Structural Dynamics Associated with Intermediate Formation in an Archetypal Conformational Disease

Abstract

SummaryIn conformational diseases, native protein conformers convert to pathological intermediates that polymerize. Structural characterization of these key intermediates is challenging. They are unstable and minimally populated in dynamic equilibria that may be perturbed by many analytical techniques. We have characterized a forme fruste deficiency variant of α1-antitrypsin (Lys154Asn) that forms polymers recapitulating the conformer-specific neo-epitope observed in polymers that form in vivo. Lys154Asn α1-antitrypsin populates an intermediate ensemble along the polymerization pathway at physiological temperatures. Nuclear magnetic resonance spectroscopy was used to report the structural and dynamic changes associated with this. Our data highlight an interaction network likely to regulate conformational change and do not support the recent contention that the disease-relevant intermediate is substantially unfolded. Conformational disease intermediates may best be defined using powerful but minimally perturbing techniques, mild disease mutants, and physiological conditions.

Update on alpha-1 antitrypsin deficiency: New therapies

“…Our NMR studies followed the polymerisation of Queen's (Lys154Asn) α 1 -antitrypsin under physiological conditions or in urea. Intermediate (M*) formation under physiological conditions was associated with highly native-like behaviour with changes in a few key motifs [14]. Global changes were observed in urea consistent with more widespread unfolding, in keeping with data from hydrogen-deuterium exchange [27].…”

“…Our work suggests that the Z mutation perturbs its local environment (breach region, Fig. 1A) to favour population of an unstable intermediate that we termed M* [8] in which β-sheet A opens [2,8] and the upper part of helix F unwinds [9,13,14]. The patent β-sheet A can then accept insertion of the reactive site loop motif.…”

“…We subsequently showed that the same process explains the profound plasma deficiency and hepatic inclusions of 3 other mutants of α 1 -antitrypsin: Siiyama (Ser53Phe) [17], Mmalton (∆Phe52) [18] and King's (His334Asp) [7]. Polymerisation also underlies the deficiency of the mild S (Glu264Val), I (Arg39Cys), Queen's (Lys154Asn) and Baghdad (Ala336Pro) alleles of α 1 -antitrypsin [12,14,19,20] but the rate of polymer formation is much slower in keeping with the absence of liver disease and only mild plasma deficiency. In many cases the reduction in the thermal stability of the native fold of α 1 -antitrypsin caused by mutations directly correlates with the polymerogenic tendency [11].…”

Reactive centre loop mutants of α-1-antitrypsin reveal position-specific effects on intermediate formation along the polymerization pathway

“…During the activation to intermediate, there is a change in far-UV circular dichroic signal, intrinsic tryptophan fluorescence, the interaction with ANS (8-anilinonaphthalene-1-sulfonic acid) or bis-ANS (4,4 -dianilino-1,1 -binaphthyl-5,5 -disulfonic acid) dyes [14,17], ion mobility mass spectrum collision crosssectional area [18] and NMR cross-peaks [19]. While denaturant can induce polymerization at concentrations that favour population of an unfolding intermediate state (I denat ) [6,20], observations indicate that there are multiple pathways favoured depending on the manner in which polymers are formed, and as a result intermediate ensembles represented by I pol and I denat could be structurally distinct [19].…”

“…While denaturant can induce polymerization at concentrations that favour population of an unfolding intermediate state (I denat ) [6,20], observations indicate that there are multiple pathways favoured depending on the manner in which polymers are formed, and as a result intermediate ensembles represented by I pol and I denat could be structurally distinct [19]. In support of this, it has recently been shown that polymers produced in the presence of denaturant lack an epitope that is expressed on polymer obtained from patient samples [18,21].…”