Superoxide Dismutase from the Eukaryotic Thermophile Alvinella pompejana: Structures, Stability, Mechanism, and Insights into Amyotrophic Lateral Sclerosis

doi:10.1016/j.jmb.2008.11.031

J. Mol. Biol. (2009) 385, 1534–1555

Available online at www.sciencedirect.com

Superoxide Dismutase from the Eukaryotic Thermophile Alvinella pompejana: Structures, Stability, Mechanism, and Insights into Amyotrophic Lateral Sclerosis David S. Shin 1 , Michael DiDonato 1 , David P. Barondeau 1 , Greg L. Hura 2 , Chiharu Hitomi 1 , J. Andrew Berglund 3 , Elizabeth D. Getzoff 1 , S. Craig Cary 4,5 ⁎ and John A. Tainer 1,2 ⁎ 1

Department of Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA 2

Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA 3

Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA 4 College of Marine Studies, University of Delaware, Lewes, DE 19958, USA 5

Department of Biological Sciences, University of Waikato, Hamilton 3240, New Zealand Received 31 July 2008; received in revised form 7 November 2008; accepted 11 November 2008 Available online 25 November 2008

Prokaryotic thermophiles supply stable human protein homologs for structural biology; yet, eukaryotic thermophiles would provide more similar macromolecules plus those missing in microbes. Alvinella pompejana is a deep-sea hydrothermal-vent worm that has been found in temperatures averaging as high as 68 °C, with spikes up to 84 °C. Here, we used Cu,Zn superoxide dismutase (SOD) to test if this eukaryotic thermophile can provide insights into macromolecular mechanisms and stability by supplying better stable mammalian homologs for structural biology and other biophysical characterizations than those from prokaryotic thermophiles. Identification, cloning, characterization, X-ray scattering (smallangle X-ray scattering, SAXS), and crystal structure determinations show that A. pompejana SOD (ApSOD) is superstable, homologous, and informative. SAXS solution analyses identify the human-like ApSOD dimer. The crystal structure shows the active site at 0.99 Å resolution plus anchoring interaction motifs in loops and termini accounting for enhanced stability of ApSOD versus human SOD. Such stabilizing features may reduce movements that promote inappropriate intermolecular interactions, such as amyloid-like filaments found in SOD mutants causing the neurodegenerative disease familial amyotrophic lateral sclerosis or Lou Gehrig's disease. ApSOD further provides the structure of a long-sought SOD product complex at 1.35 Å resolution, suggesting a unified innersphere mechanism for catalysis involving metal ion movement. Notably, this proposed mechanism resolves apparent paradoxes regarding electron transfer. These results extend knowledge of SOD stability and catalysis and suggest that the eukaryote A. pompejana provides macromolecules highly similar to those from humans, but with enhanced stability more suitable for scientific and medical applications. © 2008 Elsevier Ltd. All rights reserved.

Edited by M. Guss

Keywords: thermophile; thermostable proteins; superoxide dismutase; amyotrophic lateral sclerosis; amyloid filaments

*Corresponding authors. J. A. Tainer is to be contacted at Department of Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. E-mail addresses: [email protected]; [email protected]. Present addresses: M. DiDonato, Structural Biology, Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA; D. P. Barondeau, Department of Chemistry, Texas A&M University, College Station, TX 77842, USA. Abbreviations used: ALS, amyotrophic lateral sclerosis; ApSOD, Alvinella pompejana superoxide dismutase; BtSOD, Bos taurus superoxide dismutase; cDNA, copy or complementary DNA; DSV, deep submergence vehicle; EL, electrostatic loop; GK1, Greek key loop 1; GK2, Greek key loop 2; HsSOD, Homo sapiens superoxide dismutase; PBS, phosphatebuffered saline; S–S, disulfide region; SAXS, small-angle X-ray scattering; ScSOD, Saccharomyces cerevisiae superoxide dismutase; SmSOD, Schistosoma mansoni superoxide dismutase; SOD, Cu,Zn superoxide dismutase; SoSOD, Spinacia oleracea superoxide dismutase; VL, variable loop; XlSOD, Xenopus laevis superoxide dismutase; ZnBR, zinc binding region. 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.

Alvinella SOD Structures, Stability and Mechanism

Introduction Macromolecules from microbial extremophiles have many applications in biology, biotechnology, and industry.1 These microbes inhabit environments with extreme temperatures, pressures, pH, salinity and/or metal content. Bacteria and archaea living at high temperature frequently provide highly stable proteins.1,2 These proteins are often characterized prior to or in lieu of human homologs for three reasons: First, thermostable proteins are often better “behaved” such that individual conformations and ordered flexible regions can be kinetically trapped at mesophilic temperatures.3 This characteristic may have contributed to the higher success rate of solving thermostable protein structures in various laboratories and by high-throughput methods.3–6 Second, purification is facilitated by heat denaturation of recombinant host proteins.7–9 Third, some archaeal thermophilic proteins have closer resemblance to human proteins than those from lower-temperature unicellular model systems, such as bacteria and yeast, or offer an orthologous system with minimal complexity.3,6,10,11 These factors all aid insights into residues, domains, and subunit interactions with relevance to ubiquitous proteins in human macromolecular systems.7–9,12–15 However, the lack of complexity in microbial protein networks can limit extrapolation of multicellular eukaryotic pathways, as microbial pathways may lack orthologs of proteins that interact with the more ubiquitous central enzymes. In other cases, microbial proteins will differ enough in amino acid sequence, tertiary fold, or assembly, making comparisons to human proteins difficult. These limitations of analyzing thermophilic prokaryotic macromolecules would be overcome if a suitable multicellular eukaryotic thermophile could be found as a resource to supply more human-like thermostable macromolecules for analyses. One of the most promising such candidate thermophilic eukaryotes is the Pompeii worm, Alvinella pompejana. The Alvinellidae family of polychaetous annelids represent the most thermotolerant eukaryotes known, based on in situ temperature measurements of A. pompejana's immediate environment16,17 and laboratory observations of Paralvinella sulfincola thermotaxis.18 A. pompejana inhabits deep-sea hydrothermal vents along the East Pacific Rise, the spreading ridge axis between the Pacific and North American Plates.16,17 Here, volcanic activity causes seawater superheating plus mixing with metal ion and sulfide-rich vent fluids. The resultant metal sulfide precipitates create black smoke and help form large venting chimneys. These emit the hottest fluids (∼ 350 °C) from orifices plus hot water (b 150 °C) diffusively through the sides, where adult Pompeii worms (2– 6 cm in length) live within self-constructed tubes. This most thermotolerant, multicellular eukaryote is also eurythermal (temperature-range tolerant): an over 60 °C gradient can exist along A. pompejana's 6to 8 -cm tube length, suggesting A. pompejana has

1535 protein isoforms with different stabilities. A. pompejana thrives at pH ∼5.5 amid toxic heavy metal ions, some at concentrations 1000 times higher than that of ambient seawater,19 and is thus an outstanding candidate organism for isolating stable macromolecules resistant to high temperature, metal ions, and low pH. Mean temperatures within the worm tubes are as high as 68 °C with spikes up to 84 °C, exceeding the 55 °C upper limit predicted for eukaryotic survival.16,17,19 To test whether A. pompejana has promise as a eukaryotic resource for human-like thermophilic macromolecules, we collected A. pompejana worms with the Alvin submarine and generated copy or complementary DNA (cDNA) for sequencing, cloning and protein expression. From the available sequences encoding full-length proteins, we chose to test Cu,Zn superoxide dismutase (SOD) for three reasons. First, SOD is biologically and medically important with existing questions and missing insights regarding its catalysis and stability that might be addressed by homologous thermophilic structures. Second, SOD assembles into distinct eukaryotic and microbial dimers, providing a test of the functional homology in solution. Third, the multiple existing and reasonably high-resolution structures from many different species,20–27 including human (Homos sapiens SOD, HsSOD), provide a detailed basis for key cross-species comparisons to a first thermophilic eukaryote protein crystal structure. A. pompejana Cu,Zn superoxide dismutase (ApSOD) thus provides an important test case for A. pompejana structure determinations by X-ray crystallography for detailed active-site comparisons and solution small-angle X-ray scattering (SAXS) for examination of the assembly state in solution. Thus far, microbial SOD structures are monomeric or exhibit different dimer interfaces unlike those from eukaryotes.28–31 Therefore, using samples from a eukaryotic, rather than a prokaryotic, thermophile should yield a more relevant structure for gaining insights into human SOD assembly and stability. SOD is a master eukaryotic regulator of oxygen radicals, with relevance to brain pathology, cancer, aging, and cell biology.32 Together, superoxide and nitric oxide can initiate arachidonate and lipid peroxidation associated with cell signaling and cell killing, where the biological levels of these reactive oxygen species are precisely controlled by the SOD and nitric oxide synthase enzymes. 32–34 SOD catalyzes the disproportionation of toxic superoxide radicals (O 2·− ) to oxygen (O 2 ) and hydrogen peroxide (H2O2). SOD is also a relatively stable metalloenzyme in mesophiles, where human SOD has a two-component set of melting temperatures of 75 and 83 °C.35 Yet, in spite of this stability and multiple structures from different species, including that of human SOD, being solved over the course of N 25 years, a structure revealing any substrate or product complex has remained elusive. We hypothesized that if ApSOD was more stable, this may allow us to trap a product complex to address key questions regarding the SOD electron transfer

1536 and mechanism. Furthermore, mutations associated with SOD structural defects can cause amyotrophic lateral sclerosis (ALS), or Lou Gehrig's disease, a fatal neurodegenerative disorder.36–40 Thus far, more than 20% of inherited or familial ALS cases are correlated with SOD1 gene defects.41,42 The destabilizing SOD mutations that cause ALS result in the disease's characteristic and currently untreatable selective destruction of motor neurons leading to progressive paralysis and death. Since the protein sequence and structures of SOD proteins are conserved, we hypothesized that changes in amino acid sequence and structure in ApSOD should point to stabilizing features and provide insights into how single-site ALS mutations can cause the framework destabilization identified as the hallmark of ALS mutations,38 provided ApSOD has enhanced thermostability. We report here the sequencing, identification, and cloning of the cDNA encoding ApSOD along with the expression, purification, and combined biochemical and structural analyses of its product. We discovered ApSOD has remarkably high level of sequence identity with HsSOD and other mammalian SOD enzymes. Yet, we found ApSOD substantially more stable than HsSOD. Moreover, crystals from initial conditions diffracted just beyond 1 Å resolution, the highest yet reported for a wild-type SOD structure. Despite the high degree of sequence similarity between the structurally characterized homologs, we identified residue differences that evidently enhance stability. Furthermore, the stability and homogeneity of ApSOD protein samples were sufficient to solve a crystal structure in the presence of the reaction product H2O2 to examine catalysis and features promoting conformational stability and to generate an ab initio SAXS structure to characterize quaternary assembly in solution. These results extend knowledge of SOD stability and catalysis and also suggest that eukaryote A. pompejana offers a unique resource of macromolecules of enhanced stability for science and technology.

Alvinella SOD Structures, Stability and Mechanism

The vent fluid within worm tubes averaged 60 °C as measured by a time-lapse temperature probe, consistent with previous recordings.16,19,43 The gillbearing anterior ends of the worms project toward vent tube openings (Fig. 1a), while the posterior ends of the worms directly experience hot vent fluid. Live worms were removed from the tubes and placed in a container filled with an RNA-stabilizing solution to protect RNA during transfer from the seafloor to the ocean surface (Fig. 1b). We extracted RNA from the posterior end of the worm, which experiences the hottest vent-fluid environment, made cDNA libraries and sequenced the libraries to search for and identify the cDNA corresponding to the A. pompejana SOD1 processed mRNA transcript. The sequence encoding ApSOD was found and amplified by PCR and cloned into a bacterial expression vector. Following expression, ApSOD protein purification steps included heat denaturation at 65 °C for up to 1.5 h to remove nonthermostable bacterial proteins. The isolation of soluble protein and detection of SOD activity (Supplementary Fig. S1) verified that ApSOD is thermostable and allows for efficient expression and purification. Structure-based sequence alignment of ApSOD with the known eukaryotic structures of bovine (Bos taurus or BtSOD),25,27 human (Homo sapiens or

Results A. pompejana sample collection, cloning, and homology to human SOD Due to its 60–84 °C habitat, we chose to test the eukaryotic deep-sea hydrothermal-vent worm A. pompejana for its potential as a resource of thermostable proteins for structural studies. To initiate structural analyses of proteins isolated from this eukaryotic thermophile, we collected A. pompejana worms roughly 2–6 cm in length from worm tubes on black smoker chimneys at hydrothermal vent sites along the Axial Summit Caldera of the East Pacific Rise at a depth of approximately 2500 m using the deep submergence vehicle (DSV) Alvin.

Fig. 1. Alvinella pompejana. (a) A. pompejana worm (red arrow) projecting out of its vent tube with gills fully extended. The A. pompejana constructed vent tube is noted by the yellow arrow. (b) Ventral view of an ∼4 -cm Pompeii worm after collection (scale bar represents 1 cm) with collapsed gills on the anterior side (red structures on left) and a bulge (middle) from gas expansion due to the ∼ 250-atm change in pressure following collection.

1537

Fig. 2 (legend on next page)

Alvinella SOD Structures, Stability and Mechanism

1538 HsSOD),24,26 trematode (Schistosoma mansoni or SmSOD),20 frog (Xenopus laevis or XlSOD),21 spinach (Spinacia oleracea or SoSOD),22 and budding yeast (Saccharomyces cerevisiae or ScSOD)23 SODs revealed high conservation (Fig. 2a). At the amino acid level, ApSOD is strikingly conserved with mammalian SODs. Yet, phylogenetic analysis correctly places the ApSOD sequence relative to the mammalian SOD sequences (Fig. 2b). Among SODs with solved structures, BtSOD shares the greatest amino acid conservation with ApSOD (67% identity and 90% similarity over 151 residues), despite their genetic separation (Fig. 2a). HsSOD is also very similar to ApSOD, with 59% identity and 87% similarity over 149 residues. Most ApSOD residues located at positions corresponding with human ALS mutations either match the wild-type HsSOD residues or differ from both wild-type HsSOD and its ALS mutants. Only ApSOD Lys21 and Lys98 match human ALS Lys mutations at HsSOD Glu21 and Glu100, respectively (Fig. 2a). ApSOD crystal structure and active site To test our hypotheses that proteins from a thermophilic eukaryote will offer advantages for structure determination and identification of stability traits, we crystallized the ApSOD protein and solved its X-ray crystal structure. ApSOD proved as efficient for crystallization as it was for expression and purification. Crystals in space group P6122 grew in the first 24-well screen with ammonium sulfate as the precipitant. The initial crystals, without additional refinement of experimental conditions, diffracted X-rays beyond 1 Å resolution (Supplementary Fig. S2). Molecular replacement with the human SOD probe 1PU039 successfully provided phasing information. The resulting electron density maps showed atomic detail for residues 1–151, reflecting high order. After refinement to 0.99 Å resolution, the structure had an R-factor of 11.2% and R-free of 13.8% (Table 1). Currently, this is the highest resolution and only b1 Å resolution wild-type SOD structure in the Protein Data Bank (PDB). Significantly, the resolution and quality of our ApSOD structure allowed us to refine the active-site metal centers without geometric restraints, determine experimentally defined stan-

Alvinella SOD Structures, Stability and Mechanism

dard uncertainty errors for bond lengths and angles, and model anisotropic thermal displacement parameters (B-factors). The ApSOD structure (Fig. 2c) resembles structures of other eukaryotic SOD proteins, but crystals contain only one SOD subunit per asymmetric unit. The protein core consists of an eight-stranded antiparallel Greek key β-barrel,25 where loops β3/ β4 and β6/β7 form +3 β-strand or Greek key connections25,44 (GK1 and GK2, respectively). The structure shares the short type II′ turn between β2 and β3 with BtSOD, as opposed to longer loops. Two elongated loops extend from the β-barrel to form the metal-containing active-site channel and connections critical for structural integrity and function (Fig. 2a and c). The β7/β8 or electrostatic loop (EL) acts in the guidance of the O2·− substrate into the active site,45 accounting for the enzyme's faster than diffusion catalytic rate. In ApSOD, this loop is bounded by Cu ligand His118 and conserved Arg141, which is implicated in hydrogen bonding with O2·−.46,47 The β4/β5 loop contributes to the dimer interface in other eukaryotic SODs and is stabilized via a disulfide bond between Cys55 and β8 Cys144. This portion of β4/β5 is termed the disulfide (S–S) subloop or region. Despite prolonged exposure to the reducing effects of the synchrotron X-ray beam, the ApSOD disulfide bond remains oxidized, having a Sγ–Sγ distance of 2.248 ± 0.008 Å, matching the native left-handed spiral conformation.25 The disulfide stability in the face of reducing ionizing radiation is consistent with the framework stability of ApSOD. β4/β5 also encompasses the Zn2+-binding region (ZnBR), as it contains all Zn 2+ -liganding residues. Zn2+ ligand His61 divides the disulfide and zinc-binding subloop regions and also coordinates the catalytic Cu ion, so it is termed the bridging histidine. Since the β4/β5 loop connects the dimer interface, disulfide bond and the metal-binding sites together, their relative stabilities are interrelated. The ApSOD active-site geometry is well conserved with HsSOD. Initial 2Fo − Fc electron density for ApSOD revealed a predominantly reduced Cu(I) ion, based on its trigonal coordination. After several rounds of crystallographic refinement, Fo − Fc difference maps revealed partial occupancy for the oxidized Cu(II) ion, which was verified by R-factor

Fig. 2. ApSOD sequence conservation, fold, and residue function. (a) Structure-based alignment of ApSOD with other eukaryotic Cu,Zn SOD proteins with solved structures: Bt, B. taurus; Hs, H. sapiens; Sm, S. mansoni; Xl, X. laevis; So, S. oleracea; Sc, S. cerevisiae. Structural elements and secondary structure of ApSOD are noted above the alignment. Symbols below alignment mark ALS sites in HsSOD: green oval, ApSOD shares wild-type human residue; black square, ApSOD has a different residue; red star, ApSOD residue represents an ALS mutation. Letters below alignment: C, copper-binding ligand; D, disulfide cysteine; B, bridging histidine; Z, zinc-binding ligand; H, H2O2-liganding residue; P, stabilizing proline caps; S, stacking residue, S⁎, stacking residue hydrogen-bonding partner. Numbers below alignment represent paired interactions noted in Fig. 8 and Table 2. Underlined numbers refer to main-chain atoms involved in interactions, except Glu68, which also makes side-chain interactions. (b) Phylogenetic tree for eukaryotic SODs shows estimated evolutionary divergences. Labels at nodes represent bootstrap values and branch lengths are indicated (scale bar represents 0.1). (c) Stereo view of the ApSOD structure. Key structural elements in (a) are color coded with abbreviations (VL, variable loop; GK1 and GK2, Greek key loops 1 and 2, respectively; S–S, disulfide region; ZnBR, zinc-binding region; EL, electrostatic loop); otherwise, β-strands are cyan and loops are gray. N and C denote termini. The black bar (left) marks the potential dimer interface on the opposite side of the subunit from the active channel. Metal-liganding residues are shown as sticks with bridging histidine His61 in yellow.

Alvinella SOD Structures, Stability and Mechanism Table 1. Crystallographic data collection and analysis

Data collection and processing X-ray source Wavelength (Å) 2θ tilt (high-resolution set) (°) Detector distance (high-resolution set) (mm) Space group Unit cell lengths: a, b, c (Å) Unit cell angles: α, β, γ (°) Data range (last shell) (Å) Observations (unique) Completeness (last shell) (%) Average redundancy (last shell) Rsym (last shell)b I/σI (last shell) Refinement Resolution Reflections F N 0 (cross-validation) Data/parameter ratio Nonhydrogen protein atoms (solvent) Rcryst (%)c Rfree (%)d

Native

H2O2 complex

BL12.3.1a 0.954 20 (27)

BL12.3.1a 1.100 22

350 (250)

350

P6122 62.49, 62.49, 163.19 90, 90, 120 50–0.99 (1.03–0.99) 462,469 (98,085) 93.2 (54.0) 4.7 (1.7)

P6122 62.56, 62.56, 163.75 90, 90, 120 50–1.35 (1.40–1.35) 179,249 (39,844)

0.068 (0.29) 15.45 (2.50)

0.055 (0.32) 22.62 (2.10)

45–0.99 93,084 (4,911)

45–1.35 37,795 (1994)

6.7 1137 (400)

2.9 1138 (321)

11.2 13.8

12.8 17.6

93.6 (58.0) 4.5 (2.0)

The rmsd between the structures was 0.09 Å for 151 Cα atoms. a Advanced Light Source, Berkeley CA. b Rsym is the unweighted R value on I between symmetry mates. c Rcryst = ∑hkl||Fobs(hkl)| − |Fcalc(hkl)||/∑hkl|Fobs(hkl)|. d Rfree = the cross validation R-factor for 5% of reflections against which the model was not refined.

calculations and omit maps (Fig. 3a and b). Dual occupancy of copper resulting from a mixture of Cu(I) and Cu(II) oxidation states has been reported in other high-resolution Cu,Zn SOD structures and can result from reduction in the synchrotron X-ray beam.20,26,27 The electron density maps and refinement gave accurate Cu(I) and Cu(II) ion positions and Cu(I)– ligand bond lengths of 1.991 ± 0.008, 1.930 ± 0.007, and

1539 1.970 ± 0.008 Å for His44, His46, and His118, respectively (Fig. 3a), but alternative Cu(II) ligand positions were not modeled. The Zn ion has full occupancy and is liganded by His61, His69, His78, and Asp81 in a distorted tetrahedral arrangement, consistent with other eukaryotic SOD active sites. The final ApSOD model had refined occupancies of 80% for Cu(I) and 20% for Cu(II), with the copper positions separated by 1.06± 0.01 Å. Ordered water molecules in the active-site channel provide insights into enzyme interactions with O2·− substrate and H2O2 product (Fig. 3c and e). Final 2Fo − Fc electron density maps show that water molecule W0 resides near the expected position47 for the Cu(II)-bound oxygen atom of the superoxide radical. The B-factor of W0 is 20.7 Å2, as compared to average B-factors of ∼ 12.0 Å2 for neighboring water molecules W1–W3, consistent with this site's ability to hold a larger diatomic molecule. At high contour levels, simulated annealing 2Fo − Fc omit electron density matches 2Fo − Fc electron density, identifying the center of mass for W0. However, at lower contours, omit density for W0 extends away from the Cu ions, making a comma-shape. These results suggest that W0, with a refined occupancy of 96%, lies in a site destined for a larger molecule and may have some mobile character (see Supplementary Results R1 and Supplementary Fig. S3). H2O2 complex structure and active-site chemistry SOD catalyzes the disproportionation of superoxide to oxygen and hydrogen peroxide. In the first half reaction: O2·− + H+ + Cu(II)SOD → Cu(I)SOD + O2. In the second half reaction: O 2·− + H + + Cu(I) SOD → Cu(II)SOD + H2O2. Despite long-term efforts to characterize details of the SOD active-site structure and highly efficient reaction, the mechanism of H2O2 formation in the second half reaction remains controversial. In the originally proposed inner-sphere mechanism,47 the O2·− substrate and H2O2 product interact directly with the Cu ion, His61, Arg141, and a water molecule. Later, the second half reaction was proposed to be outer

Fig. 3. ApSOD active site. (a) Stereopair showing trigonal coordination of Cu(I) by histidine ligands in the 0.99 Å ApSOD structure. Composite omit 2Fo − Fc density contoured at 3σ clearly defines individual atoms. Bonding distances with experimentally defined standard uncertainty errors are noted by arrows. Ligand–Cu–ligand angles with errors are shown between respective bonds. (b) Rotation of (a) to show density for the two copper positions. Copper-colored spheres are scaled to reflect occupancy. (c–f) Overlay of the 0.99 Å resolution ApSOD model (light green carbon and dark green solvent atoms) and the 1.35 Å resolution ApSOD–H2O2 model (salmon-colored carbon and red solvent atoms). (c) Simulated annealing 2Fo − Fc omit electron density (light and dark green) for the solvent in the 0.99 Å noncomplexed ApSOD structure and (d) for the 1.35 Å peroxide-containing structure (light and dark red). By contouring 2Fo − Fc omit electron density at high levels, the center of mass for the constituent in the predicted active site can be located: (c) water W0 and (d) H2O2 contoured at 5σ and 3σ, respectively. (c) Lower-level contours of 3σ show a residual curved tail for W0, which suggests that while W0 lies near the hydrogen peroxide O2 position, the water molecule is mobile, as it is bound in a site reserved for a larger molecule or that a minor component of the occupancy may reside near the peroxide O1 position. (d) Lower 2σ density for the peroxide-soaked structure is cylindrical and conforms to two oxygen atoms with realistic hydrogen-bonding distances to protein and solvent. Hydrogen bonds are shown as dots. The W0/H2O2 bond to Cu(II) is shown as dashes and bonds between copper and protein as continuous lines. (e and f) Rotation of the view in (c) and (d) to show details of other solvent atoms in the active site. For clarity of position and movements (e) is contoured at 4 and 3σ and (f) at 3.5 and 2.5σ. (c and e) When water is bound in the active site, W7 is bonded to Arg141, which may play a role in maintaining the active-site channel to attract superoxide. (d and f) Arg141 then likely plays a role in catalysis through interactions with the substrates and products.

1540

Alvinella SOD Structures, Stability and Mechanism

sphere, based on the azide-binding geometry.48 This outer-sphere mechanism requires that the negatively charged electron be transferred from positive Cu(I) to negative O2·− over an intervening distance N 3 Å, yet explains neither (1) the energetics driving the rapid electron transfer nor (2) the coupling of proton

transfer with electron transfer. To resolve these paradoxes and provide additional insights into the SOD mechanism, we used ApSOD to tackle the structure of the SOD product complex. Although rapid catalysis by SOD previously thwarted trapping of substrate or product complexes

Fig. 3 (legend on previous page)

Alvinella SOD Structures, Stability and Mechanism

for structural studies, we were able to form the H2O2 complex with ApSOD. The ApSOD crystal soaked aerobically in H2O2 was isomorphous with the unsoaked crystal, diffracted to 1.35 Å resolution and allowed refinement to an R-factor of 12.8% and an R-free of 17.6% (Table 1). As expected, the ApSOD–H 2O 2 complex electron density maps revealed a structure nearly identical in global fold and side-chain orientations to the ApSOD structure, but with a diatomic molecule in the position predicted for an O2·− /H2O2 molecule within the active site as well as subtle differences in the positions and occupancies of nearby water molecules. These differences allowed detailed comparisons of active-site stereochemistry relevant to understanding the enzyme mechanism (Fig. 3c–f). The initial 2Fo − Fc maps for the ApSOD–H2O2 complex readily revealed electron density for a diatomic oxygen species with an interatomic distance of ∼ 1.5 Å in the predicted O2·− binding site. We were able to refine two oxygen atoms within the site with full occupancy and the expected 1.49 Å O–O bond length for H2O2. This ruled out the possibility of simultaneous occupancy of two water molecules in the site with combined occupancies totaling 100% (see Supplementary Results R1 and Supplementary Figs. S3 and S4). Final 2Fo − Fc omit electron density maps show the H2O2 molecule bound between Cu and Arg141 (Fig. 3d). In the structure of the ApSOD–H2O2 complex, as in the ApSOD structure, the Cu ion was found predominantly in the reduced Cu(I) position, again suggesting X-ray-induced reduction. Following refinement, occupancies were 92% for the reduced Cu(I) position and 8% for the oxidized Cu(II) position. In contrast, H2O2 occupied a single binding site that is independent of the Cu ion redox state. In our ApSOD–H2O2 complex structure, the Cu(I) position is 3.47± 0.07 Å away from the proximal H2O2 oxygen atom O2, which nearly superimposes with W0 in the noncomplexed ApSOD structure (Fig. 3c and d and Supplementary Fig. S3), and water in other high-resolution structures26,27 (Supplementary Fig. S4) as well as the proximal atoms of bound anions in the Cu(II) containing ScSOD48 and BtSOD49 structures (Supplementary Fig. S5). Together, steric restrictions (Supplementary Fig. S4) and hydrogen bonding (Figs. 3c and

1541 d and 4a and b) favor binding water, diatomic oxygen molecules, and anions in this site. Consequently, we propose a unified general mechanism for Cu,Zn SOD that takes into account this binding site, recent kinetic isotope data50 and steric restrictions at the Cu(I) site. In this structurebased mechanistic proposal, both half reactions proceed by an inner-sphere mechanism. In the first half reaction (Fig. 4c–e), the O2·− substrate binds Cu (II); Cu(II) is reduced to Cu(I), while O2·− is oxidized to O2; and the bond between the Cu ion and bridging ligand His61 Nɛ1 is broken, leaving Nɛ1 protonated. In the second half reaction (Fig. 4e–g), a proton from His61 Nɛ1 and an electron from Cu(I) are donated to O2·−; Cu(I) is oxidized to Cu(II), while O2·− is reduced to H2O2 (or HO2−); and the bond re-forms between the copper and His61. As we observed mobility for the Cu ion, yet conservation of the substrate/product site, we propose that the positively charged Cu(I) ion is attracted to and moves toward the negatively charged O2·− substrate (Fig. 4e and f). To test this, we manually moved the Cu(I) ion toward hydrogen peroxide O2 to see how closely we could position these atoms while maintaining bonding distances between 1.9 and 2.1 Å to His44, His46, and His118 (for details, see Supplementary Results R2 and Supplementary Fig. S5). During this part of the reaction, the Cu ion could transition through coordination geometry similar to that predicted for anion binding45 and observed for azide binding,48 with His44, His61, His118, and the O2·− /H2O2 intermediate as equatorial ligands and W1 and/ or His46 as axial. Significantly, we found that the Cu ion could retain a distorted trigonal coordination, while shortening the distance to the peroxide O2 to b 2.8 Å, which is reasonable for inner-sphere orbital overlap. Facile distortion of trigonal Cu(I) geometry is promoted by its filled shell d10 electronic configuration, which is not subject to geometrydependent ligand-field stabilization energy. Similar dynamic elasticity and motion in a catalytic Zn site (also d 10 ) has been proposed recently for the methionine synthases.51 For SOD, we propose that elasticity in the Cu(I) geometry, coupled with protein and substrate mobility in solution, can permit direct contact with O2·− for inner-sphere electron transfer;

Fig. 4. Unified structure-based mechanism for SOD. (a and b) Stereo views of ApSOD active sites from the (a) 0.99 Å structure and (b) the 1.35 Å H2O2 complex structure show alternative solvent positions and bonding, due to replacement of W0 with H2O2, and serve as a guide to the mechanism scheme. Hydrogen bonds are shown as purple balls and bonds to metals are colored matching the metal, where those to protein are solid. (c–g) Scheme for SOD mechanism. Green arrows denote movements and red dashed lines split the upper and lower copper positions. Atoms that form hydrogen peroxide ·− are blue. (c and d) First, an O·− 2 radical displaces active-site Cu(II) binding water W0. Then O2 hydrogen-bonds one oxygen, termed O1, to Arg141 NH1, while the other, termed O2, bonds Cu(II). (d and e) This reduces Cu(II) to Cu(I), shifting it N 1 Å deeper into the active-site channel, while simultaneously breaking its bond to His61. His61 Nɛ2 is then protonated, rotating the side chain away from Cu(I), and O2 is released. (e and f) During the second half reaction, a second O·− 2 is bound within the same site, due to sterics imposed by protein and bonds to His61, Arg141, and W4. To donate a negatively charged electron to the superoxide O2 atom, positively charged Cu(I) is attracted to and moves upward ɛ2 ·− toward O·− 2 such that orbital overlap occurs for inner-sphere electron transfer. His61 N also donates a proton to O2 prior to or simultaneously with the electron transfer to form a copper–hydroperoxide intermediate. (f and g) This intermediate is converted to Cu(II) and H2O2 following addition of another proton likely from W4 in trans to avoid steric interference to produce H2O2. After the transfers, Cu(II) is coordinated to His61, and W2 and W3 likely reorient the H2O2 orbitals to that of H2O2 in the liquid state favoring reaction equilibrium toward the product.

1542

Alvinella SOD Structures, Stability and Mechanism

Fig. 4 (legend on previous page)

1543

Alvinella SOD Structures, Stability and Mechanism

no long-distance outer-sphere electron transfer is required. Our Cu-bound peroxide position and proposed inner-sphere mechanism are consistent with kinetic isotope results.50 These data support inner-sphere interactions for the first half reaction and suggest a rate-determining proton transfer for the second half reaction at very high pH, where the bridging ligand His61 would not be protonated. For an outer-sphere mechanism, one might expect that the electron transfer would be rate limiting with relatively rapid proton transfer from His61 and/or adjacent hydrating water molecules. At physiological pH, electron transfer from the Cu(I) and proton transfer from His61 could be coupled for the inner-sphere mechanism, with superoxide being dismuted into H2O2 at the position we define in our structure. The ApSOD structures show conserved, ordered water molecules closely packed in the active-site channel (Supplementary Fig. S4). The hydrogenbonding network formed by these conserved water molecules aligns the substrate and promotes coupled proton and electron transfer (Fig. 4b and f). W4 (or the water molecule that occupies its position in other structures), which hydrogen-bonds to Arg141 Nɛ, rather than W2 (the proposed proton donor for the outer-sphere mechanism48), is the probable second proton donor for H2O2 formation (Fig. 4b and f). The His61 Nɛ2–O2–O1–W4 stereochemistry is virtually planar with a trans dihedral angle of 174°, and with 137° Nɛ2–(O2–O1) and 121° (O2–O1)–W4 bond angles minimizing steric repulsions during this reaction step. Water molecules W2 and W3 are positioned to stabilize the H2O2 leaving group (Fig. 4b and g). The W2–(O1–O2) angle is 96° and the (O1–O2)–W3 angle is 76°, both of which are near the 94.8° calculated angle for H–O–O hydrogen

peroxide bonds in the liquid or gaseous phase. The W2–(O1–O2)–W3 dihedral angle of 142° is also in range to stabilize the expected staggered anticlinical conformation for H2O2 in solution. Thus, the conserved hydrogen-bonding network of ordered water molecules in our ApSOD structures favors the binding of substrate and product in the same location and promotes coupled proton and electron transfer. The high resolution of these ApSOD structures allowed us to calculate anisotropic B-factor parameters, which provided support for Cu ion mobility described above (Fig. 5). In both ApSOD structures, most atoms were relatively isotropic; however, inspection of the active site revealed anisotropies consistent with our proposed mechanism: (1) the long axes of the Cu(II) ion thermal ellipsoids point toward the peroxide O2 atom or W0, and W1; (2) the shape of the Cu(II) ellipsoid from the H2O2 complex structure tracks the trajectory of the copper movements expected for transition from a trigonal coordination arrangement to a square planar intermediate (Fig. 5 and Supplementary Fig. S3); and (3) the ellipsoids for the His61 side chain point toward H2O2 and, furthermore, match the pivot required to bind a moving copper ion. Thus, the high resolution of these ApSOD structures allowed us to refine the proposed SOD catalytic mechanisms47,48 by visualizing how motions of the Cu(I) ion toward O2·− would allow inner-sphere electron transfer through orbital overlap. Subunit interactions in solution by SAXS The oligomeric assemblies of homologous proteins can differ between eukaryotes and prokaryotes, exemplifying another advantage of using a eukaryotic thermophile for macromolecular structural

Fig. 5. Thermal anisotropy probability ellipsoids for the ApSOD Cu-binding region. (a) The higher-resolution 0.99 Å ApSOD active-site structure. (b) The 1.35 Å ApSOD–H2O2 complex active-site structure. The thermal ellipsoids corresponding to the upper Cu ion position point between the O·− 2 /H2O2 binding site and W1. The shape of the upper Cu ion ellipsoid from the H2O2 complex structure defines the expected path of Cu ion movement during catalysis (Supplementary Fig. S5). The anisotropic B-factor ellipsoids for the His61 side chain match the motion expected to form a bond with Cu(II) in the upper position and to break the bond to Cu(I) in the lower position.

1544 studies relevant to human disease. The SOD structures from six eukaryotic species solved thus far exhibit a conserved dimeric assembly, whereas structures of prokaryotic SODs are monomers or dissimilar dimers assembled with a distinct interface.28–31 Surprisingly, only a single ApSOD subunit is found in the asymmetric unit of our crystals. The crystal packing dimer formed by crystallographic 2-fold symmetry resembles eukaryotic SOD dimers, yet only 11 out of 22 of the dimer interface residues are fully conserved with those in the other eukaryotic SODs with solved structures. So, we analyzed ApSOD's quaternary structure in solution by SAXS,52 additionally testing the suitability of A. pompejana to supply thermostable proteins that maintain human-like assemblies. Comparison of the experimental SAXS profile for ApSOD with theoretical profiles calculated from crystal structures representing a single subunit and dimer of HsSOD39 and a bacterial SOD dimer from Actinobacillus pleuropneumoniae 28 indicated a HsSOD-like dimer assembly (Fig. 6a). Further comparison of the ApSOD experimental scattering intensity and pairwise interatomic distance function

Alvinella SOD Structures, Stability and Mechanism

[P(r)] profiles with theoretical profiles calculated from structures of the ApSOD single subunit and crystallographic dimer also indicated a eukaryotictype dimer assembly in solution (Fig. 6b and c). The experimental curve of the P(r) function and Dmax value of 70 Å were in excellent agreement with the theoretical P(r) function and calculated Dmax of 68 Å for the dimer, but not the single subunit (calculated Dmax of 47 Å). Ab initio structure calculations based on SAXS data converged on a well-defined molecular envelope in which the X-ray crystal structure dimer model was readily docked (Fig. 6d and e). Thus, ApSOD is homogeneous, stable enough for solution structural analyses, and forms characteristic SOD dimers. ApSOD stability and relations to ALS mutations To evaluate if ApSOD is more stable than HsSOD, we characterized ApSOD biophysically and structurally. As SOD instability is implicated as an important factor in ALS pathogenesis,32,37–40 results on ApSOD stability may provide useful insights. We used CD to monitor denaturation of ApSOD by

Fig. 6. Determination of ApSOD dimer assembly and an ab initio structure in solution by SAXS. (a) Comparison of the experimental SAXS profile for ApSOD (black dots) to scattering profiles calculated from crystal structures of the bacterial SOD dimer from A. pleuropneumoniae (magenta line) and of HsSOD, both as a single subunit (orange line) and as a dimer (blue line), indicate that ApSOD assembles as a human-like SOD dimer. (b) Comparison of the experimental SAXS profile for ApSOD (black dots) to scattering profiles calculated from the crystallographically defined ApSOD subunit (red line) and dimer (green line) further supports ApSOD dimer assembly. (c) Comparison of the P(r) pairwise interatomic distance functions from ApSOD SAXS data (black line) with P(r) functions calculated for the single subunit (red line) and dimer (green line) crystal structure models confirms dimer assembly in solution. Scattering intensity (ordinate) is plotted against the interatomic pair distances (abscissa). (d and e) X-ray crystal structure of the crystallographic symmetry-related ApSOD dimer model (ribbon diagram) docked into an ab initio SAXS structure solution (surface).

Alvinella SOD Structures, Stability and Mechanism

Fig. 7. ApSOD high stability. Pompeii worm SOD is more stable than human SOD. Circular dichroism indicates that the unfolding midpoint for ApSOD denaturation requires nearly 1 M higher guanidine than the unfolding midpoint for HsSOD.

1545 guanidine titration. ApSOD proved exceedingly stable (Fig. 7). The unfolding midpoint of ∼5.1 M guanidine is nearly 1 M higher in concentration than the ∼ 4.25 M unfolding midpoint of HsSOD. To identify structural traits that may confer added stability, we compared the ApSOD structure with the highest-resolution wild-type SOD structures from human and five other eukaryotic species.20–23,26,27 As the overall number of residues (Fig. 2a) and fold (Fig. 8a) are well conserved among these SODs, the ApSOD dimer interface surface area and hydrophobic packing within the β-barrel were typical. We superimposed the seven structures and color-coded them by crystallographic B-factor to identify regions that are more flexible and likely less stable (Fig. 8a). The outer turns and loops had the most flexibility, consistent with results from molecular dynamic simulations53 and NMR.54 Conserved side-chain interactions among 10 crystallographically independent subunits in the HsSOD crystal structure were proposed to stabilize such protruding and flexible structural elements.24 Comprehensive inspection and analysis of the superimposed structures revealed that ApSOD has extra electrostatic interactions compared to other SODs (Fig. 8b–d and Table 2), including the most salt bridges

Fig. 8. Additional loop and termini interactions may stabilize ApSOD native fold and assembly. (a) Overlay of eukaryotic SOD structures from the seven species listed in Fig. 2a. Coloring in a spectrum according to relative Cα B-factors distinguishes more flexible, and therefore potentially less stable, parts of the molecule (red, high B-factor) from more rigid regions (dark blue, low B-factor). The most flexible parts are loops and turns localized in three areas, designated top, functional, and terminal. (b–d) Residues in the three most flexible areas of the SOD structure that form stabilizing features in ApSOD that are underrepresented in other structures (see Table 2). Subunits are colored separately. The zinc-binding region is colored the same as the zinc ion in (c) to distinguish it from the electrostatic loop (EL), and the variable loop (VL) is pink in (d).

1546

Alvinella SOD Structures, Stability and Mechanism

(Supplementary Table S1), consistent with results from a large-scale comparison of thermophilic protein structures from T. maritima with their mesophilic homologs.5 In the “top” region of the β-barrel (Fig. 8a and b), the β1/β2 turn is secured and stabilizes the dimer interface by a charged hydrogen bond between Lys9 Nζ and Asn51 Oδ1 from the adjacent subunit. β1/β2 Ser12 also hydrogen-bonds β8 Leu142. The hydrogen bond between the β2 Thr15 and β3 Thr34 side chains helps to anchor the β1/β2 and GK1 loops and may also constrain Gly35 to hydrogen-bond its N atom to the β5/β6 turn. Within the “functional” region (Fig. 8a and c), which is composed of the Zn-binding and ELs, more interloop surface interactions join Glu68 and Asn76, Asp74 and Arg126, and Gly125 and Lys133. In the “terminal” region (Fig. 8a and d), Ile2 N hydrogen-bonds to β2 Glu22 Oɛ2 across the open end of the N-terminal β-hairpin and also helps tether the adjacent β2/β3 variable loop, where only ApSOD and BtSOD share the short type II′ turn. The C-termini are stabilized by symmetric hydrogen bonds between Thr150 Oγ1 to Asp50 Oδ1 across the dimer interface, next to the symmetric Lys9 Nζ and Asn51 Oδ1 hydrogen bonds, described above. The majority of these anchoring interactions found within the β-barrel involve residues at the βstrand/turn interface, forming β–anchor–turn (βAt) and turn–anchor–β (tAβ) motifs. Altogether, 14 ApSOD residues contribute to the above hydrogen-bonding and electrostatic interactions. Of these only Asp50 and Asp74 are fully conserved among the seven species. The unusually stable bovine SOD conserves half of these tethering interactions (Fig. 2a and Table 2).

Each end of the β-barrel (Fig. 8b and d and Table 2) additionally contains “proline caps” (Pro13, Pro38, and Pro107) that may further promote protein framework stability, defined as main-chain and Cβ atom positions, by disfavoring flexibility and forming additional C–H…O bonds55 between the prolines and intra- and interloop residues. Of these turn–Pro–turn (tPt) anchoring proline cap motifs, Pro13 is conserved only within the mammalian proteins; whereas Pro38 and Pro107 are conserved within spinach and yeast, and Pro107 also in frog. There is the potential to tack down the β3/β4 GK1 loop, which contains Pro38 in ApSOD, in the bovine, human, yeast, and frog SODs by electrostatic interactions, exemplified by BtSOD's β3/β4 Glu38 and β5/β6 Lys89 charged side-chain pair. BtSOD also has unique Pro100 and Pro121 caps within β6/β7 GK2 and the β7/β8 EL. We examined the relationship of the ApSOD residues discussed above with the human ALS mutation positions. Only 3 of 14 side chains involved in hydrogen-bonding or electrostatic interactions and 1 of 3 of the proline caps lie in ALS mutation sites; none represent actual ALS mutations. However, ApSOD Lys21 and Lys98 represent the E21K and E100K ALS mutations, respectively (Fig. 2a). These lysines lie at the bottom of the β-barrel, facing outward, between the terminal region and the gap between the β-barrel and the zinc-binding subloop (Fig. 9a). In HsSOD, a charge complementary network composed of Glu21-Lys30-Glu100 links the three adjacent β-strands. Therefore, the E21K and E100K ALS mutations could destabilize HsSOD by charge repulsion or create unwanted interactions with negatively charged side chains on mutant

Table 2. Stabilizing ApSOD features Region Top 1 2 3 Functional 4 4 5 6 Terminal 7 8

ApSOD direct interaction

Observed

Potential

Not possible

ALS sitea

Lys9 Nζb–Asn51 Oδ1 Ser12 Oζ–Leu142 O Thr15 Oζ1–Thr34 Oζ1

Bt — —

Hs Soc Bt, Hs, Sm,d, So,d Xld

Sc, Sm, So, Xl Bt, Hs, Sc, Sm, Xl Sc

N, N Y, Y N, N

Glu68 N–Asn76 Oδ1 Glu68 Oɛ2–Asn76 Nδ2 Asp74 Oδ1–Arg126 NH1,2 Gly125 O–Lys133 Nζ

Bte, Xle Bte, Hse Bt, Sm, Scf Sm

Hse Soe, Xle Hsf, Sof, Xlf –

Sc, Sm, So Sc, Sm – Bt, Hs, Sc, So, Xl

N, N N, N Y, N N, N

Ile2 N–Glu22 Oɛ2 Asp50 Oδ1–Thr150 Oγ1b

Sc,g So,g Xlg Sc, So

— Xlb

Bt, Hs, Sm Bt, Hs, Sm

N, Y N, N

d

d

ApSOD proline caps

Conserved

Not conserved

ALS siteh

Pro13 Pro38 Pro107

Bt, Hs Sc, So Sc, So, Xl

Sc, Sm, So, Xl Bt, Hs, Sm, Xl Bt, Hs, Sm

N Y N

Interactions are numbered and match those in Fig. 2a; weaker water-mediated interactions are omitted. a Refers to whether the first or second residue in the interaction lies within an ALS mutation site in HsSOD, respectively. b Spans dimer interface. c One subunit bonds adjacent to Asn14 N, this interaction is prevented in ApSOD by sequestration of a N atom in Pro13 ring. d Comparable; however, short ApSOD Thr–Thr side chains ensure rigidity. e Comparable interaction is formed by alternate residues. f Comparable; however, interaction is limited to one hydrogen bond. g Comparable main chain–main chain interaction. h Refers to whether the proline lies within an ALS mutation site in HsSOD.

1547

Alvinella SOD Structures, Stability and Mechanism

HsSODs within proximity. In ApSOD, a reversal of charge complementarity provides stability through Lys21-Thr28-Lys98. Closing the “bottom” edge of ApSOD is a striking, extended hydrophobic stacking arrangement that spans from the dimer interface to the opposite subunit edge. Beginning where C-terminal Ile149 stacks with adjacent subunit GK2 Arg113 and continuing through Phe62, Pro64, and His65 of the ZnBR of β4/β5 (Fig. 9b), five side chains show favorable ∼ 4 Å van der Waals stacking. At the outside end, His65 forms an electrostatic interaction with GK2 Asp108. Only parts of these strategically placed stabilizing interactions occur in other SODs. For example, yeast SOD has the triple-ring portion, and frog SOD has a terminal electrostatic interaction. In HsSOD, Leu67 at the outside edge of the stack region (Fig. 9c) precludes an electrostatic interaction, but loosely stacks between Pro66 and Arg69 to support the Arg69 to ZnBR Glu77 salt bridge. Thus, HsSOD has weaker barrel edge interactions than those seen in ApSOD, suggesting that this edge is less restrained by favorable interactions. The most structurally important stabilizing interactions along this barrel edge represent ALS mutation sites: L67R, R115G, and I151T (Fig. 2a). As C-terminal Ile151 and its contact partner Arg115 bridge the dimer interface and Leu67 completes the end of the stack, the I151T and R115G mutations suggest that destabilization of terminal interactions and the dimer interface may play a role in ALS by reducing native SOD fold and assembly stability and hence lowering the energetic barrier to forming interactions leading to potentially toxic amyloid-like fibers and soluble aggregates.

Discussion

Fig. 9. Implications for ALS. (a) Lys21 and Lys98 in ApSOD represent charge reversal ALS mutations E21K and E100K in humans, but are likely not destabilizing in ApSOD due to charge neutralization at the structurally adjacent and intervening position Thr28 (human Lys30). HsSOD is labeled and colored in orange. (b) A stacking arrangement bridges the C-terminus and Greek key loop 2 across the ApSOD dimer interface and continues across the subunit to the zinc-binding region. In ApSOD, unconserved His65 at the end of the stack distal to the dimer interface makes a charged hydrogen or salt bridge bond with unconserved Asp108. The two subunits are colored yellow and teal, and loops within the yellow subunit are colored according to Fig. 2a. A dot surface aids identification of the stacking elements. Dimer symmetry mate residues are labeled with “-B.” (c) The stacking arrangement in HsSOD. The two subunits are colored orange and light brown. Side chains involved in ALS mutations are shown in green on one subunit and magenta on the other. The mutation sites lie at positions that bridge the dimer interface and sandwich the stack together.

Here we test and extend promising studies of the thermostability56–58 and assembly59 of proteins from A. pompejana, which inhabits one of the most physicochemically demanding environments on earth (Fig. 1). Before any macromolecule can be characterized biochemically, biophysically, or structurally, it must be sufficiently stable to allow analyses. Many planned studies on particular human proteins of medical relevance end at the stage of protein expression and purification and are replaced with characterization of homologous proteins to gain important insights into biological function. In other cases, where human protein is isolated, structural analyses may be limited by resolution, local disorder, or inability to trap certain conformations due to instability of the samples. Thus, understanding of human proteins has been substantially advanced by characterizations of their microbial homologs, and increased macromolecular stability plays a key role in these successful applications.3–10,12–15 The discovery of ApSOD, its stability, dimeric assembly, and close sequence and structural similarities to mammalian SODs support the great value of

1548 A. pompejana as a thermophilic eukaryotic macromolecule resource. We determined and present herein the high-resolution structures (Fig. 2 and Table 1), dimer assembly in solution (Fig. 6), and high stability (Fig. 7) of ApSOD. These results from the A. pompejana enzyme are relevant to the chemistry, biophysics, and physiology of its human SOD homolog and to the destabilization of HsSOD by ALS mutations. Prior to this first structural analysis test case of a thermophilic eukaryote protein, more than 70 SOD structures have been solved from a variety of different species. However, since we solved the first SOD structure over 25 years ago and predicted its substrate/product site,25,47 a structure of SOD in complex with its substrate or product had been elusive. This highly stable eukaryotic SOD, which preserved the human SOD dimer assembly, has provided active-site information absent from all previous SOD structures, suggesting A. pompejana may be a general resource for stable eukaryotic macromolecules and their assemblies. The biological importance of SOD catalysis and function as a master regulator of oxygen radicals in eukaryotic cells, the causative role of HsSOD mutants in ALS, and the many existing SOD structures, including a recent HsSOD structure at 1.07 Å resolution,27 were key reasons to choose ApSOD for these comparative analyses. These new high-resolution ApSOD and ApSOD–H2O2 structures establish conserved features, strategically positioned water molecules, and a framework for comparative analysis of active-site stereochemistry. The ApSOD active-site structures (Fig. 3 and Supplementary Fig. S4) reveal three key points: (1) the O2·−–H2O2 binding site is likely fixed, (2) the protein movements during catalysis are minor, and (3) the most significant changes are associated with His61 and the Cu ion. The high-resolution refinement of occupancies for the active-site constituents and calculation of anisotropic thermal ellipsoids delineate these Cu ion and His61 ligand movements (Fig. 5). Together, our structures and analyses support an updated and unified catalytic mechanism (Fig. 4). In contrast to Cu(I) outer-sphere electron transfer to the O2·− anion,48 our new structures and mechanism suggest that Cu(I) moves toward O2·− along a structurally facilitated trajectory for innersphere binding and orbital overlap (Supplementary Fig. S5). This inner-sphere mechanism is also consistent with prior computational analyses by density functional theory and electrostatic modeling of the energetics of SOD throughout its redox cycle, including the energetics of the coupled proton/ electron transfer.60 Our new testable model couples Cu(I)'s electron transfer to O2·− with proton transfer from His61 and formation of the His61–Cu bond. In fact, ApSOD active-site water molecules specifically suggest that O2·− protonation in the second half reaction is done in trans to avoid steric crowding, distinct from the most recently published proposal (Fig. 4).48 Furthermore, water molecules W2 and W3 are positioned to stabilize the newly formed H2O2 product, analogously to hydrogen-

Alvinella SOD Structures, Stability and Mechanism

bond recognition of H2O2 by human catalase.61 As human mitochondrial Mn SOD has a similar network of active-site water molecules, its specific roles can now be tested in the light of our proposed mechanism for the Cu,Zn enzymes.62 Thus, these ApSOD structures open the door to comparative studies relevant to all SODs, including the newly discovered Ni SOD.63 Of inherited ALS cases, N 20% involve mutations to the SOD1 gene, and SOD1 mutations have also been detected in 12% of sporadic ALS cases.41,42 Destabilization of HsSOD protein to form amyloidlike aggregates with aberrant activity is hypothesized to cause the disease.36,38,39 Our comparative structural analyses of eukaryotic SODs identified residues that may contribute to the enhanced stability of ApSOD over HsSOD and also allowed us to test and extend the idea24 that electrostatic interactions anchoring structural elements promote protein stability by maintaining the native fold and assembly. In ApSOD, specific hydrogen bonds and salt bridges secure the ends of β-strands and loops, and strategically placed tPt “proline caps” rigidify the otherwise flexible loops55 linking β-strands (Figs. 8 and 9). Significantly, the large functional loops that contribute to substrate attraction and catalysis also exhibit conspicuous stabilizing electrostatic and stacking interactions and hydrogen bonds in ApSOD. In the β-barrel core, ApSOD has some shorter hydrophobic side chains, yet retains close packing, including the Phe43 anchor buried adjacent to Cu ligand His44 to stabilize the active site. Most residues identified as contributing to the enhanced ApSOD stability lie in sequence-variable positions that do not disrupt the Greek key fold or contribute to ALS in humans (Fig. 2a and Table 2). Moreover, the majority of the electrostatic anchoring interactions involved in the Greek key fold are at the ends of β-strands forming βAt and tAβ motifs. Destabilizing mutations occur in both Cu,Zn and Mn human SODs; yet, those in Mn SOD are not associated with amyloid-like aggregation or neurodegenerative disease.64 Destabilizing ALS mutations in human Cu,Zn SOD appear to work by reducing β-barrel or dimer interface stability.32,38 Anchoring interactions at ends of α-helices were identified as important for their folding.65 Our ApSOD results suggest analogous anchoring interactions at the ends of β-strands and loops stabilize the βstructure. Based on comparisons of these anchoring interaction motifs and sites with those mutated in SOD ALS patients, such anchoring interactions may furthermore avoid the formation of the inappropriate beta-structural interactions and amyloid-like filaments associated with many neurodegenerative diseases including ALS. More generally, these ApSOD analyses suggest that functional protein stability, relevant to preservation of native fold and assembly in vivo, can be substantially improved (or degraded) by substitutions or mutations that increase (or decrease) the rigidity of the weakest or most flexible parts of the structure, without altering core fold or assembly. Such “weakest links” generated through

1549

Alvinella SOD Structures, Stability and Mechanism

mutations in loops and termini may induce local unfolding and framework destabilization that trigger nucleation of amyloid-like filaments to form aggregates (Fig. 10). These structural analyses combined with the identification of other A. pompejana proteins that show thermostability traits56–58 suggest that there is likely a structural basis, which includes extra saltbridged interactions similar to those identified in T. maritima proteins,5 for enhanced thermostability of other A. pompejana proteins, and that these proteins may aid insights into structure–function relationships in other systems. Overall, we showed here that ApSOD is more stable than HsSOD biophysically (Fig. 7) and identified interactions expected to enhance structural stability (Figs. 8 and 9). Such stable SOD forms have been sought for biotechnology and medical applications for which ApSOD may be tested.66 We furthermore showed that the human-like ApSOD dimer (Fig. 6) was able to trap a previously unobtainable H2O2 complex (Fig. 3), which extends our detailed understanding of the SOD reaction and suggests a unified general mechanism for catalysis (Fig. 4). Notably, this proposal resolves apparent paradoxes regarding the electron transfer despite restrictive active-site geometry that would seemingly

block direct binding of O2·− to Cu(I). These initial results support further analyses of the Pompeii worm's value as a thermophilic eukaryote by laboratories worldwide. For biochemists, biophysicists, and structural biologists, the Pompeii worm may provide stable full-length eukaryotic macromolecules, and for X-ray crystallography, the additional resolution to provide new insights into particular systems of interest. Indeed, A. pompejana's ability to thrive in its harsh environment suggests that its DNA, RNA, and proteins will be key resources that may prove broadly useful in biotechnology, industry, and biomedical analyses and should be tested accordingly.

Materials and Methods Sample collection and cDNA sequencing Using robotic arms housed on the DSV Alvin, we collected A. pompejana worms from hydrothermal vent sites (9°N, 50/104°W17) at a depth of ∼2500 m (Fig. 1). Temperature measurements were made with a narrow ∼ 0.3 -cm-diameter temperature probe. Worms were placed in an insulated device containing RNAlater (gift

Fig. 10. Implications for amyloid-like filament nucleation and aggregate formation. (a and b) A unified model for nucleation of amyloid-like filaments and formation of soluble aggregates. Due to flexibility in loop and termini regions, over time both (a) ApSOD and (b) HsSOD are likely subjected to reversible local structural perturbations that transiently open structures to expose regions normally sequestered by fold and assembly interactions. ALS SOD mutants that lie within loop and termini regions may cause toxicity by shifting equilibria toward more open forms with decreased barriers to nucleation and growth on nonnative, amyloid-like fiber forming contacts. Support for this hypothesis comes from (a) the increased stability of ApSOD, which has additional contacts in these regions, shown as spheres (purple, intrasubunit electrostatic and hydrogen-bonding interactions; gray, N-terminal interaction; green, cross-dimer interaction; red, Cterminal and cross-dimer interaction; blue, proline cap; see Fig. 8 and Table 2) that tether these elements and thereby may give ApSOD its higher stability to HsSOD. The interactions shared in HsSOD are also shown as spheres. ALS mutations may enhance local perturbations by removing these and other key contacts, by introducing charge repulsion, or by steric hindrance of normally favorable interactions and packing, resulting in nonnative amyloid-like-promoting interactions.

1550 from Ambion) for in situ stabilization of RNA. Worms were then frozen and transported to the laboratory in dry ice and then stored at −80 °C. Episymbiotic bacteria were later removed from the dorsal side of the worms by scraping with forceps. A single worm was dissected, and Incyte Genomics Corporation generated cDNA libraries from the worm's posterior end, which experiences higher temperatures. The cDNA were ligated into the pBluescript KS+ vector. Approximately 5300 sequences were read (Genome Therapeutics). The A. pompejana SOD gene was identified by a tblastn search using the HsSOD protein sequence as a query. Sequence analysis Using default blastp settings, we conducted a search using the predicted ApSOD protein query sequence on the National Center for Biotechnology Information database, which yielded the Crassostrea gigas (pacific oyster), Bos taurus (cattle, predicted), Oryza sativa (rice, japonica cultivar group), Pinus pinaster (maritime pine), Ixodes scapularis (black-legged tick), and Bos grunniens (domestic yak) Cu,Zn SOD proteins as the top five matches. These hits indicated that the A. pompejana sequence was conserved with other eukaryotic SODs, including those from mammals. The sequences and tertiary folds of the highest-resolution SOD proteins representing all eukaryotic species with structures deposited at the Research Collaboratory for Structural Bioinformatics—B. taurus (cattle), H. sapiens (human), S. mansoni (trematode), X. laevis (frog), S. oleracea (spinach), and S. cerevisiae (budding yeast)—were aligned and superimposed individually with ApSOD using SEQUOIA. A CLUSTALW sequence alignment was then manually edited by using the structural overlays as guides. A consensus phylogenetic tree (Fig. 2b) was generated with neighbor-joining and 100 iterations of bootstrapping. Cloning, expression, and purification A cDNA library for cloning was generated on board the R/VAtlantis. Episymbiotic bacteria were removed by briefly freezing a single worm and then scraping with forceps. The worm was then frozen and crushed with a mortar and pestle. PolyA RNAwere isolated by using the PolyA Pure kit (Ambion). cDNA were synthesized from PolyA RNA using the Marathon cDNA amplification kit (Clontech). PCR primers 5′-GAT AGG CCA TCC AGG CCG TTT GCG TCC TGA AGG GA-3′ and 5′-GCG CGG TCG ACT TAC TCC TTT GTA ATA CCA ATG ACA CCA CA-3′ were designed from the ApSOD processed mRNA transcript sequence identified by cDNA sequencing and used to amplify the coding region, with the exception of the most N-terminal residues, which were substituted with an Sfi1 restriction site. The PCR primer for the C-terminal end contained a flanking Sal1 restriction site. Following PCR amplification of the gene from the cDNA library, the PCR product was ligated into the PCR2.1 TOPO vector (Invitrogen). The ApSOD gene was removed from the PCR2.1 TOPO vector and the HsSOD gene was removed from the pPHSODC6AC111SlacIqR139 expression vector with SfiI and SalI restriction enzymes. The liberated ApSOD gene was then ligated into the linearized expression vector. The SfiI site was then mutagenized to yield the correct N-terminal sequence for ApSOD. The vector contains a leader sequence that targets expressed protein to the periplasm, where the leader is then removed. ApSOD protein was expressed and purified by following previously established protocols for other SOD

Alvinella SOD Structures, Stability and Mechanism proteins in our laboratory. Transformed SOD−/− Escherichia coli cells were cultured in Luria broth supplemented with 100 μg/ml ampicillin at 37 °C. When cells reached an optical density of 0.8 at 600 nm, 0.25 mM CuSO4 was added, and 0.4 mM IPTG was used to induce protein synthesis. Cells were cultured another 6–12 h before harvesting and resuspension in 100 ml of ice-cold Tris–Cl buffer (pH 7.5). Ice-cold 40% (w/v) sucrose (100 ml) and 0.5 M ethylenediaminetetraacetic acid (30 ml) were used to induce osmotic shock. The suspension was rocked for 30 min at 4 °C, then subjected to centrifugation. The supernatant was removed. The pellet was resuspended in 200 ml H2O and centrifuged at 4 °C. The two supernatants were combined and proteins were precipitated by the addition of ammonium sulfate to 65% (w/v). This suspension was centrifuged and the pellet was resuspended in 50 ml H2O. The protein solution was then dialyzed twice against 4 l of H2O and centrifuged. CuSO4 was added to the supernatant at a final concentration of 1 mM. The solution was heated to 65 °C for 30–90 min and centrifuged. The supernatant was dialyzed against 4 l of 100 mM Mes (pH 5.5) overnight. Following another round of centrifugation, the supernatant was applied to a POROS HS column and eluted with a NaCl gradient. ApSODcontaining fractions were pooled, dialyzed against 25 mM Tris–Cl (pH 8.0), and applied to a POROS or HiTrap HQ column from which ApSOD protein was eluted with a NaCl gradient. Pooled fractions were concentrated to 2 ml and applied to a Superdex 75 16/60 gel-filtration column. All column media were purchased from Pharmacia. ApSOD protein was metallated,27,39,67,68 following established protocols,39,67 by dialyzing against equal molar ZnSO4 in 10 mM sodium acetate (pH 5.5) buffer. Equal molar CuSO4 was then added to the dialysis solution and allowed to equilibrate. Protein was then dialyzed in H2O and concentrated to 24–31 mg/ml. The color of the protein solution was blue, indicating that the Cu ions were incorporated and oxidized. Protein samples migrated as a single band on nondenaturing polyacrylamide gels, indicative of a homogeneous protein species (Supplementary Fig. S1). Purification of SODs directly from some source organisms show that the N-terminal Met is often posttranslationally cleaved and the penultimate residue acetylated.44 Mass spectrometry analysis and N-terminal sequencing were used to verify ApSOD composition and showed that the protein's amino acid sequence starts with the second-position alanine, which was not acetylated. SOD activity was monitored with a polyacrylamide-gelbased assay (Supplementary Fig. S1).39,40,69 Protein samples (10 μg) were migrated into a nondenaturing 4– 20% Tris–glycine gel in native running buffer [50 mM Tris, 380 mM glycine (pH 8.5)] by electrophoresis. DNA double-strand break repair protein Mre118 served as a negative control, and HsSOD (prepared as described70) as a positive control. The gel was soaked in Stain I buffer (0.2% nitroblue tetrazolium) and shaken for 20 min in the dark at room temperature. After being rinsed briefly with water, the gel was soaked in Stain II buffer [0.0042% N,N, N′,N′-tetramethylethylenediamine (TEMED), 0.03 mM riboflavin, 360 mM NaKHPO4 (pH 7.8)] and shaken in the dark at room temperature for 20 min. The gel was developed by illumination on a light box for 10 min and then soaked in 10% (v/v) acetic acid for 5 min to stop development. Activity is indicated on the gel by bleaching. X-ray crystal structure determination ApSOD crystals were grown using vapor diffusion by mixing 1.5 μl of 24 -mg/ml protein solution with 1.5 μl

1551

Alvinella SOD Structures, Stability and Mechanism well solution from an ammonium sulfate grid screen. Xray diffraction data were collected at Stanford Synchrotron Radiation Laboratory (SSRL) beamline 11-1 from crystals that grew in 55% saturated ammonium sulfate, 100 mM sodium citrate buffer (pH 5.5). These crystals, grown in initial unrefined conditions, were cryoprotected in well solution supplemented with 20% (v/v) glycerol. In an effort to collect all of the high-resolution data after collecting low-resolution data, the detector was translated laterally, placing the X-ray beam center in one corner. However, the combination of beamline physics with the crystal's unit cell constants limited the resolution of the combined data set to 1.03 Å resolution. Diffraction data were processed with the use of the HKL2000 suite71 and TRUNCATE.72 Phases for this initial 1.03 Å data set were calculated by molecular replacement using AMORE72 with HsSOD (PDB code 1PU039) as a search model. An ApSOD model was then generated and refined by iterative rounds of manual fitting with XFIT73 and refinement initially with CNS74 and σ-a weighted terms, and in later rounds by SHELXL.75 Low- and high-resolution data sets with a combined range of 50–0.99 Å resolution were later collected from a single crystal at the Advanced Light Source SIBYLS beamline 12.3.1 using 2θ detector rotations (Supplementary Fig. S2). The crystal was grown under the original conditions, but cryoprotected by dehydration. Modification of the original crystal conditions or alternative cryoprotectants did not significantly alter the resolution. To dehydrate the crystal, additional saturated ammonium sulfate was added to the crystallization wells to a final 90% (v/v) concentration and the drop was allowed to equilibrate for 30 min. The initial 1.03 Å model was then combined with the 0.99 Å data set to generate new maps followed by iterative rounds of model building and refinement using SHELXL. For the H2O2 complex, a single crystal grown as described above was transferred from its mother liquor to a 3 -μl drop solution containing 90% saturated ammonium sulfate, 100 mM sodium citrate (pH 5.5), and 1 mM H2O2. Under the acidic conditions used, H2O2 with a pKa N 11 is expected to remain protonated, decreasing peroxidative reactions.76 The crystal was soaked over a 90% saturated ammonium sulfate well solution for 30 min and cryocooled. Diffraction data were again collected by using a 2θ detector rotation at the SIBYLS beamline 12.3.1 and were processed as described above, resulting in a 1.35 Å data set. The initial 1.03 Å model was fit to data for the H2O2 complex by using CNS rigid-body refinement, followed by cycles of model building and refinement, as described above. For both models, alternative main-chain and side-chain conformations and lower-occupancy metal and solvent sites found in Fo − Fc difference maps were verified by CNSgenerated simulated annealing and composite-omit maps. In later rounds of refinement, manual estimations and freeoccupancy variables were used to refine the occupancies of these atoms. In the last round of refinement, their occupancies were fixed, allowing B-factors to converge. These occupancy estimates were checked using R-factors and a variety of electron and difference density maps. Rfree calculations77 were performed by removing 5% of the data for the test set. The stereochemistry for each structure was validated using MolProbity.78 Standard errors for bond lengths and angles for both individual structures were calculated using SHELXL least-squares refinement. The number of salt-bridges in the ApSOD and other eukaryotic SOD structures was calculated with WHATIF.79 Bonds less than 4 Å5 and protein atoms involved in direct bonding to metals were not used in calculating the totals.

For the 0.99 Å resolution ApSOD structure, the clear electron density allowed modeling of residues 1–151 from a total of 152 residues, including nine alternative residue conformations, the Cu ion at two positions and one Zn ion. The model also includes two acetate, one sodium, and six sulfate ions, and 354 ordered water molecules. For the 1.35 Å ApSOD–H2O2 complex structure, residues 1–151 were also modeled, with eight alternative residue conformations, the Cu ion at two positions and one Zn ion. This model also includes a hydrogen peroxide molecule, one sodium and four sulfate ions, and 295 ordered water molecules. As expected for the mild experimental conditions employed, we did not detect any protein modifications80,81 that occur when H2O2 is converted to more reactive species.76,82,83 Small-angle X-ray scattering SAXS data sets were collected on 15 -μl ApSOD protein samples at 2, 4, and 6 mg/ml concentrations in phosphatebuffered saline (PBS, pH 7.4) at 25 °C at the Advanced Light Source SIBYLS beamline 12.3.1 using a MAR CCD 165 detector as detailed elsewhere.52 The X-ray beam was tuned to 13 keV with an incident flux of approximately 1011 photons/s. The sample-to-detector distance was 1.46 m. The detector was centered normal to the primary X-ray beam, and data were collected with a scattering vector Q (Q = 4π sin θ/λ, where 2θ is the scattering angle and λ is the wavelength) spacing range of 0.01 to 0.35 Å− 1. Short exposures of 10 s and long exposures of 100 s were taken for each sample, flanked by corresponding blank buffer samples. Short and long data sets were merged with PRIMUS84 using the 10 -s exposures for the low-Q region and the 100 -s exposures for the high-Q region. Both blank buffer data for each experiment were subtracted from the protein-scattering data and compared to ensure proper normalization due to flux variations. A concentration gradient is required to ascertain whether scattering from complex–complex interactions or small amounts of aggregation contribute to the overall scattering profile. These contributions must be eliminated in order to further analyze the scattering from the target molecule. Therefore, wider angle data out to Q = 0.64 Å− 1 were collected on ApSOD protein at a concentration of 31 mg/ml by vertically moving the detector 6 cm perpendicular to the beam. PRIMUS was used to calculate the radius of gyration (RG) using the Gunier approximation and the low resolution (QRG b 1.3) data. Using the concentration behavior of the sample, we extrapolated the scattering profile to an effective 0 concentration by scaling the superimposable data (Q b 0.1 Å− 1) and linearly fitting the intensity at each value of Q b 0.1 as a function of concentration.52 The measured RG from the 0 concentration scattering curve was 20.3 Å (Supplementary Fig. S6). The high concentrations showed long-length interactions between independent dimers, observable in the low-angle region. As the repulsion solely affects the scattering at low Q values (Q b 0.1), higher concentration data sets were merged with the 0 concentration result. Thus, the data presented in Fig. 6 are a composite of 2 -mg/ml data used in the low-angle region (up to Q = 0.1 Å− 1) merged with the data collected at the wider-angle setting (6 mg/ml results were used for data 0.15 b Q b 0.25 and 31 mg/ml at 0.2 b Q b 0.6). The P(r) functions and subsequent maximum interelectron distances Dmax were calculated with GNOM.85 Calculated curves based on the human 39 and A. pleuropneumoniae28 SODs and the 0.99 Å ApSOD X-ray crystal structure were generated with CRYSOL.86 HsSOD

1552 chain A from 1PU0 was used to represent the single subunit of HsSOD. For the ApSOD structure, the loweroccupancy alternative atoms were removed and the occupancies of the remaining atoms were set to 1.00 prior to calculations. Ab initio 3D image reconstructions were generated with GASBOR.87

Alvinella SOD Structures, Stability and Mechanism

Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2008.11.031

Denaturation assays

References Protein unfolding was monitored as previously described.39 ApSOD and HsSOD proteins were mixed with increasing amounts of guanidine HCl (0 to 7 M in 0.5 M steps) in PBS buffer (pH 7.4). The final concentration for each protein was 20 μM. Following incubation, the extents of protein unfolding were assessed by CD spectroscopy. Samples were loaded into a 0.1 -cm pathlength quartz cell and the ellipticities were measured at 218 nm with an AVIV model 2o2SF stopped-flow CD spectrometer in kinetics mode. Multiple measurements were averaged to obtain final values. To convert raw data to fraction unfolded (Fu), straight lines were fit through the upper and lower plateau regions of the plots. The following formula was then used to normalize the data: FU = ðYN
YÞ=ðYN
YD Þ UN and UD represent the fits of the lines through the upper and lower plateau regions, respectively, and U is the ellipticity value at a given guanidine concentration. Data were plotted as fraction unfolded versus guanidine concentration. A detailed analysis of the free-energy changes were precluded, as it was shown that wild-type SOD folding and unfolding are not fully reversible due to metal binding39 and illegitimate disulfide bond formation that produces aggregation.88 For accurate interspecies comparisons, we used wild-type protein rather than the less aggregation prone HsSOD Cys mutants used for in vitro studies.89 Accession numbers The ApSOD mRNA sequence has been deposited in GenBank with accession number EU178106. Coordinates and structure factors have been deposited in the PDB with accession numbers 3F7L for the ApSOD X-ray crystal structure and 3F7K for the ApSOD–H2O2 structure.

Acknowledgements We thank B. R. Chapados, D. S. Daniels, J. A. Fee, K. Henscheid, K. Hitomi, and L. Noodleman for helpful discussions and technical support. We thank the staffs of SSRL beamline 11-1 and Advanced Light Source beamline 12.3.1 for diffraction facilities. We thank the R/V Atlantis and DSV Alvin crews in aiding A. pompejana sample collection. This work was supported by the 3rd Annual Incyte Discovery Award (D.S.S., S.C.C., J.A.T.), National Institutes of Health R01 GM037684 (E.D.G.), National Sciences Foundation LExEn NSF-9907666 (S.C.C.), Biocomplexity OCE-0120648 (S.C.C.), and Department of Energy program Integrated Diffraction Analysis Technologies (J.A.T.). D.S.S. was supported in part by Skaggs Institute for Chemical Biology and Ruth L. Kirschstein NSRA Fellowships.

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