Principles of Bioinorganic Chemistry - 2004

Lecture Date Lecture Topic Reading Problems1 9/9 (Th) Intro; Choice, Uptake, Assembly of Mn+ Ions Ch. 5 Ch. 12 9/14 (Tu) Metalloregulation of Gene Expression Ch. 6 Ch. 23 9/16 (Th) Metallochaperones; Mn+-Folding, X-linkingCh. 7 Ch. 34 9/21 (Tu) Med. Inorg. Chem./MetalloneurochemistryCh. 8 Ch. 45 9/23 (Th) Mössbauer, EPR, IR Spectral FundamentalsCh. 9 Ch. 56 9/28 (Tu) Electron Transfer; Fundamentals Ch. 9 Ch. 67 9/30 (Th) Long-Distance Electron Transfer Ch. 10 Ch. 78 10/5 (Tu) Hydrolytic Enzymes, Zinc, Ni, Co Ch. 109 10/7 (Th) CO and Bioorganometallic Chemistry TBA Ch. 810 10/12 (Tu) Dioxygen Carriers: Hb, Mb, Hc, Hr Ch. 11 Ch. 911 10/14 (Th) O2 Activation, Hydroxylation: MMO, ToMOCh. 11 Ch. 1012 10/19 (Tu) Model Chemistry for O2 Carriers/ActivatorsCh. 12 Ch. 1113 10/21 (Th) Complex Systems: cyt. oxidase; nitrogenase Ch. 12 Ch. 1214 TBA Term Examination

Recitations are held on Mondays at 5 PM, or a little later on seminar days, in 18-475

Metallochaperones; Metal FoldingPRINCIPLES:

•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.

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

Copper Uptake and Transport in Cells

2O2 + 2H+ H2O2 + O2

Two Metallochaperone-mediated Cu Delivery Pathways

Two well characterized pathwaysAtx1 delivers Cu to transport ATPases in the secretory pathway,which translocates it into vesicles for insertion intomulticopper oxidases such as ceruloplasmin

Mutations in human forms of these ATPases lead toMenkes and Wilson diseases

CCS delivers copper to Cu,Zn SODHuman Cu/Zn SOD is linked to ALS

Copper Uptake and Transport in CellsThe players:

SOD, superoxide dismutase, a copper enzyme, a dimer containing two His-bridged Cu/Zn sitesCCS, a copper chaperone for superoxide dismutaseCtr, 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 motifsAtx1, the copper chaperone for Ccc2Ccc2, a cation transporting ATPase; has CXXC sitesFet3, a multicopper ferroxidase

Note the connection between Fe and Cu trafficking

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 BioinorganicChemistry (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 1.2 Å Structure, Active Site

Val 12

Thr 14 Cys 15

Ser 16

Ser 19

Cys 18Lys 65

Met 13

Ala 21

Hg2.34 Å2.33 Å

Structure of the Cu Hah1 Protein, the Human Homolog

N

C

First copper chaperone structure with Cu boundThe two molecules are primarily held together bythe bound metal ion and some hydrogen bonding

Extended H-Bonding InteractionsStabilize the Structure

T11B

M10B

T11A

M10A

C12AC15B

C12BC15A

Cu

T11B is conservedin most related domains.When it is not there it isreplaced by His, whichcould serve the samefunction.

Postulated Mechanism for MetallochaperoneHandoff of Copper to a Receptor Protein

(O’Halloran, Rosenzweig, Culotta, 2000)

HgAtx1 HgHah1 CuHah1 AgMenkes4

N

C

229CXC231

C17

C20

Domain I (Atx1-like)metal bindingnot essential

Domain II (SOD1-like)target recognition

Domain IIImetal deliverycrucial

Lamb, et al. Nature Struct. Biol. 1999, 6, 724-729

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 kDa 32 kDa 86 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 complexThe molecular weight of the complex, ~43 kDa, is most consistent with a heterodimerHigher order complexes, such as a dimer of dimers, were not detected

Biophysical and biochemical studies of complex formation

Lamb, et al. Biochem. 2000, 39, 14720-1472743 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 yCCSHeterodimer formation between Cu-yCCS and wtSOD1 in the presence of zinc is accompanied by SOD1 activationThese data suggest that in vivo copper loading occurs via a heterodimeric intermediate

Factors Affecting Heterodimer Formation

Lamb, et al. Biochem. 2000, 39, 14720-14727

Table 1 Crystallographic statistics

Data collection

Resolution range (Å) 12.0 - 2.9Unique observations 32,933Total observations 119,535Completeness (%) 98.8 (99.6)Rsym 0.109 (0.351)% > 3σ(I) 69.9(29.2)

R efineme nt

R eσolutionrange 12.0–2.9Numb erofreflectionσ 30,885Rfactor 0.217Rfree 0.260Numb er of protein, nonhydrogenatomσ

5,956

Numb erofnonproteinatom σ 25Rm σbondlength(Å) 0.007Rm σbondangleσ(° ) 1.40AverageBvalue(Å2) 27.9

Crystals of the yCCS/H48F-SOD1 heterodimeric complex

P3221 a = b = 104.1 Å, c = 233.7 ÅSolved by molecular replacement

Lamb, et al., Nature Structural Biology (2001), 8(9), 751-755.

H48F-SOD1 monomer yCCS monomer

Domain III

Domain II

Domain ISOD1 homodimer

yCCS homodimer

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 57His 120

His 48His 63

His 46

Metal Folding of BiopolymersPRINCIPLES:

•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, StructuresA. 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 CC C H HHHH

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 DomainsKd value: 2 pM5nM 2mM3mMMetal ion: Zn2+ Co2+ Ni2+ Fe3+

+ 3/5 Δo

2/5Δo

LFSE=5(2/5Δo)+2(3/5Δo)=4/5Δo+2P(σmall)

=7440cm1(σinceΔo=9300cm1)=21.3kcalmol1

For[Co(H2O)6]2+

3/5Δt

+2/5Δt

LFSE=4(3/5Δt)+3(2/5Δt)=6/5Δt+2P(σmall)

=5880cm1(σinceΔt=4900cm1)=16.8kcalmol1

For[Co(Cyσ)2(Hiσ)2]

ThuσCo2+loσeσ4.8kcalmol1ingoingfromaqueouσσolutiontothezincfingerenvironment;Zn2+doeσnot.