Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Principles of Bioinorganic Chemistry - 2004 Lecture 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Date 9/9 (Th) 9/14 (Tu) 9/16 (Th) 9/21 (Tu) 9/23 (Th) 9/28 (Tu) 9/30 (Th) 10/5 (Tu) 10/7 (Th) 10/12 (Tu) 10/14 (Th) 10/19 (Tu) 10/21 (Th) TBA Lecture Topic Intro; Choice, Uptake, Assembly of Mn+ Ions Metalloregulation of Gene Expression Metallochaperones; M n+-Folding, X-linking Med. Inorg. Chem./ Metalloneurochemistry Mössbauer, EPR, IR Spectral Fundamentals Electron Transfer; Fundamentals Long-Distance Electron Transfer Hydrolytic Enzymes, Zinc, Ni, Co CO and Bioorganometallic Chemistry Dioxygen Carriers: Hb, Mb, Hc, Hr O2 Activation, Hydroxylation: MMO, ToMO Model Chemistry for O 2 Carriers/Activators Complex Systems: cyt. oxidase; nitrogenase Term Examination Reading Ch. 5 Ch. 6 Ch. 7 Ch. 8 Ch. 9 Ch. 9 Ch. 10 Ch. 10 TBA Ch. 11 Ch. 11 Ch. 12 Ch. 12 Problems Ch. 1 Ch. 2 Ch. 3 Ch. 4 Ch. 5 Ch. 6 Ch. 7 Recitations are held on Mondays at 5 PM, or a little later on seminar days, in 18-475 Ch. 8 Ch. 9 Ch. 10 Ch. 11 Ch. 12 Metallochaperones; Metal Folding PRINCIPLES: •Metallochaperones guide and protect metals to natural sites •Chaperone and target receptor protein structurally homologou 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. 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 Two Metallochaperone-mediated Cu Delivery Pathways 2O - + 2H+ HO + O 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 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 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? Structure of the Hg(II) form of Atx1 Hg Cys 15 Cys 18 C N 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. More Details of the 1.2 Å Structure, Active Site Cys 15 Thr 14 Ser 16 Val 12 2.34 Å Hg 2.33 Å Ser 19 Met 13 Cys 18 Lys 65 Ala 21 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 Extended H-Bonding Interactions Stabilize the Structure T11B C12A C15B M10A Cu C15A C12B T11A M10B T11B is conserved in most related domains. When it is not there it is replaced by His, which could serve the same function. Postulated Mechanism for Metallochaperone Handoff of Copper to a Receptor Protein (O’Halloran, Rosenzweig, Culotta, 2000) HgAtx1 HgHah1 CuHah1 AgMenkes4 N Domain I (Atx1-like) yCCS1 Crystal Structure Domain II (SOD1-like) metal binding not essential target recognition C C20 229CXC231 C17 Domain III metal delivery crucial Lamb, et al. Nature Struct. Biol. 1999, 6, 724-729 Dimer of Dimers Model + 54 kDa SOD1 32 kDa 86 kDa homodimer is very stable yCCS and hCCS are dimeric in the crystal and in solution (yCCS under some conditions) Heterodimer Model + 54 kDa Structures 32 kDa 43 kDa indicate heterodimer formation is feasible Heterodimer formation between different SOD1s has been observed 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 Lamb, et al. Biochem. 2000, 39, 14720-14727 43 kDa 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 Crystals of the yCCS/H48F-SOD1 heterodimeric complex Table 1 Crystallogra phic statistics Data collection Resolution range (Å) Unique observations Total observations Completeness (%) Rsym % > 3 (I) 12.0 - 2.9 32,933 119,535 98.8 (99.6) 0.109 (0.351) 69.9 (29.2) Refinement Resolution range 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. Number of reflections R-factor R-free Number of protein, nonhydrogen atoms Number of nonprotein atoms Rms bond length (Å) Rms bond angles (°) Average B value (Å2) 12.0 – 2.9 30,885 0.217 0.260 5,956 25 0.007 1.40 27.9 SOD1 homodimer Domain I yCCS homodimer Domain III Domain II H48F-SOD1 monomer yCCS monomer C146 F48 C57 C229 C231 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 His 63 His 46 His 120 Cys 57 Cys 229 Transient intermonomer disulfide formation may play a role in yCCS function His 48 Cys 231 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 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 –CC – X2,4 –CC – X3 – F – X5 – L – X2H– H – X3,4 H – H – X2 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. X-ray Structure of a Zinc Finger Domain Structure of a Three Zinc-Finger Domain of Zif 268 Complexed to an Oligonucleotide Containing its Recognition Sequence The Specificity of Zinc for Zinc-finger Domains Kd value: Metal ion: 2 pM 5nM 2mM 3mM Zn2+ Co2+ Ni2+ Fe3+ For [Co(H2O)6]2+ + 3/5 o - 2/5 o LFSE = -5(2/5 o) + 2(3/5 o) = -4/5 o+ 2P (small) = -7440 cm-1 (since o = 9300 cm-1) = -21.3 kcal mol-1 For [Co(Cys)2(His)2] + 2/5 t - 3/5 t LFSE = -4(3/5 t) + 3(2/5 t) = -6/5 t + 2P (small) = -5880 cm-1 (since t = 4900 cm-1) = -16.8 kcal mol-1 Thus Co2+ loses 4.8 kcal mol-1 in going from aqueous solution 2+ does not. to the zinc finger environment; Zn