Biol. 330,1049.

  34. SufS is activated by SufE SufS Cysteine SufS+SufE SufS+SufE Selenocysteine SufS Loiseau et al., JBC 2003

  35. Biochemical analysis

  36. A B C Sulfur transfer from SufS to SufE

  37. Alanine « S2- » SufS-S364-SH SufE-S51H Cysteine DTT SufE-S51-SH SufS-S364H

  38. Cysteine [Fe-S] SufB SufA SufC S2- SufD SufE SufS Apoprotein Protein [Fe-S] Suf : a [Fe-S] insertion machinery ATP ADP Fe-S Fe 2+ ???

  39. ratio ratio +PMS +PMS Fumarase 16+/-2 13% 9+/-2 6% Glut Synthase 313+/-1 244% 283+/-3 228% PGM 36+/-9 97% 39+/-6 111% Physiological role of Suf in E. coli sufC wt 126+/-16 138+/-13 128+/-2 124+/-4 37+/-6 35+/-10

  40. MM + glycerol MM + gluconate gluconate 6PGDH (Fe-S) GND

  41. Hypothesis Suf protects oxygen labile [Fe-S] clusters

  42. Iron acquisition in E. chrysanthemi: an essential virulence factor Chr Chr Fe3+ Chr=chrysobactin

  43. Suf and iron acquisition in E. chrysanthemi MM+dipyridyl Strains - +Fe3+chryso +FeCl 3 ± ± cbs 0 19 1 20 1 ± ± sufB cbs 0 12 1 20 1 ± sufC cbs 0 0 20 1 ± sufD cbs 0 0 20 1 Hypothesis: Suf is important for iron acquisition A basis for importance of Suf in virulence ?

  44. Biogenesis of [Fe-S] proteins in Escherichia coli • Background • The IscS system: other’s story • The Suf system: our story • The Csd system: the new story?

  45. The new story Csd ygdK ygdL Cysteine desulfurase SufE-like ThiF-like

  46. O O O ThiS ThiS ThiS OH OAMP SH Thiamine biosynthesis early steps ThiF ATP ThiF+ThiI+ThiJ IscS

  47. Molecular analysis complex Csd/YgdK/YgdL Csd YgdK YgdL

  48. Biochemical analysis

  49. Biochemical analysis Sulfur transfer Targets ? YgdL S YgdK S ? Cysteine Csd YgdL active S Alanine

  50. Perspectives • Iron source ? • Mechanistic aspects • Role of SufBCD ? • Role of HscAB ? • Role of Fdx ? • Role of IscA vs IscU? • ………… • Co-translational insertion vs post-translational repair

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