Probing the Unfolding Pathway of α1-Antitrypsin

Abstract

Protein misfolding plays a role in the pathogenesis of many diseases. ␣ 1 -Antitrypsin misfolding leads to the accumulation of long chain polymers within the hepatocyte, reducing its plasma concentration and predisposing the patient to emphysema and liver disease. In order to understand the misfolding process, it is necessary to examine the folding of ␣ 1 -antitrypsin through the different structures involved in this process. In this study we have used a novel technique in which unique cysteine residues were introduced at various positions into ␣ 1 -antitrypsin and fluorescently labeled with N,N-dimethyl-N-(iodoacetyl)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-ethylenediamine. The fluorescence properties of each protein were studied in the native state and as a function of guanidine hydrochloride-mediated unfolding. The studies found that ␣ 1 -antitrypsin unfolded through a series of intermediate structures. From the position of the fluorescence probes, the fluorescence quenching data, and the molecular modeling, we show that unfolding of ␣ 1 -antitrypsin occurs via disruption of the A and C ␤-sheets followed by the B ␤-sheet. The implications of these data on both ␣ 1 -antitrypsin function and polymerization are discussed.Protein folding is the process by which the primary sequence is translated into tertiary structure. Modification of this sequence via in vivo mutation or in vitro mutagenesis often alters the ability of the protein to fold correctly. Misfolding of proteins during their synthesis within the cell can lead to loss of protein function (1), an example being ␣ 1 -antitrypsin (␣ 1 -AT) 1 deficiency (2-4). Many well characterized ␣ 1 -AT variants are associated with an ␣ 1 -AT plasma deficiency; in these cases, the variants aggregate at their site of synthesis within the liver cell (5, 6). This aggregation is via an ordered polymerization process that is initiated by protein misfolding. In the case of the ␣ 1 -AT Z mutation (Glu-342 3 Lys), the rate of protein folding is much slower than in the native state, leading to the accumulation of an intermediate, which then polymerizes (4). It has recently been shown that the heat-induced polymerization of ␣ 1 -AT also involves the formation of an unfolding intermediate (7). The actual process of polymerization has been well studied, and two mechanisms have been proposed: the loop-A-sheet and loop-C-sheet mechanisms (8 -11). Both processes involve the insertion of the reactive center loop residues of the donating ␣ 1 -AT molecule into either the A ␤-sheet (loop-A-sheet) or the C ␤-sheet (loop-C-sheet) of the acceptor molecule (11). There is evidence for the occurrence of both of these mechanisms; indeed, in vitro ␣ 1 -AT has been shown to undergo both depending upon the buffer used (12-14).␣ 1 -AT is a member of the serine proteinase inhibitor (serpin) superfamily and is composed of 394 amino acid residues arranged into three ␤-sheets (A, B, and C) and nine ␣-helices (A-I). X-ray crystallographic and biochemical data have shown that ␣ 1 -AT can adopt a number of ...

The Molecular Interactions of Heat Shock Protein 47 (Hsp47) and Their Implications for Collagen Biosynthesis

“…This first transition is consist- ent with an early unfolding event, leading to an intermediate state, and the second transition with a late unfolding event, leading to random coil. Similar transitions have been observed in urea titrations involving other members of the serpin family, most notably ␣ 1 -antitrypsin (46). These two consecutive unfolding events were observed independently in a comparable urea titration experiment performed with CD spectroscopy.…”

The High Resolution Crystal Structure of the Human Tumor Suppressor Maspin Reveals a Novel Conformational Switch in the G-helix

“…This region of the serpin scaffold was previously thought to be conformationally inert (51,52); a structural comparison of several serpin pairs does not reveal conformational mobility around the G-helix (38,53). The switch from the closed to the open form involves the G-helix rotating by approx 5°and translating ϳ3 Å on the underlying B-sheet, with corresponding 2-Å shifts in the hairpin connecting strands s1B and s2B of the B-sheet.…”

The Selective Inhibition of Serpin Aggregation by the Molecular Chaperone, α-Crystallin, Indicates a Nucleation-dependent Specificity

“…It is feasible that the non-native species of ␣ 1 -AT and ACT may be sufficiently different in conformation to explain the specificity of ␣-crystallin interaction. However, given the high sequence homology and adoption of similar folding intermediates (29,35), it seems unlikely that the relatively subtle differences in structure between ACT and ␣ 1 -AT would have a major role in determining the substrate specificity of ␣-crystallin for ACT over ␣ 1 -AT particularly because of the broad substrate specificity of ␣-crystallin and other sHsps as determined from many studies of sHsp chaperone action (6 -8). Instead, a much more tangible contribution to specificity may be the distinct mechanistic and kinetic differences between the aggregation processes of the two serpins.…”