An integrative approach combining ion mobility mass spectrometry, X‐ray crystallography, and nuclear magnetic resonance spectroscopy to study the conformational dynamics of α₁‐antitrypsin upon ligand binding

Abstract

Native mass spectrometry (MS) methods permit the study of multiple protein species within solution equilibria, whereas ion mobility (IM)-MS can report on conformational behavior of specific states. We used IM-MS to study a conformationally labile protein (α1-antitrypsin) that undergoes pathological polymerization in the context of point mutations. The folded, native state of the Z-variant remains highly polymerogenic in physiological conditions despite only minor thermodynamic destabilization relative to the wild-type variant. Various data implicate kinetic instability (conformational lability within a native state ensemble) as the basis of Z α1-antitrypsin polymerogenicity. We show the ability of IM-MS to track such disease-relevant conformational behavior in detail by studying the effects of peptide binding on α1-antitrypsin conformation and dynamics. IM-MS is, therefore, an ideal platform for the screening of compounds that result in therapeutically beneficial kinetic stabilization of native α1-antitrypsin. Our findings are confirmed with high-resolution X-ray crystallographic and nuclear magnetic resonance spectroscopic studies of the same event, which together dissect structural changes from dynamic effects caused by peptide binding at a residue-specific level. IM-MS methods, therefore, have great potential for further study of biologically relevant thermodynamic and kinetic instability of proteins and provide rapid and multidimensional characterization of ligand interactions of therapeutic interest.PDB Code(s): 4PYW

Update on alpha-1 antitrypsin deficiency: New therapies

“…Smaller, refined peptides were developed that had a similar effect with more specificity for Z, rather than M, α 1 -antitrypsin [16,[86][87][88]. However the challenge is to deliver these peptides to cells, let alone to a human liver.…”

“…1B(i)). Indeed, polymerisation is blocked by peptides that mimic the reactive loop sequence and so compete for binding to the insertion site in β-sheet A [2,16]. We subsequently showed that the same process explains the profound plasma deficiency and hepatic inclusions of 3 other mutants of α 1 -antitrypsin: Siiyama (Ser53Phe) [17], Mmalton (∆Phe52) [18] and King's (His334Asp) [7].…”

“…Importantly one such exception is the Z variant, that appears to cause a relatively mild thermal destabilisation but is highly polymerogenic [11,12,16].…”

“…Understanding the polymerogenic behaviour of the most clinically-significant variant is important as it has relevance for the readouts that may be used in screening for therapeutic agents [16,22]. Conversely, understanding the behaviour of rarer or milder variants in addition opens the way to precision medicine approaches, analogous to recent advances in cystic fibrosis [23,24].…”

Comparison of the Thermal Degradation Properties of Crystalline and Amorphous Cellulose, as well as Treated Lignocellulosic Biomass

“…0.9.4: Wojdyr 2010), which is an open-source software for non-linear curve fitting and analysis. The DTG peaks were separated by the split Gaussian method and fitted with the Levenberg-Marquardt algorithm based on the method of Nyon et al (2015).…”