Folding Catalysis by Transient Coordination of Zn²⁺ to the Cu Ligands of the ALS-Associated Enzyme Cu/Zn Superoxide Dismutase 1

Abstract

How coordination of metal ions modulates protein structures is not only important for elucidating biological function but has also emerged as a key determinant in protein turnover and protein-misfolding diseases. In this study, we show that the coordination of Zn(2+) to the ALS-associated enzyme Cu/Zn superoxide dismutase (SOD1) is directly controlled by the protein's folding pathway. Zn(2+) first catalyzes the folding reaction by coordinating transiently to the Cu ligands of SOD1, which are all contained within the folding nucleus. Then, after the global folding transition has commenced, the Zn(2+) ion transfers to the higher affinity Zn site, which structures only very late in the folding process. Here it remains dynamically coordinated with an off rate of ∼10(-5) s(-1). This relatively rapid equilibration of metals in and out of the SOD1 structure provides a simple explanation for how the exceptionally long lifetime, >100 years, of holoSOD1 is still compatible with cellular turnover: if a dissociated Zn(2+) ion is prevented from rebinding to the SOD1 structure then the lifetime of the protein is reduced to a just a few hours.

Global structural motions from the strain of a single hydrogen bond

“…5, Folding Analysis, and Table S5). Consistent with previous studies (20), holoSOD1 pwt displays elevated k u values in the presence of EDTA, exposing the apparent rate constant of metal loss, k off (Fig. 5).…”

“…An example of such long-range modulation is the observation that the common ALS-associated SOD1 mutation G93A affects the metal-binding ligands more than 18 Å away (38). For an enzyme whose stability, maturation, and cellular lifetimes are intimately coupled to the metal-binding equilibria (20), such global effects might be critical, shedding light not only on why the ALSprovoking mutations are so broadly distributed in the SOD1 structure, but also on why the SOD1 sequence is so highly conserved across divergent species (39). To this end, our data show that the evolutionary pathway to such complex functional dynamics may be very short: in this case, the change of a ubiquitous tyrosine corner into a cross-core histidine link is just one mutation away.…”

“…Removal of the H120-H43 bond also is expected to decrease metal affinity. To examine this possibility, we measured the effect of EDTA on the unfolding kinetics of the holo proteins, which specifically targets the metaloff rates (20) (Fig. 5, Folding Analysis, and Table S5).…”

“…5). This rate constant of the fully metallated protein most likely describes the simultaneous loss of both metal ions, because metal loss from the Zn site in the absence of a loaded Cu site is considerably faster (20), i.e., k off is strongly controlled by the Cu site. Upon H43F mutation, however, k u of holoSOD1 pwt H43F converges with that of the apo protein.…”

Fibrillation precursor of superoxide dismutase 1 revealed by gradual tuning of the protein-folding equilibrium

“…Non-amyloid aggregates can also be promoted without agitation in physiological buffer by simply increasing the protein concentration to 95-950 μM and extending the incubation times (10). More detailed insight into the SOD1 aggregation mechanism has so far been precluded by difficulty to identify at structural level the monomeric precursor material: Native SOD1 is a metallated dimer with a complex maturation and degradation pathway, including a plethora of intermediate states (11,12). As observed for β 2 -microglobulin (13) and TTR (14); however, it is often assumed that the precursor for SOD1 aggregation is a ruptured apo species, which either exposes sticky material through local unfolding of the central β-barrel (15, 16) cross-links oxidatively via C6/C111 (9), polymerizes via the long active site loops (17), or promotes aggregation by a mixture of mechanisms (10).…”