Dynamic local unfolding in the serpin α-1 antitrypsin provides a mechanism for loop insertion and polymerization

Abstract

The conformational plasticity of serine protease inhibitors (serpins) underlies both their activities as protease inhibitors and their susceptibility to pathogenic misfolding and aggregation. Here, we structurally characterize a sheet-opened state of the serpin α-1 antitrypsin (α₁AT) and show how local unfolding allows functionally essential strand insertion. Mutations in α₁AT that cause polymerization-induced serpinopathies map to the labile region, suggesting that the evolution of serpin function required sampling of high risk conformations on a dynamic energy landscape.

Figure 1

Design of cysteine mutations to probe low denaturant-induced strand opening in the serpin α₁AT. Comparison of structures of (a) native (PDB 1QLP) and (b) cleaved ‘loop-inserted’ (PDB 1EZX) states of α₁AT. The two light blue spheres, labeled P1 and P1′, correspond to the protease cleavage site in the RCL. Sites mutated to cysteine are indicated by yellow spheres centered on the Cβ. (c) Positions of cysteine substitutions and their secondary structural context. Structures in this and other figures were prepared using PyMol (http://www.pymol.org).

Figure 2

Accessibility of α₁AT single cysteine variants as a function of denaturant and comparison to unfolding monitored by CD. (a) Coomassie-stained SDS-PAGE results for the PEGylation of two representative single cysteine variants as a function of GdmCl concentration. Bands corresponding to the free and PEG-modified thiol are indicated by ‘-SH’ and ‘-SPEG’, respectively. (b) Comparison of the fractional accessibility of each cysteine to PEGylation (colored circles) with the unfolding of the variant α₁AT monitored by CD (black circles). The solid lines through the data are the fit to a three-state or a two-state protein unfolding model as described in data analyses (see Supplementary Tables 2 and 3 for fit data). Data are grouped and colored according to the behavior of the specific cysteine site as a function of denaturant, i.e., Class I sites, which become fully accessible at low denaturant, are shown in pink, Class II sites, which become nearly 50% accessible at low denaturant and fully accessible as the protein globally unfolds, in green, and Class III sites, which are inaccessible or only slightly accessible over the first unfolding transition and then become fully accessible when the protein globally unfolds, in orange.

Figure 3

Structural context of the cysteines belonging to the three classes with varying extents of PEGylation. Class I (pink), II (green), and III (orange) exhibit 100%, ^~50% and <20% PEGylation, respectively, in the low GdmCl intermediate state of α₁AT. The seventeen residues investigated in the current study are shown on the native structure of α₁AT (PDB ID 1QLP).

Figure 4

Solvent accessibility of single cysteines in the Z variant of α₁AT. The intermediate state of the protein was formed from the denatured state, i.e., upon dilution from high (5M GdmCl) denaturant. Bands of the PEG-modified (-SPEG) and unmodified (-SH) protein in Supplementary Fig. 4a were quantitated to obtain fraction thiol accessibility. Note that refolding of the Z variant yields a state that is not identical to native, and that this accounts for the partial exposure of 332C at the lowest GdmCl concentration.

Figure 5

Local unfolding leads to the sheet-opened intermediate of α₁AT. (a) Cysteine positions are shown on the structure of native α₁AT (PDB 1QLP) and colored by their accessibility behavior with pink, green and orange representing Class I, Class II, and III, respectively (see Fig. 2 and text for description of classes). Results for an additional six residue positions from previous work,, are indicated with appropriate coloring on the structure as well (labels are underlined) using the same color scheme. (b) The stable structural elements of the sheet-opened intermediate as inferred from cysteine accessibility data. In this structure, regions found to be accessible in the sheet-opened intermediate of α₁AT are removed and indicated schematically by a pink line connecting to the remaining structure. (c) The five naturally occurring pathogenic point mutations in α₁AT that produce full-length, polymerogenic protein and cause liver damage, along with similar polymerogenic mutations reported for other serpins (underlined labels) are represented in pink spacefill on the native structure of α₁AT. The backbone of the identified labile region is in pink.