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1 Molecular Biophysics Group, CCLRC Daresbury Laboratory, Warrington, Cheshire, WA4 4AD, United Kingdom2 Department of Biochemistry and the X-ray Crystallography Core Laboratory, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900, USA3 Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095, USA4 Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA
Reprint requests to: S. Samar Hasnain, CCLRC Daresbury Laboratory, Warrington, Cheshire, WA4 4AD, UK; e-mail: s.hasnain{at}dl.ac.uk; fax: +44-1925-603748; or P. John Hart, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA; e-mail: pjhart{at}biochem.uthscsa.edu; fax: (210) 567-6596.
(RECEIVED November 24, 2004; FINAL REVISION January 26, 2005; ACCEPTED January 26, 2005)
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Keywords: copper-zinc superoxide dismutase; familial amyotrophic lateral sclerosis; ALS; amyloid; SOD1; H46R
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041256705.
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To date, >100 FALS-inducing point mutations have been identified that map to all regions of the SOD1 molecule, including the dimer interface, 
-barrel, loop regions, disulfide bond residues, and both the copper- and zinc-binding sites (a list can be found at http://www.alsod.org). FALS SOD1 mutations were first thought to reduce SOD1 enzymatic activity and thereby cause increased oxidative damage to motor neurons (Deng et al. 1993). However, the majority of FALS mutant SOD1 proteins display SOD1 activity comparable to that of the wild-type enzyme (Borchelt et al. 1995; Rabizadeh et al. 1995), and SOD1 knockout mice do not develop ALS (Reaume et al. 1996). In contrast, transgenic mice overexpressing human FALS mutant SOD1 in addition to their own fully functional SOD1 develop symptoms characteristic of ALS, while those overexpressing the wild-type human enzyme do not (Gurney 1997). Taken together, these results strongly suggest that the pathogenic mechanism of SOD1-linked FALS involves the gain of a toxic property by the mutant SOD1 enzyme, rather than a loss of function.
H46R SOD1-mediated FALS has been described as a milder subtype of the disease compared with the majority of FALS cases, where the mean age of onset is 42.8 yr and the mean survival after diagnosis is 4.8 yr (Ceroni et al. 2001). The H46R SOD1 mutation was first observed in four Japanese families (Aoki et al. 1994) that displayed an unusual FALS pathology. The patients initially presented with weakness in the legs, while upper extremity symptoms were delayed by >5 yr and bulbar symptoms by >8 yr. The mean age of onset was 49.6 yr, and mean survival was 17.3 yr. These findings were expanded in a more recent, larger study of 17 Japanese patients that showed an earlier mean age of onset (44.3 yr) and a mean disease duration of ~12 yr (Arisato et al. 2003). This stands in sharp contrast to the A4V and I113T mutants that are associated with rapid disease progression, and for which crystal structures have recently been determined (Cardoso et al. 2002; Hough et al. 2004).
Transgenic rats expressing H46R SOD1 develop motor neuron disease (Nagai et al. 2001), and neural tissues from these rats contain intracytoplasmic Lewy-like inclusion bodies similar to those observed in spinal cord tissue of human ALS patients. G93A rats examined in the same study have neural tissues with fewer aggregates and an obvious vacuolar pathology. Although the H46R rats in this study express over twice the amount of mutant SOD1 as do the G93A rats, motor neuron disease progressed more slowly for the animals harboring H46R (24 d) compared with those expressing G93A (8 d). This finding correlates with the lengthened disease course in human patients with the H46R mutation.
Each subunit of the SOD1 homodimer binds one copper and one zinc ion and folds as an eight-stranded Greek-key -barrel that is stabilized by an intra-subunit disulfide bond near the active site (Tainer et al. 1982). Protruding from the -barrel are two major structural elements termed the "electrostatic" and "zinc" loops. The electrostatic loop (loop VII, residues 121–144) contains charged residues that help guide the negatively charged superoxide substrate toward the catalytic copper site. The zinc loop (loop IV, residues 49–84) contains two distinct substructures. The first consists of residues 49–62 and is termed the "disulfide loop," which is anchored to the -barrel through the bond formed between Cys 57 and Cys 146, the latter of which is located on the C-terminal -strand (strand 8) of the -barrel. The second consists of residues 63–84 and includes all four zinc ligands (His 63, His 71, His 80, and Asp 83) and forms the zinc-binding site. Particularly important interactions between the electrostatic and zinc loops in the wild-type enzyme come from the side chain of Asp 124 of the electrostatic loop, which accepts a hydrogen bond simultaneously from the nonliganding nitrogen atom of zinc ligand His 71 and from the nonliganding nitrogen atom of copper ligand His 46. This Asp 124–mediated linkage between the copper- and zinc-binding sites and between the electrostatic and zinc loop elements is termed the "secondary bridge." The "primary bridge" between the copper- and zinc-binding sites is also known as the "bridging imidazolate" and comes from the side chain of His 63, which binds to the two metals simultaneously in the Cu(II) form of the enzyme. Together, the electrostatic and zinc loop elements in their wild-type conformation form the substrate channel leading to the active site.
Histidine 46 is one of the four copper ligands in the SOD1 active site. As described above, Asp 124 makes simultaneous hydrogen bonds to the nonliganding imidazole nitrogen atoms of the copper ligand His 46 and zinc ligand His 71 to form the secondary bridge between the metal-binding sites that helps to stabilize and orient correctly the electrostatic and zinc loop elements. Because the pathogenic mutation occurs at the copper-binding site, H46R SOD1 has a substantially reduced ability to bind copper in vitro and possesses dramatically lower activity as compared to wild-type and other pathogenic SOD1 molecules (Carri et al. 1994).
Even when both copper and zinc are available in the growth medium, recombinant H46R SOD1 expressed in insect cells shows little detectable SOD activity and is severely metal deficient in comparison to wild-type SOD1 expressed and purified in the same manner (Hayward et al. 2002). Moreover, H46R SOD1 expressed in yeast fails to rescue the oxygen-sensitive phenotype of ySOD1
yeast, unlike other FALS mutant SOD1s with mutations outside of the metal-binding region, for example, G37R (Ratovitski et al. 1999). This metal-deficient H46R SOD1 also exhibits reduced thermal stability (as measured by differential scanning calorimetry) in comparison to recombinant metal-replete native SOD1 (Rodriguez et al. 2002). The reduction in stability relative to the wild-type protein is due primarily to metal deficiency of the mutant protein. In addition, cultured neuroblastoma cells expressing H46R SOD1 are more sensitive to paraquat than are those expressing human wild-type SODl (Gabbianelli et al. 1999).
To understand better the structural determinants of mutant SOD1 toxicity, we have determined several X-ray crystal structures of FALS SOD1 protein molecules. As part of this endeavor, we present here the structures of H46R SOD1 in the zinc-loaded and apo forms at 2.15 Å and 2.50 Å resolution, respectively. Previously, we described the modes of filamentous assembly by H46R proteins (Elam et al. 2003b). Here, we present a detailed analysis of the structures, and we report (1) that binding of zinc at the zinc site does not cause the zinc loop and secondary bridge to adopt the conformation characteristic of wild-type SOD1 that has been observed in the other metal-replete FALS mutants structurally characterized to date, and (2) that binding of zinc to the zinc site of H46R SOD1 does not prevent the formation of nonnative interactions between the protein dimers. The copper and zinc sites themselves show novel spatial arrangements that appear to compensate partially for the lack of metals and/or loop disorder. These results are discussed in light of the distinct ALS phenotype associated with H46R SOD1.
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-barrel structure of Zn-H46R is quite similar to that of wild-type SOD1. However, large stretches of the electrostatic and zinc-binding loop elements are disordered and are not visible in the electron density maps (Fig. 1
; Table 1). This comparison reveals significant differences between the Zn-H46R and native structures in residues immediately before or after the disordered loops. The zinc ligand His 71 falls within the disordered region and is absent from the electron density. Part of the zinc loop, residues 78–81 immediately following the disordered region, forms the interface to an adjacent dimer in the "helical filament" packing motif (see below) (Elam et al. 2003b).
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). Exceptions were subunits M and N that, despite high thermal parameters in this region (in the range 46–100 Å2), displayed enough electron density to trace the backbone fully, resulting in a dimer in which the conformations of these loop elements resembles those of holo wild-type SOD1. The amount of disorder in the electrostatic loop of each monomer was dependent on the exact nature of interactions occurring at the metal sites and on crystal packing interactions with symmetry-related molecules. A general trend was observed in that the more tenuous the interactions between Asp 124, Arg 46 (which replaces His 46), and His 71 that link the copper and zinc sites (see below), the more disorder was observed in the electrostatic loop adjacent to Asp 124. Despite the differences at the metal sites and the localized disorder, the overall structure of apo-H46R is similar to that of Zn-H46R and wtSOD1.
Mutation site and copper center
The copper-binding site of Zn-H46R is devoid of metal ions, and the side-chain positions are altered relative to those of wtSOD1 (Fig. 2A). A superposition of the Zn-H46R copper site with that of holo- and apo-wtSOD1 is shown in Figure 3A. The altered side-chain position of His 120 is associated with a 0.4 Å shift in the position of its C
atom, part of a concerted movement of residues Val 118–Asp 125, the latter of which is the final ordered residue in the electrostatic loop. His 63, which bridges between the copper and zinc ions in Cu(II)-wtSOD1, adopts a different conformation in the absence of copper. The new position of the His 63 imidazole ring allows a bond to form between its N
2 atom and a sulfate anion, while at the same time its conformation is sterically constrained by the close approach of Arg 46, whose C
atom comes within 3.17 Å of the His N2 atom. Sulfate occupies a position in the active site channel similar to that previously observed in the structures of native and D125H SOD1 (Elam et al. 2003a; Strange et al. 2003) such that its oxygen atoms form a number of hydrogen bonds to the protein (Fig. 2B; Table 2). Thus, the presence of sulfate in the Zn-H46R structure appears to stabilize the vacant copper site through the formation of a hydrogen-bonding network with His 120, His 63, and Arg 143.
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). One common feature in all monomers is that the copper ligands His 48 and His 120 are well-ordered and are stabilized by a hydrogen bond between their N2 atoms, adopting a similar position to that observed in Zn-H46R (see Fig. 2c). No sulfate is present in the structure of apo-H46R, although it was a minor component of the crystallization buffer. Overall, in both apo and zinc-bound H46R, Arg 46 physically blocks the site where copper binds in wtSOD1. In most of the subunits, however, the rest of the site makes only slight shifts to satisfy the hydrogen bonding donor and acceptor nature of the ligands. Parts of the active site channel are absent from the Zn-H46R and apo-H46R structures due to the disordered zinc and electrostatic loops, which exposes the metal sites to solvent.
The zinc sites
The zinc-binding sites in the Zn-H46R structure are fully occupied and yet their structures are significantly different from those observed in wtSOD1 (Fig. 3B; Table 2). The zinc atom is shifted some 0.7 Å from its position in wt-SOD1, and, only His 80 remains in a similar position in the mutant structure. His 80 forms a ~1.8–1.9 Å bond to zinc through its N1 atom, although the imidazole ring is rotated by ~20°. The new position of His 63 maintains a bond between its N1 atom and zinc at a distance of ~1.9 Å. His 71 falls in the disordered region of the H46R structure and is absent from the electron density maps. A water molecule has been modeled at ~2.5 Å from copper in the approximate direction of the missing His 71 ligand. The largest shift at the zinc site occurs in residue Asp 83. In the absence of the His 71 ligand, the side chain of Asp 83 adopts a new position such that the zinc-ligating O2 atom is shifted by ~1.6 Å relative to wtSOD1. This movement, concomitant with the shift in the position of zinc, allows Asp 83 to act as a bidentate ligand to zinc (O1 distance, 2.3–2.5 Å; O2 distance, 2.1–2.4 Å). The zinc atom in this rearranged metal center is five coordinate, but with only three ligand residues, and an additional weak water ligand. This zinc coordination has not been observed previously in any structure of SOD1 (Table 2).
In contrast, apo-H46R has no bound metal at the zinc site. The zinc ligands display a variable amount of order as in the copper site, however, all monomers show a severely disrupted zinc site. The position and stability of individual ligand residues at the zinc site in different monomers is quite variable, and different arrangements of hydrogen bonding are present in the monomers (Table 3). Residue His 71 falls within the disordered region of the zinc loop and is therefore absent from the zinc site. It is apparent that the stability of the zinc site ligands (and this portion of the zinc loop) is correlated with the relative stability of the electrostatic loop, which is in turn influenced by the presence or absence of the secondary bridge and/or packing interactions.
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). In Zn-H46R, the mutated Arg 46 forms a much weaker (~3.1 Å) hydrogen bond through its N
2 atom to Asp 124 O2. The Asp 124 C
–O2–Arg 46 N angle is ~80°–90° in Zn-H46R compared with ~130° for the Asp 124 C–O2–His 63 N2 angle in wtSOD1, indicating a conformation less preferable for hydrogen bonding. Asp 124 is shifted significantly in position. The hydrogen bond between Asp 124 O1 and Asp 125 N is maintained in monomer F but broken in the remaining monomers. Residue 125 is the final residue visible in Zn-H46R prior to the disordered region of the electrostatic loop. His 71 is also disordered and not present in the Zn-H46R structure, thus the secondary bridge is broken. Interestingly, if His 71 maintained its wild-type position, it would be within ~2.0 Å of the zinc atom in Zn-H46R. Each of the eight monomers in the apo-H46R structure exhibits some electron density for Asp 124, and in several subunits, this residue is able to make at least a tenuous hydrogen bond with the mutated residue Arg 46. The degree of disorder and the B-factor in Asp 124 for each monomer correlate with its ability to form hydrogen bonds with the backbone nitrogen atoms of Asp 125 and Leu 126.
Solvent exposed cysteine
The solvent-exposed residue Cys 111 lies in a similar position in both apo-H46R and Zn-H46R to that previously observed for wild-type SOD1. The thiol side chain of this residue from each subunit faces approximately toward the 2-fold symmetry axis at the dimer interface, such that the sulfur–sulfur separation is ~9 Å. The adjacent residues in the polypeptide, His 110 and Asp 109, face away from the interface and from Cys 111, and are therefore not in a suitable position to form a binding site for metals together with the Cys 111, as had previously been suggested by Liu et al. (2000).
Helical filament packing in Zn-H46R
The formation of helical filamentous arrays in the Zn-H46R structure and linear amyloid-like filamentous arrays in the crystal structure of apo-H46R has been described previously (Elam et al. 2003b). Each helical filament was also described as a water-filled nanotube due to the ~30 Å diameter hole in the center. Each nanotube packs side-to-side with four adjacent nanotubes (Fig. 4). The interfaces between Zn-H46R molecules in adjacent filaments consist of two salt-bridges between the Lys 36 residues of monomers W and X in one filament and the Asp 11 residues in monomers Y and Z of a second filament along with a number of more tenuous water-mediated interactions and close reciprocal hydrophobic interactions of Pro 13 (Fig. 4). This interaction is repeated for each of the four dimers forming one turn of the helical filament.
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-strands 5 and 6 as in Zn-H46R and another FALS-associated SOD1 mutant S134N. In the crystal, each linear filament interacts side-to-side with two others to form a plane of linear filaments (Fig. 5A). Similarly, each zigzag packing arrangement of dimers interacts with two others to form a separate non-intersecting plane of zigzag arrays (Fig. 5B). The two distinct planes stack in alternating layers, that lie approximately normal to the ac plane within the monoclinic crystal. A few somewhat tenuous interactions occur between these layers (Fig. 6).
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An activity assay and ESR spectroscopy study on H46R expressed in an Escherichia coli SOD1 knock-out system (Carri et al. 1994), showed no detectable SOD1 activity in cell extracts and <5% copper loading (measured by integration of the ESR spectrum). Wild-type SOD1 expressed similarly had ~80% activity and a corresponding level of copper. More recently, a combined metal binding, Raman spectroscopy and UV/visible spectroscopy study showed that Cu2+ does not bind to the copper site of apo H46R SOD1 and that Cu2+ competes with other metal ions such as Zn2+ and Co2+ for binding to the zinc site. Furthermore, Cu2+ was proposed to bind strongly to a surface residue, Cys 111, near the dimer interface of H46R SOD1, giving rise to a distinct optical absorption band at 370 nm (Liu et al. 2000).
Both the zinc-loaded and apo-crystal structures of H46R SOD1 presented here show that the copper site is completely vacant. This is consistent with measurements of copper content on the H46R protein expressed in insect cells (Hayward et al. 2002) that was used to grow the apo-H46R crystals examined in this study. The H46R SOD1 protein used to grow the Zn-H46R crystals examined here was expressed in yeast and showed a copper content of 0.23 equivalents per dimer prior to crystallization, yet the crystal structure showed no indication that copper was bound to the protein at either the copper or zinc site or near Cys 111. It is likely in the case of Zn-H46R that we are selectively crystallizing the copper-free form of the protein. The observation of a structurally different zinc site in Zn-H46R, in which only three of the usual four protein ligands participate, may indicate a higher susceptibility for metal loss at the zinc site compared with the wild-type enzyme. A decreased affinity for zinc has been determined for several FALS SOD1 mutants (Crow et al. 1997) and zinc-deficient SOD1 (either wild type or mutant) has been shown to induce apoptosis in liposomal delivery to cultured motor neurons (Estevez et al. 1999).
Several studies have shown some FALS SOD1 mutants do not properly segregate zinc and copper at their metal sites when reconstituted from their apo-forms in vitro (Lyons et al. 1996; Goto et al. 2000) and we have previously observed in crystal structures of wild-type and FALS mutant SOD1 that in the absence of sufficient copper, zinc ions will sometimes occupy the copper site (Elam et al. 2003a; S. Antonyuk, R. Strange, M. Hough, P. Doucette, J. Valentine, and S. Hasnain, in prep.). The fully occupied zinc sites in Zn-H46R suggest that ample zinc was present during protein production and crystallization, yet there was no evidence of any metal (even zinc) at the copper site in the crystal structure, consistent with the Arg side chain sterically impairing the copper site for metal binding.
The geometry of the zinc site is observed to be five coordinate, with a water ligand, His 80, His 63, and the bidentate carboxylate of Asp 83. It seems likely that Co2+ is binding in the same fashion as zinc to apo-H46R, since the visible spectrum of Co2+ H46R shows an extinction coefficient for the d-d bands near 600 nm that is somewhat lower than that of Co2+ bound to the zinc site of wt SOD1, where it is known to be four coordinate (Liu et al. 2000; Lyons et al. 2000).
Earlier studies of Cu2+ binding to apo-H46R have demonstrated the existence of a new metal-binding site on this protein, which was assigned as Cys 111 on the basis of its characteristic copper-cysteinate visible absorption and resonance Raman spectroscopic properties (Liu et al. 2000). The nature of the resonance Raman spectrum led to a tentative conclusion that both cysteines might be binding simultaneously to the copper chromophore. However, this conclusion now seems unlikely based on an examination of the crystal structure, unless a binuclear copper site is forming that bridges the two cysteines. The locations of the two Cys 111 residues are close enough, however, to explain why chemical modification using either dithiobis(2-nitro-benzoic acid) or 4-vinylpyridine gave only a 50% yield. Modification of one of the Cys 111 side chains with either reagent would be predicted to create a significant steric barrier to modification of the second Cys 111 of the protein dimer.
The secondary bridge and disordered loop regions
The disordered loop regions in the H46R crystal structures appear to be correlated with the disruption of the Asp 124 secondary bridge as a consequence of the mutation of His 46 and the subsequent disorder of the zinc ligand, His 71. In Zn-H46R, the electrostatic loop becomes disordered shortly after residue 125, adjacent to Asp 124. The loss of the bond from zinc to His 71 is related to the disorder of the zinc loop from residues 68–78 (or vice versa). In apo-H46R, the electrostatic and zinc loops are disordered in all dimers but one, in which crystal contacts appear to stabilize these loops. It is possible that the zigzag packing interaction is an intermediate to the linear, amyloid-like filament conformation, where the transition between the two could be dependent on the destabilization of the His 71-Asp 124 hydrogen bond in the secondary bridge. Overall, our results combined with other observations (Lyons et al. 2000; Banci et al. 2002; Strange et al. 2003) strengthen the idea that the hydrogen-bonding network around Asp 124 forms a crucial component in the stability and conformation of both zinc and electrostatic loops.
A significant observation is that the presence of zinc in its altered coordination environment at the zinc site in the Zn-H46R structure is not sufficient to maintain the wild type–like loop conformation. It is possible that zinc bound in this altered zinc site may be quite labile in solution, further disrupting the zinc loop as seen in apo-H46R. The observation that His 71 is ordered in apo-wtSOD1 but not in Zn-H46R is suggestive that the disruption of the secondary bridge and the zinc loop conformation is a consequence of the H46R mutation rather than of metal depletion. It is clear that the His 
Arg mutation at residue 46 compromises the correct formation and function of the secondary bridge. This is particularly significant considering that pools of stable, zinc-loaded, but copper-deficient wild-type CuZnSOD have been observed in vivo (Steinkuhler et al. 1991, 1994; Petrovic et al. 1996; Schmidt et al. 2000), and presumably, FALS mutants would also be expected to exist in that form. The H46R mutant may have highly disordered electrostatic and zinc loops prior to its interaction with the SOD1 copper chaperone CCS, if it interacts at all.
H46R and FALS pathogenesis
H46R is of particular interest in the study of SOD1-mediated FALS, as the mutant protein has severely impaired copper binding at the active site while at the same time it causes a distinctive, very slowly progressing form of FALS. Several proposed mechanisms for the pathogenicity of FALS mutant proteins involve altered copper-mediated chemistry or loss of substrate specificity (for review, see Valentine et al. 1999; Valentine and Hart 2003). H46R-associated FALS is presumably not the result of these mechanisms due to its vacant copper site, but mechanisms in which oxidative stress is caused by mishandling or release of copper from mutant SOD1 have not been entirely excluded. One possibility is that SOD1 mutants without active site copper may correlate with a milder phenotype by producing toxicity through a mechanism distinct from those mutants capable of binding active site copper ion. An alternative hypothesis has been that the SOD1 mutants, particularly in their metal-deficient forms, lose the stabilizing effects of well-ordered electrostatic and zinc loops, and become susceptible either to fibrillar or nonspecific aggregation (Elam et al. 2003b). In fact, both apo- and Zn-H46R form filamentous arrays within their crystals, which suggests a mechanism by which the mutant protein becomes susceptible to aggregation. While zinc binding is still possible in the H46R mutant, the novel arrangement at the zinc-binding site as a result of the H46R mutation is not sufficient to stabilize the two loops, despite the presence of bound zinc. Therefore, if the loss of -strand edge protection caused by loop disorder is a crucial mechanism by which SOD1 aggregates in affected tissues, then the H46R mutant protein might be expected to aggregate more readily than wild-type SOD1 or other FALS mutant SOD1 proteins that bind metals similarly to wild type. H46R SOD1 transgenic rats do in fact develop an abundance of hyaline inclusions in spinal motor neurons and supporting cells (Nagai et al. 2001). G93A transgenic rats produced from the same genetic background as the H46R rats in the same study had motor neurons with far fewer aggregates and a distinct vacuolar pathology. In spite of the increased aggregation and a 2.4-fold higher relative expression level of H46R compared to G93A SOD1, the onset and progression of motor neuron disease in H46R rats was substantially slower than for those with the G93A mutation (144 d vs. 122 d and 24 d vs. 8 d for onset and duration, respectively).
These data suggest that the distinctive H46R FALS phenotype may not result from genetic modifying factors shared by the limited number of Japanese families in which this mutation has been observed. The discrepancy that although H46R readily forms aggregates as observed from transgenic animal studies, the disease progresses much less rapidly than expected is consistent with the observation of several research groups who now believe that protofibrils rather than insoluble fibers or inclusions are toxic. The X-ray structures presented here strongly support this hypothesis because the rapid aggregation of H46R may actually reduce the amount of toxic protofibrils and hence be neuroprotective. Further support for this idea comes from recent studies showing that increasing SOD1 aggregate formation did not correlate with an increase in cell death in tissue culture (Lee et al. 2002; Takeuchi et al. 2002) or in FALS transgenic mice (Lino et al. 2002).
Conclusions
The H46R mutation in SOD1 results in an enzyme with significant structural differences relative to the wild-type enzyme. In both Zn-H46R and apo-H46R, the secondary bridge formed by His 46-Asp 124-His 71 is disrupted and the copper site is devoid of metal. Apo-H46R is also devoid of Zn, while Zn-H46R contains an altered zinc site relative to wild type due to the disordered nature of residue His71. The three remaining protein ligands form a novel five coordinate zinc environment with both O1 and O2 of Asp 83 coordinated to zinc. The coordination environment of zinc is completed by a weak water ligand. The electrostatic and zinc-loop regions of the enzyme are disordered in the H46R mutant, and notably, the presence of zinc in its altered site in Zn-H46R does not stabilize these loops. Zn-H46R dimers form so-called helical filaments in the crystals while apo-H46R forms linear, amyloid-like filaments and zigzag packing assemblies. The observation of these packing arrangements in the crystals is consistent with the increased tendency for this mutant to aggregate in motor neurons in the H46R SOD1 transgenic rat. SOD1-containing inclusions do not, however, appear to correlate with toxicity in FALS motor neurons since H46R rats exhibit much more aggregated protein than those of G93A rats, yet have a milder form of the disease. The observation that the disease progression of H46R rats correlates with the slowly progressive H46R phenotype in humans suggests that the distinctive FALS H46R phenotype may not be the result of protective genetic factors in the affected families, but rather due to the properties of the mutant SOD1 molecule itself.
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Structure solution and refinement
The Zn-H46R structure was determined by molecular replacement using MOLREP (Vagin and Teplyakov 1997) with the coordinates of a monomer of the 1.78 Å holo wild-type hSOD structure (Protein Data Bank [PDB] code 1 HL5; Strange et al. 2003) as the search model. Two SOD dimers were located in the crystallographic asymmetric unit. This initial model was refined by using the maximum likelihood method implemented in REFMAC5 (Murshudov et al. 1997) as part of the CCP4i program suite and rebuilt interactively by using 
A-weighted electron density maps with coefficients 2mFo-DFc and mFo-DFc in the program O (Jones et al. 1991). Five percent of the data (1774 reflections) was set aside to calculate a free R-factor. Each monomer was refined independently, without the application of noncrystallographic symmetry restraints. A bulk solvent correction was applied and individual solvent molecules added using ARP/WARP. In the latter stages of refinement, TLS parameters were determined. Refinement converged to a final R-factor of 20.6% (R-free = 23.4%).
The apo-H46R structure was also determined by molecular replacement using the program EPMR (Kissinger et al. 1999) with the 1.9 Å resolution structure of FALS SOD1 mutant G37R (PDB code 1AZV
[PDB]
; Hart et al. 1998) as the search model. Four SOD dimers were located in the crystallographic asymmetric unit. The structure was iteratively refined by using the program CNS (Brunger et al. 1998). When necessary, the molecular model was adjusted in the program O. NCS restraints were applied to the residues forming the -barrel core only. A bulk solvent correction was applied, and water molecules were added. Refinement converged to a final R-factor of 22.1% (R-free = 26.8%). No stereo-chemical restraints on metal-ligand bond distances or angles were applied in the refinement of either structure. The final models were evaluated by using the programs WHATCHECK and PROCHECK (Laskowski et al. 1993). The final coordinates and structure factors have been deposited in the RSCB PDB (Bernstein et al. 1977), accession codes 1OEZ (Zn-H46R) and 1OZT (apo-H46R).
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5 These authors contributed equally to this work. 
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