An Asymmetric Runaway Domain Swap Antithrombin Dimer as a Key

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An asymmetric antithrombin dimer is a key intermediate for polymerization revealed by hydrogen/deuterium-exchange mass spectrometry Morten Beck Trelle, Shona H. Pedersen, Eva Christina Østerlund, Jeppe Buur Madsen, Søren Risom Kristensen, and Thomas J.D. Jørgensen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02518 • Publication Date (Web): 26 Oct 2016 Downloaded from http://pubs.acs.org on October 26, 2016

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Analytical Chemistry

Title An asymmetric runaway domain swap antithrombin dimer is a key intermediate for polymerization revealed by hydrogen/deuterium-exchange mass spectrometry Morten Beck Trellea, Shona Pedersenbc, Eva Christina Østerlunda, Jeppe Buur Madsena, Søren Risom Kristensenb, Thomas, J.D. Jørgensena* a

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230

Odense M, Denmark b

Department of Clinical Biochemistry, Aalborg University Hospital, Hobrovej 18 , 9000 Aalborg, Denmark

c

Department of Clinical Medicine, Aalborg University, Sdr. Skovvej 15, 9000 Aalborg, Denmark

*corresponding author Thomas J.D. Jørgensen [email protected] telephone: +45 65502409

Key words: Antithrombin, polymerization, Hydrogen/deuterium-exchange mass spectrometry, serpin, conformational disease, protein dynamics

ACS Paragon Plus Environment


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Abstract Antithrombin deficiency is associated with increased risk of venous thrombosis. In certain families this condition is caused by pathogenic polymerization of mutated antithrombin in the blood. To facilitate future development of pharmaceuticals against antithrombin polymerization an improved understanding of the polymerogenic intermediates is crucial. However, X-ray crystallography of these intermediates is severely hampered by the difficulty in obtaining well-diffracting crystals of transient and heterogeneous noncovalent protein assemblies. Furthermore, their large size prohibits structural analysis by NMR spectroscopy. Here we show how hydrogen/deuterium-exchange mass spectrometry (HDX-MS) provides detailed insight into the structural dynamics of each subunit in a polymerization-competent antithrombin dimer. Upon deuteration, this dimer surprisingly yields bimodal isotope distributions for the majority of peptides, demonstrating an asymmetric configuration of the two subunits. The data reveal that one subunit is very dynamic, potentially intrinsically disordered, whereas the other is considerably less dynamic. The local subunit-specific deuterium uptake of this polymerization-competent dimer strongly supports a β4A-β5A β-hairpin runaway domain swap mechanism for antithrombin polymerization. HDX-MS thus holds exceptional promise as an enabling analytical technique in the efforts towards future pharmacological intervention with protein polymerization and associated diseases.


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Introduction Conformational diseases are a group of disorders resulting from misfolding or conformational change of particular proteins that often involves the formation of nonnative protein oligomers and polymers. Such nonnative oligomers can be formed from globular proteins by a domain-swapping mechanism.1,2 The swap occurs by an exchange of specific secondary structural elements between multiple polypeptide chains. If the swap is reciprocated between two polypeptide chains then a symmetrical self-terminating dimer is formed that is incapable of further elongation. In contrast, when the swap is propagated then a runaway domain-swapping3 scenario is possible in which one protein molecule swaps a domain into the neighboring molecule to form a growing polymer of multiple polypeptide chains. Such a continuous domain-swapping mechanism has been implicated in the formation of amyloid fibrils and polymerization of a number of globular proteins.2 In a runaway domain swap polymer only the terminal polypeptide chains are open-ended and therefore capable of elongation. In this regard, the runaway domain swap dimer represents a unique case as it is the smallest polymeric form that consists exclusively of “sticky ends”. Although several self-terminating domain swapped protein structures are known,4,5 the structure and the molecular properties of a runaway domain swap dimer is largely unknown and this is also true for the runaway domain swap dimer of the serine protease inhibitor (serpin) antithrombin.6,7 In general, the heterogeneity of nonnative protein polymers makes it exceedingly difficult to achieve structural information by crystallography. Furthermore, the analysis of transient oligomeric species such as a runaway domain swap polymer is impeded by the preponderance for their elongation during concentration and crystallization. However, hydrogen/deuterium exchange monitored by mass spectrometry8 (HDX-MS) is particularly well-suited to resolve and characterize the dynamics of transient protein oligomers as well as coexisting conformations of proteins.9-12 We have therefore utilized HDX-MS to characterize the structural dynamics of the runaway domain swap antithrombin dimer. HDX-MS probes the backbone amide hydrogen exchange rates and provides in this way direct information on the stability of the hydrogen-bonded structure of the polypeptide backbone in proteins.13 HDX-MS has proven to be an invaluable tool in the elucidation of serpin dynamics in solution.14-18 Serpins are >40 kDa proteins and it is often impossible to achieve the purity and concentration required for NMR studies, especially in the case of serpin polymers. In its natively folded state, antithrombin is a monomeric 58 kDa globular glycoprotein and it is considered to be the most important endogenous anticoagulant. Inactivation through polymerization is described for a number of antithrombin mutants.19-21 Antithrombin modulates blood coagulation by inhibiting a wide spectrum of serine proteases such as thrombin (FIIA), factors IXa and Xa, and thereby correctly controlling and preventing inappropriate clot formation, which otherwise may cause thrombotic disorders.22 Hereditary deficit in the activity



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of antithrombin significantly increases the risk of venous thromboembolism23 and complete deficiency is lethal in mice.24 Antithrombin is composed of 3 β-sheets (named A, B and C) and 9 alpha-helices (Fig. 1A). Natively expressed antithrombin folds to an active inhibitory conformation in which the reactive center loop (RCL) is exposed to the solvent. The target protease binds and cleaves the reactive center loop, which triggers insertion of the protease-linked N-terminal part of the reactive center loop as an extra β-strand in the middle of β-sheet A. This insertion of the reactive center loop, and translocation of the protease, distorts the active site in the protease and thereby traps it in the inhibitory complex with the serpin. The inherent structural flexibility of active antithrombin makes the molecule prone to mutational induced conformational change and inactivation, for instance through conversion to a “latent” form, in which the reactive center loop is inserted without prior cleavage by protease (Fig. 1B),25 or through polymerization (Fig. 1C and 1D).19-21,25 Efforts are being made to develop pharmaceuticals that can prevent inactivation of serpin mutants,26,27 but this endeavor is impeded by an incomplete understanding of the molecular mechanism behind conformational change in serpins. Huntington and coworkers purified and crystallized a heat-generated symmetric antithrombin dimer with a reciprocated β4A-β5A β-hairpin domain swap7 (Fig. 1C). This is a hyper-stable self-terminating dimer (denoted “D1”7) which is unable to participate in further elongation. In fact, another dimer (denoted “D2”7) was observed on native gels at intermediate time points during polymerization and then consumed after prolonged polymerization at pH 5.7.7 We speculate that the D2 dimer is an “open-ended” (not self-terminating) runaway domain-swapped form with a propagated β4A-β5A domain-swap (Fig. 1D), however, the precise structural arrangement of the D2 dimer remains uninvestigated. If this hypothesis is correct and the β4A-β5A domainswap polymerization mechanism is valid for higher order antithrombin polymers also, then the D2 dimer would be an early and critical intermediate structure in the polymerization process, i.e., the first building block in the runaway domain-swapping mechanism. A characterization of the structure and dynamics of a polymerizationcompetent antithrombin dimer is of major importance in the attempt to understand the precise mechanism of antithrombin polymerization and the molecular dynamic properties of a runaway domain-swap dimer in general.

Experimental section Antithrombin dimer purification and characterization The native and active α-glycoform of antithrombin was purified from donor plasma by heparin affinity chromatography followed by anion exchange chromatography.19,28 Inactive latent form of antithrombin was prepared by incubation of 0.2 mg/ml of active antithrombin (20 mM Tris/HCl, pH 7.4) at 45°C for 33 days. Polymerization of antithrombin was induced by incubation of 0.5 mg/ml of the prepared antithrombin (20 mM Tris/HCl, pH 7.4) at elevated temperature (65 and 53 °C) or at pH 5.7 (10 mM Sodium acetate buffer, 37°C) and


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the extent of polymerization was monitored using native gel electrophoresis (4-15% Tris/HCl from Biorad) followed by silver staining. 0.5 mg/ml of the prepared antithrombin (20 mM Tris/HCl, pH 7.4) was heated at 65°C for 15 minutes to produce the antithrombin dimers followed by purification on a Superdex 200 10/300 GL size-exclusion column (GE healthcare). The pooled dimer fractions were concentrated by Microcon Ultracel YM-30 (Millipore) and purity was confirmed by native gel electrophoresis (4-15% Tris/HCl from Biorad) followed by silver staining. The availability of a free solvent-exposed reactive center loop in monomeric native and purified dimer antithrombin was assessed using a non-reducing SDS-PAGE of 0.15 mg/ml monomeric and dimeric antithrombin in the absence and presence of thrombin (BIOFAC A/S, Denmark) in 5-fold molar excess for 0 hours (no incubation time) or 24 hours.

Hydrogen/deuterium-exchange mass spectrometry Thirty pmol of active, latent or dimeric antithrombin (2.2 µM stock solutions) samples were diluted 10-fold in deuterated PBS (pD 7.8, corrected value) to reach a final concentration of 0.22 µM protein in 90% D2O. Following incubation at 37°C for 0.1, 1, 10, 100 and 1000 minutes the labeling reaction was quenched by lowering the pD to 2.5 (through addition of formic acid to a final concentration of 0.65%) and snap-freezing the sample in liquid N2. A fully deuterated control of antithrombin was prepared by diluting 40 pmol antithrombin in 8 M Guanidine-d5 deuteriochloride (Aldrich) dissolved in deuterated PBS. Online proteolysis and LC-MS analysis of both undeuterated and deuterated antithrombin samples was done using a cooled nanoACQUITY UPLC system with HDX technology coupled to a Synapt G2 mass spectrometer (Waters). The samples were injected into a loop wherefrom they were flushed through a 2 × 5 mm column packed with immobilized pepsin (Pierce) and desalted on an ACQUITY UPLC BEH C18 VanGuard Pre-column, (130Å, 1.7 µm, 2.1 × 5 mm, Waters) using a solution of 0.23 % formic acid. The desalting flow was ramped from 60-400 µL/min in 2 minutes and maintained at 400 µL/min for another minute. The peptides were eluted off the precolumn and separated on a ZORBAX C18 analytical column (130Å, 3.5 µm, 1 × 50 mm column, Agilent) using a gradient of solvent A (0.23 % formic acid) and solvent B (acetonitrile + 0.23 % formic acid) from 5%-35% B in 8 minutes and from 35%-90% in 1.4 minutes at a flow of 40 µL/min. The mass spectrometer was operated with a capillary voltage of 3.60 kV, cone voltage of 20 V, ion source block temperature at 100°C with a desolvation gas flow of 800 L/h at 250°C. Peptic peptides from non-deuterated antithrombin were identified using data-independent MS/MS acquisition (Waters MSE) as well as data-dependent MS/MS acquisition of the 3 most intense peptide signals in each MS spectrum. All samples were analyzed in triplicate.

Data analysis



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Raw mass spectral data of undeuterated samples were processed using PLGS 2.5 (Waters) and searched against a custom made database including the antithrombin sequence for identification of peptic peptides using PLGS 2.5 (Waters) or MASCOT (Matrix Science). A total of 89 peptides were identified, covering 79% of the antithrombin sequence. Data from a representative selection of 19 peptides covering most of the antithrombin sequence is shown in this report. Raw peptide mass spectra of deuterated dimer samples were analyzed using Masslynx (Waters) and isotopic distributions showing visually distinct bimodality were fitted to two binomial distributions using HXexpress229 for calculation of the deuterium content of each population. Raw mass spectra of deuterated monomer samples (active and latent, all time points) as well as dimer samples which did not show visually distinct bimodality (some or all time points in certain peptides) were analyzed using DynamX 3.0 (Waters) and the deuterium content was calculated from the average mass of the isotopic distributions. Bimodality in isotopic distributions as a consequence of carry-over 30 was ruled out by control blank injections and the time evolution of the observed bimodality is inconsistent with EX1 kinetics.16,31 The deuterium content measured in the full deuteration control samples is in several cases slightly lower than that measured for regular samples. This is a known phenomenon17 most likely attributed to slightly increased back-exchange during quench conditions in the presence of high concentration of guanidinium hydrochloride. Crystal structure images and models were prepared using The PyMOL Molecular Graphics System, Version 1.5 (Schrödinger, LLC.).

Results Purification of an antithrombin dimer

Antithrombin was purified from citrated plasma and polymerization was induced by either heating (Fig. 2A) or low pH (Fig. S1). Irrespective of the method, we consistently observed distinct reproducible ladders of polymeric antithrombin on native gels. While the balance between monomeric active (the native state), monomeric latent and polymeric species depends on the temperature, pH and incubation time, the relative electrophoretic mobility of dimer and trimer bands are highly reproducible, suggesting similar mechanisms of antithrombin polymerization at elevated temperature and acidic pH. Polymerization of antithrombin using heat is in competition with the process of latency transition.32 This balance is shifted towards polymerization when antithrombin is incubated at 65°C (Fig. 2A) and this temperature was therefore chosen for production of polymeric antithrombin for further studies. Following incubation at 65°C and pH 7.4 for 15 minutes, the dimer fraction was purified by size-exclusion chromatography and the purity of the fractions was investigated using native gels (Fig. 2B). The ability of purified monomeric and dimeric antithrombin to react with thrombin and form a covalent complex was tested to assess the availability of a free


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solvent-exposed reactive center loop and an accessible β-sheet A. While monomeric antithrombin reacted rapidly with thrombin to produce the inhibitory complex, no complex formation was observed with dimeric antithrombin (Fig. 2C). Instead, a new band, migrating slightly above active monomeric antithrombin, was observed. A similar band was observed when the D2 dimer previously produced by Huntington and coworkers was subjected to thrombin, and it was attributed to an antithrombin molecule with a cleaved reactive center loop.7 The presence of two bands after incubation with thrombin indicated an asymmetric configuration of the two antithrombin subunits in the dimer, in which one of the subunits have a cleavable reactive center loop and the other does not. The absence of a thrombin-antithrombin complex, despite the presence of a cleavable reactive center loop in one of the antithrombin subunits, suggests that this subunit already has a 6-stranded β-sheet A, presumably as a consequence of the dimerization process, and therefore cannot accept the protease linked reactive center loop as the sixth strand.

HDX-MS reveals pronounced conformational asymmetry in the purified dimer

The purified dimeric antithrombin was diluted 10-fold into deuterated buffer at 37 °C, pD 7.8, and incubated for 0.1, 1, 10, 100 and 1000 minutes. The isotopic exchange reaction was subsequently quenched by lowering the pD to 2.5 and the temperature to 0°C. The antithrombin samples were cleaved into peptides using porcine pepsin and the deuterium content was measured using mass spectrometry (as exemplified for peptide 63-86 in Fig. 3). Active and latent monomeric antithrombin samples were analyzed in a similar manner as control. Antithrombin unfolded by addition of 8 M guanidinium hydrochloride (GndHCl) was also labeled with deuterium and included as a full (maximum) deuteration control. Hydrogen/deuterium-exchange on homogenous preparations of stable monomeric proteins typically results in mass spectra with bell-shaped isotopic distributions, which can be fitted accurately using single binomial distributions.33,34 This type of exchange behavior is clearly dominant in the case of the monomeric active and monomeric latent preparations of antithrombin (Fig. 3A and 3B). Surprisingly, examination of the antithrombin dimer data revealed bimodality of the observed isotopic envelopes (Fig. 3C and Fig. S2). If a polypeptide chain is present in two different conformations in the sample, with sufficiently different structural protection against deuterium uptake, it will lead to bimodal isotope distributions. We know the dimer preparation is free of any monomeric protein, and that only one dimer band is observed on native gels (Fig. 2B). If more than one type of dimer was present in the preparation we would expect to also see more than one dimer band on the native gel. The bimodality in isotope distributions must therefore be caused by conformational asymmetry between the two antithrombin subunits that constitute the dimer. The observations of bimodality for nearly all



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antithrombin peptides indicate that the duality in structural flexibility of the two subunits in the dimer is widespread (Fig. S2).

One subunit of the dimer has monomer-like stability, while the other is extremely dynamic

HDX-MS data from different conformational states of a protein are typically compared on the basis of the deuterium content in a given peptide as a function of labeling time (Fig. 3D). The difference in deuterium uptake between conformational states reflects primarily differences in protein structure and dynamics, i.e. the number and stability of protecting hydrogen bonds involving back-bone amide hydrogens. The deuterium content is typically calculated from the average mass of the deuterated isotope distribution, but in the case of bimodal isotope distributions each isotopic envelope pertains to a distinct population that should be analyzed separately. To obtain population specific data, we have fitted two binomial distributions (Fig. 3C) for the calculation of their deuterium contents (Fig. 3D) using the HXexpress2 software.29 This allows for a direct comparison of the deuterium content of each subunit in the dimer to the deuterium content in the monomeric active and latent conformations. In the case of peptide 63-86, one subunit of the dimer (corresponding to the low-mass population) is rather protected against deuterium uptake in this region, while the other (corresponding to the high-mass population) is fully exchanged already after 6 seconds (Fig. 3D, compare “high and “low”). Considering the location of this peptide (C-terminal half of helix A, β6B and the N-terminal half of helix B, see Fig. 3E), in the hydrophobic core of the native antithrombin structure it is remarkable to find a population of this peptide in the dimer with no detectable protection against deuterium uptake. Such exchange behavior is typically limited to solvent exposed loops, very dynamic secondary structures or intrinsically disordered regions. The protected dimer population has a deuterium uptake profile which, apart from slightly lower deuterium content, resembles the profile of the latent form (Fig. 3D). A similar analysis to that described for peptide 63-86 above was conducted for a representative set of peptides covering most of the antithrombin sequence (Fig. S2). Distinct bimodality was observed, at one or more time points in all but 3 peptides (38-51, 106-113 and 114-121) from the dimer sample (Fig. S2). In certain dimer HDX mass spectra the bimodal isotope distributions were only observed at some of the earlier time points. The deuterium uptake at later time points, where bimodality was not observed, was calculated based on the average mass of the isotopic distribution and indicated by the blue “dimer (average)” line (Fig. S2). In general, doublebinomial fits to the bimodal isotopic distributions revealed a high-mass population, with deuterium contents close to the full deuteration levels, and a low-mass population, with deuterium contents in the range of the monomeric active and latent forms (Fig. S2). This strongly suggests that the dimer consists of one subunit with a


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folded monomer-like deuterium uptake (“stable” subunit) and another subunit with surprisingly little structural protection against deuterium uptake (“unstable” subunit).

Only β-sheet B is protected against isotopic exchange in the “unstable” subunit of the dimer

Although the high-mass populations in the dimer HDX mass spectra were similar to the full-deuteration level in many peptides, a slight protection against exchange was observed in some peptides at the earliest time points. Peptides with this “phenotype” are mapped on the structure of the subunit with an open β-sheet A in the proposed model of the dimer (Fig. 4A and S3). These peptides represent regions of the “unstable” subunit in the dimer, with detectable (although modest) structural protection against isotopic exchange. Interestingly, these peptides map predominantly to β-sheet B (blue β-sheet in Fig. 4A and S3). β-sheet B is generally the most protected region against isotopic exchange in monomeric serpins14-18,35 and although the “unstable” subunit of the dimer possess rather little structural protection against isotopic exchange it is still β-sheet B that is the most stable region in that subunit.

The “stable” subunit of the dimer has an active-like upper half and a latent-like lower half

Conversion of antithrombin from the active to the latent form implies dislocation of β-strand 1C from βsheet C, passage of the reactive center loop through the “gate region” and insertion of the reactive center loop as β-strand 4A in β-sheet A (Fig. 1B). This transition results in an increased stability of the lower half and a decreased stability of the upper half of antithrombin, as manifested by accordingly lower and higher rates of deuterium uptake (Fig. S2). The increased stability of the lower half is likely imparted by expansion of β-sheet A upon reactive center loop insertion, whereas the reduced stability of the upper half is likely imparted by the detachment of β-strand 1C and a strained conformation of the reactive center loop down the left side of the molecule. We investigated the deuterium uptake profiles of the low-mass populations and compared them to the deuterium uptake profiles of the active and latent monomeric proteins. Peptides, for which the low-mass population show an “active-like” deuterium uptake profile mapped surprisingly well to the upper half of antithrombin (Fig. 4B) whereas peptides which show a “latent-like” deuterium uptake profile map equally well to the lower half of antithrombin (Fig. 4C). These data suggest that the “stable” subunit of the dimer has an upper-half with active-like stability and a lower half with latent-like stability. Importantly, the deuterium uptake profiles of the low-mass populations are generally closest to the monomeric form (active or latent) with the



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lowest deuterium uptake. This means that the overall deuterium uptake of the “stable” subunit in the dimer is lower than that of both active and latent monomeric form.

Discussion Through HDX-MS investigation of an “open-ended”, and thus polymerization-competent, antithrombin dimer in solution we provide strong experimental support for the β4A-β5A domain swap mechanism in antithrombin polymerization. This runaway domain swap dimer has two important features in common with the elusive D2 dimer produced by Huntington and coworkers7: It is consumed after prolonged incubation at 53°C, pH 7.4 (Fig. 2a and S1) and two antithrombin bands are observed on native gels following incubation with thrombin (Fig. 2C). Furthermore, the observation of bimodality in the isotopic distributions following hydrogen/deuteriumexchange of the dimer clearly argues for an asymmetric configuration of the two antithrombin subunits in the dimer. We can thus exclude the possibility that our dimer has a self-terminating symmetric intermolecular linkage, i.e., with a reciprocated domain swap, such as the D1-type.7 Fitting of the bimodal isotope distributions to two binomial distributions allowed us to decode the deuterium content of each subunit of the dimer individually. This analysis identified one subunit as being highly “unstable”, potentially intrinsically disordered, with only β-sheet B showing detectable protection against isotopic exchange, and another “stable” subunit as having an active-like upper half and a latent-like lower half. The identification of a rather unstable subunit in our purified dimer was unexpected. The presence of such an unstable subunit is inconsistent with both the active/latent reactive center loop-β1C-linked heterodimer mechanism36 as well as the loop-sheet mechanism37,38, as neither of these dimerization mechanisms accounts for such an unstable subunit. However, the combined hydrogen/deuterium-exchange profiles of the two subunits in our dimer are in excellent agreement with the structure of a runaway domain swap and thus polymerogenic dimer that has a propagated β4A-β5A domain swap in contrast to the reciprocated β4A-β5A domain swap observed in crystallized self-terminated antithrombin dimer7 (Fig. 1D and S3). One subunit of the runaway domain swap dimer has the full sequence from helix I and to β1C, including β5A and the reactive center loop, pulled out of the structure (Fig. 1D). A large number of native contacts will be disrupted in this subunit, which expectedly would result in significant destabilization of that subunit. The other subunit has a six-stranded β-sheet A, which agrees with the observed latent-like stability of the lower half of the “stable” subunit. The active-like stability of the upper half can also be reconciled with the fact that this subunit in the runaway domain swap


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dimer model does not have a true latent-like conformation of the reactive center loop, where the N-terminal half of the reactive center loop is positioned down the left side of the molecule. Serpin polymerization was for long assumed to occur through insertion of the reactive center loop from one monomer into β-sheet A of another monomer (loop-sheet polymerization), partly based on the ability to block polymerization using reactive center loop mimicking peptides.37,38 The absence of crystal structure data to support this hypothesis, and the presence of crystal structure data showing other types of linkages, has strongly questioned the relevance of the loop-sheet polymerization mechanism. A crystal structure of heterodimeric antithrombin has been solved in which the reactive center loop of an active conformation antithrombin molecule was inserted as β-strand 1C into a latent conformation antithrombin molecule.36 This heterodimer is, however, not capable of further polymerization.39 There has thus been much debate over the exact mechanism of serpin polymerization in the scientific community, and there could easily be more than one involved, depending on the particular serpin in question. Interestingly, polymerization of the serpin α1-antitrypsin in 0.75M GndHCl could be prevented by disulfide-trapping of β5A to β6A, arguing strongly for a similar β4A-β5A domain swap polymerization mechanism in this serpin upon GndHCl treatment in vitro.7 Subsequent investigations, aided by a specific monoclonal antibody with reactivity towards hepatocellular inclusion of polymeric α1-antitrypsin40, as well as crystal structure analysis41 and disulfide trapping experiments42, has indicated that another C-terminal domain swap (including β4B-β5B) is probably the favored mechanism of pathological polymerization of α1antitrypsin in vivo. In contrast to the observed heterogenous polymer ladders of α1-antitrypsin on native gels upon heat or GndHCl treatment41, antithrombin produces rather discrete and consistent polymer ladders on native gels under a range of polymerization promoting conditions, including heat7, low pH7, and mutation.19,21,25 While α1-antitrypsin appears to facilitate different mechanisms of polymerization in vitro dependent on how polymerization is promoted, there is no clear indication of such ambiguity in the mechanism of antithrombin polymerization. The β4A-β5A domain swap thus remains a plausible model for the physiologically relevant polymerization mechanism of antithrombin. The observation of bimodality in the isotopic distributions of our antithrombin dimer also excludes the possibility of a dimeric version of the self-terminating β4B-β5B domainswapped α1-antitrypsin trimer.41 The β4B-β5B domain-swap model for propagated polymers imply a subunit where the β4B-β5B hairpin is pulled out, but at the same time the reactive center loop is inserted as β-strand 4A in β-sheet A.41,42 What the overall stability of such a subunit would be is difficult to predict, but instability would logically be expected in the upper half around β-sheet B where the β4B-β5B hairpin is missing. This does not agree with the observation that β-sheet B is the only area where protection against isotopic exchange is detected altogether in the “unstable” subunit of our asymmetric antithrombin dimer. Furthermore, the β4B-β5B domain swap mechanism would also require breakage of the disulfide bond between C247 and C430 in the antithrombin



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monomer. Based on these arguments we conclude that of the various polymerization mechanisms evaluated here, the β4A-β5A domain swap is clearly the mechanism best supported by the data acquired on our dimer. While the β4B-β5B domain-swap mechanism may be dominant in the polymerization of α1-antitrypsin there is little evidence that this mechanism is also dominant in the disease-causing polymerization of antithrombin mutants.1921

Based on the crystal structure analysis performed by Huntington and coworkers,7 together with the in-solution

HDX-MS characterization reported here, we argue that the β4A-β5A domain swap is the most likely mechanism of antithrombin polymerization. The present data provides insight to the structural dynamics of a runaway domain swap dimer but the precise process of dimer formation remains to be investigated. We have previously detected unexpected transient unfolding in plasminogen activator inhibitor 1 (PAI-1), a serpin which spontaneously changes conformation to the inactive latent form at physiological conditions.16 Activated precursors with destabilized or partially unfolded structures must also be involved in the process of antithrombin polymerization. Similarly, locally or globally unfolded states have been implicated in the formation of other domain swapped globular proteins. 2As polymerization is a spontaneous process it must be driven by a decrease in free energy. At a first glance, it is thus surprising that one subunit has undergone substantial unfolding as this would usually entail an increase in free energy. It is important to note, however, that the “stable” subunit of the runaway domain swap dimer actually has a lower overall deuterium uptake compared to both the active and latent monomeric forms. This suggests that the increase in free energy contribution from the “unstable” subunit is compensated for by a decrease in free energy contribution from the “stable” subunit.

Conclusions Characterization of polymerogenic protein material in solution is of paramount importance for future development of effective treatment strategies against conformational diseases in which pathogenic protein aggregation or polymerization plays a role. Only with information about the exact polymerization mechanism do we have a proper basis for designing future pharmaceuticals. However, detailed biophysical characterization of polymerogenic protein material in solution is extremely challenging due to its heterogenous nature and preponderance for further elongation when concentrated. Here we report a promising example of the enabling utility HDX-MS offers in this respect, with a detailed characterization of a polymerogenic antithrombin dimer Our data reveals bimodal isotope distributions in the HDX-MS spectra of the purified dimer indicative of an asymmetric configuration of the subunits. Detailed subunit-specific analysis of these data reveals an excellent agreement with the runaway β4A-β5A domain swap model for antithrombin polymerization.


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Acknowledgements: We thank Peter Andreasen and Jan Jensen (Aarhus University) for fruitful discussions and Alice Østergaard (Aalborg University) for excellent technical assistance. This project was funded by a grant from The Danish Council for Independent Research | Natural Science (4002-00405B) and a grant from the Danish Research Council for Technical Sciences and Production (09-072885). MBT and TJDJ were additionally supported by grants from the Carlsberg Foundation (2012_01_0369 to MBT; 2012_01_0332 to TJDJ) and MBT was supported by a grant from the Lundbeck Foundation (R18-A11217).

Supporting Information: Supplementary Figure S1, S2 and S3 are associated with this manuscript. Figure S1 shows antithrombin polymerization data at 53 °C, pH 7.4 and at 37 °C, pH 5.7. Figure S2 shows hydrogen/deuterium-exchange data for a representative set of antithrombin peptides, Figure S3 shows a phenotypic characterization and mapping of dimer peptide deuterium uptake profiles on the hypothetical “open” version of the β5A-β4A domain swap dimer. Figure S4 shows hydrogen/deuterium-exchange data from blank injections following a real sample injection.

References (1) Bennett, M. J.; Sawaya, M. R.; Eisenberg, D. Structure 2006, 14, 811-824. (2) Zerovnik, E.; Stoka, V.; Mirtic, A.; Guncar, G.; Grdadolnik, J.; Staniforth, R. A.; Turk, D.; Turk, V. The FEBS journal 2011, 278, 2263-2282. (3) Guo, Z.; Eisenberg, D. Proc Natl Acad Sci U S A 2006, 103, 8042-8047. (4) Liu, Y.; Eisenberg, D. Protein Science 2002, 11, 1285-1299. (5) Shameer, K.; Shingate, P. N.; Manjunath, S. C. P.; Karthika, M.; Pugalenthi, G.; Sowdhamini, R. DatabaseOxford 2011. (6) Huntington, J. A. Trends Biochem Sci 2006, 31, 427-435. (7) Yamasaki, M.; Li, W.; Johnson, D. J.; Huntington, J. A. Nature 2008, 455, 1255-1258. (8) Engen, J. R.; Wales, T. E. Annu Rev Anal Chem 2015, 8, 127-148. (9) Wang, G. B.; Abzalimov, R. R.; Bobst, C. E.; Kaltashov, I. A. Proceedings of the National Academy of Sciences of the United States of America 2013, 110, 20087-20092. (10) Paslawski, W.; Mysling, S.; Thomsen, K.; Jorgensen, T. J. D.; Otzen, D. E. Angew Chem Int Edit 2014, 53, 7560-7563. (11) Mysling, S.; Betzer, C.; Jensen, P. H.; Jorgensen, T. J. D. Biochemistry 2013, 52, 9097-9103.



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Figure legends:

Fig. 1. Conformations of antithrombin. A) Crystal structure of antithrombin in the active conformation (pdb ID: 2ANT43). β-sheet A is colored red and reactive center loop residues yellow. B) Crystal structure of antithrombin in the inactive “latent” conformation (pdb ID: 1ATH44). The dashed yellow line represents missing reactive center loop residues in the 1ATH44 crystal structure. C) Crystal structure of the domain-swap selfterminated antithrombin dimer (D1) (pdb ID: 2ZNH7). D) A proposed model of an “open-ended” and polymerization-competent antithrombin dimer (D2) with a propagated runaway domain swap. The rendition of the subunits in this model is made using The PyMOL Molecular Graphics System to highlight, or make invisible, specific parts of the 2ZNH structure7 and amino acid sequences connecting the two subunits are sketched as broken lines. Each subunit has the full sequence from helix I and to β1C, including β5A and the reactive center loop, pulled out of the structure. β5A together with the N-terminal half of the reactive center loop from one subunit forms a β-hairpin (red-yellow β-hairpin), which is inserted into β-sheet A of another subunit (green). Thereby this hairpin becomes β-strand 5A and 4A of the subunit it is inserted into. This insertion completes β-sheet A as an antiparallel 6-stranded β-sheet, similar to that observed in the inactive latent form B). The subunit with β5A pulled out thereby has an open β-sheet A, which can accept a β-hairpin from another antithrombin molecule. The precise structure, or ensemble of structures, this model of a polymerizationcompetent antithrombin dimer would facilitate is obviously unknown.

Fig. 2. Polymerization, purification and characterization of dimeric antithrombin. A) Native gel electrophoresis of antithrombin following incubation at 65 and 53 °C at pH 7.4 for the indicated times. B) Native gel electrophoresis of dimeric antithrombin purified by size-exclusion chromatography. C) Non-reducing SDSPAGE of monomeric and dimeric antithrombin (AT) in the absence and presence of thrombin (T) for 0 and 24 hours as indicated. The thrombin-antithrombin complex is denoted TAT and the reactive center loop-cleaved form of antithrombin is denoted c-AT.

Fig. 3. Hydrogen/deuterium-exchange data of antithrombin peptide 63-86. Mass spectra of the (M+3H)3+ charge state of peptide 63-86 following incubation in 90% D2O for the indicated times are shown for A) monomeric active, B) monomeric latent and C) dimeric antithrombin. Single binomial distributions are fitted to


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the data of monomeric active (red line) and monomeric latent (green line). Two binomial distributions are fitted to the bimodal isotope distributions observed in the dimer data (blue lines, circle and square markers). D) Deuterium uptake is plotted as a function of time for monomeric active, monomeric latent and each of the two mass populations in the dimer data (“low” and “high”). The full deuteration level is indicated by the broken black line. Error bars indicate the standard deviation between independent replicates (n=3). E) Structure of antithrombin in the active conformation (pdb ID: 2ANT43) with the location of peptide 63-86 highlighted in blue.

Fig. 4. Phenotypic characterization and mapping of dimer peptide deuterium uptake profiles. The high and low-mass population in the dimer HDX mass spectra were characterized based on their deuterium uptake profiles and mapped accordingly to the two subunits in the proposed dimer model (Fig. 1D). A) All peptides for which the high-mass populations show protection against isotopic exchange are colored blue on the structure of the subunit with an open β-sheet A in the dimer model. All peptides for which the low-mass population show active-like and latent-like deuterium uptake profile are colored orange in B) and purple in C), respectively, on the structure of the subunit with a competed 6-stranded β-sheet A in the dimer model. Peptides associated with each phenotypic group are listed below the structures in A), B) and C) for reference. The β-strands of β-sheet A are colored red in A). Only β-strand 5A is colored red in B) and C) and the reactive center loop/β4A is colored yellow. A concatenated depiction of the phenotypic mapping of the data on the proposed polymerizationcompetent runaway domain swap dimer is provided in Fig. S3. D), E) and F) shows example deuterium uptake plots of one of the peptides colored on the structures in A), B) and C), respectively. Error bars indicate the standard deviation between independent replicates (n=3).



Fig. 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

RCL

B

D β5A- β4A hairpin

Active form

Latent form

C

Self-terminated domain swapped dimer (D1)

Model of ”open” polymerogenic dimer (D2)


Page 18 of 22

Page 19 of 22


B 65 °C pH 7.4 5 10 30 60

53 °C pH 7.4 0 5 10 30 60 480

Dimer fraction

1 2 A 3 4 5 min. 6 7 8 Trimer Dimer 9 10 Monomer 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

AT polymers

Fig. 2 C

Monomer

Dimer

AT + + + + + + T + - + + - + + Inc. 0 24 M 0 24 M

TAT c-AT AT T



Page 20 of 22

Fig. 3 B Monomer Active

Monomer Latent

Undeuterated

D

C

Mono - Active Mono - Latent Dimer (low) Dimer (high)

Dimer

Undeuterated

0.1 min

0.1 min

1 min

12.0

Undeuterated Deuterium content (Da)

A

0.1 min

1 min

1 min

10.0 8.0 6.0 4.0 2.0 0.0

10 min

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

10 min

10 min

0.1

1.0

10.0

100.0

1000.0

Time (min)

E

888

100 min

100 min

100 min

1000 min

1000 min

1000 min

Full D

Full D

Full D

893

888

893

888

893

m/z


Peptide 63-86 YQHLADSKNDNDNIFLSPLSISTA (M+3H)3+ = 888.436 m/z

Page 21 of 22


Fig. 4

Deuterium content (Da)

1 2 3 A 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 D 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

B

C

E

412-422

7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

0.1

1.0

51-58, 63-86, 87-92,164-175, 216-225, 330-340, 363-373, 388-402

226-239 240-260 282306 316-323 403-411

269-281, 282-306, 316-323, 403-411, 412-422

10.0 100.0 1000.0

F

226-239 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

216-225

7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.1

1.0

10.0 100.0 1000.0

Time (min)

Mono - Active Dimer (low) Dimer (average)

0.0

0.1

1.0

10.0 100.0 1000.0

Mono - Latent Dimer (high) Full D

7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

0.1

1.0

10.0

100.0

1000.0



Table of content graphic (TOC)

H2O D2O

D-content

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

m/z


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An Asymmetric Runaway Domain Swap Antithrombin Dimer as a Key

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