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Structural and Biophysical Properties of the Pathogenic SOD1 Variant H46R/H48Q
†
Duane D. Winkler,
‡,§
Jonathan P. Schuermann,
‡,§
Xiaohang Cao,
‡,§
Stephen P. Holloway,
‡,§
David R. Borchelt,
|
Mark C. Carroll,
⊥
Jody B. Proescher,
⊥
Valeria C. Culotta,
⊥
and P. John Hart*
,‡,§,#
 Department of Biochemistry and X-ray Crystallography Core Laboratory, the Uni
V
ersity of Texas Health Science Center,San Antonio, Texas 78229-3900, Department of Neuroscience, McKnight Brain Institute, Uni
V
ersity of Florida,Gaines
V
ille, Florida 32610, Department of En
V
ironmental Health Sciences, Bloomberg School of Public Health, The Johns Hopkins Uni
V
ersity, Baltimore, Maryland 21218, and Department of Veterans Affairs, Audie Murphy Di
V
ision, Geriatric Research, Education, and Clinical Center, South Texas Veterans Health Care System, San Antonio, Texas 78229 Recei
V
ed No
V
ember 24, 2008; Re
V
ised Manuscript Recei
V
ed February 16, 2009
ABSTRACT
: Over 100 mutations in the gene encoding human copper
-
zinc superoxide dismutase (SOD1)cause an inherited form of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Twopathogenic SOD1 mutations, His46Arg (H46R) and His48Gln (H48Q), affect residues that act as copperligands in the wild type enzyme. Transgenic mice expressing a human SOD1 variant containing bothmutations develop paralytic disease akin to ALS. Here we show that H46R/H48Q SOD1 possesses multiplecharacteristics that distinguish it from the wild type. These properties include the following: (1) an ablatedcopper-binding site, (2) a substantially weakened affinity for zinc, (3) a binding site for a calcium ion, (4)the ability to form stable heterocomplexes with the copper chaperone for SOD1 (CCS), and (5) compromisedCCS-mediated oxidation of the intrasubunit disulfide bond in vivo. The results presented here, togetherwith data on pathogenic SOD1 proteins coming from cell culture and transgenic mice, suggest thatincomplete posttranslational modification of nascent SOD1 polypeptides via CCS may be a characteristicshared by familial ALS SOD1 mutants, leading to a population of destabilized, off-pathway foldingintermediates that are toxic to motor neurons.
The homodimeric antioxidant enzyme copper
-
zinc su-peroxide dismutase (SOD1
1
) has been studied for nearly fourdecades. In 1993, interest in the molecule intensified whenmutations in the gene encoding SOD1 were linked to thelethal neurodegenerative disease amyotrophic lateral sclerosis(ALS) (
1, 2
). Since then,
∼
100 distinct pathogenic mutationshave been documented (reviewed in ref 
3
), with mostresulting in single amino acid substitutions and a few intruncations in the C-terminal portion of the polypeptide.Landmark studies in transgenic mice established thatpathogenic SOD1 proteins elicit motor neuron dysfunctionthrough the acquisition of a deleterious property and not aloss of enzymatic function (
4
-
6 
). SOD1-enriched inclusionsare observed in cell culture model systems, ALS-SOD1transgenic mice, and fALS patients, suggesting that SOD1-linked ALS pathology is related to misfolding or aggregation(reviewed in refs
7 
-
9
). However, the precise molecularmechanism underlying SOD1 toxicity to motor neurons isunknown, and it remains to be clarified whether the observedinclusions are causal or symptomatic of motor neurondysfunction.Given the observations described above, ALS mutantSOD1 proteins likely possess properties distinct from thoseof the wild type enzyme that facilitate their aggregation invivo. Initial structural studies suggested that metal-deficient,pathogenic SOD1 dimers might be ideal candidates for self-assembly because they have been observed to engage in non-native SOD1
-
SOD1 interactions that propagate bidirection-ally to form linear and helical filamentous arrays (
10
).Subsequent biochemical studies suggested that dissociated,monomeric pathogenic SOD1 may aggregate (
11, 12
), andthis suggestion focused attention on the structural elements
* To whom correspondence should be addressed. E-mail: pjhart@biochem.uthscsa.edu. Tel: 210-567-0751. Fax: 210-567-6595.
†
This work was supported by grants NIH-NINDS R01-NS39112(P.J.H.), P01-NS04913 (Joan S. Valentine, D.R.B., and P.J.H.), NIH-GMS GM50016 (V.C.C.), and the JHU NIEHS center (V.C.C.). D.D.W.was supported in part by the American Foundation for Aging Research(AFAR). J.P.S. was supported by NIH T32AG021890-03. J.B.P. wassupported by NIEHS training grant ES 07141. X.C. was supported inpart by the William and Ella Owens Medical Research Foundation andthe Judith and Jean Pape Adams Charitable Foundation. Support forthe X-ray Crystallography Core Laboratory and the Center for Analyti-cal Ultracentrifugation of Macromolecular Assemblies by the UTH-SCSA Executive Research Committee and the San Antonio CancerInstitute is also gratefully acknowledged.
‡
Department of Biochemistry, the University of Texas HealthScience Center.
§
X-ray Crystallography Core Laboratory, the University of TexasHealth Science Center.
|
University of Florida.
⊥
The Johns Hopkins University.
#
South Texas Veterans Health Care System.
1
Abbreviations: SOD1, copper
-
zinc superoxide dismutase; hSOD1,human copper
-
zinc superoxide dismutase; ySOD1, yeast copper
-
zincsuperoxide dismutase; ALS, amyotrophic lateral sclerosis; fALS,familial amyotrophic lateral sclerosis; H46R/H48Q, copper
-
zincsuperoxide dismutase with His46 and His48 substituted with Arg andGln, respectively; CCS, copper chaperone for SOD1; hCCS, humancopper chaperone for SOD1; yCCS, yeast copper chaperone for SOD1;EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonylfluoride; TCEP, tris(2-carboxyethyl)phosphine; PCR, polymerase chainreaction; TEV, tobacco etch virus; MES, 2-(
 N 
-morpholino)ethane-sulfonic acid; PEG, poly(ethylene glycol); PAR, 2-pyridylazo-resor-cinol; ICP-MS, inductively coupled plasma mass spectrometry; MME,monomethylether.
 Biochemistry
2009,
48,
3436–34473436
10.1021/bi8021735 CCC: $40.75
2009 American Chemical SocietyPublished on Web 02/19/2009
 
responsible for stabilization of the homodimer (
13
). Morerecent studies highlighted the critical roles of both the metalbinding and the presence of an oxidized
intrasubunit 
disulfidebond to SOD1 dimer stability (
14
-
16 
). These findings turnedattention to a helper protein, the copper chaperone for SOD1(CCS), which specifically recognizes newly translated SOD1and activates it by inserting the catalytic copper ion andcatalyzing the oxidation of the SOD1 intrasubunit disulfidebond (
17 
-
19
). Because these two posttranslational modifica-tions are integral to SOD1 stability, pathogenic SOD1mutations that might (directly or indirectly) interfere withmetal binding, disulfide bond oxidation, and/or interactionwith CCS are increasingly under scrutiny.Of the
∼
100 distinct disease-causing mutations that havebeen identified, two (H46R and H48Q) map to copper ligandsin the active site. In rodent models of the disease, ratsexpressing H46R SOD1 (
20
), mice expressing the H46R/ H48Q double mutant (
21
), and mice expressing a quadrupleSOD1 mutant that disrupts all four copper ligands (
22
) alldevelop motor neuron disease characterized by the appear-ance of fibrillar SOD1-containing aggregates. Because theoverexpressed human SOD1 proteins in these models allharbor a compromised copper-binding site, these studiestogether strongly suggest that copper binding is not arequirement in SOD1-linked ALS etiology.Here we report crystal structures of human H46R/H48QSOD1 in two distinct crystal systems and characterize thebiophysical properties of this pathogenic SOD1 variant andits complexes with CCS. The structural and biochemical datapresented here, together with results from previous studiesin cell culture and in transgenic mice, suggest that acharacteristic common to all fALS mutant SOD1 proteinsmay be that a fraction of the newly translated mutants “failto mature”. That is, for each pathogenic mutant, a subset of the newly translated SOD1 proteins are not stabilized viaposttranslational modification by CCS, leading to what arein essence off pathway folding intermediates that are toxicto motor neurons.
EXPERIMENTAL PROCEDURES
 Materials.
Monobasic and dibasic potassium phosphate,acetonitrile (Optima grade), formic acid, sodium hydroxide,yeast extract, peptone, dextrose (glucose), EDTA, sodiumchloride, and sodium acetate were obtained from FischerScientific. Ammonium sulfate was purchased from USBiological. Polyethelyne glycol 1000 came from Fluka. Triswas purchased from Research Products International. Primerscame from Invitrogen. Agarose glycerol, (diethylamino)ethyl(DEAE) Sephadex, and PMSF were obtained from Sigma.
Pfu
DNA polymerase and deoxyribonucleotides were pur-chased from Stratagene. Glass beads were obtained fromBiospec. Tris(2-carboxyethyl)phosphine (TCEP), crystalliza-tion screening kits, and crystal growth trays were purchasedfrom Hampton Research. Phenyl Sepharose and SephadexG-75 came from Pharmacia. All solutions unless otherwisenoted in the text were prepared using deionized water passedthrough a Millipore ultra-purification system. In experimentsusing a reducing agent, TCEP was added as a bufferedsolution with the pH adjusted to the values indicated.
SOD1 Cloning, Expression, and Purification.
DNA frag-ments encoding the human H46R/H48Q SOD1 doublemutant were amplified by PCR and ligated into the YEP351-hSOD plasmid, where expression of the SOD1 protein isdirected under the control of its own promoter. The proteinwas expressed, purified, and characterized as previouslydescribed (
15
) with the addition of a DEAE-Sephadexchromatography step between the hydrophobic interactionchromatography and the gel filtration column steps. Metalcontent of purified SOD1 proteins was determined usinginductively coupled plasma mass spectrometry (ICP-MS).
CCS Cloning, Expression, and Purification.
DNA frag-ments encoding wild type yeast and human CCS weregenerated by PCR from plasmids generously supplied by J.S.Valentine (UCLA). CCS constructs were cloned into apkA6H vector, which contains an inducible
LacZ 
promoter,a 6x-N-terminal His-tag, and a tobacco etch virus (TEV)cleavage site. CCS proteins were expressed in
Escherichiacoli
BL21 (DE3). Cells containing these expression plasmidswere grown in Luria-Bertani media at 37
°
C to an OD
600
of 0.6
-
0.8. The hCCS expression medium was supplementedwith ZnSO
4
to a final concentration of 200
µ
M. Afterinduction with isopropyl-
 β
-
D
-thiogalactopyranoside, the cellswere transferred to 30
°
C for an additional 4 h before beingharvested. CCS proteins were purified using a HisTrap high-performance nickel column purchased from Amersham. Afterpurification, the hexa-His-tag was removed from the CCSproteins using TEV protease produced in-house and engi-neered to contain its own noncleavable hexa-His tag. Afterdigestion, TEV protease was removed from the CCS sampleby a final pass through the nickel column. This procedureleaves a two residue (Gly-His) extension on the CCSN-termini. Metal content of purified CCS proteins wasdetermined using ICP-MS at the Chemical Analysis Labora-tory at the University of Athens, Georgia.
Crystallization, Structure Determination, and Refinement.
Purified H46R/H48Q SOD1 was concentrated to 15 mg/mLin 2.25 mM potassium phosphate, pH 7.0 (protein solution).Crystals were grown by the hanging drop vapor diffusionmethod. For crystals grown in space group
P
2
1
, proteinsolution was mixed with an equal volume of precipitatingsolution containing 25% (v/v) poly(ethylene glycol) (PEG)550 MME, 0.1 M 2-(
 N 
-morpholino)ethanesulfonic acid(MES) at pH 5.9, and 0.01 M ZnSO
4
. Rectangular platesappeared after about a week of incubation at 22
°
C. Forcrystals grown in space group
C 
222
1
, protein solution wasmixed with an equal volume of precipitating solutioncontaining 20% (v/v) PEG 1000, 0.1 M imidazole at pH 8.0,and 0.2 M calcium acetate. Rectangular prisms grew afterabout a week of incubation at 22
°
C. Both crystal forms werecryoprotected in a solution of the mother liquor plus 10%(v/v) PEG 200 and flash frozen in liquid nitrogen prior todata collection.X-ray diffraction data were acquired on a Rigaku FR-Drotating copper anode X-ray generator equipped with RAXIS-HTC image plate detectors. The oscillation range was 1
°
and0.5
°
for crystals in space groups
P
2
1
and
C 
222
1
, respectively,and the crystal-to-detector distance was 150 mm for bothcrystal forms. Crystals were cooled during data acquisitionusing an X-STREAM low-temperature system. Diffractiondata were processed using HKL2000 (
23
). Five percent of the reflections were selected at random as a function of resolution shell and set aside during refinement for cross-validation (
24
). The structure of the human G37R variant of Structures of Pathogenic H46R/H48Q SOD1
Biochemistry
,
Vol. 48, No. 15, 2009
3437
 
SOD1 (pdb code 1AZV) (
25
) was used as the search modelin molecular replacement performed by the program MOL-REP (
26 
), which positioned the SOD1 dimers in the unitcells of both crystal forms. Rigid-body refinement usingmonomers as individual rigid elements was followed byiterative cycles of positional and atomic displacementparameter (B-factor) refinement in REFMAC (
27 
) andmanual model building using the program COOT (
28
).Translational, librational, and screw (TLS) parameters (
29
)were implemented in the final refinement cycles.
 Nati
V
e Gel-Shift.
A total of 10
-
15
µ
g each of wild typeand H46R/H48Q SOD1 and y/hCCS in 50 mM MES pH6.0 and 150 mM NaCl with or without 10 mM TCEP (areducing agent) was run on 12% nondenaturing polyacryl-amide gels. SOD1/CCS proteins were mixed in a 1:1stoichiometric ratio, immediately loaded onto the native gel,and run at 120 mV. The running buffer was 25 mM Tris pH8.0 and 200 mM glycine.
 Zinc Release Assay.
The relative affinities of H46R/H48Qand wild type SOD1 proteins for zinc ion were comparedusing a slightly modified 2-pyridylazo-resorcinol (PAR) assay(
30
). All measurements were acquired in triplicate in assaybuffer containing 100
µ
M PAR, 50 mM Tris pH 8.1, and150 mM NaCl with and without 10 mM TCEP on a BeckmanDU7400 spectrophotometer. The extinction coefficient forthe zinc
-
PAR
2
complex at 500 nm (
ε
500nm
) was 84 000 M
-
1
cm
-
1
, as determined experimentally from standard curvesderived from known concentrations of ZnSO
4
in assay buffer.A 50
-
60
µ
g portion of human wild type and H46R/H48QSOD1 and equal amounts of yeast and human CCS wereadded to a quartz cuvette containing assay buffer with andwithout TCEP, and absorbance readings were taken at 1 minintervals for 1 h at room temperature. After 60 min, 0.5 mMEDTA was added to strip the zinc from PAR, quenchingthe absorbance at 500 nm.
 Analytical Ultracentrifugation.
Sedimentation velocityexperiments were performed with a Beckman XL-A analyti-cal ultracentrifuge using absorbance optics. Sedimentationvelocity experiments were performed at 60 000 rpm over 4 hat 4
°
C. The concentration of the protein samples cor-responded to OD
280nm
readings of 0.8 (SOD1
)
∼
2.4 mg/ mL, yCCS
)
∼
0.8 mg/mL, and hCCS
)
∼
1.9 mg/mL).Sedimentation velocity data were analyzed using the methodof van Holde and Weischet (
31
) to determine the sedimenta-tion coefficient distribution
G
(s), followed by 2-D Spectrumand genetic algorithm analyses to acquire estimates of themolecular weights of the protein components as implementedin the ULTRASCAN software package (
32
).
Gel Filtration Chromatography.
A Superdex 200 column(Amersham) was equilibrated with a solution containing 50mM MES at pH 6.0 and 150 mM NaCL with or without 10mM TCEP and calibrated with purified human SOD1 (32kDa) and human CCS (58 kDa) proteins plus proteins fromhigh and low molecular weight gel filtration calibration kits(Amersham). These proteins included thyroglobulin (669kDa), Ferritin (440 kDa), albumin (65 kDa), ovalbumin (43kDa), and ribonuclease A (13.7 kDa). H46R/H48Q SOD1and y/hCCS proteins were mixed in a 1:1 stoichiometric ratioand immediately loaded onto and run through a Superdex200 column at constant rate of 0.5 mL/min.
 Determination of SOD1 Disulfide Status in Cells.
4-Ac-etamido-4
′
-maleimidylstilbene-2,2
′
-disulfonic acid (AMS),which covalently modifies cysteine thiolates, was employedto monitor the status of the SOD1 intrasubunit disulfide bondin yeast (
33
) and in human embryonic kidney (HEK 293)cells (
34
) as described previously.
RESULTS
Structures of the H46R/H48Q SOD1 Double Mutant.
Crystal structures of H46R/H48Q SOD1 were determinedand refined in space groups
P
2
1
and
C 
222
1
to resolutions of 2.3 and 2.2 Å, respectively. X-ray diffraction data collectionand protein structure refinement statistics are shown in Table1. The H46R/H48Q SOD1 subunits in space group
P
2
1
aredesignated A, B, C, and D with AB and CD representingdistinct SOD1 homodimers. The H46R/H48Q SOD1 subunitsin space group
C 
222
1
are designated E, F, G, H, I, and J,with EF, GH, and IJ representing homodimers. In the wildtype enzyme, the metal-binding sites are formed by H46,H48, and H120 coming from the exterior of the
β
-barrel andby H63, H71, H80, and D83 coming from loop IV. LoopIV (residues 49
-
83), also known as the “zinc loop”, containsa substructure we designate the “disulfide loop” (residues49
-
60). Loop VII (residues 121
-
142), also known as the
Table 1: X-ray Diffraction Data and Refinement Statistics for H46R/ H48Q Crystals Used in This Study
PDB accessioncode
2NNX 3GQF
space group
P
2
1
C 
222
1
Diffraction Datawavelength (Å) 1.5418 1.5418space group
P
2
1
C 
222
1
unit celldimensions (Å)
a
)
76.5,
b
)
63.5,
c
)
85.1
a
)
112.6,
b
)
194.3,
c
)
143.2
R )
90.0
°
,
 β
)
116
°
,
γ
)
90.0
°
R )
β
)
γ
)
90.0
°
diffractionresolution (Å)50
-
2.30 50
-
2.20no. of observations 139 672 361 783no. of uniquereflections32 846 79 657completeness (%)
a
100.0 (100.0) 99.8 (100.0)mean I/ 
σ 
I
15.8 (2.6) 14.7 (2.9)
 R
sym
0.092 (0.531) 0.088 (0.503)Structure Refinementno. protein atoms 4440 6660no. watermolecules308 504no. Zn ions 8 6no. sulfate ions 1 0no. Ca
2
+
ions 0 5
 R
cryst
0.186 (0.217) 0.204 (0.239)
 R
free
b
0.241 (0.313) 0.241 (0.264)rmsd bondlengths (Å)0.011 0.011rmsd bondangles (deg)1.4 1.3Ramachandran Plot
c
favored (%) 88.0 90.2allowed (%) 11.8 9.4generous (%) 0.2 0.4disallowed (%) 0.0 0.0Average B-Factors (Å
2
)protein 28 31water 33 38Zn 43 28sulfate 24 N/ACa
2
+
N/A 56
a
Values in parentheses are for the highest resolution shell.
b
5% of reflections were chosen randomly in each resolution shell forcross-validation.
c
Generated using the program PROCHECK (
58
).
3438
Biochemistry
,
Vol. 48, No. 15, 2009
Winkler et al.
of 00

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