Lucia Banci CERM and Department of Chemistry University of Florence

1. NMR in SPINEStructural proteomics of metalloproteins Lucia Banci CERM and Department of Chemistry University of Florence

2. Structural Genomics From the sequence…to the function through structure Structure Sequence Function Gene knock out + protein localization + bioinformatic tools + biochemical assays Structural genomics: from gene to structure of the proteins, a complete coverage of the genomes

3. Searching for metalloproteins in genebanks A bioinformatic analysis based on Metal Binding Patterns (MBP) and sequence identity around the MBP found Copper Zinc 610 Zn-binding proteins structures from any organism in PDB They provide 490 distinct patterns 116 Cu-binding proteins structures from any organism in PDB They provide 97 distinct patterns ~2300Human zinc-binding proteins ~850Human copper-binding proteins

4. Structural biology by NMR in Florence Metalloproteinstructures solved: ca. 15 iron-sulfur proteins ca. 30 heme-proteins ca. 50 copper + zinc + calcium proteins Start of SPINE

5. Today I talk of… • Copper in Cytochrome c Oxidase assembly

6. Proteins involved in the assembly of copper centers of CCO Cox23 Cu(I) Cox23 Ctr1 Cox17 Cox17 CCO Sco1 Sco2 Cox11 Surf1 Cox19 Cox19 Mitochondria Cu(I) O2-., HO-. O2, H2O2 Cu(II), Fe(III) Cu(I), Fe(II) Fre1 Cu(I)

7. Sco1: in vivo data • Sco1 cells are respiratory-deficient. Excess Cu(II), or overexpression of either Cox17 or Sco2, cannot correct the Sco1-associated deficiency • In the absence of Sco1, Cox2 subunit is unstable and degraded • Deletion of Sco in B. subtilis depresses expression of CcO but not menaquinol oxidase (no CuA site) • Sco1 contains a potential metal-binding motif CXXXC, and both cysteines are essential for function • Eukaryotic organisms have two homologous proteins, Sco1 ad Sco2, both dimeric, while they are monomeric in prokaryotes

8. Solution structure of B. subtilis Sco1 homologue Cys 45 3 a1 2 6 1 a2 5 a3 4 His 135 Cys 49 + Cu(I) 7 1 8 C Cys 49 Cys 45 a4 His 135 N Sco1 also binds type2 Cu(II) (also EPR and EXAFS data) Banci L., Bertini I., Ciofi-Baffoni S., Cantini F., Balatri E., Structure, 2003

9. Comparison with proteins having a similar fold Apo BsSco1 TlpA, thiol:disulphide oxidoreductase HBP23, peroxiredoxin Banci L., Bertini I., Ciofi-Baffoni S., Cantini F., Balatri E., Structure 2003

10. Which is the functional role of Sco1? • Copper binding ability suggests that Sco1 is copper chaperone • Structural data suggest that Sco1 can have a disulfide reductase activity (thioredoxin fold) on the protein partner CuA site • maybe both??

11. Role of Cox17 as a Mitochondrial Copper Chaperone Sco1 inserts two copper ions to CuA site Cox11 inserts one copper ion to CuB site in vitro and cytosolic data confirm Cu transfer to Sco1 and Cox11 Yeast cells lacking the COX17 gene are respiratory deficient. Cell respiration is recovered by copper addition Horng, Cobine, Maxfield, Carr, Winge JBC 2004

12. Cox17, 10 mM DTT Cox17, 1 mM DTT Cox17 red: 6 SH on the basis of 13C carbon b shifts Cox17 ox:2 S-S bonds and 2 SH on the basis of 13C carbon b shifts Thefolding properties of apoCox17 Mixture of oxidized and reduced protein Cox17, no DTT

13. 13Ca,b 13C-13C CBCACO 13CO Proton-less 13C direct detection spectra are essential for assignment of partially unfolded proteins

14. The reduced apoCox17 apoCox17 contains a coil-helix-coil-helix (CHCH) domain and behaves as a molten globule In the reduced statethe helical secondary structure is retained Cox17 red Cytoplasm Arnesano,Balatri,Banci, Bertini, Structure 2005 CCO Sco2 Sco1 Cox11 Cu(I) Mitochondria Beers, Glerum, Tzagaloff, J Biol Chem 1997

15. X The oxidized apoCox17 In the oxidized state the cysteines form two disulfide bonds It can bind 1 eq of Cu(I) Cox17 ox Cytoplasm CCO Sco2 Sco1 Cox11 Mitochondria Arnesano, Balatri, Banci, Bertini, Structure ,2005

16. The disulfide isomerization of ox Cox17 upon Cu(I) binding CX9C Arnesano, Balatri, Banci, Bertini, Winge, Structure 2005

17. The Cu(I)4Cox17 Reduced Cox17 binds 4 Cu(I) ions in a Cu4(m-S-Cys)62-cluster and exists in a dimer/tetramer equilibrium with a 20 M Kd Cu(I)4Cox17 Palumaa,Kangur,Voronova, Sillard, Biochem. J. 2004 Cytoplasm CCO Sco2 Sco1 Cox11 Cu(I)Cox17 Arnesano,Balatri,Banci, Bertini, Structure 2005 Mitochondria

18. Cox17 mitochondrial import and copper binding S S S S SH SH IM matrix S S S S S S S S S S Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu Cu S S S S S S S S S S S S S S S S S S S S isomerization and copper binding S S S S S apoCox17 reduced S oxidative folding Cu SH Cu1Cox17 apoCox17 oxidized import SH SH SH SH TOM SH SH SH SH SH SH SH copper binding Arnesano,Balatri,Banci, Bertini, Structure 2005 Cu4Cox17 OM cytosol IMS multimerization

19. Searching Cox17 in gene-bank At variance with Sco1, Cox17 orthologs are found only in eukaryotes ! We browsed bacterial genomes to find a protein functionally equivalent to Cox17

20. Gene neighborhood analysis of cytochrome c oxidase accessory proteins CuACuB PA SO,Mdeg,Avin PP Pflu Bcep RS Reut Aq Psyr Cyt c PSPTO VV VPA Cox1 XCC XAC Cox2 CC Magn Cj, VCA Cox11 NMA, NMB DR1886 DR1885 DR Sco1 Sco1/Hyp1: - Co-occurrence - Conserved neighborhood Hyp1 A gene neighboring search identifies (Hyp1) a potential Sco1 protein partner in bacteria with a consensus motif H(M)X32HXM: a good candidate to substitute Cox17 Arnesano, Banci, Bertini, Martinelli, J. Proteom Research, 2005

21. Met 110 b5’ His 108 Cu(I) b4 Met 75 Met 86 A Sco1-related copper protein A gene neighboring search identifies a potential Sco1 protein partner in bacteria with a consensus motif H(M)X32HXM: it may substitute Cox 17? Cu(I) form apo form Cu(I) Met 110 His 108 Met 75 Met 86 The charge-state distribution of the ESI-MS peaks and CD spectra indicates that copper(I) binding produces a more compact conformational state than the apo form Banci L., Bertini I., Ciofi-Baffoni S., Katsari E., Kubicek K., PNAS 2005

22. C b5 b5’ b2 b4 b1 b4’ b3 b7 b6 N Comparison with proteins with similar fold DR1885 b1 DR1885 adopts a fold reminiscent of other bacterial extra-cytoplasmic copper proteins N b2 HxM b5 b5’ b4’ b4 M M b3 b6 b7 C Banci, Bertini, Ciofi-Baffoni, Kubicek, et. al. PNAS, 2005 cupredoxin fold CopC b1’ MxxMxHxxM b4’ b1 N b5 DR1885 might be an extra-cytoplasmic chaperone specific for copper(I) b2 b4 b5 b3 C b2 b4’ b4 b6 b1 b3 b1’ b6 b7 b7 C Cu(I) site Cu(II) site N Arnesano, Banci, Bertini, Mangani, Thompsett, PNAS, 2003

23. Cu(I) Cu(I) Cox17 Sco1-partner In prokaryots In eukaryotes Sco2 Assembly of the CuA center of CCO Sco1 CuA

24. Cox11 has an immunoglobulin-like fold with a novel type of b-strand organization ApoCox11 A linker domain of a bacterial sialidase A motile major sperm protein of Ascaris suum b- strands (gray colored) are common to all Ig-like domains.

25. Cu(I) binding in Cox11 Copper binding induces protein dimerization The importance of EXAFS in NMR structure determination of metalloproteins Cu(I)Cox11 Cys 99 3S atoms at 2.27 Å and a second copper ion located at 2.71 Å Cys 101 Banci L., Bertini I., Cantini F., Ciofi-Baffoni S., Gonnelli L., Mangani S. J. Biol. Chem. 2004

26. Assembly of the CuB center of CCO Cox17 Cu(I)Cox 11 Cu(I) CuB Cu(I)

27. The Florence contribution within SPINE project From gene to function through structure Gram-negative Bacteria Cox17 Gram-positive Bacteria SOD-like ATP ADP CadA Toxic Cadmium

28. a-Parvalbumin Calmodulin apoS100A13 MMP12 b-Parvalbumin CaS100A13 Sco1 The Florence contribution within SPINE project From gene to function through structure Cox17 Mitoch Cu8MT Cox17

29. THE END I THANK YOU FOR YOUR INTEREST AND ATTENTION!

30. Cu(I) Cys 101 Cys 99 Solution structure of Cox11 homologue from S. meliloti Cys 101 Cys 99 PDB code: 1SO9 Banci L., Bertini I., Cantini F., Ciofi-Baffoni S., Gonnelli L., Mangani S. J. Biol. Chem. 2004