Journal of Molecular Biology
Identification and Characterization of a Misfolded Monomeric Serpin Formed at Physiological Temperature
Graphical Abstract
Research Highlights
► Wild-type antichymotrypsin (ACT) can adopt the delta conformation under physiological conditions. ► Temperature determines which misfolded state ACT can adopt. ► Delta ACT is a misfolded end point rather than an intermediate on the misfolding pathway.
Introduction
Large proteins fold to their native conformation via a rugged free-energy landscape, which contains many nonnative minima regions where a protein can become trapped. Identifying these nonnative states and understanding how a folding molecule avoids these traps are important. The serpin superfamily represents a biomedically important family of large proteins that need to navigate a complex energy landscape—with several nonnative conformations present in energy minima—to fold.1, 2, 3 Several of these nonnative states have been structurally characterized and play a role in diseases such as emphysema4, 5 liver cirrhosis,6 dementia,7 and thrombosis.8 The native serpin fold is metastable, possessing an inherent strain that is utilized during proteinase inhibition. Upon interaction with a target proteinase, the exposed reactive center loop (RCL) is cleaved and inserts into the central five-stranded A β-sheet, resulting in a cleaved structure with an expanded six-stranded A β-sheet.9 During this process, the proteinase is dragged from one pole of the molecule to another, resulting in inactivation of both the proteinase and the serpin.9, 10, 11
The cleaved serpin conformation is highly stable; however, there are similar stable conformations accessible on the folding landscape that do not require cleavage. These include the latent and delta conformations, which have differing degrees of RCL insertion.12, 13 The latent conformation, crystallographically characterized for a number of serpins, possesses a fully incorporated RCL that requires the removal of one strand from the C β-sheet.14, 15 The energetic barrier between the native conformation and the latent conformation differs between members of the serpin family. Antithrombin and plasminogen activator inhibitor-1 can adopt the latent conformation under near-physiological conditions,14, 15 while other serpins such as α1-antitrypsin must be subjected to extreme chemical environments or high temperatures for them to adopt this conformation.16 The delta conformation, so far only described for a disease-linked variant of antichymotrypsin (ACT),12 also represents a noncleaved inactive conformation. As observed in the latent conformation, the A β-sheet of the delta structure is composed of six β-strands; however, the central fourth strand is provided by a different part of the molecule. While the RCL completely inserts as the fourth strand in latent serpins, this strand is formed by two separate structural elements in the delta conformation: the RCL partially inserts in the upper space, while the F helix partially unwinds to occupy the remaining space. A previous study proposed that the delta conformation is an intermediate on the polymerization and complex formation pathway.12 The promiscuity of the serpin RCL lends itself to misfolding via self-insertion, as observed in the latent and delta conformations; however, the RCL can also interact with other molecules, leading to a polymer chain propagated through β-sheet linkages.17, 18 Several models describing how each monomer interacts within the polymer chain have been proposed, with the most recent suggesting that the RCL forms part of a β-hairpin that inserts into the A β-sheet of another molecule.18 Polymers are the main cause of many serpin-related disorders and can be readily formed in vivo by proteins carrying mutations, or in vitro by modifying the temperature or pH of the solution. Each of these conformations is present on the serpin folding landscape; however, the way in which these conformations relate to one another is poorly understood.
The native serpin conformation sits precariously between other more stable conformations such as the latent and polymeric forms. In this study, we probed the energy landscape of ACT, an inhibitory member of the serpin family. The majority of mutations within ACT, identified from patients with emphysema and liver disease,19 lead to the formation of polymers. Additionally, a misfolded monomeric form of wild-type ACT has been found in the lung lavage fluid of healthy individuals, highlighting that an inappropriate conformational change can occur even under physiological conditions.20 Through a combination of crystallographic and biophysical techniques, we have explored the relationship between native ACT and several of these nonnative conformations. We have isolated wild-type ACT in the delta conformation, formed under physiological conditions, and have proposed that this conformation is an end point on a misfolding pathway rather than a representative of a structural folding intermediate.
Section snippets
ACT forms two distinct misfolded species
Serpins have an inherent tendency to misfold into inactive conformations, which can result in disease in vivo. Our current understanding indicates that there are two major misfolding pathways for ACT, as with other serpins: (1) the formation of long-chain polymers, and (2) the formation of misfolded monomers, generally in the latent conformation (Fig. 1). Factors influencing which of these misfolding pathways are favored, or to what extent these pathways are shared, are of great importance due
Discussion
Protein misfolding is a key causative factor in a wide range of diseases encompassing many neurological diseases such as Alzheimer's disease and emphysema associated with α1-antitrypsin deficiency.26 These diseases are caused primarily by the self-association of specific proteins, resulting in the detrimental accumulation of proteinaceous material and causing damage to various cells and organs.27 Protein aggregation can also lead to disease through the loss of function of the aggregated
Expression and purification
ACT was expressed and purified as previously described,28, 29 with the following modifications. Following SP Sepharose separation, fractions containing ACT were mixed with buffer (4 M NaCl) to a final NaCl concentration of 3.5 M. This solution was then loaded onto a 5-ml HiTrap Phenyl Sepharose column (GE Healthcare). The column was washed with 4 M NaCl, and a 60-ml linear gradient from 4 M to 0 M NaCl was run to elute all bound proteins. Those fractions containing inhibitory activity against
Acknowledgements
This work was supported by the Australian Synchrotron Research Program, which is funded by the Commonwealth of Australia under the Major National Research Facilities Program. Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Energy Research. GM/CA CAT has been funded, in whole or in part, with US Federal funds from the National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Science (Y1-GM-1104). S.C.F.
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2016, Cell Chemical BiologyCitation Excerpt :This conformation has a partial insertion of the RCL into the central β-sheet A, with P14–P12 buried, the RCL bending outward at position P11, and β-strand 1C remaining associated to β-sheet C. Instead of full insertion of the RCL into β-sheet A, the lower part of the space between β-strand 3A and β-strand 5A is filled by a short β strand formed by the α-helix F-β-strand 3A loop and the last turn of α-helix F, which is partially unwound (Gooptu et al., 2000) (Figure 1). The inactive monomeric δ conformation of α1-ACT is stable in solution and is not an intermediate step toward polymerization (Pearce et al., 2010). We have now isolated an RNA aptamer stabilizing the L55P mutant of α1-ACT in a monomeric active conformation, while leaving the protease inhibitory activity intact.
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2011, Methods in EnzymologyCitation Excerpt :In this way, it is possible to separate native, latent, cleaved, and polymeric forms of the same protein with good resolution. A method for separation of native ACT from the delta form (Pearce et al., 2010) is presented here and depicted graphically in Fig. 2.3. Materials
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Joint first authors.