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 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.

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 Intro Why copper is important-key cofactor in many enzymes etc Copper chaperones deliver copper to specific target proteins in cells by direct protein-protein interactions Copper chaperones are needed to reserve copper for their targets since there is little free copper, also to protect against damage by copper 2 well characterized pathways shown here Atx1 delivers Cu to transport ATPases in the secretory pathway which translocate it into vesicles for insertion into multicopper oxidases 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 *These two chaperones are not interchangeable but are specific for their target proteins 2O2- + 2H+ H2O2 + O2

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

What are the details of copper binding by 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 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

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

Structure of the Cu Hah1 Protein, the Human Homolog 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 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

yCCS1 Crystal Structure Domain I (Atx1-like) Domain II (SOD1-like) metal binding not essential Domain II (SOD1-like) target recognition C C20 229CXC231 The N-terminal domain is about 70 residues and is referred to as Domain I or the Atx1-like domain We have no metal bound in the yCCS structure but Domain I contains a conserved loop that we know binds metal ions in Atx1, Hah1, and their target domains-- the metal binding residues on this loop are 2 cysteines Only essential under conditions of strict copper limitation The middle domain is about 150 residues and is referred to as Domain II or the SOD1-like domain Resembles SOD1 in overall structure but lacks metal binding sites,believed to function in target recognition Involved in dimerization of CCS, in fact one key observation from this structure is that CCS and SOD1 dimerize in the same way There are ~30 residues at the C-terminus that are disordered in the crystal structure but have been shown to be essential for SOD1 activation by CCS, proposed to play a role in metal delivery C17 Domain III metal delivery crucial Lamb, et al. Nature Struct. Biol. 1999, 6, 724-729

Dimer of Dimers Model + SOD1 homodimer is very stable 54 kDa 32 kDa 86 kDa Based on the structural data, we and others have proposed 2 models for how CCS and SOD1 interact In the first, a dimer of CCS interacts with a dimer of SOD1 to make a dimer of dimers in which copper delivery to each subunit of SOD1 occurs simultaneously In support SOD1 homodimer is very stable yCCS and hCCS are dimeric in the crystal and in solution (yCCS under some conditions)

Heterodimer Model + 54 kDa 32 kDa 43 kDa Alternatively, a monomer of CCS could form a heterodimer with a monomer of SOD1 What makes this model attractive This model exploits a conserved dimer interface between CCS and SOD1 In support Structures 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 To test these models, we investigated protein-protein complex formation between yCCS and yeast SOD1 86 kDa 43 kDa Lamb, et al. Biochem. 2000, 39, 14720-14727

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 Mutations at dimer interfaces of either CCS or SOD1 in yeast cells prevent interaction of the 2 proteins and SOD1 activation Zinc binding is known to cause conformational changes in loop regions of SOD1 and likely stabilizes both the zinc subloop and the S-S subloop The affinity of the 2 SOD1 monomers for one another is influenced by both the occupancy of the metal sites and the integrity of a disulfide bond between the S-S subloop and the beta barrel structure By analogy, the affinity of SOD1 for yCCS might depend on conformational priming of the SOD1 dimer interface region by zinc binding Binding of copper is not a prerequisite for interaction in vivo Binding of copper at a site involving Domains I and III might not affect complex formation Physiological? No higher molecular weight complexes are formed Activity appears Complex with H48F is more stable, can see wt in crosslinking but not AY Lamb, et al. Biochem. 2000, 39, 14720-14727

Crystals of the yCCS/H48F-SOD1 heterodimeric complex Used the information from the biophysical and biochemical studies to prepare a stable complex between yCCS and H48F-SOD1 for crystallization Structure solved by molecular replacement to 2.9 Å resolution 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.

Domain I Domain III Domain II H48F-SOD1 monomer yCCS monomer SOD1 homodimer yCCS homodimer Domain III Domain II Unambiguously establishes that the very stable SOD1 homodimer can dissociate to interact with its chaperone Conserved interface residues form interactions as we predicted main chain hydrogen bonds and hydrophobic interactions The conserved residues are the key elements in recognition and docking Domain III is present H48F-SOD1 monomer yCCS monomer

C146 F48 C57 C229 Instead Cys 57 forms a disulfide with Cys 299 from yCCS domain III - completely unexpected This new disulfide alters the conformation of the S-S subloop and opens up the SOD1 active sote Might be part of mechanism-- SOD1 activation by CCS does require oxygen This disulfide would have the added benefit of propping the active site open to facilitate metal ion insertion Oxidation of the cysteines might be necessary to promote release of the metal ion from Domain III into the active site Copper loading of SOD1 by CCS might be regulated by intracellular reaction oxygen levels This disulfide is not required for stable heterodimer formation Mass spec analysis of the solution heterodimer indicated no covalent linkage Lots of reductant present in solution expts and in crystallization Could have happened in month it took crystal to grow, but other cysteines in Domain I are still reduced Sulfate ion interacts with Arg 143 which plays an important role in guiding superoxide to the active site in the SOD1 homodimer Here Arg 143 no longer projects into the active site 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 Transient intermonomer disulfide formation may play a role in yCCS function His 63 His 48 His 46 His 120 This mechanism has been proposed before but the heterodimer structure establishes its feasibility for the first time Cys 229 is within 10 Å of the copper ion and SOD1 His 120 A combination of conformational flexibility and reduction of the Cys 229 - Cys 57 disulfide bond could bring both yCCS domain III Cys residues within 5 Å of the His ligands Cys 57 Cys 229 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 – 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.

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: 2 pM 5nM 2mM 3mM Metal ion: Zn2+ Co2+ Ni2+ Fe3+