Cutting Off Functional Loops from Homodimeric Enzyme Superoxide Dismutase 1 (SOD1) Leaves Monomeric β-Barrels

Abstract

Demetallation of the homodimeric enzyme Cu/Zn-superoxide dismutase (SOD1) is known to unleash pronounced dynamic motions in the long active-site loops that comprise almost a third of the folded structure. The resulting apo species, which shows increased propensity to aggregate, stands out as the prime disease precursor in amyotrophic lateral sclerosis (ALS). Even so, the detailed structural properties of the apoSOD1 framework have remained elusive and controversial. In this study, we examine the structural interplay between the central apoSOD1 barrel and the active-site loops by simply cutting them off; loops IV and VII were substituted with short Gly-Ala-Gly linkers. The results show that loop removal breaks the dimer interface and leads to soluble, monomeric ␤-barrels with high structural integrity. NMR-detected nuclear Overhauser effects are found between all of the constituent ␤-strands, confirming ordered interactions across the whole barrel. Moreover, the breathing motions of the SOD1 barrel are overall insensitive to loop removal and yield hydrogen/deuterium protection factors typical for cooperatively folded proteins (i.e. the active-site loops act as a "bolt-on" domain with little dynamic influence on its structural foundation). The sole exceptions are the relatively low protection factors in ␤-strand 5 and the turn around Gly-93, a hot spot for ALS-provoking mutations, which decrease even further upon loop removal. Taken together, these data suggest that the cytotoxic function of apoSOD1 does not emerge from its folded ground state but from a high energy intermediate or even from the denatured ensemble.Cu/Zn-superoxide dismutase (SOD1) is a ubiquitous radical scavenger that is observed to misfold and aggregate into small intracellular inclusions in the motor neurons of patients with both familial and sporadic amyotrophic lateral sclerosis (ALS) 2 (1-3). As with other protein misfolding diseases (e.g. Alzheimer disease, Parkinson disease, and the prion diseases), the structural events underlying the pathological aggregation (4, 5) and also the role of this aggregation in the neurodegeneration are still obscure (6). Native SOD1 is a thermodynamically stable (7), long lived (8, 9), and structurally robust (10) homodimer that coordinates one redox-active Cu 1ϩ/2ϩ ion and one structural Zn 2ϩ ion in each subunit (7,(11)(12)(13). The Cu 1ϩ/2ϩ ion is coordinated directly to the protein's immunoglobulin-like scaffold, whereas the neighboring Zn 2ϩ ion is coordinated more peripherally by the long loop IV (Fig. 1). Loop IV also stretches across the SOD1 surface to support the dimer interface, contains the conserved disulfide bond between Cys-57 and Cys-146, and forms together with loop VII the rigid encasement of the metallated active site (Fig. 1). Upon dissociation of the metal ions, however, the active-site loops loosen up and become more dynamic (14 -18), coupled to a radical loss of protein stability (7, 9, 18) and increased propensity to aggregate (19 -22). Consistently, several in vivo studies (23) and fo...

Global structural motions from the strain of a single hydrogen bond

“…1), in good agreement with previous CPMG measurements of apoSOD1 pwt (11) and H/D exchange analysis (16). Besides some loss of signal at positions that previously interfaced the dynamic loop-IV structure, the main effect of loop removal overall is lower CPMG amplitudes.…”

“…An advantage of this variant is that it eliminates interference from the active-site loops, which otherwise leads to NMR line broadening at low temperatures, at which the barrel motions are most pronounced. In most other respects, apoSOD ΔIVΔVII behaves as the wild-type monomer (apoSOD1 pwt ) (16). Measurements of 15 N Carr-Purcell-Meiboom-Gill (CPMG) relaxation profiles show here that 34 of the 110 apoSOD ΔIVΔVII residues are involved in dynamic exchange ( Fig.…”

“…As a sharp outlier in this trend, the CPMG amplitude of I99 in β6 displays a nearly eightfold increase upon loop removal, rendering this position the strongest signal in apoSOD ΔIVΔVII . Judging by the unaffected H/ D exchange rates of β6 and the neighboring β3, this signal increase likely is not the result of the increased dynamics of β6 itself (16). Rather it seems induced by altered fluctuations of β5 against which the I99 side chain packs (Fig.…”

“…The dynamic motions of the apoSOD1 barrel are localized mainly to the β-strands facing the active site, and are indicated experimentally by diminished NMR NOE couplings, relaxation-dispersion measurements (11), and backbone hydrogen-exchange [hydrogen/deuterium (H/D)] kinetics (16). To enhance the structural resolution of these dynamic motions as much as possible, we focus here on a SOD1 variant with truncated loops IV and VII (apoSOD ΔIVΔVII ) (16). An advantage of this variant is that it eliminates interference from the active-site loops, which otherwise leads to NMR line broadening at low temperatures, at which the barrel motions are most pronounced.…”

SOD1 aggregation in ALS mice shows simplistic test tube behavior

“…A general concern in studies of protein aggregation is that the data show high intrinsic variability and require good statistics to pin down (2,21). The phenomenon is here illustrated by the aggregation of the SOD1 barrel (SOD1 barrel ) (22,23) in pure buffer and 5 M urea (Fig. 1D).…”