Local cooperativity in the unfolding of an amyloidogenic variant of human lysozyme

Abstract

Hydrogen exchange experiments monitored by NMR and mass spectrometry reveal that the amyloidogenic D67H mutation in human lysozyme significantly reduces the stability of the beta-domain and the adjacent C-helix in the native structure. In addition, mass spectrometric data reveal that transient unfolding of these regions occurs with a high degree of cooperativity. This behavior results in the occasional population of a partially structured intermediate in which the three alpha-helices that form the core of the alpha-domain still have native-like structure, whereas the beta-domain and C-helix are simultaneously substantially unfolded. This finding suggests that the extensive intermolecular interactions that will be possible in such a species are likely to initiate the aggregation events that ultimately lead to the formation of the well-defined fibrillar structures observed in the tissues of patients carrying this mutation in the lysozyme gene.

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“…The additional peak (coloured in yellow) has been shown to result from a locally cooperative unfolding event, identified as the unfolding of the b-domain and the C-helix of the variant protein molecule [117]. From this result, and those of previous studies [18,117,118], a mechanism of fibril formation has been proposed for lysozyme (red box). As a result of its reduced stability and global cooperativity, the D67H variant protein populates transiently a partially unfolded intermediate (species I) in which the three helices that form the core of the a-domain still have native-like structure, whereas the b-domain and the C-helix are substantially disordered.…”

“…Four single-point mutants (I56T, F57I, D67H and W64R) and a double mutation (F57I/T70N) of human lysozyme are associated with systemic amyloid disease [114][115][116]. The effects of the D67H mutation on the properties of the lysozyme molecule have been studied at the molecular level using a variety of techniques including pulse labelling hydrogen/deuterium exchange analysed by mass spectrometry and NMR spectroscopy [28,117]. In the electrospray mass spectra of the D67H variant pulse labelled in the absence of the antibody fragment, two peaks are observed (Panel a), whereas the spectrum of the wild type protein under similar conditions shows a single peak [117].…”

“…The effects of the D67H mutation on the properties of the lysozyme molecule have been studied at the molecular level using a variety of techniques including pulse labelling hydrogen/deuterium exchange analysed by mass spectrometry and NMR spectroscopy [28,117]. In the electrospray mass spectra of the D67H variant pulse labelled in the absence of the antibody fragment, two peaks are observed (Panel a), whereas the spectrum of the wild type protein under similar conditions shows a single peak [117]. The additional peak (coloured in yellow) has been shown to result from a locally cooperative unfolding event, identified as the unfolding of the b-domain and the C-helix of the variant protein molecule [117].…”

“…In the electrospray mass spectra of the D67H variant pulse labelled in the absence of the antibody fragment, two peaks are observed (Panel a), whereas the spectrum of the wild type protein under similar conditions shows a single peak [117]. The additional peak (coloured in yellow) has been shown to result from a locally cooperative unfolding event, identified as the unfolding of the b-domain and the C-helix of the variant protein molecule [117]. From this result, and those of previous studies [18,117,118], a mechanism of fibril formation has been proposed for lysozyme (red box).…”

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“…3D). In this species, the b-domain and the C-helix (referred to as the amylotope [44]) are cooperatively unfolded while the rest of the adomain remains native [81,82] (Fig. 3A).…”