An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behaviour.

Neda Motamedi-Shad,Arjun Scott Nanda,James A Irving,Alistair M Jagger,Maximilian Liedtke,Enrico Salvadori,Juan Perez,David A Lomas,Adriana Ordóñez,Sarah V Faull,Christopher W.M Kay,Narinder Heyer-Chauhan,Imran Haq,Joshua L Wort,Elena Miranda

doi:10.1042/bcj20160159

Abstract

Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A — on the opposite face of the molecule — more liable to adopt an ‘open’ state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin–enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the ‘open’ state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation.

Highlights

Α1-Antitrypsin (α1-AT) is the most abundant circulating protease inhibitor and a member of the serpin superfamily
It is unsurprising that varied models of the α1-AT polymer have been proposed, based on biophysical [28,29] and crystallographic [30] data. These models differ in the nature of the domain swap that forms the basis of the polymer chain, but one common feature predicted by all extant models is an expanded 6-stranded β-sheet A, with reactive centre loop (RCL) residues accommodated in equivalent positions to the canonical cleaved, and hyperstable, conformation (Supplementary Figure S1)
Our recent work described the 4B12 monoclonal antibody as a suppressor of α1-AT polymerisation. This involved the use of the monomeric form of Z α1-AT as the antigen to generate a hybridoma library, and its use in a primary screen that selected antibodies able to recognise this form of the protein [44]

Summary

Introduction

Α1-Antitrypsin (α1-AT) is the most abundant circulating protease inhibitor and a member of the serpin superfamily. The stark increase in thermal stability of antibody-bound α1-AT (Figure 1E, left) was used as a further tool to evaluate the effect of mutations on binding and the polymerisation suppression mechanism.

Results

Conclusion