Supporting Information Human cytoplasmic copper chaperones

Supporting Information
Human cytoplasmic copper chaperones Atox1 and CCS exchange
copper ions in vitro
Svenja Petzoldt, Dana Kahra, Michael Kovermann, Artur PG Dingeldein, Moritz S Niemiec,
Jörgen Åden, and Pernilla Wittung-Stafshede
Figures S1-S4
Table S1
Figure S1.
Homology modeling of Atox1-Cu-CCS1 hetero-protein complex. A homology-based structural
model for the Atox1–Cu–CCS1 complex, created using PyMol (PyMOL Molecular Graphics
System, Version Schrödinger, LLC.) that is based on the structure of the Atox1–Cu(I)
dimer (PDB ID: 1FEE). The position of one Atox1 monomer (blue) and the Cu ion remain as
in 1FEE (Wernimont, Huffman et al. 2000); the structure of CCS1 (PDB ID: 2RSQ;
DOI:10.2210/pdb2rsq/pdb) (pink) was then aligned onto the second Atox1 monomer of 1FEE
with the PyMol alignment tool. Using the PyMol sculpting wizard tool, the loop region of
CCS1 with the motif MTCXXC was backbone aligned separately, to mimic the Cu-bound
state, followed by optimization to avoid steric clashes. Both cysteine residues in the Cubinding motif were screened against the provided PyMol rotamer database for optimal side
chain configurations.
Figure S2. CD spectra of CCS1 in apo and holo forms. Far-UV CD spectra of apo and holo
forms of CCS1 demonstrate that CCS1 is folded and that there is no significant structural
change associated with Cu binding.
Figure S3A. The effect of copper on the 1D proton NMR spectrum of CCS1. For CCS1 in the
apo state (n = 0) a disperse NMR spectrum is observed, as expected for a well-folded 10 kDa
protein. Addition of copper leads to a decrease of the observed signal intensity (up to n = 3),
whereas the chemical shifts remain as in the apo state. Further increase of copper (n = 5) leads
to a different NMR spectrum with no dispersion in the amide proton signal region and loss of
separate high field signals lower than 0.7 ppm. This indicates an increase in molecular weight
at these conditions.
Figure S3B. CCS1 oligomerization in presence of excess copper. The lower marker
corresponds to a hydrodynamic radius of CCS1 of 18.0±0.6 Å. This value is found for the apo
form (n=0) and for copper additions up to stoichiometric amounts (n=0.5, n=1) when inserting
the diffusion data into the Stokes-Einstein relation (Einstein 1905):
with the diffusion coefficient D, Boltzmann’s constant kB=1.38x10-23 JK-1, temperature T=298
K and the viscosity η=1 mPa·s. This radius agrees with the calculated rH of 17.4 Å for a
sphere-like protein of the size of CCS1, i.e., 85 residues (Wilkins, Grimshaw et al. 1999), and
supports that CCS1 is monomeric in apo and holo (1 eq. Cu) forms. The measured diffusion
coefficient of D=0.9x10-10 m2s-1 for n = 2, 3 and 5 added copper equivalents corresponds to an
apparent hydrodynamic radius of 24.2±1.6 Å (upper marker). This value matches that for a
CCS1 trimer (3·85 residues) which would have a predicted hydrodynamic radius of 23.9 Å in
case of a sphere. The dashed line represents the expected rH for increasing degrees of
oligomerization calculated for a sphere of 85 residues times x (x= integer numbers from 1 to
Figure S4. Cu-transfer between full-length CCS and Atox1. The elution of protein mixtures
were analyzed by absorbance at 254 nm (red) and 280 nm (black). CCS elutes in different
fractions at about 7.8 ml (tetrameric), 8.3 ml (dimeric) and 9.4 ml (monomeric). Atox1 elutes
at about 14.1 ml. The top panels show the apo forms of both proteins (left) and the holo form
of Atox1 (right). The apo forms show lower absorbance signals at 254 nm than at 280 nm.
The holo forms of Atox1 and CCS (middle panels, in presence of 1 eq. and 2 eq. Cu) show
absorbance signals at 254 nm which are higher (Atox1) or nearly equal (CCS) relative to the
280 nm absorbance. In addition, the holo data for CCS indicate that with increasing number of
Cu equivalents the absorbance at 254 nm increases and, in general, the amount of dimeric and
tetramic CCS rises. For the mixing experiment (bottom panels) CCS was loaded with 1 eq.
(left) and 2 eq. (right) Cu and then apo Atox1 was added (1:1 protein-to-protein ratio).
Transfer of Cu from CCS to Atox1 is evident from the decreased 254 nm signal for CCS and
the increased 254 nm signal for Atox1 (as compared to the expected holo and apo signals if no
reaction had taken place). In the 2 eq. Cu transfer experiment, it appears that Atox1 gets
completely loaded with Cu and the remaining CCS fractions roughly match that found in the
individual run of 1 eq. loaded CCS. In all chromatograms, the absorption of Atox1 at 280 nm
has been normalized to 1 (whereas the absorbance signals of the holo CCS runs have been
normalized to 1 at the CCS maximum 280 nm absorption peak).
Table S1. Cu content in SEC elution peaks of CCS1 and Atox1 probed by ICP-MS. Individual
apo and holo forms of both proteins were analyzed after SEC elution. For the mixing
experiment, CCS1 was preloaded with 1 eq. Cu before apo-Atox1 was added (1:1 protein-toprotein ratio) and after SEC elution of the mixture both protein peaks were analyzed
individually. As expected, the apo forms of CCS1 and Atox1 contained negligible amounts of
Cu, whereas the holo forms contain significant amounts of Cu. The mixture contained Cu in
both the CCS1 and the Atox1 peaks, indicating that Cu had been transferred from CCS 1 to
Atox1. A control of pure CuCl2 was also included among the ICP-MS samples; for a solution
of 10.2 M CuCl2 the ICP-MS data reported a Cu concentration of 11.4 M. This implies that
measurement errors can reach 10 %.
Mixture -
Mixture -
c(protein) [µM]
c(Cu) [µM]
Einstein, A. (1905). "Ü ber die von der molekularkinetischen Theorie der Wärme geforderte
Bewegung von in ruhenden Flü ssigkeiten suspendierten Teilchen." Ann. Phys. 17: 549-560.
Wernimont, A. K., D. L. Huffman, A. L. Lamb, T. V. O'Halloran and A. C. Rosenzweig (2000). "Structural
basis for copper transfer by the metallochaperone for the Menkes/Wilson disease proteins." Nat
Struct Biol 7(9): 766-771.
Wilkins, D. K., S. B. Grimshaw, V. Receveur, C. M. Dobson, J. A. Jones and L. J. Smith (1999).
"Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR
techniques." Biochemistry 38(50): 16424-16431.