Structural and dynamic aspects related to oligomerization of apo SOD1 and its mutants

Abstract

The structural and dynamical properties of the metal-free form of WT human superoxide dismutase 1 (SOD1) and its familial amyotrophic lateral sclerosis (fALS)-related mutants, T54R and I113T, were characterized both in solution, through NMR, and in the crystal, through X-ray diffraction. We found that all 3 X-ray structures show significant structural disorder in 2 loop regions that are, at variance, well defined in the fully-metalated structures. Interestingly, the apo state crystallizes only at low temperatures, whereas all 3 proteins in the metalated form crystallize at any temperature, suggesting that crystallization selects one of the most stable conformations among the manifold adopted by the apo form in solution. Indeed, NMR experiments show that the protein in solution is highly disordered, sampling a large range of conformations. The large conformational variability of the apo state allows the free reduced cysteine Cys-6 to become highly solvent accessible in solution, whereas it is essentially buried in the metalated state and the crystal structures. Such solvent accessibility, together with that of Cys-111, accounts for the tendency to oligomerization of the metal-free state. The present results suggest that the investigation of the solution state coupled with that of the crystal state can provide major insights into SOD1 pathway toward oligomerization in relation to fALS.amyotrophic lateral sclerosis ͉ NMR ͉ X-ray ͉ mobility ͉ H2O/D2O exchange M ore than 100 different variants of human copper-zinc superoxide dismutase (Cu 2 Zn 2 SOD) have been identified and linked to the neurodegenerative disease familial amyotrophic lateral sclerosis (fALS) by a gain-of-function mechanism (1, 2). Although the mechanism of the toxicity is unknown, aberrant SOD1 protein oligomerization has been strongly implicated in disease causation (3,4). Several recent publications (5, 6) have presented compelling evidence that in vivo abnormal disulfide cross-linking of ALS mutant SOD1 plays a role in this oligomerization, and disulfide-linked SOD1 multimers have been detected mainly in mitochondria of neuronal tissues of SOD1-linked fALS patients and transgenic mice (7-9).WT human SOD1 is an exceptionally stable, homodimeric 32-kDa protein, located mainly in the cytoplasm, but it is also present in the peroxisomes, the mitochondrial intermembrane space, and the nucleus of eukaryotic cells (10, 11). Each subunit of the dimer binds 1 copper and 1 zinc ion and folds as an 8-stranded Greek-key ␤-barrel that is stabilized by an intrasubunit disulfide bond (Cys-57, Cys-146) near the active site (12). In vivo, in the highly reducing cytoplasm environment, the existence of this intrasubunit disulfide bond points to its very low reduction potential.In addition to the 2 cysteines involved in the formation of the intramolecular disulfide bond, 2 reduced cysteines, Cys-6 and Cys-111, are located on ␤-strand 1 and loop VI of WT human SOD1, respectively. Among the loops connecting the 8 ␤-strands, 2 have structural and functional roles. The ...

Transient structural distortion of metal-free Cu/Zn superoxide dismutase triggers aberrant oligomerization

“…That fact that regions of apo-SOD1 are highly dynamic has been recognized for years (25), but it is not clear whether the observed variables, such as NMR relaxation rates and amide hydrogen exchange with solvent deuterium (18,19,25,33), imply that SOD1 experiences a conformational continuum within a single basin in the energy landscape, large-scale unfolding of the affected regions, or exchange to a relatively well-defined excited state. Our present results provide evidence for the latter scenario, and further show that the excited state is a unifying feature of all SOD1 variants investigated herein, despite their significant variation in stability toward global unfolding.…”

“…It also is known that reduction of the C57-C146 disulfide promotes dissociation of the native dimer (11,22), but contrasting data have been reported regarding the role of intermolecular disulfide bonds in the aggregation of SOD1 (8,15,16,23,24). Previous reports have identified a flexible region in monomeric apo-SOD1, including the metalbinding loops and neighboring ␤-sheet, which has been suggested as the primary candidate for forming an aggregation-prone interface (18,19,25).…”

“…SOD1 undergoes complex posttranslational maturation, including formation of the intramolecular C57-C146 disulfide bond, binding of Zn, chaperone-assisted insertion of Cu, and dimerization (10,11). However, several lines of evidence indicate that it is apo-SOD1, not the metal-bound states, that plays a key role in aberrant oligomerization (11)(12)(13)(14)(15)(16)(17)(18)(19). In vitro studies of the aggregation kinetics have implicated the monomeric apo state as the origin for aggregation (12)(13)(14).…”

Global structural motions from the strain of a single hydrogen bond

“…As a consequence, the dimerization of SOD1 becomes tightly controlled by metal binding via the structure of loops IV and VII, and the conserved C57-C146 disulfide linkage (9): if the metals and disulfide bond are lost, the protein dissociates into free monomers. Metal loss also unleashes characteristic dynamic motions of the central barrel scaffolds (10)(11)(12)(13) that are most pronounced in the β-sheets facing the metal-binding sites. Together, these features form an intricate network of structural communication within the SOD1 molecule, spanning from the dynamic motions of the monomeric scaffold, via metal binding and the active-site loops, to the dimer interface.…”

“…As a clue to the functional role of the communication, the binding of a single Zn 2+ ion to one subunit of the dynamic apoSOD1 dimer is found to alter the structure and stability of the entire molecule (14). Based on the general view that globular proteins with compromised structural rigidity are at increased risk of misfolding (5,6), the dynamic apoSOD1 molecule also has drawn attention as a possible precursor for pathological aggregation in ALS (12,13,15). In this study, we show by a combination of NMR, X-ray crystallography, and protein engineering that the origin of these dynamic motions is the built-in strain of a conserved hydrogenbond linkage through the hydrophobic core.…”