Loop-Sheet Mechanism of Serpin Polymerization Tested by Reactive Center Loop Mutations

Abstract

The serpin mechanism of protease inhibition involves the rapid and stable incorporation of the reactive center loop (RCL) into central ␤-sheet A. Serpins therefore require a folding mechanism that bypasses the most stable "loop-inserted" conformation to trap the RCL in an exposed and metastable state. This unusual feature of serpins renders them highly susceptible to point mutations that lead to the accumulation of hyperstable misfolded polymers in the endoplasmic reticulum of secretory cells. The ordered and stable protomer-protomer association in serpin polymers has led to the acceptance of the "loop-sheet" hypothesis of polymerization, where a portion of the RCL of one protomer incorporates in register into sheet A of another. Although this mechanism was proposed 20 years ago, no study has ever been conducted to test its validity. Here, we describe the properties of a variant of ␣ 1 -antitrypsin with a critical hydrophobic section of the RCL substituted with aspartic acid (P8 -P6). In contrast to the control, the variant was unable to polymerize when incubated with small peptides or when cleaved in the middle of the RCL (accepted models of loop-sheet polymerization). However, when induced by guanidine HCl or heat, the variant polymerized in a manner indistinguishable from the control. Importantly, the Asp mutations did not affect the ability of the Z or Siiyama ␣ 1 -antitrypsin variants to polymerize in COS-7 cells. These results argue strongly against the loop-sheet hypothesis and suggest that, in serpin polymers, the P8 -P6 region is only a small part of an extensive domain swap.

Mutagenesis of the bovSERPINA3‐3 demonstrates the requirement of aspartate‐371 for intermolecular interaction and formation of dimers

“…Indeed, the P8-P6 Asp mutations do not prevent the in vitro and in vivo polymerization. 33 Our in vitro study, performed on SERPINA3-3s extracted from the bovine skeletal muscle or produced as a bacterial recombinant protein, allows to propose another modeling of dimerization without swap of b-strands. In this modeling, the D371 residue, localized in the C-terminus loop, is essential for this mechanism of dimerization in denaturing conditions.…”

Structures of multidomain proteins adsorbed on hydrophobic interaction chromatography surfaces

“…Antitrypsin also is known to unfold via a multistate mechanism in solution [35]. The partially unfolded intermediate is critical for antitrypsin biological function and exists as a small, but significant, subpopulation under physiological conditions [36]. In the presence of low concentrations of GdnHCl, antitrypsin has a well characterized unfolding intermediate structure where at ∼1 M, several ␤ sheet strands and an ␣ helix unfold.…”

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

“…This variant has been found to behave in an equivalent fashion to wild-type in the previous studies (for example [22]). Reactive loop mutants were prepared on the C232S background using aspartic acid-scanning mutagenesis [12] to generate T345D, A347D, G349D, M351D, L353D, A355D, P357D and S359D substitutions (corresponding with RCL positions P 14 , P 12 , P 10 , P 8 , P 6 , P 4 , P 2 and P 1 in subsite notation [28]), yielding in combination the variants P 14 P 12 , P 10 P 8 , P 6 P 4 , P 2 P 1 , P 6 and P 4 . The pcDNA plasmid containing the Z (E342K) α 1 -antitrypsin allele was used as the basis for the mutants in cell culture experiments [29].…”

“…. , P 1 ) with aspartic acid [12] (Figure 1A). We determined the effect of these mutations on the rate of activation to the intermediate state by monitoring changes in tryptophan fluorescence, CD and binding to bis-ANS, and we report a novel FRET (Förster resonance energy transfer)-based polymerization assay.…”

“…Polymers can be induced in vitro at elevated temperatures [6,7], by proteolytic digestion following the P 6 , P 7 or P 10 residues [8][9][10], by low concentrations of denaturant [6,7], by a peptide mimetic of the P 14 -P 9 residues of the RCL [11,12] or at low pH [13]. A mechanism has been proposed, which describes the transition from monomer (M) to polymer (P) in which the monomer is 'activated' to an intermediate state (I pol ), which then (A) The location of the mutations used in this study are indicated against a cartoon representation of wild-type α 1 -antitrypsin (prepared using PyMol and PDB entries 1QLP [42] and 1EZX [43]).…”