Principles of Bioinorganic Chemistry - 2003

Lecture Date Lecture Topic Reading Problems1 9/4 (Th) Intro; Choice, Uptake, Assembly of Mn+ Ions Ch. 5 Ch. 12 9/ 9 (Tu) Metalloregulation of Gene Expression Ch. 6 Ch. 23 9/ 11 (Th) Metallochaperones; Metal Folding, X-linkingCh. 7 Ch. 34 9/16 (Tu) Metals in Medicine; Cisplatin Ch. 8 Ch. 45 9/18 (Th) Electron Transfer; Fundamentals Ch. 9 Ch. 56 9/23 (Tu) Long-Distance Electron Transfer Ch. 9 Ch. 67 9/25 (Th) Hydrolytic Enzymes, Zinc, Ni, Co Ch. 10 Ch. 78 9/30 (MU) Model Complexes for Metallohydrolases Ch. 109 10/2 (MU) Dioxygen Carriers: Hb, Mb, Hc, Hr Ch. 1110 10/7 (Tu) O2 Activation, Hydroxylation: MMO, P-450, R2Ch. 11 Ch. 811 10/9 (Th) Model Chemistry for O2 Carriers/Activators Ch. 11 Ch. 912 10/16 (Th) Complex Systems: cyt. oxidase; nitrogenase Ch. 12 Ch. 1013 10/21 (Tu) Metalloneurochemistry/Medicinal Inorg. Chem.Ch. 12 Ch. 1114 10/23 (Th) Term Examination Ch. 12 Ch. 12

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 Mr 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

IRP

Components of the Metalloregulatory System

Stem-loop

structure in the

mRNA

Iron-responsive

protein (IRP)

Fe

IRP

IRP

Regulation eventsHigh Fe, low TfR, high FtLow Fe, high TfR, low Ft

Message translated Message degraded

Message blocked Message translated

Ferritin Transferrin

Fe

IRP1 is the Cytosolic AconitaseContains an Fe4S4 Cluster

Cluster assembled inprotein, which then dissociates

frommRNA

S

SFe

SFe

Fe

SR

RS

RS

SR

Fe

S

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•Ca2+, a second messenger and sentinel at the synapse•Cisplatin, an anticancer drug

2O2 + 2H+ H2O2 + O2

Copper Uptake and Transport in Cells

The players:SOD, superoxide dismutase, a copper enzyme, a dimer containing two His-bridged Cu/Zn sitesCCS, a copper chaperone for superoxide dismutaseLys7, the gene encoding yCCS in yeast; CCS and SOD1 co-localize in human tissueCtr, 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 freeHow 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 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

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 1.2Å 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 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, 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 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, 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)

Refinement

Resolutionrange 12.0–2.9Numberof reflections 30,885-R factor 0.217-R free 0.260

Number of protein, nonhydrogenatoms

5,956

Numberof nonprotei n atoms 25Rmsbondlength( )Å 0.007Rmsbondangle s (°) 1.40Average B valu e(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 Struct. Biol. 2001, in press.

H48F-SOD1 monomer yCCS monomer

Domain III

Domain II

Domain ISOD1 homodimer

yCCS homodimer

Domain III

Domain II

Domain I

Loop 7 Loop 7

Two heterodimers in the asymmetric unit

Domain III

Domain II Domain I

C17

C20

C17

C20C229

C231

C57

C146

C57SO4

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 57His 120

His 48His 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•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 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 Domains

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

+ 3/5 Δo

-2/5Δo

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

=-7440cm-1( sinceΔo=9300cm-1)=-21.3 kcal mol-1

[ (For Co H2 )O 6]2+

-3/5Δt

+2/5Δt

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

=-5880cm-1( sinceΔt=4900cm-1)=-16.8 kcal mol-1

[ (For Co Cys)2(His)2]

Thus Co2+ loses4.8 kcal mol-1 in going from aqueous solution

;to the zinc finger environment Zn2+ .does not