1. Principles of Bioinorganic Chemistry - 2004
Recitations are held on Mondays at 5 PM, or a little later on seminar days, in

2. 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,
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,

3. 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

4. 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
2O H H2O2 + O2

5. 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

6. 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

7. 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?

8. 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

9. 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

10. 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

11. 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.

12. Postulated Mechanism for Metallochaperone
Handoff of Copper to a Receptor Protein
(O’Halloran, Rosenzweig, Culotta, 2000)
HgAtx1
HgHah1
CuHah1
AgMenkes4

13. 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,

14. 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)

15. 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

16. 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,

17. 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,

18. 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 = Å, c = Å
Solved by molecular replacement
Lamb, et al., Nature Structural Biology (2001), 8(9),

19. 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

20. 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

21. 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 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

22. 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

23. 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 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.

24. X-ray Structure of a Zinc Finger Domain

25. Structure of a Three Zinc-Finger Domain of Zif 268 Complexed to an Oligonucleotide Containing its Recognition Sequence

26. The Specificity of Zinc for Zinc-finger Domains
Kd value: 2 pM 5nM 2mM 3mM
Metal ion: Zn2+ Co2+ Ni2+ Fe3+