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.

Mun Peak Nyon,Imran Haq,John Christodoulou,Geraldine Levy,Konstantinos Thalassinos,Bibek Gooptu,Jemma Day,Ganesh N Sivalingam,Tanya Prentice,James A Irving,David A Lomas,John Kirkpatrick

doi:10.1002/pro.2706

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

Highlights

IntroductionAberrant conformational behavior of proteins during and after folding is recognized as the basis for an increasing number of chronic diseases, including Alzheimer’s, Parkinson’s, and Huntington’s disease.[1] a1-Antitrypsin deficiency is a conformational disease associated with severe lung (early-onset, panacinar emphysema) and liver (hepatic cirrhosis, hepatocellular carcinoma) disease.[2,3] The Z (Glu342Lys) mutation causes a1-antitrypsin, the major circulating human antiprotease, to misfold and selfassociate into polymers within the endoplasmic reticulum of hepatocytes, with toxic gain-of-function effects.[4,5,6] The concomitant deficiency of circulating protein renders lung tissue vulnerable to destructive and proinflammatory effects of neutrophil elastase, that is the physiological target of a1-antitrypsin, and a clinical association with early-onset emphysema.[7,8,9] In populations of North European descent, the heterozygote frequency for the Z-variant is as high as 1 in 27.10 the risk of severe disease is strongly associated with the homozygous state, a1-antitrypsin deficiency is one of the most common monogenic disorders
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 show the ability of Ion mobility coupled to mass spectrometry (IM-MS) to track such disease-relevant conformational behavior in detail by studying the effects of peptide binding on a1-antitrypsin conformation and dynamics

Summary

Introduction

Aberrant conformational behavior of proteins during and after folding is recognized as the basis for an increasing number of chronic diseases, including Alzheimer’s, Parkinson’s, and Huntington’s disease.[1] a1-Antitrypsin deficiency is a conformational disease associated with severe lung (early-onset, panacinar emphysema) and liver (hepatic cirrhosis, hepatocellular carcinoma) disease.[2,3] The Z (Glu342Lys) mutation causes a1-antitrypsin, the major circulating human antiprotease, to misfold and selfassociate into polymers within the endoplasmic reticulum of hepatocytes, with toxic gain-of-function effects.[4,5,6] The concomitant deficiency of circulating protein renders lung tissue vulnerable to destructive and proinflammatory effects of neutrophil elastase, that is the physiological target of a1-antitrypsin, and a clinical association with early-onset emphysema.[7,8,9] In populations of North European descent, the heterozygote frequency for the Z-variant is as high as 1 in 27.10 the risk of severe disease is strongly associated with the homozygous state, a1-antitrypsin deficiency is one of the most common monogenic disorders. Its disease mechanisms are among the best characterized of any human disease, and extensive efforts are underway to translate these scientific insights into novel therapeutic strategies.[11,12]

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Conclusion