From the Department of Chemistry and the
§ Department of Biochemistry, Molecular Biology and Cell
Biology, Northwestern University, Evanston, Illinois 60208-3113
Received for publication, September 1, 2000, and in revised form, October 2, 2000
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ABSTRACT |
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The mechanism for copper loading of the
antioxidant enzyme copper, zinc superoxide dismutase (SOD1) by its
partner metallochaperone protein is not well understood. Here we show
the human copper chaperone for Cu,Zn-SOD1 (hCCS) activates either human
or yeast enzymes in vitro by direct protein to protein
transfer of the copper cofactor. Interestingly, when denatured with
organic solvents, the apo-form of human SOD1 cannot be reactivated by
added copper ion alone, suggesting an additional function of hCCS such
as facilitation of an active folded state of the enzyme. While hCCS can
bind several copper ions, metal binding studies in the presence of
excess copper scavengers that mimic the intracellular chelation
capacity indicate a limiting stoichiometry of one copper and one zinc
per hCCS monomer. This protein is active and unlike the yeast protein,
is a homodimer regardless of copper occupancy. Matrix-assisted laser
desorption ionization-mass spectrometry and metal binding
studies suggest that Cu(I) is bound by residues from the first and
third domains and no bound copper is detected for the second domain of
hCCS in either the full-length or truncated forms of the protein.
Copper-induced conformational changes in the essential C-terminal
peptide of hCCS are consistent with a "pivot, insert, and release"
mechanism that is similar to one proposed for the well characterized
metal handling enzyme, mercuric ion reductase.
Eukaryotic and prokaryotic cells accumulate essential first row
transition metals for various cellular functions. Copper for instance,
is maintained at a total intracellular concentration in the
10 A class of metal ion trafficking proteins is emerging that act in
distribution of essential transition metal cofactors to specific
targets in the cytoplasm, to the cell surface or integral membranes, or
to various intracellular compartments. Several newly discovered
metal-receptor proteins called metallochaperones are now known to serve
as key agents in cellular trafficking of these cofactors (2-4). These
cytosolic proteins are characterized by a metal-exchange partnership
with one or more specific intracellular target proteins and by a
metal-specific binding activity.
A prototypical member of the metallochaperone family, Atx1 of
Saccharomyces cerevisiae, is now well characterized at the
genetic, biochemical, and structural levels (5-8). This cytosolic
8-kDa protein functions to deliver copper ions to a P-type ATPase
cation transport protein (Ccc2) localized in membranes of trans-Golgi vesicles. A human homologue of Atx1, HAH1 (or Atox1), has also been
identified (9). Crystallographic and NMR solution structures of Atx1
(10)1 and HAH1 (12), as well
as the cytosolic domains of their ATPase target proteins including the
human Wilson and Menkes Disease proteins (13, 14), reveal that they
adopt a common "ferridoxin-like" Physiological incorporation of copper into the eukaryotic antioxidant
enzyme Cu,Zn-superoxide dismutase
(SOD1)2 requires the CCS
metallochaperone (copper chaperone for
SOD1) (1, 15, 16). Both yeast and human CCS proteins have
been identified, as well as potential homologues in plants and insects (17, 18). These 26-30-kDa proteins possess an Atx1-like sequence at
the N terminus, complete with the MXCXXC
metal-binding motif, which is fused to a sequence homologous to its
SOD1 target. The C-terminal region of CCS has high sequence homology
with CCS proteins from other species, and also contains two highly
conserved cysteine residues. While the latter region is essential for
CCS activity in vivo, a requirement for the Atx1-like region
is only apparent under copper-limiting conditions (17). The three
regions correspond to domain I (Atx1-like), domain II (SOD1-like), and
domain III (unique CCS sequence). Recent crystallographic structures
for yeast and human CCS show that domain II from yeast and human fold in a "Greek key" Based on a combination of genetic and biochemical experiments with
yeast CCS, mechanistic functions for each of the three domains in yeast
SOD1 activation have been hypothesized (17). An isolated peptide
corresponding to domain III of yCCS is capable of binding Cu(I) ions
and is hypothesized to function independently of domain I, which is
thought to act in copper acquisition from the cellular source. Domain
III is proposed to carry out insertion of the copper ion into
apo,(Zn)-ySOD1 (17). Domain II, with highly conserved SOD1-like dimer
interface residues (17, 19, 20), is assumed to provide a specific
interaction with SOD1 to target the delivery of copper ions. Recent
studies of the metal-binding environments in purified human and tomato
CCS support the involvement of both domains I and III in metal ion
binding (18, 22). Although these studies contend that both domains bind
metal ions simultaneously, the spectroscopic data on isolated CCS may
describe only "transport stage" of the metallochaperone as it
diffuses through the cytoplasm.
To elucidate the mechanism of SOD1 activation by CCS, we have
investigated the biochemical basis of copper transfer for human CCS
protein. Direct activation of both human and yeast SOD1 by human CCS is
demonstrated by in vitro assays with denatured apo-SOD1. Intriguingly, the denatured human SOD1 enzyme always requires the
chaperone for optimal SOD activity. Under the same experimental conditions, the yeast SOD1 can be activated by simple copper salts (1).
Furthermore, conformational changes in the human CCS protein observed
upon binding of copper support the proposal that domains I and III
carry out independent functions, with domain III directing the exchange
of copper ion to the active site of SOD1. The differences between the
biochemistry of the human and yeast SOD1/CCS partnership thus reveal
several new insights into how one family of metallochaperones execute
the direct transfer of copper ions.
Human CCS (hCCS) and hCCS Domain Truncations--
The gene for
the full-length, wild-type human CCS protein was cloned into the
expression vector pET24d (Novagen) and transformed into
Escherichia coli strain BL21(DE3). Several liters of this transformed strain were grown to A600 = 0.6 in
LB media with 40 µg/ml kanamycin and induced with 0.5 mM
isopropyl-1-thio-
Human CCS polypeptides corresponding to the Atx1-homolous
segment, residues 9-79 (referred to as the DI polypeptide), the SOD1-homologous region from
Gly77-Lys241 (DII
polypeptide), or to a truncation missing the C-terminal-most domain
Ala2-Lys241 (DI, II polypeptide) were isolated
as follows. DNA fragments corresponding to the appropriate regions
of the human CCS gene were amplified by polymerase chain
reaction and inserted into complementary sites NcoI and
EcoRI in pET 24d overexpression vector (Novagen). The
plasmid products were transformed into E. coli strain
BL21(DE3). Cultures were grown to A600 = 0.6 in
LB media with 40 µg/ml kanamycin and induced with 0.5 mM
isopropyl-1-thio- Analytical Gel Filtration Chromatography--
Human CCS proteins
were injected on Superose 12 gel filtration column (Amersham Pharmacia
Biotech) calibrated with 100 µM samples of protein
standards. Nitrogen-purged and Chelexed 50 mM Tris, pH 8.0, buffer with 100 mM NaCl was used as the elution buffer.
Standards included blue dextran (5000 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), and cytidine
(243 Da).
Metal Incorporation of hCCS and hCCS Domain Constructs in
Vivo--
The expression strains for full-length hCCS or hCCS domain
polypeptides were grown as described above, with the exception that
after 30 min of protein induction, the culture was spiked with
CuSO4 to a final media concentration of 1 mM.
After 4 h of induction, the cells were separated by centrifugation
and cell pellets were resuspended in Chelex-treated buffer (50 mM Tris, 200 mM NaCl, pH 8.0) to remove
residual media and extracellular CuSO4. Purification of
copper-bound proteins was accomplished through a combination of
freeze-thaw extraction, 0.5% streptomycin sulfate treatment, 40%
(NH4)2SO4 precipitation, and
preparative gel filtration chromatography. All buffers used during
these preparations contained fresh 1 mM DTT. Initial freeze
thaw extraction buffers also contained 0.2 mM
4-(2-aminoethyl)benzenesulfonyl fluoride to inhibit protease activity.
Ion exchange chromatography was removed from this purification
procedure to avoid stripping of metal ions from the hCCS protein constructs.
Metal Incorporation of hCCS in Vitro--
All stages of the
in vitro metal complexation of purified hCCS were carried
out under anaerobic atmosphere in a VAC Atmospheres glove box. Buffers
were thoroughly treated with Chelex to remove all trace metal
impurities prior to use. A solution of 80 µM hCCS protein
in 50 mM Tris, 200 mM NaCl, 10 mM
DTT, 10 mM reduced glutathione (GSH), 10 mM
histidine, pH 7.8, was mixed slowly with an excess of ZnSO4
and Cu(I)(CH3CN)4PF6 (final
concentration of each was 240 µM, corresponding to a
3-fold molar excess to hCCS protein monomer) followed by overnight
incubation at 15 °C. The excess metal ions, either free in solution
or bound to nonspecific sites on the protein, were first removed by
five exchanges with the same Tris/NaCl/DTT/GSH/histidine buffer in an
Amicon ultrafiltration stir cell, followed by five exchanges with a
buffer containing only 50 mM Tris, pH 8.0, to remove all
metal-binding competitors from the protein solution.
Human and Yeast Cu,Zn-Superoxide Dismutase--
Human SOD1,
holo-wild type form, was kindly donated by the laboratory of I. Bertini, University of Florence. Yeast SOD1 was overexpressed in
E. coli BL12(DE3) and purified as described previously (1).
The apo-form of hSOD1 for CCS activation assays was prepared by first
removing the bulk (about 80-85%) of the metal by dialysis against 50 mM ETDA in 100 mM NaOAc, pH 3.8, followed by
dialysis versus 100 mM MgCl2 in 100 mM NaOAc, pH 3.8, and then 100 mM NaOAc, pH
5.5. Since the apo-hSOD was found to retain 10-15% of its native activity, a further treatment was employed to reduce the residual activity of the SOD1 and therefore enhance the sensitivity for detection of SOD activation by CCS or copper controls. The
"apo"-hSOD1 obtained after dialysis demetallation (3 mg/ml, 100 µM) was incubated with a mixture of 10 mM
EDTA, 1 mM BCS, and 20 mM sodium ascorbate in
15% acetonitrile, 10% methanol, and 0.03% trifluoroacetic
acid for 3 h at 4 °C. The apo, denatured hSOD1 protein
was purified of the chelating reagents and residual metal by reverse
phase high performance liquid chromatography separation through a Vydac C4 column with H2O/CH3CN/trifluoroacetic
acid gradient elution. The protein-containing eluent was
stripped of solvent under vacuum and resuspended in Chelex-treated
potassium phosphate buffer (50 mM), pH 7.8, immediately
prior to use in SOD1 activation assays.
The yeast SOD1 protein purified from E. coli contained only
10-20% of its holo-metal content. This protein sample was therefore directly applied to the second stage demetallation/denaturation with
organic solvents and high performance liquid chromatography clean-up
for use in the activation assays.
SOD1 Activation Assays--
Design of an in vitro
assay for CCS activity poses several challenges. Based on EXAFS data
and metal-complexation methods, the copper bound to hCCS is concluded
to be in the reduced, +1 oxidation state as applied in the assay. Given
the possibility of copper-catalyzed air oxidation of the metal-binding
cysteine thiol ligands, the metal-loading and transfer events are
evaluated under strictly anaerobic atmosphere. Copper ion transfer to
apo-SOD1 in this assay is conveniently assessed by the gain of SOD
activity in classic enzyme assays, yet these require aerobic buffers to produce superoxide substrate. Another challenge inherent in
determination of CCS function is the well documented ability of
apo-SOD1 to acquire uncomplexed, or "free" copper ions from
solution without the aid of a chaperone protein. Because of this
self-activating ability of SOD1 with copper ions from solution,
additional considerations were made to distinguish between SOD1
activation by direct copper insertion mechanisms (facilitated
activation), and SOD1 activation by simple co-equilibration mechanisms
(incidental activation).
To address these challenges, an SOD1 activation assay for CCS function
has been designed with partitioned anaerobic and aerobic stages in
combination with the application of appropriate metal chelates in each
stage (schematic illustrated in Fig. 5A). The copper source
as either Cu(I)-glutathione complex or Cu-bound CCS was combined with
ZnSO4 and reduced GSH in one tube, the apo-SOD1 and BCS
chelate competitor in another. Both solutions were prepared anaerobically in deoxygenated and Chelex-treated, 50 mM
potassium phosphate buffer, pH 7.8, and the total copper concentration
was normalized for all reaction samples. The two solutions were then combined and incubated in a heating block at 34 °C for 2 h. An aliquot of each reaction mixture was extracted from the anaerobic chamber and diluted into an aerobic 50 mM potassium
phosphate buffer, pH 7.8, for assay of SOD activity by standard in-gel
assay with NBT staining (23) or cytochrome c assay (24). The
aerobic buffers (both loading and running buffers for in-gel assay)
included 1 mM EDTA to scavenge any Cu(II) formed from
oxidation of any Cu(I) source.
Proteolytic Analyses of hCCS--
The multidomain structure of
hCCS was established by partial proteolysis with trypsin. A 400-µl
solution with 1 µg/µl apoZn-hCCS was proteolyzed by 1 µg of
trypsin in 50 mM Tris, pH 8.0, for 30 min at 15 °C in an
anaerobic chamber. Proteolysis was halted after 30 min with 2 µl 6 M phenylmethylsulfonyl fluoride. Prior to extracting the
sample from the anaerobic chamber for analysis by MALDI-TOF MS, 2 µl
of 1 M DTT solution was added to prevent disulfide
cross-linking of cysteine thiols on the peptide fragments. A 3-µl
aliquot of the whole protein digest was added to a standard sinapinic
acid/H2O/CH3CN/trifluoroacetic acid matrix
solution for spotting on a MALDI-TOF sample plate. MALDI-TOF spectra of the digest mixture were collected with delayed extraction (300 ns) in
linear mode on a Voyager DE Pro spectrometer from PE Biosystems. An
internal standard of myoglobin (horse skeletal muscle, Sigma) was used
in a parallel MALDI-TOF sample spot for accurate calibration of the
digest fragment masses. Fragment masses were matched to predicted
tryptic sequences of hCCS with the MS Digest program on the
ProteinProspector web site authored by the UCSF Mass Spectrometry Facility.
To examine copper-induced conformational changes and localization of
the copper-binding site in hCCS, limited trypsin proteolysis of
E,Zn-hCCS, and Cu,Zn-hCCS was carried out with similar methods as
above. For these studies, the trypsin digest was monitored as a
function of time to elucidate relative proteolytic rates. Aliquots of
the proteolysis mixture were extracted and terminated with
phenylmethylsulfonyl fluoride at digest times ranging from 5 min to
3 h. For examination of metal content in the proteolytic fragments, the digest aliquots were mixed in a matrix solution without
trifluoroacetic acid.
Thiol Quantification/Mapping--
5,5'-Dithiobis(nitrobenzoic
acid) assay for quantifying the reduced cysteine thiols of E,Zn-hCCS
was carried out according to the published procedures of Riddles
et al. (25). Localization of the thiol and disulfide
cysteines was accomplished by first alkylating a 20 µM
hCCS protein sample (no DDT, anaerobically) with 10 mM
iodoacetamide in 6 M guanidine-HCl, 50 mM
potassium phosphate, adjusted to pH 7.2. After removal of excess
iodoacetamide and guanidine-HCl with several buffer exchanges in an
Amicon ultrafiltration stir cell, the alkylated protein was subject to
total proteolysis with 10 µg/µl trypsin at 34 °C for
20 h. The alkylated cysteine residues, indicating those not
involved in disulfide linkages on the original protein, were identified
with MALDI-TOF MS by a mass increase of 58 Da for the predicted mass of
a specific tryptic peptide. Disulfide-linked peptides were first
identified by a covalent combination of two predicted peptide masses
and then confirmed by the disappearance of the combined-peptide mass signal in a digest sample treated with DTT.
CCS Notation--
The metal-complexed forms of human CCS are
abbreviated in this report according to a similar system as that used
for Cu,Zn-SOD (26). Unlike the yeast form of the CCS protein, human CCS
binds both copper and zinc. We have designated the copper-deficient protein as E,Zn-hCCS rather than apo-hCCS, and the copper-occupied as
Cu,Zn-hCCS rather than holo-hCCS. The Cu,Zn-hCCS designation does not
represent the protein with copper bound to the potential SOD1 site
within domain II of hCCS. As shown below, the copper ions in Cu,Zn-hCCS
in fact bind to sites other than in domain II.
Human CCS Consists of Three Protein Domains in Solution--
The
human CCS protein consists of three proteolytically separable domains
in solution that closely correlate to the independent domain structure
predicted for yCCS (17, 19). MALDI-TOF mass spectrometry of a limited
trypsin digest mixture of hCCS isolated with no bound copper (E,Zn-hCCS
form) reveals three protease-resistant fragments corresponding closely
to the predicted Atx1-like domain I (dI, residues 2-76), SOD1-like
domain II (dII, residues 77-241), and the fusion of domains I and II
(dI,II, residues 2-241) (Fig. 1).
Domains determined by proteolytic susceptibility are denoted by
lowercase (i.e. dI,dII, etc.) while expressed and purified proteins that correspond to given domains are denoted by the uppercase (i.e. DI). For example, the expressed protein corresponding
to the Atx1-like region of hCCS was constructed for amino acids 9-79 while the observed trypsin fragment of hCCS corresponding to the domain
I consists of amino acids 2-76. Both position 76 and 79 are within the
expected 8-12 amino acid junction between domains I and II of hCCS
based on the crystallographically characterized yeast protein (19). The
trypsin-accessible domain junctions in hCCS are also equivalent to the
trypsin cleavage sites observed for yeast CCS protein (17).
Unlike domains I and II, the third domain (dIII) is rapidly cleaved
from the human CCS protein with either trypsin or chymotrypsin proteases, indicating that this C-terminal peptide segment has little
conformational stability in the absence of copper. There are only two
trypsin sites near the dI-dII junction (Fig. 1A): site A is
adjacent to Arg71 and site B follows Lys76.
Under a variety of proteolytic conditions, hCCS is preferentially cleaved at Lys76 between dI and dII, although the
consistent detection of a small portion of domain II fragment with
residues 72-241 indicates appreciable cleavage at Arg71.
Trypsin proteolysis at Lys76 in hCCS is homologous to the
cleavage junction between domains I and II that we previously reported
for yCCS. In tryptic digests of E,Zn-hCCS the stable dII and dI,II
fragments both terminate with the Lys241 proteolytic site
(Fig. 1A, site C).
The hCCS protein (E,Zn-hCCS) was also analyzed for cysteine disulfide
linkages with a combination of thiol quantification by
5,5'-dithiobis(nitrobenzoic acid) assay and sequence localization with
iodoacetamide alkylation and tryptic peptide mapping. Prior to
examination of thiol/disulfide content, samples of freshly purified
hCCS were transferred into a anaerobic glove box and DTT (typically at
5 mM concentration) reductant that was used in all buffers
during purification was removed by several buffer exchanges in an
ultrafiltration stir cell. 5,5'-Dithiobis(nitrobenzoic acid)
colorimetric assay of the guanidine-denatured hCCS revealed 5-6 thiols
per protein from the total 9 cysteines residues in hCCS. Alkylation of
all free thiols in hCCS with iodoacetamide followed by MALDI-TOF MS of
a total tryptic digest allows the localization of cysteine
thiol and disulfide sequence sites. The expected disulfide in the
SOD-like domain II (between Cys141 and
Cys227) is identified by a covalent combination of
peptides 113-163 and 225-232 for assignment of a fragment mass signal
at 6,301 Da (with alkyl groups for the two nondisulfide cysteines in
the 113-163 peptide). No other disulfide-linked peptides were detected between separate tryptic peptides, however, a significant population of
peptides 2-23 and 113-163, each containing three cysteine residues, were identified with only one alkylated cysteine, implicating possible
intra-fragment disulfides. A dominant mass signal of 1,769 Da for
peptide 242-255 with two alkyated cysteines indicates that the
cysteines of the CXC motif of hCCS-dIII remain entirely in
the reduced state.
In addition to the full-length hCCS protein, the biochemical properties
of polypeptides corresponding to isolated domains were examined. Human
CCS domain I, domain II, and domain I,II proteins were each expressed
and purified from E. coli to a yield of ~20 mg/liter LB
media. In contrast to the yeast CCS protein, both the copper-depleted
and copper-bound forms of full-length hCCS exhibit an apparent
molecular mass of 61.1 kDa corresponding to the dimeric species in
analytical gel filtration chromatography experiments (Table
I). The isolated hCCS-DII and hCCS-DI,II
proteins elute with apparent molecular masses of 33.2 and 46.7 kDa,
respectively, also near the expected values for dimeric species. The
isolated hCCS-DI protein, however, migrates as a monomeric polypeptide at an apparent molecular mass of 9.7 kDa.
Metal Binding Properties of hCCS--
Human CCS protein is
isolated with one equivalent of zinc (0.95 eq) per protein monomer and
no significant amount of copper (0.04 eq) when E. coli
expression is carried out under standard growth conditions (no metal
supplementation of LB media). Isolated constructs of hCCS-dI,II (DI,II)
and hCCS-dII (DII) also contain 1 eq of zinc per protein monomer (0.98 eq for DI,II and 0.92 eq for DII), whereas hCCS-dI does not. As
additional evidence for the localization of the zinc ion in expressed
full-length hCCS, the MALDI mass spectrum of a limited tryptic digest
of E,Zn-hCCS in matrix solution without trifluoroacetic acid shows a
significant +65 Da adduct on both +1 and +2 mass signals for the dII
fragment, whereas no such adduct is seen for the dI fragment (Fig.
2).
Copper loaded forms of hCCS were obtained in two ways, one of which
involved expression of the human gene in E. coli grown in
copper supplemented medium. Supplementation of the growth media with 1 mM final concentration of CuSO4 during hCCS
expression yields protein bound with 1-1.5 eq copper per isolated
protein monomer. Similar in vivo copper binding techniques
applied during expression of hCCS-dI and hCCS-dI,II resulted in
comparable copper contents in the isolated proteins. In contrast,
isolated hCCS-dII expressed with CuSO4 did not retain a
significant amount of copper.
The metal binding capacity of hCCS was also examined in
vitro with the isolated protein. Since copper ions can readily
occupy many types of sites, including those that are physiological
zinc-binding sites, a competitive introduction of an equal amount of
each metal was introduced to hCCS and incubated with excess
small-molecule metal ligands that also act as potent competitors.
Anaerobic incubation of E,Zn-hCCS with 3 eq each of
Cu(I)(CH3CN)4PF6 and
ZnSO4 was carried out under stringent competition with
excess DTT (10 mM), histidine (10 mM),
glutathione (10 mM), and NaCl (200 mM),
followed by several buffer exchanges to remove the unbound metal ions
and nonprotein reagents. After thorough buffer exchanges with and without competitor reagents, the hCCS protein retained slightly more
than 1 eq of copper (1.2 Cu per protein monomer) and nearly 1 eq of
zinc ion (0.90 Zn/protein monomer). Gel filtration chromatography of
copper loaded forms of Cu,Zn-hCCS prepared by either in
vitro or in vivo methods indicated that most of the
protein remained a dimer, although a small fraction repeatedly migrated
with a higher apparent mass (molecular mass = 118 kDa) consistent
with a tetrameric hCCS species (molecular mass = 115.6 kDa) (Fig.
3). The higher apparent mass fractions
were found to contain approximately twice as much copper as monomer
protein, whereas the dimeric fractions contained closer to 1 eq of
copper per protein monomer. Zinc concentrations in these fractions
followed a 1:1 Zn:monomer protein stoichiometry throughout all gel
filtration fractions. The higher and lower mass fractions were
collected separately for the SOD1 activation assays described
below.
Samples of in vitro Cu-loaded hCCS (1.2 equivalents per
monomer) were separated into domains by limited trypsin proteolysis and
analyzed by MALDI-TOF MS in the absence of added acid. Under these
conditions, metal-protein interactions were observed. As in the
MALDI-TOF mass spectrum of trypsin digested E,Zn-hCCS, the spectrum for
the trypsin digest of Cu,Zn-hCCS reveals a zinc adduct for the domain
II fragment and no additional adduct for copper (Fig. 2B).
Control samples of holo bovine-SOD1 indicate that both the copper and
zinc complexes with the protein can be observed by MALDI-TOF MS (Fig.
2C). Therefore, none of the entire 1.2 eq of bound copper in
the in vitro reconstituted hCCS protein is bound in the
canonical copper site of SOD1. It is thus presumed that all the copper
is complexed in sites that incorporate residues of domain I and/or III.
Copper-induced Conformational Changes in hCCS--
A clear
difference in hCCS protein conformation was observed with comparison of
time-resolved proteolytic maps of E,Zn-hCCS and Cu,Zn-hCCS. As we have
reported for the yeast CCS protein (17), domain III is more slowly
cleaved from hCCS by either trypsin or chymotrypsin when copper is
bound to hCCS (data not shown). Two specific copper-induced
conformational changes in hCCS are observed by comparison of tryptic
maps from E,Zn-hCCS and Cu,Zn-hCCS (Fig.
4). First of all, cleavage after
Arg71 is significantly inhibited upon copper loading,
implicating that this segment of hCCS is likely to be important in
copper manipulation by domain I. The second difference in the tryptic
profiles is the appearance of a stabilized 77-255 fragment in the
time-resolved digest of Cu,Zn-hCCS. This fragment, corresponding to dII
and much of dIII, is never a dominant species in the time-resolved digest of E,Zn-hCCS (trypsin cleavage at site D, Fig. 1A).
The stabilized portion of hCCS-dIII with copper binding includes the CXC potential metal binding motif and the segment of dIII
with highest sequence homology to CCS proteins from various other
organisms (17).
Human CCS Activates Human and Yeast SOD1 in Vitro--
An SOD1
activation assay was designed to measure the activity of the purified
hCCS metallochaperone in vitro. The assay procedure is
summarized by the scheme in Fig.
5A and described in detail under "Experimental Procedures." The ability of the various copper donors to activate apo-hSOD1 in this reaction system was assessed by
the increase in SOD1 activity as measured by the standard native PAGE,
in-gel NBT qualitative assay and by the more quantitative cytochrome
c kinetic assay. Both in vivo and in
vitro reconstituted Cu,Zn-hCCS can activate hSOD1 in this in
vitro assay in the presence of excess chelate competitor, BCS
(Fig. 5). As expected from previous studies with yCCS, the reduced
glutathione complex of Cu(I) (Cu-GSH) is incapable of hSOD1 activation
with BCS competitor present. Furthermore, the visible absorbance of the
Cu-GSH/apo-hSOD1 reaction mixture with BCS at 483 nm
(
A significant difference between human and yeast SOD1 enzymes in this
activation assay is the low activity that resulted from apo-hSOD1
exposed to Cu-GSH complex without BCS competitor present (Fig. 5, B and C, sample 3). Yeast SOD1 that had
been inactivated through denaturation, demetallation, and subsequent
high performance liquid chromatography purification demonstrated the
ability to reactivate with an unchallenged Cu-GSH supply to a level
comparable to that from Cu-yCCS activation (1). However, human SOD1
that was inactivated with similar denaturing procedures for these
experiments did not show this ability to efficiently reactivate with a
simple Cu(I) complex.
To further test the correspondence of these biochemical results to
features of the CCS activity observed in the cell, hCCS was tested for
the ability to activate the yeast SOD1 (ySOD1) enzyme in
vitro. As was previously shown by yeast complementation studies
in vivo (15), Cu,Zn-hCCS is capable of activating this nonphysiological partner protein in vitro (Fig.
6). Efficient ySOD1 activation by
Cu,Zn-hCCS in the presence of excess BCS demonstrates that this
activation must also occur via direct transfer of copper ion.
Human CCS Itself Does Not Have SOD Activity--
The remarkable
similarity of the second domain of hCCS to the SOD1 target (15, 20)
raises the possibility that this metallochaperone itself can act as an
SOD catalyst. Recent studies from Culotta and co-workers (28) have
shown that lysates from yeast expressing wild-type hCCS do not show SOD
activity attributable to the hCCS protein, although a D201H
mutation of hCCS yields an SOD-active protein (28). SOD activity assays
of both purified Cu,Zn-hCCS and purified hCCS-DII incubated with
CuSO4 and ZnSO4 in vitro indicate
that neither are SOD active (Fig. 7),
corroborating the in vivo observations. It is thus unlikely
that the SOD1-like domain of hCCS functions physiologically as an
SOD-like enzyme.
Human CCS Activates SOD1 by Direct Insertion of the Copper Ion
Cofactor--
The in vitro activation results demonstrate
direct transfer of copper from the hCCS metallochaperone to hSOD1 and
furthermore, reveal that transfer occurs through an intermediate in
which the copper is bound by both proteins. When low
Mr copper donors such as the copper-glutathione
complex are mixed with apo-SOD1 in the presence of stringent copper
chelating agent BCS, any released or loosely bound copper ion is
quickly scavenged in this reaction system. Based on this control, it is
clear that any copper released to solution by Cu,Zn-hCCS would also be
effectively trapped as the Cu(BCS)2 complex. We conclude
that activation of hSOD1 by Cu,Zn-hCCS in this in vitro
reaction system must occur without release of copper by either protein
during the transfer process. A direct transfer process has also been
shown in yeast. This allows for protection of the cofactor from
competing intracellular chelators that otherwise maintain free copper
concentrations below the level of one atom per cell (1).
The lack of significant activation of hSOD1 by the Cu-GSH in the
absence of BCS competition stands in contrast to the yeast SOD1
system (1). As with most SOD1 enzymes, hSOD1 demetallated by
nondenaturing methods such as dialysis against excess EDTA is readily
reactivated by simple copper and zinc salts. However, hSOD1 cannot be
reactivated in this manner when it has been denatured with organic
solvents and reducing agents. This unexpected result implies that the
complete function of the human CCS metallochaperone may be more complex
than copper delivery alone. The human SOD1 enzyme is notably less
stable to standard chloroform-ethanol purification procedures than the
yeast and bovine forms of the protein (29, 30). Given that organic
solvents were employed in these experiments for optimal inactivation of
the apo-enzyme prior to application in the in vitro assay,
it is likely that apo-hSOD1 prepared in this manner cannot refold
properly upon addition of copper salts alone. The fact that Cu,Zn-hCCS
is able to activate this pre-denatured form of apo-hSOD1 may indicate
that hCCS serves an additional mechanistic role. This raises the
possibility that hCCS also functions to stabilize an optimal folding
state of hSOD1 or catalyze the formation of the conserved disulfide
within the hSOD1 enzyme.
Chemical mechanisms for direct copper transfer from a metallochaperone
have been reported for Atx1 and its physiological partners (5, 7, 10,
12). We have previously proposed that CCS metallochaperones transfer
copper ions to apo-SOD1 through a similar series of ligand exchange
steps wherein the metal is never required to be released into solution
as a free ion (1, 17, 19). Below we discuss two aspects of how the
direct transfer of copper ion might occur: 1) the location and ligand
environment of the copper bound to CCS, and 2) the orientation of this
copper site relative to the active site in SOD1 when docked to CCS.
Interaction of Copper Ion with Human CCS--
The copper binding
and proteolytic protection data for hCCS are consistent with the
two-hand mechanism of copper manipulation previously proposed for yeast
CCS (17). Metal binding experiments indicate that in the presence of
stringent physiological competitors, copper can bind to the cysteine
motifs of both domains I and III. Furthermore, structural studies
indicate that the substitution of an aspartate for a single
SOD1-homologous copper ligand (histidine) may greatly decrease the
affinity of the dII site in hCCS for copper ions (20).
Data for the copper bound state of human CCS described here
corroborates our earlier results with yeast CCS (17), and are also
consistent with the conclusions from Co(II)-substitution experiments of
tomato CCS (18) and Cu-EXAFS studies human CCS (22); however, there
remains a question of whether hCCS carries only one copper ion at a
time, or two (perhaps more) copper ions as seen in some samples
prepared for EXAFS studies (22). Although observations from all of
these studies are consistent with the involvement of both domain I and
domain III in copper ion manipulation by hCCS, none resolve the
possibility that CCS proteins may adopt several different
copper-binding modes to carry out the individual stages of its function
including: (a) copper-acquisition from source,
(b) protection of copper during transport, and
(c) insertion of copper into hSOD1 target (Scheme
1).
Key insights into the mechanism of hCCS interaction with copper ions
are also revealed by the differential proteolytic protection experiments. First of all, protection of Arg71 upon copper
binding to hCCS is consistent with a movement of this residue to a site
near the metal ion. A similar copper-induced change is observed for the
homologous residue (Lys65) in the NMR structural studies of
the Cu(I) complex of Atx1(11) and the x-ray structure of Hg-Atx1(10).
This highly conserved basic residue is found in both families of copper
chaperones but is substituted with a hydrophobic aromatic residue in
homologous domains of the copper-acceptor proteins (13). Furthermore,
mutation of this residue can disrupt the function of Atx1 in
vivo (8). These observations have led us to conclude that the
basic residue at this position can protect the copper before the
protein docks with partner and play a switching role in the release
steps (12). Without a metal ion bound at this site in dI, the loop
containing Arg71 must either be significantly more flexible
in conformation or oriented in a more solvent-exposed position. The
occlusion of Arg71 backbone induced by metal ion binding to
this site suggests similar movement to that observed in the apo and
Cu-Atx1 solution structures (11).
Copper Binding Alters Domain III Structure--
Although very
little is known about the mechanistic function of the third domain of
CCS, it is essential to its activity in vivo. Copper binding
to hCCS induces stabilization of the dII-dIII junction along with a
portion of domain III extending through the CXC motif (Fig.
4). Interestingly, this stabilization does not require the presence of
dI, suggesting that dIII is capable of binding copper independently of
dI. This observation may also reflect an association of the
CXC-containing portion of dIII with dII upon copper binding. Given the
high sequence homology and very similar overall structure between
hCCS-dII and the hSOD1 target (20), we speculate that such an
interaction of dII and dIII upon copper binding emulates the
interaction of copper-loaded dIII with the SOD1 target enzyme. In this
model, dIII would be the sole portion of the CCS protein responsible
for insertion of the copper ion into SOD1. This copper insertion
mechanism is further supported by in vivo studies in yeast
that showed a yCCS-dII,III truncated protein is still capable of ySOD1
activation in vivo, whereas the yCCS-dI,II is not (17).
Models of a docked yeast CCS·SOD1 complex reveal that a domain
III extension from the C terminus of domain II to the CXC
motif is capable of spanning the distance to the CXXC copper
site in domain I as well as to the SOD1 copper active site (19).
Precedent for Metal Insertion by Cysteine Residues of a C-terminal
Domain--
Enzymatic and structural studies of Hg(II) transfer into
the flavin-containing active site of mercuric ion reductase (MerA) (31-33) provide a framework for understanding metal transfer between CCS and SOD1. While this protein shows little overall homology to CCS,
analysis of its domain structure reveal several striking similarities
(Fig. 8A). Like CCS,
biochemical and structural studies of MerA distinguish three separate
regions in the protein structure. The N-terminal most domain is
homologous to MerP (35% identity), which has the same fold as Atx1 and
includes the MXCXXC metal binding motif. The next
domain or "core" is highly homologous to glutathione reductase and
has a pair of mercury-binding cysteine residues in the active site.
Finally, the homodimeric protein has a 15-amino acid C-terminal
extension from the core of one monomer that is embedded into the
mercury-binding active site of the partner monomer (Fig. 8,
B and C) (34). As with CCS, the N-terminal domain
is not essential for activity and the C-terminal extension contains a
pair of highly conserved cysteines that are essential for activity. In
the case of MerA, these residues are involved in steady state turnover
of the MerA enzyme (35). This C-terminal domain of MerA exhibits
significant similarities to the CCS-dIII sequence, including a
conserved spacing from the end of the large second domain to the
proposed metal binding motifs: Cys-X-Cys for copper in CCS
or Cys-Cys for mercury in MerA (Fig. 8B).
The function of the C-terminal protein domain of MerA in metal ion
transfer has been demonstrated most recently in a study by Engst and
Miller (31) that concluded the C-terminal cysteines facilitate the
movement of Hg(II) into the buried active site of an adjacent monomer
(Fig. 8D). If the cysteine residues in the C-terminal domain
of MerA are mutated, cells lose resistance to mercury and only Hg(II)
complexes with small ligands such as chloride can serve as substrates.
Even low molecular weight exogenous thiols are too bulky and prevent
entry of the Hg(II) into the active site of truncated MerA. In
contrast, the full-length protein can readily obtain Hg(II) from most
donors, presumably by initial binding to the CC motif in this
C-terminal extension which subsequently hands off the metal to the
active site thiols. The CXC motif of domain III of CCS is
likely to play a similar role in delivering copper to the buried active
site of SOD1. These and other features of the biochemistry, metal
binding, structure, mutagenesis, and function of the Hg-MerA homodimer
provide precedents for the mechanism of CCS proposed below.
Proposed Mechanism of SOD1 Activation by hCCS--
We have
established that hCCS activates either human or yeast apo-SOD1 by
direct insertion of the copper ion cofactor, and propose further that
the transfer process occurs through the cysteine pair of domain III via
ligand exchange with copper-binding residues of the SOD1 active site.
Since the CXXC region of domain I can also be involved in
copper binding to hCCS and is proposed to act in the acquisition of
copper ions from an unknown source (17), domain III could interact
alternatively with the dI site and the SOD1 target site in a presumed
switch-like translocation of the copper ion (Fig.
9A). A key issue yet to be
resolved is the orientation of interaction between the CCS and SOD1
proteins that allows this direct copper transfer to occur.
One significant difference in the solution properties of the human and
yeast CCS proteins is that hCCS is a dimer regardless of copper content
or protein concentration. The apo-form of yeast CCS protein is
monomeric, but forms a mixture of monomers and dimers upon binding
copper ion (17). Despite the difference in oligomerization state of the
metallochaperone itself, hCCS is able to act as yCCS in activation of
ySOD1 in the in vitro assay. Taken together, these
observations leave open two mechanistic possibilities after the
homodimeric Cu,Zn-hCCS encounters the homodimeric apo-hSOD1: 1)
Cu,Zn-hCCS simply activates apo-hSOD1 enzyme through a dimer-to-dimer
transfer of copper ion, or 2) the Cu,Zn-hCCS and apo-SOD1 dimers
"swap" monomers to yield a pair of heterodimers or a heterotetramer
prior to copper transfer (17, 18, 20, 21) (Fig. 9B). In the
first scenario, disruption of the stable dimeric form of apo-SOD1 is
unnecessary, and localization of a dI-dIII co-complex of copper can be
envisioned at a site adjacent to the copper-binding site of SOD1 in a
model tetrameric complex (21). The second mechanistic possibility is
supported by the conservation of residues responsible for SOD1
dimerization in all CCS proteins, and modeling studies demonstrating
that dIII can access both dI and the active site of the adjacent SOD1
in a docked heterodimer that uses this interface (20).
The thermodynamic stability of the interaction between SOD1 monomers
has been argued to support the first scenario, yet it is not
necessarily grounds for eliminating the second. Although the interface
between monomers of SOD1 is known to be very strong in a thermodynamic
sense, exchange of monomers between dimeric proteins can nonetheless be
kinetically facile, especially if the two have similar dimerization
interfaces. It has been shown that tight homodimers of similar Greek
key
The necessity for the involvement of metal binding residues of domain I
in direct ligand exchange with residues of the SOD1 active site is also
unfounded. This proposed exchange is incompatible with the ability of
the yCCS lacking domain I to activate ySOD1 in vivo (17).
The requirement for dI of yCCS is only observed when copper
concentrations are limiting in the growth media. In addition, a close
inspection of the putative CCS sequence from Drosophila
melanogaster (gene product CG17753) reveals that this protein
lacks the CXXC motif in domain I while maintaining high homology to hCCS throughout the remaining portions of the protein.
Given the data in hand, we favor the second mechanism wherein monomers
are swapped prior to copper transfer and the resulting heterodimer
interface is established that involves the most highly conserved
residues between SOD1 and CCS (19, 20). The domain III peptide in this
docked complex will have access to, but not does not require the
presence of, the metal-binding site of domain I. A pivoting action at
the domain II,III junction would allow movement of this key metal
binding peptide to the active site of the adjacent SOD1 enzyme active
site. Repositioning of the copper in the CXC motif of CCS
domain III proximal to a histidine residue in the SOD active site could
then account for the direct ligand exchange transfer of copper from
metallochaperone to target. The MerA system provides precedence for the
extension of a metal-binding domain from one protein into another and
leads to several testable postulates. The first conserved aromatic
residue in the third domain of each protein (corresponding to
Phe237 in hCCS or Phe619 in RC607 MerA) is
positioned to serve as an anchor around which the remainder of the
flexible C-terminal peptide might pivot and extend into the active site
of the adjacent monomer (Fig. 9A). In the case of CCS, the
dIII peptide would bind copper, pivot toward the adjacent partner,
insert into the active site in a "pivot, insert, and release"
mechanism. It remains to be seen how the copper might be released from
a thiol-rich site in domain III to the nitrogen-rich site in SOD1.
Conclusions--
Results from these biochemical investigations on
the human CCS protein suggest that this metallochaperone can adopt
several conformations to fulfill its role in delivery of copper to
SOD1. The "copper acquisition state," wherein CCS obtains copper
from a yet to be identified donor, most likely involves domain I since copper-rich growth conditions render this domain unnecessary in vivo (17). The "transiting state," in which CCS protects the copper from metal-binding scavengers in the cellular environment, most
likely corresponds to the copper loaded forms of the holo protein
examined in these in vitro studies. Here, in the absence of
the donor or target enzyme, the copper is most likely bound by the
cysteine motifs of both dI and dIII. Last, a "copper insertion state" is induced upon capture of the target protein which releases the copper ion to dIII and subsequently to the SOD1 active site. The
observed conformational changes upon copper binding to hCCS and the
mechanistic precedents in the analogous MerA metalloprotein support a
proposal that dIII is independently responsible for the ligand-exchange
transfer of copper to the SOD1 active site.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4-10
5 M range. Despite this
abundance, it has recently been shown that copper ions are typically
unavailable in the cytoplasm for direct substitution into
metalloenzymes. In fact, the steady state concentration of the free or
labile form of copper ion in the cytoplasm of yeast is far less than
one ion per cell (1). This finding suggests that the intracellular
milieu has a significant overcapacity for metal chelation, and raises
the issue of how apo-proteins acquire the correct metal cofactor.
fold. In each
structurally characterized case, the metal ion is bound by two cysteine
thiolate moeities in a highly conserved MXCXXC sequence motif. A mechanism has
been proposed that allows for rapid metal exchange between the
otherwise tightly bound Cu(I)-protein complexes involving formation and
decay of two- and three-coordinate complexes of the Cu(I) ion with the
cysteines of both proteins (5, 7, 12). Both acquisition and delivery of
the copper ion between these proteins is expected to occur at the
single copper-binding site, although this may not necessarily be the case for the more complex metallochaperones.
-barrel conformation that is quite similar to its target, SOD1 (19-21). The x-ray structure of yeast CCS solved by
Rosenzweig and co-workers (19) also reveals a fold for domain I that is
similar to Axt1. To date, no structural data is available for either
domain III or a copper-bound form of any CCS protein.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-D-galactopyranoside. Purification of hCCS was accomplished through freeze-thaw extraction with 0.2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride
protease inhibitor in the extraction buffer, followed by precipitation with 40% ammonium sulfate, salt removal with several buffer exchanges, anion chromatography through Uno-Q12 (Bio-Rad), and gel filtration chromatography through Superdex 75 26/60 column (Amersham Pharmacia Biotech). The purified full-length hCCS protein was confirmed by a mass
of 28,906.4 ± 4.7 Da determined by ESI mass spectrometry, which
corresponds to the mass predicted for hCCS missing the N-terminal methionine residue (28,909.4 Da). In this report, residue numbering is
maintained with methionine as residue 1, giving a sequence of 2-274
for the isolated protein we refer to as full-length human CCS. A
correction factor for the Bradford protein assay,
[hCCS]actual = {[hCCS]apparant/1.85}
versus IgG protein standard, was applied to calculations of
hCCS concentrations for all analyses.
-D-galactopyranoside. Purification of
hCCS DI, II, or DII was accomplished through freeze-thaw extraction
followed by precipitation with 40% ammonium sulfate, salt removal with
several buffer exchanges, anion chromatography through Uno-Q12
(Bio-Rad), and gel filtration chromatography through Superdex 75 26/60
column (Amersham Pharmacia Biotech). Purification of the domain I
polypeptide was accomplished through freeze-thaw extraction followed by
salt removal with several buffer exchanges and gel filtration
chromatography through Superdex 75 26/60 column (Amersham Pharmacia
Biotech). The masses of the purified hCCS polypeptides were confirmed
by ESI mass spectrometry (hCCS-DI ESI/MS = 7,555.6 ± 0.26, predicted = 7,555.7; hCCS-DI, II ESI/MS = 25,320.9 ± 0.97, predicted = 25,322.3; hCCS-DII ESI/MS = 17,365.3 ± 1.30, predicted = 17,371.4).
RESULTS
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ABSTRACT
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DISCUSSION
REFERENCES
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Fig. 1.
Human CCS consists of three structural
domains in solution. A, human CCS sequence
homologies to human Atx1, human SOD1, and yeast CCS. Known or suspected
metal-binding residues are boxed. Discussed tryptic
proteolysis sites are marked with triangles. Alignment of
hCCS domain II to human SOD1 is based on structural data from x-ray
crystallography (20). B, MALDI-TOF mass spectrum of a
limited trypsin digest of E,Zn-hCCS. Masses determined: polypeptide
2-241 (dI,II) MALDI-TOF MS = 25,327.3 Da, predicted 25,323.5 Da;
polypeptide 77-241 (dII) MALDI-TOF MS = 17,372.5 Da,
predicted = 17,371.4 Da; polypeptide 2-76 MALDI-TOF MS = 7,965.8 Da, predicted = 7,971.1 Da.
Characterization of the oligomeric state of hCCS and its domains by
analytical gel filtration
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Fig. 2.
Metal content in trypsin-separated domains of
hCCS. A, portions of the MALDI-TOF mass spectrum of
trypsin-proteolyzed E,Zn-hCCS from a nonacidified matrix solution (no
trifluoroacetic acid). B, portions of the MALDI-TOF mass
spectrum of trypsin-proteolyzed Cu,Zn-hCCS (1.2 eq Cu:protein) in a
nonacidified matrix solution. C, MALDI-TOF mass spectrum of
holo-SOD1 (bovine) in a nonacidified matrix solution. All adduct
signals assigned to metal-protein ions were separated by 64 ± 2 mass units from the apo-protein ions.
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Fig. 3.
Cu,Zn-hCCS metal content through gel
filtration chromatography. 100 µM Cu,Zn-hCCS was
analyzed with a calibrated gel filtration column as described (Table
I). The protein eluted as two species, tetrameric and dimeric. The
fractions containing each species were confirmed as hCCS by
SDS-polyacrylamide gel electrophoresis and the protein content was
measured by Bradford assay using a standard correction factor. Copper
concentrations were measured by graphite furnace atomic absorption
(GFAA) spectroscopy and zinc concentrations were measured by ICP AES.
Human CCS protein concentrations are displayed as molarity of protein
monomer.
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Fig. 4.
Copper binding to hCCS induces conformational
changes in the protein structure. A, yeast CCS
x-ray crystal structure highlighting significant tryptic sites between
domains I and II and potential metal-binding residues of domain I. In
parentheses are the homologous residues in human CCS.
B, the domain II mass region of the MALDI-TOF spectrum of
E,Zn-hCCS versus Cu,Zn-hCCS. Labeled masses in both spectra
were stabilized fragments throughout the time course-sampled trypsin
digest of each protein. Spectra shown are of the 30-min time point
aliquots of each protein digest.
483 = 12, 250 M
1
cm
1 for Cu(BCS)2 (27)) yields a concentration
for Cu(BCS)2 complex of 9.7 µM, indicating
that essentially all the Cu(I) ion supply from the original Cu(I)-GSH
complex was acquired by BCS rather than the apo-SOD1 target. Cu,Zn-hCCS
collected from gel filtration fractions at either the apparent dimeric
mass or tetramer mass (Fig. 3) activated apo-hSOD to comparable levels.
Cu,Zn-hCCS prepared by in vitro copper loading of the
isolated protein also activated apo-hSOD to a similar extent (not
shown).
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Fig. 5.
Human CCS activates human SOD1 in
vitro. A, reaction scheme for the
in vitro assay of CCS activity. Concentrations listed are
those for each component in the final SOD1 activation reaction
solution. B, human SOD1 activation assayed by NBT staining
of the reaction mixtures on native-PAGE. Sample 5 included Cu,Zn-hCCS
from larger-sized gel filtration chromatography fractions
(~tetramer), sample 6 included the smaller-sized Cu,Zn-hCCS fractions
(~dimer). The holo-hSOD1 marker lane contained ~75 ng of enzyme.
The total hSOD1 protein applied in lanes 2-6 was equal and
estimated at 300 ng. C, human SOD1 activation assayed by
cytochome c kinetic assay of the reaction mixtures. Residual
activity from the apo-hSOD1 control (sample 2) is subtracted from all
SOD1 activity values so that the values shown reflect hSOD1
activation by each copper source.
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Fig. 6.
Human CCS actives yeast SOD1 in
vitro. Native polyacrylamide gel
electophoresis/nitro blue tetrazolium assay of apo-ySOD1
activation by the indicated copper sources. Approximately 50 ng of
holo-ySOD was applied to lane 1 as a marker. Identical
amounts of all reaction mixtures were applied to lanes 2-4,
and the gel load of total ySOD1 protein in these lanes was estimated at
300 ng.
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Fig. 7.
Human CCS is not an SOD catalyst.
Gel filtration chromatography fractions corresponding to tetrameric or
dimeric Cu,Zn-hCCS were evaluated for SOD acticity by native
polyacrylamide gel electophoresis/nitro blue tetrazolium assay.
Samples were incubated with all supplemental reagents used for the
in vitro SOD1 activation reactions (10 µM
ZnSO4, 1 mM GSH, and 200 µM BCS).
The purified hCCS-DII construct shown in the last lane is ~200 ng of
protein that was incubated at 4 °C for 24 h with 1 equivalent
each CuSO4 and ZnSO4 per protein monomer.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
View larger version (19K):
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Scheme 1.
Possible copper ion binding modes for
CCS.
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Fig. 8.
Model CCS domain III function: the C-terminal
domain of MerA. A and B, sequence lineups
of CCS domain III from various sources with MerA from Bacillus
sp. RC607 and Pseudomonas aeruginosa Tn501.
C, the homodimeric interface in the three-dimensional
crystal structure of the MerA from Bacillus sp. RC607 (34)
highlighting the C-terminal from one monomer folded within the active
site of the partner monomer. D, illustration
depicting the reported role of the MerA C-terminal peptide in mercury
acquisition for the reductase active site of the enzyme (31).
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Fig. 9.
Proposed mechanism of CCS activation of
SOD1. A, proposed mechanism of CCS copper ion
insertion into apo-(E,Zn)SOD1. B, possible monomer-swap or
rearrangement mechanisms for the docked transfer of copper from human
CCS to human or yeast SOD1 utilizing the conserved dimer interface
between CCS-dII and SOD1.
-barrel proteins can readily exchange monomers under
physiological conditions. For example, the
2- and
3-crystallin
proteins of the mammalian eye lens have been shown to display this
monomer-swapping property, even though their homodimeric interactions
have dissociation constants measured in the micromolar range (36).
Furthermore, the dimeric interaction in SOD1 is known to be
significantly weakened with removal of the metal ions as well as with
reduction of the conserved disulfide (11).
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ACKNOWLEDGEMENTS |
---|
We thank Anja Herrnreiter, Mary Kimmel, and Chris Rhee for assistance in protein sample preparations, the laboratory of Ivano Bertini for donation of the overexpression plasmid for wild-type human SOD1, and Dr. Emil Pai for the MerA (RC607) x-ray crystal structure coordinates. We also thank Val Culotta and Amy Rosenzweig for helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM 54111 (to T. V. O.) and GM 19457 (to T. D. R.), the Illinois Minority Graduate Incentive Program (to A. S. T.), and the ALS Association. Mass spectrometry data from MALDI-TOF and electrospray MS were acquired with instruments purchased through National Institutes of Health Grants S10 RR13810 and S10 RR11320, respectively.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Northwestern University, 2145 Sheridan Rd., Evanston, IL 60208-3113. Tel.: 847-491-5060; Fax: 847-491-7713; E-mail: t-ohalloran@nwu.edu.
Published, JBC Papers in Press, October 3, 2000, DOI 10.1074/jbc.M008005200
1 F. Arnesano, L. Banci, I. Bertini, D. L. Huffman, and T. V. O'Halloran, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: SOD, superoxide dismutase; ySOD, yeast superoxide dismutase; CCS, copper chaperone for SOD1; hCCS, human copper chaperone for SOD1; DTT, dithiothreitol; BCS, bathocuproine sulfonate; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight.
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---|
1. |
Rae, T. D.,
Schmidt, P. J.,
Pufahl, R. A.,
Culotta, V. C.,
and O'Halloran, T. V.
(1999)
Science
284,
805-8 |
2. |
O'Halloran, T. V.,
and Culotta, V. C.
(2000)
J. Biol. Chem.
275,
25057-25060 |
3. | Harrison, M., Jones, C., Solioz, M., and Dameron, C. (2000) Trends Biochem. Sci. 25, 29-32[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Valentine, J. S.,
and Gralla, E. B.
(1997)
Science
278,
817-818 |
5. |
Pufahl, R. A.,
Singer, C. P.,
Peariso, K. L.,
Lin, S.-J.,
Schimdt, P.,
Culotta, V. C.,
Penner-Hahn, J. E.,
and O'Halloran, T. V.
(1997)
Science
278,
853-856 |
6. |
Lin, S.-J.,
Pufahl, R. A.,
Dancis, A.,
O'Halloran, T. V.,
and Culotta, V. C.
(1997)
J. Biol. Chem.
272,
9215-9220 |
7. |
Huffman, D. L.,
and O'Halloran, T. V.
(2000)
J. Biol. Chem.
275,
18611-18614 |
8. |
Portnoy, M. E.,
Rosenzweig, A. C.,
Rae, T.,
Huffman, D. L.,
O'Halloran, T. V.,
and Culotta, V. C.
(1999)
J. Biol. Chem.
274,
15041-15045 |
9. |
Hung, I.,
Casaren, R. L. B.,
Labesse, G.,
Matthews, F. S.,
and Gitlin, J. D.
(1998)
J. Biol. Chem.
273,
1749-1754 |
10. | Rosenzweig, A. C., Huffman, D. L., Hou, M. Y., Wernimont, A. K., Pufahl, R. A., and O'Halloran, T. V. (1999) Struct. Fold Des 7, 605-617[Medline] [Order article via Infotrieve] |
11. |
Hartz, J. W.,
and Deutsch, H. F.
(1972)
J. Biol. Chem.
247,
7043-7050 |
12. | Wernimont, A. K., Huffman, D. L., Lamb, A. L., O'Halloran, T. V., and Rosenzweig, A. C. (2000) Nature Struct. Biol. 7, 766-771[CrossRef][Medline] [Order article via Infotrieve] |
13. | Banci, L., Bertini, I., Simone, C., Huffman, D., and O'Halloran, T. (2001) J. Biol. Chem. 276, in press |
14. | Gitschier, J., Moffat, B., Reilly, D., Wood, W. I., and Fairbrother, W. J. (1998) Nat. Struct. Biol. 5, 47-54[Medline] [Order article via Infotrieve] |
15. |
Culotta, V. C.,
Klomp, L. W. J.,
Strain, J.,
Casereno, R. L. B.,
Krems, B.,
and Gitlin, J. D.
(1997)
J. Biol. Chem.
272,
23469-23472 |
16. |
Wong, P. C.,
Waggoner, D.,
Subramaniam, J. R.,
Tessarollo, L.,
Bartnikas, T. B.,
Culotta, V. C.,
Price, D. L.,
Rothstein, J.,
and Gitlin, J. D.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
2886-91 |
17. |
Schmidt, P. J.,
Rae, T. D.,
Pufahl, R. A.,
Hamma, T.,
Strain, J.,
O'Halloran, T. V.,
and Culotta, V. C.
(1999)
J. Biol. Chem.
274,
23719-23725 |
18. | Zhu, H., Shipp, E., Sanchez, R. J., Liba, A., Stine, J. E., Hart, P. J., Gralla, E. B., Nersissian, A. M., and Valentine, J. S. (2000) Biochemistry 39, 5413-5421[CrossRef][Medline] [Order article via Infotrieve] |
19. | Lamb, A. L., Wernimont, A. K., Pufahl, R. A., Culotta, V. C., O'Halloran, T. V., and Rosenzweig, A. C. (1999) Nat. Struct. Biol. 6, 724-729[CrossRef][Medline] [Order article via Infotrieve] |
20. | Lamb, A. L., Wernimont, A. K., Pufahl, R. A., O'Halloran, T. V., and Rosenzweig, A. C. (2000) Biochemistry 39, 1589-95[CrossRef][Medline] [Order article via Infotrieve] |
21. | Hall, L. T., Sanchez, R. J., Holloway, S. P., Zhu, H., Stine, J. E., Lyons, T. J., Demeler, B., Schirf, V., Hansen, J. C., Nersissian, A. M., Valentine, J. S., and Hart, P. J. (2000) Biochemistry 39, 3611-23[CrossRef][Medline] [Order article via Infotrieve] |
22. | Eisses, J. F., Stasser, J. P., Ralle, M., Kaplan, J. H., and Blackburn, N. J. (2000) Biochemistry 39, 7337-7342[CrossRef][Medline] [Order article via Infotrieve] |
23. | Flohe, L., and Otting, F. (1984) Methods Enzymol. 105, 93-104[Medline] [Order article via Infotrieve] |
24. | Fridovich, I. (1985) in CRC Handbook of Methods for Oxygen Radical Research (Greenwald, R. A., ed) , pp. 213-215, CRC Press, Boca Raton, FL |
25. | Riddles, P. W., Blakeley, R. L., and Zerner, B. (1983) Methods Enzymol. 91, 49-60[Medline] [Order article via Infotrieve] |
26. | Valentine, J. S., and Pantoliano, M. W. (1982) in Copper Proteins (Spiro, T. G., ed) , pp. 292-358, John Wiley and Sons, Inc., New York |
27. | Blair, D., and Diehl, H. (1961) Talanta 7, 163-174[CrossRef] |
28. |
Schmidt, P. J.,
Ramos-Gomez, M.,
and Culotta, V. C.
(1999)
J. Biol. Chem.
274,
36952-36956 |
29. |
Stansell, M. J.,
and Deutsch, H. F.
(1965)
J. Biol. Chem.
240,
4306-4311 |
30. | Jabusch, J. R., Farb, D. L., Kerschensteiner, D. A., and Deutsch, H. F. (1980) Biochemistry 19, 2310-2316[Medline] [Order article via Infotrieve] |
31. | Engst, S., and Miller, S. M. (1999) Biochemistry 38, 3519-3529[CrossRef][Medline] [Order article via Infotrieve] |
32. | Distefano, M. D., Au, K. G., and Walsh, C. T. (1989) Biochemistry 28, 1168-1183[Medline] [Order article via Infotrieve] |
33. | Moore, M. J., and Walsh, C. T. (1989) Biochemistry 28, 1183-1194[Medline] [Order article via Infotrieve] |
34. | Schiering, N., Kabsch, W., Moore, M. J., Distefano, M. D., Walsh, C. T., and Pai, E. F. (1991) Nature 352, 168-172[CrossRef][Medline] [Order article via Infotrieve] |
35. | Miller, S. M., Moore, M. J., Massey, V., Williams, C. H., Distefano, M. D., Ballou, D. P., and Walsh, C. T. (1989) Biochemistry 28, 1194-1205[Medline] [Order article via Infotrieve] |
36. | Hejtmancik, J. F., Wingfield, P. T., Chambers, C., Russell, P., Chen, H. C., Sergeev, Y. V., and Hope, J. N. (1997) Protein Eng. 10, 1347-1352[Abstract] |