Fully Metallated S134N Cu,Zn-Superoxide Dismutase Displays Abnormal Mobility and Intermolecular Contacts in Solution

Lucia Banci,Ivano Bertini,Nicola D'Amelio,Elena Gaggelli,Elisa Libralesso,Irena Matecko,Paola Turano,Joan Selverstone Valentine

doi:10.1074/jbc.m506637200

Abstract

S134N copper-zinc superoxide dismutase (SOD1) is one of the many mutant SOD1 proteins known to cause familial amyotrophic lateral sclerosis. Earlier studies demonstrated that partially metal-deficient S134N SOD1 crystallized in filament-like arrays with abnormal contacts between the individual protein molecules. Because protein aggregation is implicated in SOD1-linked familial amyotrophic lateral sclerosis, abnormal intermolecular interactions between mutant SOD1 proteins could be relevant to the mechanism of pathogenesis in the disease. We have therefore applied NMR methods to ascertain whether abnormal contacts also form between S134N SOD1 molecules in solution and whether Cys-6 or Cys-111 plays any role in the aggregation. Our studies demonstrate that the behavior of fully metallated S134N SOD1 is dramatically different from that of fully metallated wild type SOD1 with a region of subnanosecond mobility located close to the site of the mutation. Such a high degree of mobility is usually seen only in the apo form of wild type SOD1, because binding of zinc to the zinc site normally immobilizes that region. In addition, concentration-dependent chemical shift differences were observed for S134N SOD1 that were not observed for wild type SOD1, indicative of abnormal intermolecular contacts in solution. We have here also established that the two free cysteines (6 and 111) do not play a role in this behavior.

Highlights

Ual mutations have been shown to exert their pathological effects by a gain of function mechanism, implying that the copper-zinc superoxide dismutase variant (Cu,Zn-SOD) expressed from the mutated gene has in some way become toxic
S134N wtSOD1 is partially metal deficient when isolated from its yeast or baculovirus expression systems and has been observed to crystallize in linear, amyloid-like filaments in which disorder in the loop regions has resulted in a deprotection of exposed ␤-strands and formation of abnormal intermolecular protein-protein contacts [20]
These loops are found to be significantly disordered in the x-ray structure, and residues 125–131 play an important role in filament formation as they participate to H-bonds and apolar interactions with the ␤-barrel of a neighboring protein molecule. This structure suggests that similar intermolecular contacts might be forming in solution as the first step in the protein oligomerization that leads to the toxicity associated with the disease

Summary

Introduction

Ual mutations have been shown to exert their pathological effects by a gain of function mechanism, implying that the copper-zinc superoxide dismutase variant (Cu,Zn-SOD) expressed from the mutated gene has in some way become toxic. Many of the ALS-linked Cu,Zn-SOD variant proteins have been expressed, purified, and characterized in an effort to identify properties that they hold in common that differ from the same properties of wild type Cu,Zn-SOD [5] These studies have revealed a wide variability in their biophysical and biochemical properties. Those of the Cu,Zn-SOD variant proteins with modifications far from the metal binding region have normal SOD activities and spectroscopic characteristics. Their three-dimensional crystal structures are overall quite similar to those of wild type Cu,Zn-SOD and give no clues as to possible mechanisms of misfolding and aggregation [14, 15]. The former was used to ensure that any intermolecular contacts observed in solution were not due to formation of intermolecular disulfide bonds by air oxidation of the sample

Methods

Results

Conclusion