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 β-barrels with high structural integrity. NMR-detected nuclear Overhauser effects are found between all of the constituent β-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 β-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.
Highlights
Tural events underlying the pathological aggregation [4, 5] and the role of this aggregation in the neurodegeneration are still obscure [6]
The results show that the loop removal is energetically favorable in the sense that it increases the thermodynamic stability of the apoSOD1 monomer but has limited effect on the structure and dynamic motions of the barrel to which they are anchored
To examine how the active-site loops influence the structural properties of the apoSOD1 molecule, we truncated them by protein engineering (Fig. 1)
Summary
Gene Design—The monomeric reference protein, SOD1pwt, was obtained from the human SOD1 wild-type sequence by introduction of the following mutations: C6A/C111A, which. The reason for using C111S, rather than C111A, in SOD1⌬IV,⌬VII is that this mutation yields an unfolding rate constant indistinguishable from that of the parent protein SOD1pwt, which offers a considerable advantage in the interpretation of H/D exchange data (see below). The protein was eluted by a linear 0 –1 M NaCl gradient in 50 mM Tris-HCl, pH 7.5, and purity was analyzed using the Ready Gel SDS-PAGE system from Bio-Rad and by electrospray ionization mass spectrometry performed at the Protein Analysis Center (Karolinska Institute, Solna, Sweden) after dialysis against milliQ H2O and centrifugation at 18,000 rpm in a Beckman Avanti J-25 centrifuge, JA-25.50 rotor. The decay of the peak intensities was fitted to a single exponential decay with base line with a linear slope
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