principles of bioinorganic chemistry - 2003. metalloregulation of iron uptake and storage bacteria:...
DESCRIPTION
IRP Components of the Metalloregulatory System Stem- loop structure in the mRNA Iron- responsive protein (IRP) FeTRANSCRIPT
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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
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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.
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IRP
IRP
Components of the Metalloregulatory System
Stem-loop
structure in the
mRNA
Iron-responsive
protein (IRP)
Fe
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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
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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
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Metallochaperones; Metal FoldingPRINCIPLES:
•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
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2O2 + 2H+ H2O2 + O2
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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
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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
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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)
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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.
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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
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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
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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.
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Postulated Mechanism for MetallochaperoneHandoff of Copper to a Receptor Protein
(O’Halloran, Rosenzweig, Culotta, 2000)
HgAtx1 HgHah1 CuHah1 AgMenkes4
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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
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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
+
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Heterodimer Model
Structures indicate heterodimer formation is feasible
Heterodimer formation between different SOD1s has been observed
43 kDa32 kDa54 kDa
+
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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
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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
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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 Struct. Biol. 2001, in press.
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H48F-SOD1 monomer yCCS monomer
Domain III
Domain II
Domain ISOD1 homodimer
yCCS homodimer
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Domain III
Domain II
Domain I
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Loop 7 Loop 7
Two heterodimers in the asymmetric unit
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Domain III
Domain II Domain I
C17
C20
C17
C20C229
C231
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C57
C146
C57SO4
2-
S-S subloop
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C231
C229
C57
C146
F48
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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
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Metallochaperones; Metal FoldingPRINCIPLES:
•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
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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.
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X-ray Structure of a Zinc Finger Domain
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Structure of a Three Zinc-Finger Domain of Zif 268 Complexed to an Oligonucleotide Containing
its Recognition Sequence
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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.