1 Introduction

The last decade has witnessed an extension to the protein structure-function paradigm, with the progressive understanding of the functional importance of intrinsically disordered proteins (IDPs) or regions (IDRs). These proteins or protein regions lack ordered secondary and tertiary structure under physiological conditions and exist in solution as dynamic and heterogeneous conformational ensembles [1–3]. Approximately 40% of the human proteins are predicted to contain at least one disordered segment of at least 30 amino acids, with as many as 25% of them likely to be disordered from start to end [4]. Predictions on representative genomes from the three kingdoms of life (i.e., bacteria, archaea, and eukaryotes) confirm the ubiquitous character of structural disorder, in spite of significant differences in its relative amount in the three domains [5]. The extent of protein structural disorder tends to increase with biological complexity. This trend could be related to the typical involvement of IDPs in signaling and regulation [6–8].

The structural plasticity of IDPs allows for recognition and binding of multiple partners, resulting in pleiotropic roles of these proteins. Many cases have been described in the literature, in which IDPs acquire ordered conformations upon binding to partners or ligands. Folding coupled to binding can pertain either to specific segments or to the whole protein [9]. Complete folding can lead to well-structured complexes that can be analyzed by conventional techniques such as X-ray crystallography [10–12]. However, increasing evidence shows that many IDPs retain a high degree of structural disorder even in the bound state. These “fuzzy” complexes [13] are stabilized by short, ordered recognition elements, referred to as molecular recognition elements (MoREs), and a large number of highly unstable contacts, leading to a cloud of interconverting conformations around a structured core [9, 10, 13–16]. This staccato-type of interactions is much more difficult to characterize than stable interactions of folded complexes. Yet, it is thought to be relevant for biological function. Fuzzy regions within complexes can harbor regulatory post-translational modification sites, or can mediate interactions with additional partners. They can even, directly or indirectly, interfere with recognition elements, promoting or inhibiting binding. In addition, fuzziness provides a way to reduce the entropic penalty, thereby affording enhanced affinity [15, 17].

Hence, description of the conformational ensembles characterizing fuzzy IDP complexes is essential to the understanding of the molecular processes by which IDPs establish their functional networks. However, the highly heterogeneous nature of IDPs and the fuzziness that is often observed in their bound state makes their structural characterization very challenging. Such investigation demands the combined application of various biophysical methods capable of capturing conformational heterogeneity and identifying metastable states.

In this regard, native mass spectrometry based on nano-ESI sources has emerged as a powerful approach, allowing detection of coexisting conformers with distinct global compactness [18, 19]. The average charge state of each component yields an estimate of SASA for the structure in solution, at the moment of transfer to the gas phase [20, 21]. Hyphenation with IM measurements adds a further dimension to species separation and offers estimates of the CCS for each detected structure in the gas phase [22–25]. These techniques conjugate the exceptional analytical power of mass spectrometry with structural description and, therefore, are particularly well suited to the challenges posed by conformational studies on IDPs [23, 26, 27]. The development of atomic-resolution models requires, however, complementation of experimental data by computational simulations. Theoretical approaches have been improved to effectively model disordered conformational ensembles based on structural information derived from native mass spectrometry and other biophysical analyses [28–30].

We herein combine ESI-MS, ESI-IM-MS and computational methods to describe the NTAIL–PXD complex from measles virus (MeV). While NTAIL is an intrinsically disordered domain of 125 residues [31–34], PXD is folded into an ordered three-helix bundle [35]. Interaction between NTAIL and PXD is crucial for MeV replication, as it allows recruitment of the viral RNA-dependent RNA polymerase onto the nucleocapsid template [36, 37]. Upon binding to PXD, NTAIL undergoes induced folding localized in an 18-residue α-helical region. This α-helical molecular recognition element (α-MoRE) [33, 34, 38, 39] associates to PXD forming a four-helix bundle [35, 40]. The NTAIL–PXD interaction mostly relies on hydrophobic contacts [33, 40, 41] and is characterized by a KD in the submicromolar range, as consistently indicated by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) experiments [38, 42, 43]. Previous studies on the NTAIL–PXD complex by small-angle X-ray scattering (SAXS) and NMR showed that the majority of NTAIL remains disordered in the bound state, thus supporting the fuzzy and heterogeneous nature of this complex [33, 38, 44]. Although hydrophobic complexes could be expected to dissociate in the gas phase, detection of such complexes by MS has been reported in some instances [45–47]. The results of this study show that the MeV NTAIL–PXD complex is amenable to MS analysis and that distinct conformational states can be detected. Experimental data are used to validate structural modeling. The results highlight the conformational freedom of the complex and the hydrophobic nature of its interface.

2 Materials and Methods

2.1 Expression and Purification of MeV NTAIL and PXD Proteins

The expression constructs in the pDEST14 vector, allowing expression of N-terminally (NTAIL) or C-terminally (PXD) hexahistidine tagged forms of the MeV proteins under the control of the T7 promoter, have already been described [38]. Expression and purification of MeV NTAIL and PXD were carried out as described [38], except that the final gel filtration step was carried out using 10 mM ammonium acetate pH 6.5 as elution buffer.

2.2 Mass Spectrometry

Nano-ESI-MS analyses were performed on a hybrid quadrupole time of flight (Q-TOF) mass spectrometer (QSTAR Elite; AB Sciex, Framingham, MA, USA) equipped with a nano-electrospray ionization sample source. Metal-coated borosilicate capillaries (Proxeon, Odense, Denmark), with medium-length emitter tips of 1-μm internal diameter, were used to infuse the samples. The instrument was calibrated by the standard Renin-inhibitor solution (AB Sciex, Framingham, MA, USA) on the intact molecular ion [M +2H]2+ (879.97 Da) and its fragment [M + H]1+ (110.07 Da). Data were acquired with ion spray voltage 1200 V and declustering potential 80 V, and were averaged over 2-min acquisitions. The interface was kept at room temperature (interface heater off). Pure preparations of the NTAIL and PXD protein samples in 10 mM ammonium acetate pH 6.5 were stored at –20 °C and diluted to the indicated final concentrations in the same buffer before analyses. Samples were incubated at room temperature for 10 minutes before measurements.

Mass spectra from Figure 1a and Figure 2a were transformed from x = m/z to x = z as abscissa axis, and data points were fitted by Gaussian functions. The analysis was performed by the software Origin7 (Originlab, Northampton, MA, USA) [48, 49].

Figure 1
figure 1

Nano-ESI-MS spectra of MeV NTAIL (AB Sciex QSTAR Elite instrument). (a) 10 μM MeV NTAIL in 10 mM ammonium acetate, pH 6.5; (b) Gaussian fitting of the spectrum reported in panel (a); (c) 10 μM MeV NTAIL in 10 mM ammonium acetate, 1% formic acid (pH ~3); (d) 10 μM MeV NTAIL in 10 mM ammonium acetate, 50% acetonitrile, 1% formic acid (pH ~3)

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Figure 2
figure 2

Nano-ESI-MS spectra of MeV PXD. (AB Sciex QSTAR Elite instrument). (a) 10 μM MeV PXD in 10 mM ammonium acetate, pH 6.5; (b) Gaussian fitting of the spectrum reported in panel (a); (c) 10 μM MeV PXD in 10 mM ammonium acetate, 1% formic acid pH ~3); (d) 10 μM MeV PXD in 10 mM ammonium acetate, 50% acetonitrile, 1% formic acid (pH ~3)

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Nano-ESI-IM-MS analyses were performed on a Synapt G2 HDMS instrument (Waters, Milford, MA, USA) by direct infusion, under nondenaturing conditions. The samples were injected at the indicated protein concentrations in 10 mM ammonium acetate pH 6.5 at room temperature. Instrument parameters were carefully optimized to minimize disruption of noncovalent interactions. The following instrumental settings were applied: spray voltage 1400 V, sampling cone voltage 50 V, trap collision energy 4 V, transfer collision energy 0 V, trap DC bias 45 V, backing pressure 2.98 mbar, trap pressure 5 × 10–2 mbar, IMS pressure 3.09 mbar. Instrument calibration for CCS measurements was performed using concanavalin A, albumin, β-lactoglobulin, and cytochrome c as standards while referring to the absolute CCS values determined on a modified Synapt G1 as described by Bush et al. [50].

2.3 Structural Modeling

Homology modeling was performed to build an initial structure of the NTAIL–PXD complex for molecular simulations. The folded core was modeled on the crystallographic structure of the chimeric protein (PDB 1T6O) [40]. The missing regions (residues 401-485 and 505-525 of NTAIL) were split into fragments and used to screen the structure database searching for suitable templates, following the approach by Bowie et al. [51, 52]. Ten templates (Supplementary Figure S1), showing from a minimum of 28% sequence identity over 41 residues to a maximum of 56% over 17 residues to the target, were identified, reaching a cumulative 96.5% sequence coverage of the NTAIL disordered arms. These homology-derived restraints were used to generate 2000 models of the NTAIL–PXD complex by the MODELLER 9v9 package [53]. Most (94.5%) of the resulting structures have favorable backbone conformations [54] for more than 80% of the residues, according to the Procheck program [55]. The lowest-DOPE (discrete optimized protein energy) score [56] model was selected as the starting structure for the following molecular simulations.

Replica-exchange Monte Carlo (REMC) simulations were carried out by the PROFASI package (PROtein Folding and Aggregation SImulator) [57–59], with eight replicas performed at distinct temperatures between 298 and 348 K. The energy distributions at these temperatures (Supplementary Figure S2) show optimal overlap between consecutive temperatures of the explored range (Supplementary Figure S2). Such an observation is relevant in order to guarantee a sufficient number of replica exchanges between neighboring replicas, thus allowing an efficient sampling of the conformational space of the protein [60]. Dihedral and distance restraints [61] were applied to the regions represented in the crystallographic structure [40]. A total of 5.0 × 106 cycles of simulations were performed, according to a previously described protocol [28]. Models obtained at 298 K were extracted, constituting an ensemble of 2831 conformations.

The chemical shifts (CSs) of the NTAIL moiety for each conformation of the ensemble were calculated using the SHIFTX package [62] (Supplementary Figure S3). The values of solvent accessible surface area (SASA) were calculated by the tools implemented in GROMACS 4.5.5 [63]. The structures showing the best correlation with either experimental CSs or experimental SASA were identified. The circular dichroism (CD) spectra for each conformation were calculated by the DichroCalc webserver [64], using the parameters derived from both ab initio [65] and semi-empirical calculations [66]. All the figures for structure visualization were drawn using PyMOL (Molecular Graphics System, ver. 1.3, Schrödinger LLC, New York , NY, USA).

3 Results and Discussion

3.1 NTAIL

The MeV NTAIL protein was analyzed by nano-ESI-MS under non-denaturing conditions (Figure 1a). The spectrum shows a very broad, bimodal charge-state distribution (CSD), indicating structural heterogeneity consistent with the intrinsically disordered nature of this protein. Nonetheless, a compact conformation can be identified in the high m/z region of the spectrum, centered on the 9+ charge state. Such an extent of ionization approaches the behavior of normally folded proteins of the same size. The rest of the spectrum is characterized by a broad peak envelope that spans the region between the 10+ and the 24+ ions, with main charge state 16+. This component corresponds to disordered conformations, characterized by low compactness. Deconvolution by Gaussian fitting is shown in Figure 1b. The results indicate that, in addition to the compact and disordered conformations, a third component of intermediate compactness can be identified. Although the relative amounts of the distinct components depend somewhat on the type of instrument and the parameter settings (as is generally true), the intermediate species was detected with good reproducibility under the conditions described here.

The compact and the intermediate conformations are depleted under acidic conditions (Figure 1c) and disappear upon further addition of 50% acetonitrile (Figure 1d), wheras the disordered component slightly shifts to higher charge states. This response to denaturing conditions suggests that the NTAIL protein populates collapsed and partially folded states that can be destabilized by acids and organic solvents. The isotopically averaged molecular weight by mass deconvolution (data not shown) yields a value of 14,630.94 ± 0.17 Da, in close agreement with the value calculated on the basis of the amino acid sequence, including the initial methionine (14631.7 Da).

3.2 PXD

Figure 2 shows the nano-ESI-MS spectra of PXD preparations. Although PXD folds into a helical domain, it displays a bimodal CSD under nondenaturing conditions, with main charge states 6+ and 8+ (Figure 2a). Gaussian fitting of the two components is reported in Figure 2b. This result suggests that the three-helix bundle of the isolated PXD fragment tends to open to a less compact structure, at least under the here employed conditions. The 8+ component does not represent the fully denatured state, since it converts progressively into a more highly charged component (9+) by the addition of formic acid and acetonitrile (Figure 3c and d). The experimental mass (6554.28 ± 0.04 Da) corresponds to the value calculated from the amino acid sequence of the protein without the initial methionine (6555.66 Da). The conformational heterogeneity adopted by MeV PXD is in agreement with previous biophysical and structural studies on the homologous phosphoprotein X domains from members of the closely related Rubulavirus genus, indicating that these domains span a structural continuum, ranging from compact to largely disordered states in solution [67, 68].

Figure 3
figure 3

Nano-ESI-MS spectra of MeV NTAIL and PXD mixtures. (AB Sciex QSTAR Elite instrument). Equimolar mixtures in 10 mM ammonium acetate, pH 6.5; (a) 10 μM of each protein; (b) 20 μM of each protein. Squares, PXD; circles, NTAIL; stars, complex

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3.3 NTAIL–PXD

The interaction between NTAIL and PXD was investigated by mixing equimolar amounts of the two proteins and infusing the sample after 10-min incubation at room temperature without agitation. The results obtained with concentrations of either 10 or 20 μM of both proteins are shown in Figure 3. The spectra are dominated by the peaks of the free proteins, but signals specific to the NTAIL–PXD complex become evident at the higher protein concentration. The measured mass of the complex (21,187.4 ± 0.61 Da) corresponds closely to the sum of the theoretical values expected for NTAIL with the initial methionine and PXD without the initial methionine (21,187.46 Da), indicating a 1:1 stoichiometry.

The complex disappears upon applying denaturing solvent conditions or high declustering potentials (data not shown). These results show that the MeV NTAIL–PXD complex, which is stabilized in solution by hydrophobic interactions, is preserved, at least partially, during the electrospray process and gas-phase ion separation, allowing detection by ESI-MS in a concentration-dependent manner. The expected fraction of bound protein at the concentrations shown in Figure 3b, based on solution affinity data [38, 42, 43], is 93%. Although relative amounts of different species cannot be quantified by ESI-MS, and although the buffer conditions are not identical to previous solution studies, these results suggest that significant dissociation of the complex occurs under electrospray conditions.

The CSD of the complex-specific peaks is quite broad and, itself bimodal, suggesting coexistence of at least two distinct conformational states of the bound species. This conformational heterogeneity likely reflects the persistence of a conspicuous degree of disorder in the complex [33, 34, 38]. The high-charge component of the complex (18+) would then correspond to an “open” conformation, in which the disordered arms of NTAIL, upstream and downstream to the α-MoRE, fluctuate maintaining high solvent accessibility. The low-charge component (11+), instead, likely represents a compact or “closed” conformation of the complex, in which the NTAIL arms collapse onto the surface of the folded partner, as suggested by the low extent of ionization, which approaches the expected value for folded globular proteins of the same mass (9.46+) [20]. The average SASA value of the “open” and “closed” states can be calculated from the empiric relation with the average charge state [20], resulting in estimated values of 16,100 Å2 and 9500 Å2, respectively.

It is worth noting that this dual nature of the NTAIL–PXD complex did not emerge from previous experimental studies [37]. Thus, the question arises as to whether this result reflects a real conformational heterogeneity in solution, captured only by the exquisite sensitivity of native MS, or an altered conformational ensemble induced by the electrospray conditions. To this regard, it should be noted that CSDs generally reflect structural compactness and conformational heterogeneity in solution, before or during the transfer to the gas phase. Thus, hypothetical conformational rearrangements leading to the low-charge component in the CSDs should take place at the level of ESI droplets. The fact that CSDs of IDPs detected by nano-ESI-MS and their response to solvent conditions are protein specific, as for instance, indicated by opposite pH dependence and distinct effects of organic solvents, [20, 69–72] suggests that they reflect real conformational properties rather than artifacts induced by the experimental conditions. However, it cannot be ruled out that poorer kinetic trapping of disordered conformations, compared with folded structures, exposes IDPs to more significant conformational changes inside ESI droplets, affecting in turn CSDs. Even more relevant structural rearrangements could be expected to take place in the gas phase, after electrospray and downstream to protonation reactions. Such structural rearrangements would not affect the CSDs but could be captured by ion-mobility measurements [73].

In order to get further insight on the compact state of the complex, we further investigated the conformational space of the complex by ion mobility on a Waters Synapt G2 HDMS instrument under nondenaturing conditions comparable to the MS measurements reported above. The IM-MS spectrum obtained with a mixture of 20 μM NTAIL and 20 μM PXD is reported in Figure 4. The CSDs are similar to those observed on the QSTAR Elite instrument, although with higher relative amounts of the compact form. The additional separation of analytes by IM allows detection of distinct structural species even at the same charge state. In this case, two slightly different conformations are found to populate the compact state of the NTAIL–PXD complex (charge states 8+ to 10+). Average CCS of 1326 and 1422 Å2 can be derived for the two main peaks detectable for the 8+ charge state. This result suggests that the compact state of the complex is characterized by further structural heterogeneity. Thus, although its overall ionization suggests a collapsed structure, its arrival-time distribution reveals distinct peaks rather than the single peak typically observed for normally folded proteins. The presence of multiple conformers, although with different CCS values, is observed also for the 9+ and 10+ ions. The predominant conformer displays progressively larger CCS values (1422, 1466, 1607 Å2) as the charge state increases from 8+ to 10+. This trend is consistent with the lowest charge state (8+ in this case) corresponding to the most compact species in the original ensemble and to the species with lowest interference of Coulomb repulsions upon transfer to the gas phase. Interestingly, at higher charge states (Figure 4c), the structurally heterogeneous nature of the complex is maintained, whereas for a fully disordered protein such as alpha-synuclein this converges into a single arrival-time distribution for higher charge states [74]. Altogether, these results suggest that both the open and closed states of the complex should be described as conformational ensembles. The high variability of CCS values revealed by the IM profiles could represent a distinct feature of IDPs compared with normally folded, globular structures. Further studies will be needed to clarify to which extent this variability reflects solution and/or gas-phase properties.

Figure 4
figure 4

Nano-ESI-IM-MS spectrum of a MeV NTAIL and PXD mixture (Waters Synapt G2 HDMS instrument). (a) Excerpt of the higher m/z region showing complex formation of PXD-NTAIL for an equimolar ratio of 20 μM protein in 10 mM ammonium acetate (pH 6.5); (b) The PXD–NTAIL complex displays a significant degree of structural heterogeneity, based on the arrival times of the low charge (8–10+) states. The 8–10+ charge states of the PXD–NTAIL complex show at least two different conformations, with corresponding CCSs values that vary over a wide range; (c) Experimentally determined collision cross-sections of the PXD–NTAIL complex reveal that it maintains its structurally heterogeneous character over a broad range of charge states. Colored lines indicate the theoretical collision cross sections for the structural models obtained from MD simulations as discussed in Figure 5, with model 1, 2, and 3 represented as red, blue, and green lines, respectively

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3.4 Modeling the NTAIL–PXD Complex

The structural models of the NTAIL–PXD complex were generated by combining homology modeling and an all-atom Monte Carlo based simulation tool, PROFASI [57–59], which allows exploring the conformational ensemble of the complex in implicit solvent (see Methods for details). The quality of the conformational sampling was established by the agreement with available experimental data, namely NMR CSs of NTAIL bound to PXD [33] and CD of the NTAIL–PXD complex [42]. Quite good agreement can be observed between calculated CSs and those measured by solution NMR measurements [33] (Supplementary Figure S3).

The calculated CD spectra of the conformational ensemble are similarly close to the experimental ones [42], reproducing the minimum at 208 nm, a shoulder at 222 nm, and a positive maximum at 192 nm for the complex, although a well-defined local minimum at 222 nm is missing (Supplementary Figure S4). Finally, the SASA mean value calculated from the models is 17,920 ± 690 Å2, indicating that the conformational ensemble well describes the open state of the complex detected by ESI-MS.

Three models, namely Model 1, Model 2, and Model 3, were selected from the conformational ensemble based on best agreement with the CSs from NMR or the SASA from ESI-MS (Figure 5a, see Methods for selection criteria). Although these models are not representative of the entire NTAIL–PXD conformational ensemble, they are useful to inspect distinct interaction networks that could drive protein compaction and can also provide hints for targeting the complex in a conformation-specific manner by small molecules. Thus, contact analysis was performed, in order to investigate the nature of the interactions involving either the α-MoRE or the disordered regions that were missing from the crystallographic structure. Schematics of the interface between NTAIL and PXD are shown in Figure 5b. The interface between the α-MoRE and PXD shows minor rearrangements relative to the X-ray structure, which could reflect structure dynamics in solution and/or differences between the chimeric protein and the intermolecular complex. Nevertheless, the interface within the four-helix bundle remains almost exclusively hydrophobic in Model 1 and Model 2, whereas it shows some additional electrostatic interactions in Model 3. Analysis of the contacts established by the fuzzy regions (see Figure 5b) unveiled their prevalently hydrophobic nature, suggesting that the interactions between PXD and NTAIL are dominated by hydrophobic interactions, even outside the folded, globular core. A few hydrogen bonds and salt-bridges seem to give additional contribution to the binding of the two proteins. Thus, it seems that not only the helical core of the complex but also the surrounding disordered cloud is dominated by hydrophobic interactions, although hydrogen bonding and electrostatic interactions seem to play a role in stabilizing the more compact states. The prevalently hydrophobic nature of the NTAIL–PXD complex is in agreement with previous reports highlighting that although IDPs are characterized by a low content in hydrophobic residues not sufficient to drive formation of a hydrophobic core, hydrophobic residues often play a dominant role in mediating physiologically relevant protein-protein interactions [75].

Figure 5
figure 5

Structural models of the MeV NTAIL–PXD complex. (a) Cartoon representation of selected models obtained by PROFASI (Model 1, Model 2, and Model 3) for the disordered component. Blue, NTAIL; orange, PXD; grey, superimposed X-ray structure (PDB ID: 1T6O) [40]. The comparison between the calculated and experimental CSs of the N, C, Cα, and Cβ atoms of NTAIL bound to PXD in each selected model is shown in Supplementary Figure S3; (b) Schematic, 2D representation of the NTAIL–PXD interface, for the X-ray structure (PDB ID: 1T6O) [40] and for each of the three models shown in panel (a), generated by the LIGPLOT package [76]. Hydrophobic interactions are indicated by spokes (pink, NTAIL; red, PXD) and hydrogen bonds by dashed green lines. Bond lengths (Å) are also shown. The residues forming hydrogen bonds are shown by ball-and-stick representations (blue, NTAIL; orange, PXD). The NTAIL residues belonging to the α-MoRE (488-499) are labeled by stars

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4 Conclusions

This study combines information derived from biophysical investigation and molecular simulations, in order to develop high-resolution structural models for the fuzzy complex between NTAIL and PXD. The results show that the staccato-type interactions characterizing this molecular ensemble are mainly of hydrophobic nature. While previous atomic models focused only on the α-MoRE region of NTAIL, this study combines experimental data and computational modeling to generate models of the entire NTAIL–PXD complex at atomic resolution. By including the disordered arms in the molecular modeling, it was possible to extend interaction analysis to the dynamic regions of the complex, which are likely involved in biological function. The information that can be derived from this analysis sheds light onto the nature of the interactions that drive protein compaction and sets the basis for targeting specific conformations of the complex by small-molecule inhibitors.

The good agreement between SASA values derived from CSD analysis and solution models for the open state of the NTAIL–PXD complex suggests that the high-charge component of MS spectra reflects structural properties of the heterogeneous conformational ensemble and gives, in turn, further support to the structural details derived from the models. The MS results reported here also point out, for the first time, a compact state of the complex. IM/MS data indicate that this component is characterized by high structural heterogeneity in the gas phase. The broad arrival time distributions observed for the low charge-state component could be a distinct feature of collapsed states of IDPs, as opposed to natively folded, globular structures. Since compact conformations were not detected by previous experimental investigation, further studies will be required to tell whether they represent a minor population of the solution ensemble or a collapsed species induced by the electrospray process. Normally, charge states and CCS are closely linked to each other, but if the protein can restructure after the charge states are generated (i.e., after ESI), there can be a discrepancy between the two. Systematic comparison of solution and gas-phase conformational ensembles of IDPs will be needed to clarify this point.