The Structural Basis of Serpin Polymerization Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry

Abstract

The serpinopathies are a group of inherited disorders that share as their molecular basis the misfolding and polymerization of serpins, an important class of protease inhibitors. Depending on the identity of the serpin, conditions arising from polymerization include emphysema, thrombosis, and dementia. The structure of serpin polymers is thus of considerable medical interest. Wild-type ␣ 1 -antitrypsin will form polymers upon incubation at moderate temperatures and has been widely used as a model system for studying serpin polymerization. Using hydrogen/deuterium exchange and mass spectrometry, we have obtained molecular level structural information on the ␣ 1 -antitrypsin polymer. We found that the flexible reactive center loop becomes strongly protected upon polymerization. We also found significant increases in protection in the center of ␤-sheet A and in helix F. These results support a model in which linkage between serpins is achieved through insertion of the reactive center loop of one serpin into ␤-sheet A of another. We have also examined the heat-induced conformational changes preceding polymerization. We found that polymerization is preceded by significant destabilization of ␤-sheet C. On the basis of our results, we propose a mechanism for polymerization in which ␤-strand 1C is displaced from the rest of ␤-sheet C through a binary serpin/serpin interaction. Displacement of strand 1C triggers further conformational changes, including the opening of ␤-sheet A, and allows for subsequent polymerization.2 is a member of the serpin class of protease inhibitors. Serpins inhibit their target proteases via a unique mechanism ( Fig. 1) (1). Cleavage of a serpin's reactive center loop (RCL) by a protease triggers a massive conformational change in which the RCL inserts into the central ␤-sheet A, becoming a sixth strand. This process disrupts the protease active site and traps the acyl-enzyme intermediate, rendering the protease inactive. The active, loop-expelled form of ␣ 1 -AT is significantly less stable than the loop-inserted form, and as a result of this metastability, the serpin structure is readily disrupted by mutations that result in inappropriate conformational changes. It has been established that a number of serious diseases associated with serpin mutations are caused by the formation and accumulation of serpin polymers (2). The details of the polymerization mechanism are therefore of considerable interest.Serpin polymerization has been studied extensively by a variety of methods, including circular dichroism, fluorescence spectroscopy, electron microscopy, and x-ray crystallography (3-5). The most generally accepted model of pathological serpin polymerization is one in which the RCL of one serpin anneals between ␤-strands 3 and 5A of another (1). Two major lines of evidence support this model. The first is that RCLmimicking peptides have been shown to anneal between strands 3 and 5A and have also been shown to block polymerization in vitro (6). The second comes from fluorescence spectroscopy. Dist...

Functional Unfolding of α1-Antitrypsin Probed by Hydrogen-Deuterium Exchange Coupled with Mass Spectrometry

“…Although we could not analyze the peptides spanning s1C due to the absence of a common peptide between D1, D2, and D3, the current results suggest that ␤-sheet C maintains its structure during complex formation. This is quite a contrast to the recent report of Wintrode and co-workers (39) in which ␤-sheet C appears to be destabilized first during polymerization. Our previous mutational studies on ␣ 1 AT (6) showed that destabilization of ␤-sheet C caused the molecule to convert into the latent form (RSLinserted monomer) and suggested that the integrity of ␤-sheet C is crucial for maintaining the kinetic trap of the native form.…”

“…The overall H/D-EX level of ␣ 1 AT was slightly higher than that of a previous report (38,39) presumably because of the nonsalt condition of the current H/D-EX study. It was reported that the presence of salts stabilized protein conformation and suppressed H/D-EX (40).…”

“…Because complete opening of the two strands is required for the full insertion of RSL and complex formation, the strand opening of s3A and s5A seemingly does not involve solvent exposure. In a recent study of serpin polymerization using H/D-EX, the increase in deuterium exchange was not observed at these sites during polymerization (39). The serpin polymerization also accompanies the opening event of the ␤-strands (s3A and s5A) with RSL insertion of one molecule into another.…”

Defining the mechanism of polymerization in the serpinopathies

“…Endopeptidase cleavage data showed that helix I remained protected from digestion in monomeric Z α 1 -antitrypsin, in polymers of Z α 1 -antitrypsin formed under physiological conditions in vitro and in polymers isolated from the endoplasmic reticulum of hepatocytes of a Z α 1 -antitrypsin homozygote. These data are consistent with hydrogen/deuterium exchange studies that showed no change in the helix I of α 1 -antitrypsin upon formation of the polymerogenic intermediate or chains of polymers (30).…”

Deficiency Mutations of Alpha-1 Antitrypsin. Effects on Folding, Function, and Polymerization

“…It is likely that this difference is also manifest in intermediate ensembles induced by the two approaches. Indeed, the heatinduced alpha-1 antitrypsin intermediate shows evidence for a more compact state than that observed in 1 M guanidinium hydrochloride (41,42,44). However, regional differences in s5A behavior are similar in both the denaturant-mediated and heat-induced polymerization (Table 2; Figure 4C, right).…”