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 Kiley and Beinert, 2003 Trouver differents types de clusters Kiley and Beinert, 2003

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

[Fe-S] cluster containing proteins FNR PJ Kiley

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

[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

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

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

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

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

Enzymatic activity of Cysteine desulfurases

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

[Fe-S] cluster assembly scafold 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

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

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

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

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

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

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

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

Takahashi et al., JBC, 2002

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

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

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 0,5 1 1,5 2 [ATP] (mM)

Interactions SufB-SufC SufD-SufC Yeast Two-Hybrid SufB SufC SufD SufB SufC SufD LexA

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

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

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

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.

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

Biochemical analysis Vm (units/mg) SufS 0.02 Csd 1.1 IscS 0.38 Units : µmol Ala / min

Structural studies Flexible loop Black: IscS from T. maritima Cys324 Flexible loop 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.

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

Biochemical analysis Vm (units/mg) SufS 0.021 SufS+SufE 0.750 SufS(C369S) 0.0006 SufS(C369S)+SufE 0.001

Sulfur transfer from SufS to SufE B C

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

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

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

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

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

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

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

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?

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

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

complex Csd/YgdK/YgdL Molecular analysis complex Csd/YgdK/YgdL Csd YgdK YgdL

Biochemical analysis Vm (units/mg) Csd 1.1 Csd+YgdK 2.5 Csd(C61)+YgdK ND

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

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

Perspectives Substrate and/or Environmental specificity of each system ? Suf Isc Csd [Fe-S] Enz-SSH Thiamin Fe S O Fe S Fe S IscS-S-S ThiS Fe S

Apoprotein [Fe-S] Protein Oxidized [Fe-S] ROS * [Fe-S] Suf Isc

Genetic interaction between Isc and Suf Synthetic lethality iscS - DsufABCSDE Outen et al., Mol Mic, 2004 Suppression iscS - psufABCSDE Takahashi et al., JBC, 2002 Redundancy?

Apoprotein [Fe-S] Protein Oxidized [Fe-S] ROS * [Fe-S] Suf Isc

Imminoaspartate + DHAP Genetic Suppression Imminoaspartate + DHAP Quinolinic acid NAD iscRSUA NAD- /pcsdAygdK NAD+ NadA [4Fe-4S] Redundancy?

Apoprotein [Fe-S] Protein Oxidized [Fe-S] ROS * [Fe-S] Csd Suf Isc

Growth under iron limitation Strains Growth under iron limitation (Dipyridyl 0.32mM) sufA + sufB - sufC sufC + psufCK40A sufD sufS sufE sufE + psufEC51S Suf required under iron limitation

Strains Growth under iron limitation iscS + sufS + piscS - csdA ygdK sufS + pcsdA-ygdK Suf is specifically required under iron limitation

Substrate specificity Suf Isc Iron assimilation Thionucleosides Fe-S Thiamin Molybdopterin Csd

Environmental specificity Suf Isc H2O2 OxyR IscR [2Fe-2S] Fur Csd Nachin et al., 2001,, Schwartz et al., 2001, Zheng et al., 2001, Hantke, 2002 Lee et al., 2004

Sandrine Ollagnier de Choudens Actors LCB, Marseille Laurent Loiseau Laurence Nachin Collaborators INAPG, Paris Dominique Expert CEA, Grenoble Sandrine Ollagnier de Choudens Marc Fontecave