Metal-free Superoxide Dismutase-1 and Three Different Amyotrophic Lateral Sclerosis Variants Share a Similar Partially Unfolded β-Barrel at Physiological Temperature

Armando Durazo,Bryan F Shaw,Madhuri Chattopadhyay,Kym F Faull,Aram M Nersissian,Joan Selverstone Valentine,Julian P Whitelegge

doi:10.1074/jbc.m109.052076

Abstract

The structure and unfolding of metal-free (apo) human wild-type SOD1 and three pathogenic variants of SOD1 (A4V, G93R, and H48Q) that cause familial amyotrophic lateral sclerosis have been studied with amide hydrogen/deuterium exchange and mass spectrometry. The results indicate that a significant proportion of each of these proteins exists in solution in a conformation in which some strands of the beta-barrel (i.e. beta2) are well protected from exchange at physiological temperature (37 degrees C), whereas other strands (i.e. beta3 and beta4) appear to be unprotected from hydrogen/deuterium exchange. Moreover, the thermal unfolding of these proteins does not result in the uniform incorporation of deuterium throughout the polypeptide but involves the local unfolding of different residues at different temperatures. Some regions of the proteins (i.e. the "Greek key" loop, residues 104-116) unfold at a significantly higher temperature than other regions (i.e. beta3 and beta4, residues 21-53). Together, these results show that human wild-type apo-SOD1 and variants have a partially unfolded beta-barrel at physiological temperature and unfold non-cooperatively.

Highlights

There are Ͼ120 known Amyotrophic lateral sclerosis (ALS) mutations of SOD1 that give rise to a similar clinical pathology
We describe our investigation of the solution structure of three ALS variants of SOD1 (A4V, G93R, and H48Q) and human wild-type SOD1 by measuring their rates of amide hydrogen/deuterium exchange (HDX) with mass spectrometry [13,14,15,16]
We compared homogenous samples of SOD1 apoproteins (i.e. Zn0Cu0), and we report that the electrostatic loop is not destabilized in A4V, G93R, and H48Q mutant apo-SOD1 compared with human wild-type (hWT) apo-SOD1

Summary

Introduction

There are Ͼ120 known ALS mutations of SOD1 that give rise to a similar clinical pathology. Our goal was to test the hypothesis that ALS-linked mutations that are located in different regions of the protein can lead to a common region of structural perturbation. This similarity has been observed for various pathogenic variants of lysozyme that cause amyloidosis [13]. Recent work from our laboratory has shown that SOD1 can convert to amyloid fibrils under physiologically relevant conditions and that a small amount of disulfide-reduced hWT apoSOD1 can initiate fibrillation in apo and partially metalated forms of SOD1 [17]. Partial Unfolding of Apo-SOD1 at Physiological Temperature tiate aggregation do, become incorporated into the propagating fibril after initiation

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