Principles of Bioinorganic Chemistry

Metalloregulation of Iron Uptake and Storage Bacteria: A single protein, Fur (for iron uptake regulator), controls the transcription of genes involved in siderophore biosynthesis. Fur is a dimer with subunits of M r 17 kDa. At high iron levels, the Fur protein has bound metal and interacts specifically with DNA repressing transcription. Mammals: Expression of ferritin and the transferrin receptor is regulated at the translational level.

IRP Components of the Metalloregulatory System Stem- loop structure in the mRNA Iron- responsive protein (IRP) Fe

IRP Regulation events High Fe, low TfR, high Ft Low Fe, high TfR, low Ft Message translatedMessage degraded Message blockedMessage translated FerritinTransferrin Fe

IRP1 is the Cytosolic Aconitase Contains an Fe 4 S 4 Cluster Cluster assembled in protein, which then dissociates from mRNA Apoprotein stays associated with mRNA

Metallochaperones; Metal Folding PRINCIPLES: Metallochaperones guide and protect metals to natural sites Chaperone and target receptor protein structurally homologous Metal-mediated protein structure changes affect transcription Metal-mediated protein structure changes affect translation Metal-induced protein structure changes also activate enzymes Metal-induced bending of DNA affects function Metal ionic radii and M–L water bridging are used to advantage ILLUSTRATIONS: Copper insertion into metalloenzymes Zinc finger proteins control transcription Ca 2+, a second messenger and sentinel at the synapse Cisplatin, an anticancer drug

2O 2  + 2H + H 2 O 2 + O 2

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 Lys7, the gene encoding yCCS in yeast; CCS and SOD1 co-localize in human tissue Ctr, family of membrane proteins that transport copper across the plasma membrane, delivering it to at least three chaperones: CCS, Cox17, Atx1 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 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

 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?  What are the details of copper binding by these proteins, including stoichiometry and coordination geometry? Key Questions Address by Structural Bioinorganic Chemistry (Rosenzweig, O’Halloran, Culotta)

C N Cys 15 Cys 18 Hg Structure of the Hg(II) form of Atx1 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 1.2Å Structure, Active Site Val 12 Thr 14 Cys 15 Ser 16 Ser 19 Cys 18 Lys 65 Met 13 Ala 21 Hg 2.34 Å 2.33 Å

Structure of the Cu Hah1 Protein, the Human Homolog N C 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) HgAtx1HgHah1CuHah1AgMenkes4

N C 229 CXC 231 C17 C20 Domain I (Atx1-like)  metal binding  not essential Domain II (SOD1-like)  target recognition Domain III  metal delivery  crucial Lamb, et al. Nature Struct. Biol. 1999, 6, yCCS1 Crystal Structure

Dimer of Dimers Model  SOD1 homodimer is very stable  yCCS and hCCS are dimeric in the crystal and in solution (yCCS under some conditions) 54 kDa32 kDa86 kDa +

Heterodimer Model  Structures indicate heterodimer formation is feasible  Heterodimer formation between different SOD1s has been observed 43 kDa32 kDa54 kDa +

 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 Biophysical and biochemical studies of complex formation Lamb, et al. Biochem. 2000, 39, kDa86 kDa

 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 Factors Affecting Heterodimer Formation Lamb, et al. Biochem. 2000, 39,

Crystals of the yCCS/H48F-SOD1 heterodimeric complex P a = b = Å, c = Å Solved by molecular replacement Lamb, et al. Nature Struct. Biol. 2001, in press.

H48F-SOD1 monomeryCCS monomer Domain III Domain II Domain I SOD1 homodimer yCCS homodimer

Domain III Domain II Domain I

Loop 7 Two heterodimers in the asymmetric unit

Domain III Domain II Domain I C17 C20 C17 C20 C229 C231

C57 C146 C57 SO 4 2- S-S subloop

C231 C229 C57 C146 F48

 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 Mechanism of metal ion transfer Cys 231 Cys 229 Cys 57 His 120 His 48 His 63 His 46

Metallochaperones; Metal Folding PRINCIPLES: Metallochaperones guide and protect metals to natural sites Chaperone and target receptor protein structurally homologous Metal-mediated protein structure changes affect transcription Metal-mediated protein structure changes affect translation Metal-induced protein structure changes also activate enzymes Metal-induced bending of DNA affects function Metal ionic radii and M–L water bridging are used to advantage ILLUSTRATIONS: Copper insertion into metalloenzymes Zinc finger proteins control transcription Ca 2+, 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. Zn 2+ not removed by EDTA. 9 tandem repeats Zn/protein. Y or F – X – C – X 2,4 – C – X 3 – F – X 5 – L – X 2 – H – X 3,4 – H – X 2,6 CCCHHHHH 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. T d 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 K d value:2 pM5nM2mM3mM Metal ion:Zn 2+ Co 2+ Ni 2+ Fe 3+