Candida albicans SOD5 represents the prototype of an unprecedented class of Cu-only superoxide dismutases required for pathogen defense Julie E. Gleasona,1, Ahmad Galaleldeenb,1, Ryan L. Petersona, Alexander B. Taylorc, Stephen P. Hollowayc, Jessica Waninger-Saronib, Brendan P. Cormackd, Diane E. Cabellie, P. John Hartc,f,2, and Valeria Cizewski Culottaa,2 a Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205; bDepartment of Biological Sciences, St. Mary’s University, San Antonio, TX 78228; cDepartment of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229; dDepartment of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205; eChemistry Department, Brookhaven National Laboratories, Upton, NY 11973-5000; and fDepartment of Veterans Affairs, Geriatric Research, Education, and Clinical Center, South Texas Veterans Health Care System, San Antonio, TX 78229

Edited by Sabeeha S. Merchant, University of California, Los Angeles, CA, and approved March 5, 2014 (received for review January 3, 2014)

The human fungal pathogens Candida albicans and Histoplasma capsulatum have been reported to protect against the oxidative burst of host innate immune cells using a family of extracellular proteins with similarity to Cu/Zn superoxide dismutase 1 (SOD1). We report here that these molecules are widespread throughout fungi and deviate from canonical SOD1 at the primary, tertiary, and quaternary levels. The structure of C. albicans SOD5 reveals that although the β-barrel of Cu/Zn SODs is largely preserved, SOD5 is a monomeric copper protein that lacks a zinc-binding site and is missing the electrostatic loop element proposed to promote catalysis through superoxide guidance. Without an electrostatic loop, the copper site of SOD5 is not recessed and is readily accessible to bulk solvent. Despite these structural deviations, SOD5 has the capacity to disproportionate superoxide with kinetics that approach diffusion limits, similar to those of canonical SOD1. In cultures of C. albicans, SOD5 is secreted in a disulfide-oxidized form and apo-pools of secreted SOD5 can readily capture extracellular copper for rapid induction of enzyme activity. We suggest that the unusual attributes of SOD5-like fungal proteins, including the absence of zinc and an open active site that readily captures extracellular copper, make these SODs well suited to meet challenges in zinc and copper availability at the host–pathogen interface.

host immunity, and the intracellular SOD1 of C. albicans has been shown to be important for virulence (14). C. albicans also harbors three genes for expressing SOD1-related GPI-anchored proteins, namely, SOD4, SOD5, and SOD6. Of these genes, SOD5 was shown to be critical for combating the oxidative burst of infection (15–17). C. albicans mutants lacking SOD5 are more susceptible to killing by macrophages and neutrophils, and are associated with increased reactive oxygen species production by macrophages and neutrophils (16, 18, 19). Most notably, sod5Δ/Δ mutant fungi exhibit decreased virulence in a mouse model for infection (15). A similar GPI-anchored protein known as SOD3 was reported for the respiratory human fungal pathogen Histoplasma capsulatum (20). As with C. albicans SOD5, SOD3 helps protect H. capsulatum from killing by macrophages and is important for virulence in a mouse model of infection (20). Despite the importance of these EC-SOD1–like proteins in fungal pathogenesis, virtually nothing is known regarding their mechanism of action. In this study, we investigated the nature of these molecules by focusing on C. albicans SOD5. The 3D structure of SOD5 was solved by single-crystal X-ray diffraction, and the reactivity of SOD5 with superoxide was examined in vitro and in C. albicans cultures in vivo. We report here that SOD5 represents an Significance

E

ukaryotes are known to express two highly related classes of copper-containing superoxide dismutase (SOD) enzymes that play widespread roles in oxidative stress resistance and signaling. These two classes include an intracellular, largely cytosolic SOD1 (1) and an extracellular SOD (EC-SOD) (2), both of which are bimetallic enzymes with copper and zinc cofactors. The redox active copper catalyzes the disproportionation of superoxide anion to oxygen and hydrogen peroxide, whereas the zinc helps stabilize the protein (3–5) and promotes pH independence of the reaction (6–8). Additionally, all eukaryotic copper and zinc SODs contain an active site channel that consists of a cluster of charges in loop VII that culminate with an invariant arginine adjacent to the copper site. This element, commonly known as the electrostatic loop, is proposed to provide long- and short-range guidance for the superoxide substrate, thereby facilitating the remarkable kinetics of the SOD reaction (2, 9, 10). SOD1 is among the fastest enzymes known, with rates (109 M−1 s−1) that approach diffusion limits (9, 11, 12). Very recently, certain pathogenic fungi have been reported to express a class of extracellular proteins that are homologous to SOD1 and are covalently attached to the cell wall through GPI anchors. These SODs were first described for the opportunistic fungal pathogen Candida albicans. C. albicans is a common commensal microbe of the human gut, but under conditions of a weakened immune system, the organism can become invasive and pathogenic, with infections ranging from mild mucosal candidiasis to life-threatening systemic disease (13). As with many pathogens, C. albicans must evade the oxidative burst of

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Candida albicans is the most prevalent human fungal pathogen. To combat the host immune response, C. albicans expresses superoxide dismutase 5 (SOD5), a cell wall protein related to Cu/Zn SODs. We find that SOD5 structure markedly deviates from Cu/Zn SOD molecules. It is a monomeric copper-only SOD that lacks a zinc site and electrostatic loop. In spite of these anomalies, SOD5 disproportionates superoxide at remarkably rapid rates. When expressed in C. albicans, SOD5 can accumulate outside the cell in an inactive form that can subsequently be charged for activity by extracellular copper. SOD5-like molecules are present in many fungal pathogens and appear to be specialized for the metal and oxidative challenges presented by the host immune system. Author contributions: J.E.G., A.G., R.L.P., B.P.C., D.E.C., P.J.H., and V.C.C. designed research; J.E.G., A.G., R.L.P., S.P.H., and J.W.-S. performed research; J.E.G. and A.B.T. analyzed data; and J.E.G., A.G., R.L.P., D.E.C., P.J.H., and V.C.C. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: Coordinates and diffraction data for SOD5 structures coming from crystal forms A and B have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 4N3T (CuI) SOD5 and 4N3U (CuII) SOD5]. 1

J.E.G. and A.G. contributed equally to this work.

2

To whom correspondence may be addressed. E-mail: [email protected] or [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1400137111/-/DCSupplemental.

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Results SOD5-Like Proteins Do Not Conform to Traditional Cu/Zn SOD Enzymes.

Upon inspection of the primary sequences, we noted striking deviations between the fungal EC-SODs and canonical SOD1, an example of which is shown with C. albicans SOD5 in Fig. 1A. Although SOD5 possesses the copper binding histidines (H75, H77, H93, and H153), the disulfide cysteines (C87 and C162), and the active arginine (R159) of SOD1, it is missing two of four zinc binding histidines as well as 17 residues of the all-important electrostatic loop (Fig. 1A). These aberrations are not unique to SOD5 but are also seen with C. albicans SOD4 and SOD6 and with H. capsulatum SOD3 (Fig. 1B). Moreover, upon inspection of available databases, SOD5-like proteins are predicted to occur throughout Ascomycota and Basidiomycota fungi, each retaining the SOD1-like copper site and disulfide but missing ligands for zinc and the electrostatic loop region (examples are provided in Fig. 1B and Table S1). Three-Dimensional Structure of C. albicans SOD5. To explore the structure and mechanistic action of these curious fungal molecules, we used C. albicans SOD5 as a model. Recombinant SOD5 lacking the N- and C-terminal signals for protein secretion and GPI anchorage, respectively, was expressed in Escherichia coli and reconstituted with copper to an occupancy of ∼80%. Extensive attempts to reconstitute similarly with zinc or iron were unsuccessful, which was an unexpected finding, given the avidity of

Fig. 1. Sequence of SOD5 and SOD5-like proteins. (A) Alignment of the C. albicans (Ca) SOD5 against C. albicans and S. cerevisiae (Sc) SOD1. Copper ligands, blue; zinc ligands, green; His bridges Zn(II) and Cu(II) in SOD1, blue and green boxed; disulfide Cys, purple; active site Arg, cyan; N- and C-terminal secretion and GPI anchorage extensions, yellow. Blue and black arrows demark recombinant SOD5 from C. albicans and E. coli. A SOD1 electrostatic loop is underlined and a single dagger (†) marks the invariant Asp in copper-containing SODs. (B) SOD5 residues F72–N164 aligned against similar domains from select fungi. ΔEL, missing electrostatic loop; Hc, Ascomycota human pathogen H. capsulatum SOD3 (20); Pg, Basidiomycota plant pathogen Puccinia graminis XP_00336004; Pt, Ascomycota plant pathogen Pyrenophora teres XP_003305710.

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zinc for the copper site in SOD1 (21) (Discussion). We determined and refined the crystal structures of Cu(I)SOD5 and Cu(II)SOD5 to resolutions of 1.4 and 1.75 Å, respectively. The 1.4-Å structure (crystal A) was determined using short-wavelength, high-flux synchrotron radiation, whereas the 1.75-Å structure (crystal B) was determined using longer wavelength, in-house X-rays in an experiment designed to minimize photoreduction by the X-ray beam. Table S2 summarizes the diffraction data and protein structure refinement statistics. The overall architecture of SOD5 in the same orientation as Saccharomyces cerevisiae SOD1 is shown in Fig. 2. Superimposition of the two molecules (Fig. 2C) reveals that the key β-barrel and zinc loop IV elements of SOD1 are largely retained in SOD5. The two molecules superimpose with an rmsd of 0.9 Å for target backbone atoms. SOD5 cysteines C87 and C162 form a disulfide that spatially coincides with that of SOD1 (Fig. 2). As notable distinctions, SOD5 contains an additional nine residues at the C terminus for GPI anchorage and an expanded disulfide subloop (Fig. S1), and it lacks zinc. With no electrostatic loop, the copper site in SOD5 appears more open than that of SOD1 (Fig. 2). In addition, there is no evidence for a dimer interface in the crystal structure or for self-association in the analytical ultracentrifugation sedimentation velocity profile (Fig. S2). Unlike dimeric and tetrameric SOD1 and EC-SOD, SOD5 is a monomer. Molecular modeling strongly suggests that the expansion of the disulfide loop of SOD5 precludes dimer formation through steric considerations (Fig. 2C and Fig. S1). The architecture and coordination geometries of Cu(I) and Cu(II) bound to SOD5 are similar to those observed for S. cerevisiae SOD1 (22, 23). In Cu(I) SOD5, the copper is coordinated by residues H75, H77, and H153 in a pseudotrigonal planar arrangement and there is an absence of connecting electron density between the copper and the epsilon nitrogen of H93 located ∼3.5 Å away (∼3.2 Å in SOD1). These observations are consistent with protonation of the H93 side chain during the reduction of Cu(II) to Cu(I) (22, 23) (Fig. 3 A and B). Conversely, in Cu(II) SOD5, this distance shortens to 2.8 Å (2.7 Å in yeast SOD1) and clear connecting electron density is observed between the copper ion and the epsilon nitrogen atom of H93 (Fig. 3C). These findings are consistent with deprotonation of H93, permitting the epsilon nitrogen of H93 to act as a ligand to Cu(II) (22, 23) (Fig. 3D). In the absence of spectroscopic data taken from a crystal, however, care should be taken when correlating redox state with geometries. For example, in contrast to the trigonal planar Cu(I) coordination geometries observed in SOD5 and yeast SOD1, crystallographic determination of bovine SOD1, in which the copper had been forcefully reduced with exogenous reducing agents, revealed a four-coordinate copper with an intact imidazolate bridge. This finding was attributed to the influence of crystal packing forces and the SOD rack on copper oxidation state (24). Overall, however, the (His)3 coordinate copper observed in crystal A and the (His)4 coordinate copper in observed crystal B are as expected for a copper-containing SOD enzyme during the two half-reactions of superoxide disproportionation. A remnant of the electrostatic loop in SOD5 is R159. In SOD1, the equivalent R143 helps attract superoxide to the copper site (12) and is anchored via carbonyl oxygens from G61 and C57. These same interactions are preserved in SOD5, and SOD5 R159 is additionally stabilized by H-bonding with T90 (Figs. 2A and 3 A and C). This positioning of R159 near the copper site, together with the geometry of bound Cu(I) and Cu(II), is highly reminiscent of the active site of Cu/Zn SOD molecules, despite no zinc and no electrostatic loop. Rapid Kinetics of SOD5 Activity. Thus far, the only evidence supporting a SOD function for SOD5 has emerged from macrophage and neutrophil infection studies, where the oxidative burst was dampened by C. albicans expressing SOD5 (18, 19). To measure SOD activity directly, we used pulse radiolysis, the definitive method for obtaining rate constants in SOD enzymes. We observed that SOD5 rapidly reacts with superoxide and that PNAS | April 22, 2014 | vol. 111 | no. 16 | 5867

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unprecedented class of Cu-only monomeric SODs that function without a zinc cofactor and an electrostatic loop. When secreted from C. albicans, SOD5 is rapidly charged with extracellular copper and the enzyme has the capacity to remove superoxide at diffusion-limited rates. SOD5-like proteins appear widely spread throughout pathogenic fungi, poised for the unique challenges at the host–pathogen interface.

Fig. 2. Overall structure of SOD5 and comparison with SOD1. The 3D structure of C. albicans SOD5 is compared with S. cerevisiae SOD1, where yellow, blue, and green spheres correspond to disulfide bonds, Cu atoms, and Zn atoms, respectively. (A) C. albicans SOD5. The β-barrel is blue, loop IV is green, loop VII is yellow, and His93 is orange. H-bonds and Cu–ligand interactions are yellow dashes. (B) S. cerevisiae SOD1. The β-barrel is pink, loop IV is blue, loop VII is red, and His63 is orange. H-bonds and metal–ligand interactions are yellow dashes. (C) Superimposition of SOD5 and SOD1. The boxed region of the disulfide loop (a subelement of loop IV) prevents homodimerization of SOD5 (Fig. S1).

reactivity is proportional to enzyme concentration (Fig. 4A). The kcat calculated from the linear regression of these data was found to be 1.1 × 109 M−1 s−1 at pH 7.25 (Fig. 4B), a rate that closely approximates that determined per metal site in SOD1 (9). Thus, Cu-only SOD5 is a bona fide SOD enzyme with rates that approach diffusion limits. The zinc cofactor in SOD1 promotes pH independence of kcat from pH 6–9.5 (6–8). Although lacking zinc, SOD5 is relatively unaffected up to pH 8.0, with maximal activity at pH 6.0, where kcat = 1.8 × 109 M−1 s−1 (Fig. 4C). The precipitous drop in activity at pH ≥ 8.5 was readily reversible upon lowering the pH, even in the presence of metal chelators and under conditions when SOD5 was pulsed with superoxide and generated micromolar H2O2 (Fig. 4C). Hence, there is minimal peroxide damage to the enzyme at high pH, and the copper cofactor is retained. It is noteworthy that in the human host, C. albicans is not expected to experience pH conditions that exceed 8.0 and, moreover, the organism is capable of neutralizing the pH of its environment (25). SOD1 activity decreases under conditions of high ionic strength, presumably due to ionic shielding of the steering forces in the electrostatic loop (9, 10, 12). Surprisingly, SOD5 also exhibited a decrease in kcat at increasing ionic strength with slopes of −0.6 and −0.35 at pH 6.0 and pH 7.25, respectively (Fig. 4D). Thus, even without an electrostatic loop, steering forces may participate in guiding substrate to the SOD5 active site (Discussion). SOD5 Functions as a Copper-Dependent SOD in C. albicans. Although recombinant SOD5 expressed in E. coli is clearly an efficient SOD, it was critical to evaluate SOD5 expressed from its native 5868 | www.pnas.org/cgi/doi/10.1073/pnas.1400137111

host, where the protein is processed through the secretory pathway. SOD5 is normally anchored in the cell wall, but the harsh treatments needed to extract SOD5 protein from the cell wall (17) would prohibit recovery of active enzyme. Also, the abundant cell surface reductases in C. albicans interfere with SOD assays in intact cells (26). To circumvent these issues, we expressed SOD5 lacking the GPI anchor, such that secreted SOD5 could be recovered in the growth medium, precisely as was done with Histoplasma SOD3 (20). Recombinant SOD5-170 missing the C-terminal GPI anchor signal was secreted from C. albicans in a heavily glycosylated form, as indicated by strong reactivity with endoglycosidases (Fig. 5A). By a native gel assay for SOD enzymes, SOD5-170 activity was strongly induced by supplementing cultures with copper (Fig. 5B) but not zinc (Fig. 5C), and the same was true for a larger version of SOD5, namely, SOD5-204, which more closely approximates full-length SOD5 anchored to the cell wall at position 205 (Fig. S3B). We also examined the disulfide status of SOD5 using a redox Western blot (27). In the case of fungal SOD1, disulfide oxidation is typically coupled with copper insertion (27, 28). However, with SOD5, the disulfide appears oxidized under all conditions, even without the copper supplements needed to activate the enzyme maximally (Fig. 5D). Disulfide oxidation in SOD5 does not appear to be dependent on copper. Eukaryotic Cu/Zn SODs are regulated posttranslationally through the controlled insertion of copper and oxidation of the disulfide bond. With intracellular SOD1, both modifications are accomplished through the copper chaperone for SOD1 (CCS) (28–30), whereas EC-SOD matures inside the secretory pathway using protein disulfide isomerases in the endoplasmic reticulum to oxidize disulfides (31) and copper derived from ATP7a, a Golgi copper-transporting ATPase (32). We expected maturation of C. albicans SOD5 to rely likewise on secretory pathway copper and the fungal ATP7a. As predicted, SOD5 activity did not require the cytosolic CCS copper chaperone because activity was not affected in a ccs1Δ/Δ deletion mutant (33) (Fig. 6A). Surprisingly, however, there were no deleterious effects of mutating

Fig. 3. Copper coordination in SOD5. (A) Sigma-A (s-A) 1.4-Å weighted electron density with coefficients 2mFo-DFc contoured at 1.2 s on the Cu (I) SOD5 structure in divergent stereo. The color coding is as in Fig. 2, except His93 is tan and a Tris molecule is pink. In lieu of Zn, a hydrogen bond is observed between the nonliganding imidazole nitrogen of H93 and E110. Select H-bonds and Cu(I)–ligand interactions are orange and blue dashes, respectively. (B) Cu(I) pseudotrigonal planar geometry (yellow dashes) with axial ligands (red dashes). The H93 imidazole nitrogen is ∼23° off-axis. (C ) s-A 1.75 Å weighted electron density for Cu(II) SOD5 as calculated as in A. Note connecting electron density between Cu (II) and H93. (D) Cu(II) coordination by four His ligands (blue dashes) with longer pseudoaxial interactions (red dashes).

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Fig. 4. Pulse radiolysis measurements of SOD5 reactivity (A) Time-dependent disappearance of superoxide at pH 7.25 as a function of SOD5 concentration: black (7.7 μM), light blue (3.8 μM), red (1.9 μM), green (0.97 μM), blue (0.48 μM), pink (0.24 μM). (B) Linear regression of the data from A was used to calculate pseudo–first-order rate constants. (C) Calculated second-order rate constants as a function of pH. Blue and black stars, SOD5 preincubated at pH 9.5 and pulsed with superoxide at pH 10.0 before analysis of activity at pH 7.3. (D) Experimentally determined rate constants for the loss of superoxide as a function of varying ionic strength (NaCl) at pH 6.0 (red) and pH 7.25 (blue). A slope of −0.6 (pH 6.0) and −0.35 (pH 7.25) is observed.

Based on our analysis of C. albicans SOD5, the absence of an extended electrostatic loop results in a copper site that does not lie at the bottom of a tapering funnel as in SOD1. Instead, the copper site is more open, which might promote reactivity with substrates other than superoxide or could enhance capture of copper from the extracellular environment. We show here that SOD5 can accumulate outside the yeast cell in a disulfide-oxidized apo-state, primed for rapid activation by extracellular copper. With an open copper site, the secreted enzyme appears to acquire copper on its own, independent of an accessory copper chaperone. In its natural setting, C. albicans SOD5 relies on the animal host for copper, and one intriguing source is the macrophage. During the innate immune response, macrophages release copper in an attempt to kill invading microbes through copper toxicity. The levels of copper in the macrophage phagolysosome can be extraordinarily high (100–300 μM), sufficient to induce copper stress responses in invading microbes and inhibit invasive growth of copper-sensitive C. albicans mutants (35–40). Based on our studies of secreted SOD5, this high level of host copper should be favorable for SOD5 activation. In essence, SOD5 could “turn the sword” on the copper attack, exploiting host copper to transform itself into an antioxidant defense for the pathogen. This concept of using host copper for oxidative stress defense has also been proposed for uropathogenic E. coli that secretes a copper binding siderophore (yersiniabactin) with SOD mimetic activity (41). C. albicans SOD5 demonstrates an inability to bind zinc. Not only is a zinc site missing, but zinc does not readily bind the copper site. In SOD1, zinc can occupy either the copper or zinc site (21). Upon close inspection of the SOD5 copper site, a backbone carbonyl oxygen (from V151) that is absent in SOD1 sits 4 Å axially

Discussion Fungi such as C. albicans express a previously unidentified class of copper-only SOD enzymes. To our knowledge, these are the first copper-containing SODs from any organism (prokaryotic or eukaryotic) reported to lack the extended loop VII, which constitutes the electrostatic loop in eukaryotic SOD1 and EC-SOD. Despite this anomaly, our studies of C. albicans SOD5 show reactivity with superoxide at rates that approach diffusion limits, similar to SOD1. The fungal enzyme also exhibits special metal cofactor attributes, including an open copper site and no zinc site, which are structural signatures of these fungal enzymes that could represent prime targets for future drug design strategies. Gleason et al.

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CCC2 encoding the ATP7a for secretory pathway copper (34). The same was true whether we tested SOD5-170 that had accumulated over 16 h in culture (Fig. 6B) or SOD5-170 that was freshly secreted over 1 h (Fig. 6C). Mutations in ccc2 also did not inhibit oxidation of the SOD5 disulfide (Fig. 6D). Disulfide oxidation may occur in the secretory pathway, as is the case with mammalian EC-SOD (31). However, unlike EC-SOD, SOD5 does not require the Golgi ATP7a for copper. If copper loading occurs in the secretory pathway, there must be a non-ATP7a source for the metal; alternatively, the enzyme may be charged with copper outside the cell. To study SOD5 activation by extracellular copper, cells were removed from 16-h cultures, where a large fraction of the accumulated SOD5-170 is inactive but disulfide-oxidized (as in Fig. 5 B and D). In this cell-free milieu, SOD5-170 activity was rapidly induced by copper (Fig. 6E) and the same results were obtained with full length SOD5-204 (Fig. S3C). Thus, SOD5 can accumulate outside the cell as an apodisulfide–oxidized protein that is primed for rapid activation by extracellular copper. The capture of extracellular copper by SOD5-like molecules may be particularly relevant during the “copper burst” of the host immune response (35) (Discussion). Fig. 5. Copper activation of SOD5 expressed in C. albicans. Growth medium from 16 h C. albicans cultures expressing SOD5-170 or transformed with empty vector (EV) (B) was subjected to PNGase endoglycosidase treatment before immunoblot analysis for SOD5 [A, C (Lower), and D] or SOD activity by the native gel assay using samples not treated with PNGase [B and C (Upper)]. (A) Collapse of SOD5 upon PNGase treatment (+) indicates extensive N-linked glycosylation. − indicates untreated samples. C. albicans transformed with empty vector or expressing SOD5-170 was treated with 10 μM CuSO4 for the indicated times (B) or with the indicated concentrations of CuSO4 or ZnCl2 for 1 h (C). (D) Redox Western blot, where upward shift in mobility with DTT (+) indicates oxidized disulfide (27). Red and Ox, positions of disulfide-reduced and disulfideoxidized SOD5, respectively. Lanes 1–2, recombinant disulfide-oxidized SOD5 from E. coli as a control; lanes 3–5, SOD5-170 from C. albicans cultures, where +Cu indicates cells treated with 10 μM CuSO4 for 1 h before SOD5170 analysis.

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neutralized by mutagenesis (45). In addition to substrate guidance, the extended electrostatic loop of SOD1 helps stabilize the zinc site, and the reverse is true: Zinc helps position the electrostatic loop (3, 4, 21). Because the fungal Cu-only SODs lack both features, it is possible that in Cu/Zn SODs, the electrostatic loop coevolved with zinc binding properties, features built onto a primordial SOD5-like molecule. Materials and Methods Recombinant SOD5 from E. coli. Recombinant SOD5 spanning sequences 27–181 was expressed in E. coli and purified as described in SI Materials and Methods. To reconstitute with metals, the SOD5 sample was dialyzed against 50 mM sodium acetate (pH 5.5) and 0.25 mM CuSO4, followed by dialysis against the same buffer without copper to remove adventitiously bound metal ions. The reconstituted protein was subsequently dialyzed against 25 mM Tris (pH 8.0) and concentrated to 3.2 mg/mL for use in pulse radiolysis experiments (as described in SI Materials and Methods) and to 20 mg/mL in preparation for analytical ultracentrifugation experiments (as described in SI Materials and Methods) and crystallization trials. This protocol was repeated using ZnCl2 and FeCl3 instead of CuSO4. All three SOD5 samples were used in crystallization trials.

Fig. 6. Activation of SOD5 by extracellular copper. SOD5-170 secreted from WT or the indicated mutants of C. albicans was analyzed for SOD activity and protein levels (A–C and E) as in Fig. 5C and for disulfide oxidation (D) as in Fig. 5D. Cu (+), samples treated with 10 μM CuSO4 for 1 h (A–D) or for the indicated times (E). (A and B) SOD5 was secreted for 16 h before copper addition. The high level of SOD5 accumulated over this time frame requires additional copper for maximal activity. (C and D) SOD5 secreted for only 1 h, where the medium has ample copper to activate this low level of enzyme maximally. Fourfold more sample was analyzed in C compared with B. (E) Cells were removed from 16-h cultures, and medium was either reconstituted with cells (+ cells) or not reconstituted (− cells) before copper addition.

from the pseudotrigonal plane formed by the Cu binding ligands H75, H77, and H153 (Fig. 3B). Such a trigonal planar copper site with an outer-sphere axial oxygen atom may be incompatible with the tetrahedral and octahedral coordination preferences of zinc. During infection, zinc levels can greatly fluctuate, because the host either starves pathogens of zinc or attacks the microbe with toxic doses of the metal (42, 43). Because SOD5 neither requires zinc nor allows zinc to misincorporate into the copper site, this SOD is resistant to such extremes in zinc. It is worth noting that the pathogenic Mycobacterium tuberculosis expresses a copper-containing SodC that has retained the extended loop VII (equivalent to the electrostatic loop) but, like SOD5, is missing ligands for zinc binding (44). Remarkably, the same two zinc ligands are missing in bacterial SodC and fungal SOD5, yet both retain the Asp that would represent the fourth zinc ligand in Cu/Zn SODs (44) (Fig. 1B). In the absence of binding zinc, this Asp H-bonds to copper binding H90 in SodC, and to copper binding H75 in SOD5 (Fig. S4). It is remarkable that nature has preserved this Asp to connect to the copper site but through different routes in Cu/Zn vs. Cu-only SODs (Fig. S4). Despite lacking the extended electrostatic loop, we observed an effect of ionic strength on kinetics of SOD5 reactivity with superoxide, indicative of an electrostatic guidance system for substrate. The remnant of the electrostatic loop in SOD5 includes the active site R159 that, together with the Cu(I) or Cu(II) ion, may account for the effects observed. It is also possible that the fungal family of Cu-only SODs evolved with an alternative electrostatic guidance system. In fact, an auxiliary electrostatic guidance system was previously proposed for SOD1, where the reaction rate of Xenopus laevis SOD1 was seen to increase rather than decrease when all of the electrostatic loop charges were 5870 | www.pnas.org/cgi/doi/10.1073/pnas.1400137111

Structure Determination. Purified SOD5 proteins that had been dialyzed against CuSO4, ZnCl2, and FeCl3-containing buffer solutions were concentrated to 20 mg/mL and set up in crystallization experiments using an Art Robbins Instruments Phoenix crystallization robot and commercially available crystallization kits in the X-ray Crystallography Core Laboratory at University of Texas Health Science Center at San Antonio. Initial hits were optimized at 20 °C using the hanging drop vapor diffusion method. Isomorphous crystals were grown from two different crystallization conditions. These isomorphous crystals are designated as crystal forms A and B, respectively, to reflect the differences in reservoir solutions. For crystal form A, SOD5 was mixed with an equal volume of reservoir solution containing 2.4 M ammonium sulfate and 0.1 M bicine (pH 9.0). For crystal form B, the reservoir solution was 1.6 M sodium citrate (pH 6.5) and 0.1 M Hepes (pH 7.5). Prism-shaped crystals appeared within 3 d in both conditions. Suitable specimens were extracted with nylon loops, and the excess liquid was carefully wicked away before flash-cooling in liquid nitrogen. Initial datasets were collected in-house, and the structures were determined by molecular replacement as implemented in the program PHASER (46), using the human G37R variant of SOD1 [Protein Data Bank (PDB) ID code 1AZV] as the search model. The structures were refined with the PHENIX suite of programs (47), and manual model adjustments were performed after each round of refinement in the program Coot (48). Crystals grown from SOD5 protein that had been dialyzed against ZnCl2 and FeCl3 were completely devoid of metal ions and were not studied further. Copper-containing crystal forms A and B were subsequently taken to beamline 24-ID-E at the Advanced Photon Source, Argonne National Laboratory, to obtain the highest resolution possible. X-ray fluorescence scans on the crystals revealed a strong signal for copper with the expected absorption edge at 1.38 Å. Diffraction data were indexed and scaled using the X-ray Detector Software (XDS) program. The coordinates of the SOD5 protein structures determined in-house were refined against the high-resolution data from the synchrotron as described above. The copper ion coordination was pseudotrigonal planar, consistent with the presence of Cu(I) in both crystal forms, possibly as a result of the photoreduction that often occurs using synchrotron radiation of short wavelength and very high flux. The highest resolution dataset at a resolution of 1.4 Å came from a form A crystal. In an effort to avoid photoreduction, 120° of diffraction data from a fresh form B crystal were measured in-house at a resolution of 1.75 Å using oscillation images at 1° with an exposure time of 1 min (one oscillation image per degree per minute). Connecting density from the copper to His93 is present in this form B structure, indicating it contains a significant fraction of four-coordinate Cu(II). All structural figures were created using the PyMol (Molecular Graphics System, version 1.5.0.4; Schrödinger, LLC) program. Coordinates and diffraction data for SOD5 structures coming from crystal forms A and B have been deposited into the PDB with ID codes 4NT3 and 4NTU, respectively. Biochemical Analyses of SOD5 Secreted from C. albicans. The growth medium from C. albicans strains secreting SOD5-170 and SOD5-204 was concentrated as described in SI Materials and Methods. Samples for immunoblots were treated with peptide-N-glycosidase f before denaturing gel electrophoresis and probing with an anti-SOD5 antibody. Samples for SOD activity by the native gel assay (27) were assayed without prior PNGase treatment. Details are provided in SI Materials and Methods. Redox Western blots of SOD5

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ACKNOWLEDGMENTS. We thank D. Kornitzer for kind gifts of yeast strains, and B. Demeler and V. Schirf for assistance in analytical ultracentrifugation experiments. This work was funded by National Institutes of Health Grants R21 AI097715 (to V.C.C.), R37 GM50016 (to V.C.C.), and R01AI046223 (to B.P.C.) and by Veterans Administration Grant VA 1I01BX000506 and Robert A. Welch Foundation Grant AQ-1399 (to P.J.H.). J.E.G. and R.L.P. were supported by Grant T32 CA009110, and A.G. and J.W.-S. were supported by a St. Mary’s University grant and the Biaggini Research Program. The work at Brookhaven National Laboratory was carried out at the Accelerator Center for Energy Research, supported by the US Department of Energy Office of Science, Division of Chemical Sciences, Geosciences, and Biosciences under Contract DE-AC0298CH10886. The Center for Analytical Ultracentrifugation of Macromolecular Assemblies is supported by the Office of the Vice President for Research at the University of Texas Health Science Center.

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BIOCHEMISTRY

were carried out using nonreducing gel electrophoresis as described (27). Concentrated medium samples were first treated with PNGase in the absence of DTT, followed by inactivation of PNGase by incubation at 75 °C for 10 min. Samples were then incubated at 70 °C for 10 min with SDS gelloading buffer in the absence or presence 100 mM DTT before SDS gel electrophoresis. Under these conditions, 0.5 μg of recombinant disulfideoxidized SOD5 from E. coli exhibits a small upward shift in mobility upon reduction of the disulfide with DTT (Fig. 5D, lanes 1 and 2), as has been shown for SOD1 (27). The same upward shift was seen with SOD5 secreted from C. albicans. Because the disulfide cysteines are the only cysteines in SOD5, this reactivity with DTT is presumed to reflect reduction of a preexisting disulfide, as has been shown for SOD1.