NMR in SPINE Structural proteomics of metalloproteins Lucia

NMR in SPINE Structural proteomics of metalloproteins Lucia Banci CERM and Department of Chemistry University of Florence

Structural Genomics From the sequence…to the function through 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

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

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

Today I talk of… Copper in Cytochrome c Oxidase assembly

Proteins involved in the assembly of copper centers of CCO O 2 , H 2 O 2 -. , HO-. Cu(II), Fe(III) Cu(I), Fe(II) Fr e 1 Cu(I) Ctr 1 Cox 23 CCO Sco 1 Sco 2 Cox 11 Surf 1 Cox 19 Cox 17 Cox 19 Mitochondria Cu(I)

Sco 1: in vivo data 1. Sco 1 cells are respiratory-deficient. Excess Cu(II), or overexpression of either Cox 17 or Sco 2, cannot correct the Sco 1 -associated deficiency 2. In the absence of Sco 1, Cox 2 subunit is unstable and degraded 3. Deletion of Sco in B. subtilis depresses expression of Cc. O but not menaquinol oxidase (no Cu. A site) 4. Sco 1 contains a potential metal-binding motif CXXXC, and both cysteines are essential for function 5. Eukaryotic organisms have two homologous proteins, Sco 1 ad Sco 2, both dimeric, while they are monomeric in prokaryotes

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

Comparison with proteins having a similar fold Apo Bs. Sco 1 Tlp. A, thiol: disulphide oxidoreductase HBP 23, peroxiredoxin Banci L. , Bertini I. , Ciofi-Baffoni S. , Cantini F. , Balatri E. , Structure 2003

Which is the functional role of Sco 1? v Copper binding ability suggests that Sco 1 is copper chaperone v Structural data suggest that Sco 1 can have a disulfide reductase activity (thioredoxin fold) on the protein partner Cu. A site v maybe both? ?

Role of Cox 17 as a Mitochondrial Copper Chaperone Sco 1 inserts two copper ions to Cu. A site Cox 11 inserts one copper ion to Cu. B site in vitro and cytosolic data confirm Cu transfer to Sco 1 and Cox 11 Horng, Cobine, Maxfield, Carr, Winge JBC 2004 Yeast cells lacking the COX 17 gene are respiratory deficient. Cell respiration is recovered by copper addition

The folding properties of apo. Cox 17, no DTT Mixture of oxidized and reduced protein Cox 17, 10 m. M DTT Cox 17, 1 m. M DTT Cox 17 red: 6 SH on the basis of 13 C carbon shifts Cox 17 ox: 2 S-S bonds and 2 SH on the basis of 13 C carbon shifts

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

The reduced apo. Cox 17 contains a coil-helix-coil-helix (CHCH) domain and behaves as a molten globule In the reduced state the helical secondary structure is retained Cytoplasm CCO Sco 2 Sco 1 Cox 11 Cu(I) Cox 17 red Arnesano, Balatri, Banci, Bertini, Structure 2005 Mitochondria Beers, Glerum, Tzagaloff, J Biol Chem 1997

The oxidized apo. Cox 17 In the oxidized state the cysteines form two disulfide bonds It can bind 1 eq of Cu(I) Cox 17 ox Cytoplasm CCO Sco 2 Sco 1 Cox 11 Mitochondria Arnesano, Balatri, Banci, Bertini, Structure , 2005 X

The disulfide isomerization of ox Cox 17 upon Cu(I) binding CX 9 C Arnesano, Balatri, Banci, Bertini, Winge, Structure 2005

The Cu(I)4 Cox 17 Reduced Cox 17 binds 4 Cu(I) ions in a Cu 4( -S-Cys)62 - cluster and exists in a dimer/tetramer equilibrium with a 20 M Kd Cu(I)4 Cox 17 Palumaa, Kangur, Voronova, Sillard, Biochem. J. 2004 Cytoplasm CCO Sco 2 Sco 1 Cox 11 Cu(I)Cox 17 Mitochondria Arnesano, Balatri, Banci, Bertini, Structure 2005

Cox 17 mitochondrial import and copper binding isomerization and copper binding apo. Cox 17 reduced SH SH SH oxidative folding SH SH SH imp ort TO M SH SH SH S S SH SH cytosol S S Cu 1 Cox 17 apo. Cox 17 oxidized SH copper binding Arnesano, Balatri, Banci, Bertini, Structure 2005 S OM S Cu 4 Cox 17 Cu S Cu Cu S S IMS multimerization S Cu S Cu Cu S S Cu S S IM matrix

Searching Cox 17 in gene-bank At variance with Sco 1, Cox 17 orthologs are found only in eukaryotes ! We browsed bacterial genomes to find a protein functionally equivalent to Cox 17

Gene neighborhood analysis of cytochrome c oxidase accessory proteins PA SO, Mdeg, Avin PP Pflu Bcep RS Reut Aq Psyr PSPTO VV VPA Cu. B Cyt c Cox 1 XCC XAC CC Magn Cox 2 Cj, VCA NMA, NMB DR Cox 11 DR 1886 DR 1885 Sco 1/Hyp 1: - Co-occurrence - Conserved neighborhood Sco 1 Hyp 1 A gene neighboring search identifies (Hyp 1) a potential Sco 1 protein partner in bacteria with a consensus motif H(M)X 32 HXM: a good candidate to substitute Cox 17 Arnesano, Banci, Bertini, Martinelli, J. Proteom Research, 2005

A Sco 1 -related copper protein A gene neighboring search identifies a potential Sco 1 protein partner in bacteria with a consensus motif H(M)X 32 HXM: it may substitute Cox 17? Cu(I) form apo form Cu(I) Met 110 His 108 5’ Cu(I) 4 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

Comparison with proteins with similar fold DR 1885 C 1 N 2 M 5 5’ 4 Hx. M M 3 5 5’ 2 1 4 6 7 C 4’ 3 7 6 cupredoxin fold Banci, Bertini, Ciofi-Baffoni, Kubicek, et. al. PNAS, 2005 N Cop. C DR 1885 might be an extracytoplasmic chaperone specific for copper(I) DR 1885 adopts a fold reminiscent of other bacterial extracytoplasmic copper proteins N 1’ Mxx. Mx. Hxx. M 1 2 5 4’ C 4 4 1’ 3 7 3 2 6 1 7 6 Arnesano, Banci, Bertini, Mangani, Thompsett, PNAS, 2003 4’ 5 C Cu(I) site Cu(II) site N

Assembly of the Cu. A center of CCO Cu(I) Sco 1 -partner Cox 17 In prokaryots In eukaryotes Sco 2 Cu. A

Cox 11 has an immunoglobulin-like fold with a novel type of -strand organization Apo. Cox 11 A linker domain of a bacterial sialidase A motile major sperm protein of Ascaris suum - strands (gray colored) are common to all Ig-like domains.

Cu(I) binding in Cox 11 Copper binding induces protein dimerization The importance of EXAFS in NMR structure determination of metalloproteins Cu(I)Cox 11 Cys 99 Cys 101 Banci L. , Bertini I. , Cantini F. , Ciofi-Baffoni S. , Gonnelli L. , Mangani S. J. Biol. Chem. 2004 3 S atoms at 2. 27 Å and a second copper ion located at 2. 71 Å

Assembly of the Cu. B center of CCO Cox 17 Cu(I)Cox 11 Cu(I) Cu. B Cu(I)

The Florence contribution within SPINE project From gene to function through structure Gram-negative Bacteria Cox 17 Gram-positive Bacteria SOD-like ATP Cad. A ADP Toxic Cadmium

The Florence contribution within SPINE project From gene to function through structure Ca. S 100 A 13 Cox 17 apo. S 100 A 13 -Parvalbumin Calmodulin a-Parvalbumin MMP 12 Cu 8 MT Mitoch Sco 1 Cox 17

THE END I THANK YOU FOR YOUR INTEREST AND ATTENTION!

Solution structure of Cox 11 homologue from S. meliloti Cys 99 Cys 101 Cys 99 Cu(I) Cys 101 PDB code: 1 SO 9 Banci L. , Bertini I. , Cantini F. , Ciofi-Baffoni S. , Gonnelli L. , Mangani S. J. Biol. Chem. 2004