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Principles of Bioinorganic Chemistry - 2004

Principles of Bioinorganic Chemistry - 2004. Recitations are held on Mondays at 5 PM, or a little later on seminar days, in 18-475 . Metallochaperones; Metal Folding. PRINCIPLES:. Metallochaperones guide and protect metals to natural sites

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Principles of Bioinorganic Chemistry - 2004

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  1. Principles of Bioinorganic Chemistry - 2004 Recitations are held on Mondays at 5 PM, or a little later on seminar days, in 18-475

  2. Metallochaperones; Metal Folding PRINCIPLES: • Metallochaperones guide and protect metals to natural sites • Chaperone and target receptor protein structurally homologous ILLUSTRATION: • Copper insertion into metalloenzymes Useful references: • Copper Delivery by Metallochaperone Proteins. A. C. Rosenzweig,Acc. Chem. Res., 2001, 34, 119-128. • Perspectives in Inorganic Structural Genomics: A Trafficking Route for Copper. F. Arnesano, L. Banci, I. Bertini, and S. Ciofi-Baffoni, Eur. J. Inorg. Chem., 2004, 1583-1593.

  3. Copper Uptake and Transport in Cells The puzzles: The total cellular [Cu] in yeast is 0.07 mM, none free. How does copper find its way into metalloproteins? The implications: Mn, Fe, Zn have similar systems; understanding one in detail has implications for all

  4. Two Metallochaperone-mediated Cu Delivery Pathways 2O2- + 2H+ H2O2 + O2

  5. Two well characterized pathways Atx1 delivers Cu to transport ATPases in the secretory pathway, which translocates it into vesicles for insertion into multicopper oxidases such as ceruloplasmin Mutations in human forms of these ATPases lead to Menkes and Wilson diseases CCS delivers copper to Cu,Zn SOD Human Cu/Zn SOD is linked to ALS

  6. Copper Uptake and Transport in Cells The players: SOD, superoxide dismutase, a copper enzyme, a dimer containing two His-bridged Cu/Zn sites CCS, a copper chaperone for superoxide dismutase Ctr, family of membrane proteins that transport copper across the plasma membrane, delivering it to at least three chaperones: CCS, Cox17, Atx1 N-terminus has 8 putative Cu motifs (MXMXXM) C-terminus has 2 CXC motifs Atx1, the copper chaperone for Ccc2 Ccc2, a cation transporting ATPase; has CXXC sites Fet3, a multicopper ferroxidase Note the connection between Fe and Cu trafficking

  7. Key Questions Address by Structural Bioinorganic Chemistry (Rosenzweig, O’Halloran, Culotta) What are the details of copper binding by ¨ these proteins, including stoichiometry and coordination geometry? How do these chaperones interact with their ¨ copper receptor proteins? What features of the copper binding and ¨ protein-protein interactions render each chaperone specific for its target protein?

  8. Structure of the Hg(II) form of Atx1 Cys 15 Hg Hg(II) is exposed at the surface of the protein, which is reasonable for a protein that functions in metal delivery-- metal sites in enzymes are more buried. Hg(II) coordinated by the 2 cysteines. The apo protein has same structure but with a disulfide bonds between the cysteine residues. Cys 18 C N

  9. More Details of the 1.2 Å Structure, Active Site Cys 15 Thr 14 Ser 16 Val 12 2.33 Å Hg 2.34 Å Ser 19 Met 13 Cys 18 Lys 65 Ala 21

  10. Structure of the Cu Hah1 Protein, the Human Homolog C N First copper chaperone structure with Cu bound The two molecules are primarily held together by the bound metal ion and some hydrogen bonding

  11. Extended H-Bonding Interactions Stabilize the Structure T11B is conserved in most related domains. When it is not there it is replaced by His, which could serve the same function.

  12. Postulated Mechanism for Metallochaperone Handoff of Copper to a Receptor Protein (O’Halloran, Rosenzweig, Culotta, 2000) HgAtx1 HgHah1 CuHah1 AgMenkes4

  13. N 229CXC231 yCCS1 Crystal Structure • Domain I (Atx1-like) • metal binding • not essential • Domain II (SOD1-like) • target recognition C C20 C17 • Domain III • metal delivery • crucial Lamb, et al. Nature Struct. Biol. 1999, 6, 724-729

  14. Dimer of Dimers Model + 54 kDa 32 kDa 86 kDa • SOD1 homodimer is very stable • yCCS and hCCS are dimeric in the crystal and in solution (yCCS under some conditions)

  15. Heterodimer Model + 54 kDa 32 kDa 43 kDa • Structures indicate heterodimer formation is feasible • Heterodimer formation between different SOD1s • has been observed

  16. Biophysical and biochemical studies of complex formation • According to gel filtration chromatography, dynamic light scattering, analytical ultracentrifugation, and chemical crosslinking experiments, yCCS and SOD1 form a specific protein-protein complex • The molecular weight of the complex, ~43 kDa, is most • consistent with a heterodimer • Higher order complexes, such as a dimer of dimers, were • not detected 86 kDa 43 kDa Lamb, et al. Biochem. 2000, 39, 14720-14727

  17. Factors Affecting Heterodimer Formation • The heterodimeric complex formed with a mutant of SOD1 that cannot bind copper, H48F-SOD1, is more stable • Heterodimer formation is facilitated by zinc • Heterodimer formation is apparently independent of whether • copper is bound to yCCS • Heterodimer formation between Cu-yCCS and wtSOD1 in • the presence of zinc is accompanied by SOD1 activation • These data suggest that in vivo copper loading occurs via a • heterodimeric intermediate Lamb, et al. Biochem. 2000, 39, 14720-14727

  18. Crystals of the yCCS/H48F-SOD1 heterodimeric complex P3221 a = b = 104.1 Å, c = 233.7 Å Solved by molecular replacement Lamb, et al., Nature Structural Biology (2001), 8(9), 751-755.

  19. Domain I SOD1 homodimer yCCS homodimer Domain III Domain II H48F-SOD1 monomer yCCS monomer

  20. C146 F48 C57 C229 C231

  21. Mechanism of metal ion transfer • yCCS Domain I probably does not directly deliver the metal ion • yCCS Domain III is well positioned in the heterodimer to insert the metal ion • Transient intermonomer disulfide formation may play a role in yCCS function His 63 His 48 His 46 His 120 Cys 57 Cys 229 Cys 231

  22. Metal Folding of Biopolymers PRINCIPLES: • Metal ions organize the structures of biopolymers • In binding proteins, metal ions typically shed water molecules • In binding nucleic acids, aqua ligands remain for H-bonding • Metal-mediated biopolymer folding facilitates interactions • Cross-link formation underlies metallodrug action • High coordination numbers are used for function ILLUSTRATIONS: • Zinc finger proteins control transcription • Ca2+, a second messenger and sentinel at the synapse • Cisplatin, an anticancer drug

  23. Zinc Fingers - Discovery, Structures A. Klug, sequence gazing, proposed zinc fingers for TFIIIA, which controls the transcription of 5S ribosomal RNA. Zn2+ not removed by EDTA. 9 tandem repeats. 7-11 Zn/protein. Y or F – X – C – X2,4 – C – X3 – F – X5 – L – X2 – H – X3,4 – H – X2,6 C C C H H H H H The coordination of two S and 2 N atoms from Cys and His residues was supported by EXAFS; Zn–S, 2.3 Å; Zn–N, 2.0 Å. Td geometry. The protein folds only when zinc is bound; > 1% of all genes have zinc finger domains.

  24. X-ray Structure of a Zinc Finger Domain

  25. Structure of a Three Zinc-Finger Domain of Zif 268 Complexed to an Oligonucleotide Containing its Recognition Sequence

  26. The Specificity of Zinc for Zinc-finger Domains Kd value: 2 pM 5nM 2mM 3mM Metal ion: Zn2+ Co2+ Ni2+ Fe3+

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