Compaction and binding properties of the intrinsically disordered C-terminal domain of Henipavirus nucleoprotein as unveiled by deletion studies

Abstract

Henipaviruses are recently emerged severe human pathogens within the Paramyxoviridae family. Their genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). We have previously shown that in Henipaviruses the N protein possesses an intrinsically disordered C-terminal domain, N_TAIL, which undergoes α-helical induced folding in the presence of the C-terminal domain (P_XD) of the P protein. Using computational approaches, we previously identified within N_TAIL four putative molecular recognition elements (MoREs) with different structural propensities, and proposed a structural model for the N_TAIL–P_XD complex where the MoRE encompassing residues 473–493 adopt an α-helical conformation at the P_XD surface. In this work, for each N_TAIL protein, we designed four deletion constructs bearing different combinations of the predicted MoREs. Following purification of the N_TAIL truncated proteins from the soluble fraction of E. coli, we characterized them in terms of their conformational, spectroscopic and binding properties. These studies provided direct experimental evidence for the structural state of the four predicted MoREs, and showed that two of them have clear α-helical propensities, with the one spanning residues 473–493 being strictly required for binding to P_XD. We also showed that Henipavirus N_TAIL and P_XD form heterologous complexes, indicating that the P_XD binding regions are functionally interchangeable between the two viruses. By combining spectroscopic and conformational analyses, we showed that the content in regular secondary structure is not a major determinant of protein compaction.

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Introduction

Henipaviruses are recently emerged human pathogens. Hendra virus (HeV) came to light in 1994 as the causative agent of a sudden outbreak of acute respiratory disease in horses in Brisbane (Australia) with a fatal human case. HeV infections currently pose a serious threat to livestock in Australia, where sporadic and lethal transmission to humans has also occurred. Nipah virus (NiV), the second known member of the Henipavirus genus, was recognized in 1998–1999 as the etiologic agent of an outbreak of disease in pigs and humans in Malaysia. 265 human cases of encephalitis with 105 deaths were reported and 1 million pigs were culled to stop the epidemic. Since 2001, and almost every year, including 2011,¹ outbreaks of encephalitis caused by NiV occurred in Bangladesh with a case fatality rate approaching 75% (see ref. 2 and references therein cited). Fruit-eating bats are the natural reservoir of Henipaviruses (see ref. 3 and references therein cited). Occasional person-to-person NiV transmission is observed,^4–7 thus further extending the potential of NiV to cause deadly outbreaks in humans. In addition to acute infection, these viruses cause asymptomatic infections in up to 60% of exposed people and may lead to late-onset of disease or relapse of encephalitis years after initial infection, as well as persistent or delayed neurological sequelae.⁸

A few distinctive properties, including their much larger genome size, led to the classification of NiV and HeV within the Henipavirus genus of the Paramyxoviridae family.⁹ A number of Henipavirus strains have been isolated from humans, bats, horses and pigs over a wide geographic area in the last decade. Noteworthy, Henipaviruses are also found outside Australia and Asia, thus extending the endemic area of one of the most pathogenic virus genera known in humans.³ The susceptibility of humans, their high pathogenicity, the wide host range and interspecies transmission led to the classification of HeV and NiV as biosecurity level 4 (BSL-4) pathogens.¹⁰ To date, no therapeutic agents nor vaccines are available to fight against these severe pathogens.

As in all Mononegavirales members, the negative-strand, non-segmented RNA genome of Henipaviruses is encapsidated by the nucleoprotein (N) within a helical nucleocapsid. This helical N:RNA complex, and not naked RNA, is the substrate used by the polymerase complex during both transcription and replication. The helical nucleocapsid, made of genomic RNA encapsidated into a linear non-covalent polymer of the N protein, has the characteristic herringbone-like structure typically observed in other Paramyxoviridae members.^11–17 When expressed in heterologous hosts, NiV N was found to form nucleocapsid-like particles that contain RNA.^18–21

Minigenome replicon studies showed that in Henipaviruses, the N protein, the phosphoprotein (P) and the large protein (L) are necessary and sufficient to sustain replication of viral RNA.²² By analogy with other Paramyxoviridae members, the polymerase complex is thought to consist of the L protein and of the P protein, with the latter serving as a tether for the recruitment of L onto the nucleocapsid template. As in all Mononegavirales members, Henipavirus N and P proteins were shown to interact with each other, being able to form both homologous and heterologous N–P complexes.^23,24

So far, high-resolution structural information is only available for the fusion (F) and attachment (G) glycoproteins.^25–28 For the N and P proteins, the only molecular data come from our previous studies.^21,29–31 Those studies showed that the N-terminal region of P (PNT, aa 1–404 or 1–406) and the C-terminal region of N (N_TAIL, aa 400–532) belong to the family of intrinsically disordered proteins (IDPs), although they both contain short order-prone segments.²⁹ The occurrence of some residual, transient secondary and/or tertiary structure within N_TAIL and PNT led to their classification within the premolten globules (PMG) subfamily within the family of IDPs.^32–37 IDPs are ubiquitous, functional proteins that lack highly populated secondary and tertiary structure under physiological conditions and in the absence of a partner/ligand (for recent reviews on IDPs, see ref. 38–43). In contrast, we showed that the C-terminal X domain of the Henipavirus P proteins (P_XD, aa 660–709 of NiV P and aa 657–707 of HeV P) is folded, and adopts an α-helical conformation.²¹Henipavirus N_TAIL and P_XD domains were shown to form a 1 : 1 stoichiometric complex that is stable up to 1 M NaCl, and whose K_D is within the μM range.²¹ Using bioinformatics approaches, we identified within both HeV and NiV N_TAIL domains four putative molecular recognition elements (MoREs), where these latter are short order-prone regions within intrinsically disordered regions that have a propensity to undergo induced folding upon binding to partner(s).^44–47 Among these predicted MoREs, at least two of them (aa 408–422 and 473–493) exhibit a clear α-helical nature, and spectroscopic studies carried out in the presence of trifluoroethanol (TFE) unveiled that both HeV and NiV N_TAIL do possess α-helical propensities.²⁹ In agreement, using far-UV circular dichroism (CD) and heteronuclear NMR we showed that binding to P_XD triggers α-helical folding of N_TAIL, with the resonance behavior suggesting a role for the predicted α-MoRE spanning residues 473–493 in binding to P_XD.²¹ Using fluorescence spectroscopy, we showed that P_XD has no impact on the chemical environment of a Trp residue introduced at position 527 of Henipavirus N_TAIL, thus arguing for the lack of stable contacts between the C-terminus of N_TAIL and P_XD.²¹ Based on all these observations, and also by analogy with the related measles virus (MeV),^48,49 we proposed a tentative structural model of the Henipavirus N_TAIL–P_XD interaction where the α-MoRE encompassing residues 473–493 adopt an α-helical conformation and are embedded between helices α2 and α3 of P_XD, leading to a relatively small interface dominated by hydrophobic contacts.²¹

The objective of the present study was to directly assess the role of this α-MoRE in binding to P_XD and to investigate the possible contribution of other short order-prone N_TAIL segments to complex formation and folding. To this endeavor, for both HeV and NiV N_TAIL proteins, we designed four constructs encoding truncated N_TAIL proteins bearing various combinations of the four predicted MoREs. We report their bacterial expression, purification and characterization and show that removal of the α-MoRE spanning residues 473–493 does indeed abrogate binding to P_XD. In contrast, removal of the other MoREs does not significantly affect the affinity of the binding reaction, although it impacts the folding propensities to various extents. Spectroscopic studies allowed us to shed light on the structural state of the various MoREs, showing that the MoRE encompassing residues 444–464 are unlikely to transiently populate a β structure. By combining spectroscopic and conformational analyses, we showed that the content in regular secondary structure is not a major determinant of protein compaction. Finally, we estimated the cross-reactivity between the HeV and NiV N_TAIL–P_XD interacting pairs and show that the P_XD-binding regions are functionally interchangeable between the two viruses.

Results and discussion

Design, expression and purification of Henipavirus N_TAIL deletion constructs

In order to provide direct experimental support for the ability of the α-MoRE encompassing residues 473–493 of Henipavirus N_TAIL to bind to P_XD and to assess the possible involvement of other short order-prone N_TAIL fragments in P_XD binding and in the formation of local transiently populated secondary structure elements, we have designed four deletion constructs bearing various combinations of the predicted MoREs²⁹ (see Fig. 1A). For the sake of clarity, the four predicted MoREs are hereafter referred to as Box1, Box2, Box3 and Box4, with Box3 corresponding to the α-MoRE that has been modeled at the P_XD surface.²¹

Fig. 1 (A) Schematic representation of N_TAIL deletion proteins. (Top) Structural organization of Henipavirus N proteins according to ref. 29 showing that they are composed of an N-terminal structured region (N_CORE, aa 1–399) and a C-terminal disordered region (N_TAIL, aa 400–532). The two putative α-MoREs (aa 408–422 and aa 473–493), with the second one corresponding to the region modeled at the P_XD surface,²¹ are shown. A putative irregular MoRE (I-MoRE) (aa 523–532) and a putative MoRE of dubious state (aa 444–464, see region contoured by a dashed line)²⁹ are also shown. The N_TAILΔ1, N_TAILΔ1Δ2, N_TAILΔ4 and N_TAILΔ3Δ4 deletion proteins are devoid of Box1, Box1 plus Box2, Box4 or Box3 plus Box4, respectively. They all possess an N-terminal hexahistidine tag. (B) 15% SDS-PAGE of purified deletion proteins followed by Coomassie blue staining.

The gene fragments encoding the different N_TAIL deletion proteins were cloned into the pDEST17OI vector to yield N-terminally histidine-tagged recombinant products, the expression of which is under the control of the T7 promoter being tightly controlled by IPTG. In all cases, the N_TAIL proteins were readily expressed in E. coli and their solubility was high, thus allowing their recovery from the soluble fraction of the bacterial lysates. The proteins were purified to homogeneity (>95%) by immobilized metal affinity chromatography (IMAC) followed by preparative size exclusion chromatography (SEC) (Fig. 1B). The identity of the final purified proteins was confirmed by mass spectrometry analysis of the tryptic fragments obtained after digestion of the purified proteins excised from sodium dodecyl sulfate (SDS)-polyacrylamide gels: those analyses yielded for all N_TAIL proteins a high sequence coverage attesting their identity and integrity (data not shown).

All the N_TAIL proteins migrate in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular mass higher than expected (see Fig. 1B and Table S1, ESI‡). Indeed the apparent molecular masses of the HeV and NiV N_TAILΔ1 proteins are 18 and 19 kDa, respectively, while their expected molecular mass is 14 kDa (see Fig. 1B and Table S1, ESI‡). Likewise, the apparent molecular masses of HeV and NiV N_TAILΔ4 proteins are 18 and 19 kDa, respectively, while their expected molecular mass is 15.5 kDa (see Fig. 1B and Table S1, ESI‡). The HeV and NiV N_TAILΔ3Δ4 proteins both have an apparent molecular mass of approximately 12 kDa, while their expected mass is 10 kDa. As for the HeV and NiV N_TAILΔ1Δ2 proteins, which have an expected molecular mass of 10 kDa, they display an even more abnormal electrophoretic mobility, with apparent molecular masses of approximately 14 kDa (HeV) and 17 kDa (NiV) (Fig. 1B and Table S1, ESI‡).

This abnormal migratory behavior has already been documented for both HeV and NiV full-length N_TAIL proteins, where mass spectrometry analyses gave the expected results.²⁹ This anomalous electrophoretic mobility is frequently observed in IDPs (for a few illustrative examples see ref. 12, 21, and 50–52) and has been ascribed to a rather high content of acidic residues as compared to globular proteins.⁵³ The high negative charge is thought to lead to a larger Stokes radius of the protein–detergent complex together with a reduction in detergent binding.⁵⁴ It should be noted however that the content in acidic residues of those N_TAIL proteins that display the most aberrant migration, namely NiV and HeV N_TAILΔ1Δ2 (see Fig. 1B and Table S1, ESI‡), is 11.1%, a value lower than the average acidic content (11.8%) of globular proteins⁵³ (see Table S2, ESI‡). In addition, the content in acidic residues of the Henipavirus N_TAILΔ1Δ2 proteins is also lower than that of the HeV and NiV N_TAILΔ3Δ4 proteins (13.6% and 11.5%, respectively), where these latter have the same expected molecular mass as the N_TAILΔ1Δ2 proteins and yet migrate less abnormally than these latter (see Tables S1 and S2, ESI‡). This observation suggests that other parameters, beyond the content in acidic residues, are responsible for the abnormal electrophoretic migration of IDPs. That the occurrence of PP and/or PXP motifs may be a possible determinant of the observed aberrant migration is unlikely. Indeed, while the HeV N_TAILΔ1, N_TAILΔ1Δ2 and N_TAILΔ4 proteins possess a PP and a PKP motif, the NiV proteins, including the NiV N_TAILΔ1Δ2 that displays the most aberrant migration, are all devoid of such motifs.

Sequence properties of N_TAIL deletion proteins

Sequence analysis of N_TAIL deletion proteins by the MeDor metaserver for the prediction of disorder⁵⁵ and by the PONDR VL-XT predictor^56,57 showed that they are all predicted to be mostly disordered (data not shown and Table S2, ESI‡). Consistent with these predictions, the mean hydrophobicity (〈H〉) value of N_TAIL deletion proteins was found to be quite low and close to the value calculated for the full-length forms (see Table S2, ESI‡). Such a low hydrophobicity places the N_TAIL proteins, including the full-length forms, in the IDP region of the mean net charge/mean hydrophobicity plot developed by Uversky and co-workers,⁵⁸ with the only exception of NiV N_TAILΔ3Δ4 that lies on the border between IDPs and folded proteins and is thus predicted to adopt a more compact conformation (Fig. 2). According to the distance from the boundary (referred to as CH-distance), HeV N_TAILΔ1Δ2 is predicted to be the least ordered N_TAIL protein, and HeV N_TAIL proteins are predicted to be more disordered than NiV N_TAIL proteins (Fig. 2 and Table S2, ESI‡). For both HeV and NiV N_TAIL proteins, the extent of disorder follows the order N_TAILΔ1Δ2 > N_TAILΔ1 > N_TAIL full-length > N_TAILΔ4 > N_TAILΔ3Δ4. Thus, for both HeV and NiV proteins, removal of Box1 plus Box2 and, to a minor extent, of Box1 alone leads to forms that are predicted to be less ordered (i.e. more extended) than the parental, full-length form. By contrast, removal of Box4 either alone or in conjunction with Box3 leads to forms that are predicted to be less extended.

Fig. 2 (A) Charge–hydropathy (CH) plot of Henipavirus N_TAIL proteins. The mean net charge (R) is plotted against the mean hydropathy (H). In the left part of the CH plot, a protein is predicted to be intrinsically disordered, whereas it is predicted to be structured if it falls in the right part of it (see Experimental).

Recent analyses of IDPs by the group of Pappu indicated sequence polarity as a determinant of IDP compaction: indeed polar IDPs were found to favor collapsed ensembles in water despite the absence of hydrophobic groups.⁵⁹ To evaluate sequence polarity, we therefore calculated for each N_TAIL protein the net charge per residue (NCR) as described by Pappu and co-workers⁵⁹ (see Table S2, ESI‡). For all N_TAIL proteins, the NCR is negative and well below the well-recognized threshold of 0.2, discriminating collapsed and extended IDPs.⁵⁹ This suggests that the N_TAIL proteins would preferentially populate collapsed states. According to Pappu and co-workers, these proteins would belong to the category of weak polyelectrolytes/polyampholytes, where |f+ − f−| ≤ 0.2 and each of f+ and f− are small.

Conformational properties of N_TAIL deletion proteins

All the N_TAIL proteins are monodisperse, as indicated by the elution profile observed in analytical SEC coupled to multiple angle laser light scattering (SEC-MALLS) and in preparative SEC (Fig. 3 and data not shown). The molecular masses inferred from SEC-MALLS were found to be in close agreement with the expected values (see Table 1), thus showing that the proteins are monomeric while confirming their integrity and ruling out possible proteolytic degradation. The Stokes radii (R_S) of the N_TAIL proteins, as derived by SEC-MALLS, are shown in Table 1. In all cases, the experimentally observed values were found to be higher than those expected for natively folded forms, as expected for proteins that are truncated forms of an IDP. Note that these elution profiles were found to be independent from ionic strength, as the same R_S values were obtained irrespective of the NaCl concentration. For all N_TAIL proteins, the observed R_S values were found to be closer to the values expected for IDPs of the same molecular mass (as calculated based on the simple power-law model)⁶⁰ than to those expected for fully unfolded proteins (in urea)⁶¹ (see Table 1). Interestingly, the experimentally observed Stokes radii were found to be in better agreement with the values expected for IDPs derived from the simple power-law model (see eqn (5) in Experimental) than with those derived from the sequence-based model (see eqn (6) in Experimental). The observed Stokes radii of HeV N_TAILΔ1Δ2 and HeV N_TAILΔ4 were found to be unexpectedly lower and higher, respectively, than those of the corresponding NiV proteins (see Table 1). Aside from these two exceptions, the other HeV N_TAIL proteins were found to have R_S values quite similar to those of the corresponding NiV N_TAIL proteins. By comparing each experimentally determined R_S with the theoretical values of the Stokes radius expected for various conformational states (R^NF_s: monomeric natively folded protein; R^U_s: fully unfolded state in urea; R^PMG_s: PMG conformation, see eqn (2)–(4) in Experimental), the HeV N_TAILΔ1Δ2 and N_TAILΔ3Δ4 proteins, and the NiV N_TAILΔ4 and N_TAILΔ3Δ4 proteins were found to have Stokes radii similar to the values expected for IDPs adopting a PMG-like conformation (see Table 1), as already observed in the case of both HeV and NiV full-length N_TAIL proteins.²⁹ Conversely, both HeV and NiV N_TAILΔ1 proteins were found to have R_S values closer to those expected for fully unfolded forms, while HeV N_TAILΔ4 and NiV N_TAILΔ1Δ2 have borderline R_S values between PMG-like and random coil-like forms (see Table 1). In order to quantitatively estimate the degree of compaction of the various N_TAIL proteins, we used the compaction index (CI) as described by Brocca and co-workers.⁶² This parameter (see eqn (7) in Experimental) does not mathematically depend on the residue number and hence allows comparison between proteins of different lengths. The CI value can vary between 0 and 1, with 0 indicating minimal compaction and 1 maximal compaction. For each N_TAIL protein, we therefore calculated the CI using the reference R_S values for unfolded or natively folded proteins as defined by either Uversky⁶¹ or Marsh and Forman-Kay.⁶⁰ These two calculation methods yielded slightly different CI values. Irrespective of the calculation method used to derive the CI, for both NiV and HeV the least compact form was found to be N_TAILΔ1 and the most compact form N_TAILΔ3Δ4. Thus, this latter form has a CI that is even higher than that of the parental full-length protein, indicating that removal of Box3 plus Box4 leads to a more compact form. That removal of Box3 plus Box4 was to lead to a more compact form was rather unexpected, given the relative enrichment in hydrophobic clusters within these regions and the occurrence of a predicted α-helix within Box3 (see Fig. 1 in ref. 29). These latter features were indeed the basis for the speculation that those regions could correspond to MoREs, with the third MoRE (Box3) corresponding to a putative α-MoRE involved in binding to P_XD.²¹ Notably, the unexpected high degree of compaction of N_TAILΔ3Δ4 is in agreement with the sequence analyses shown in Fig. 2, which predict this protein to be the most compact. Strikingly, similar, although reciprocal, unexpected observations were done in the case of the intrinsically disordered Sic1 protein, where removal of a region predicted to be highly disordered led to a truncated form that was found to be less compact than the parental, full-length form.⁶²

Fig. 3 SEC-MALLS-RI analysis of HeV (A) and NiV (B) N_TAIL proteins. The right y-axis represents the molecular mass and the left axis represents the differential refractive index. The horizontal traces show the molecular masses calculated from light-scattering intensity at different angles and differential refractive indexes as a function of the elution volume. The concentrations of the injected samples were as follows: HeV N_TAILΔ1: 18.5 mg mL⁻¹, HeV N_TAILΔ4: 23.3 mg mL⁻¹; HeV N_TAILΔ1Δ2: 14.5 mg mL⁻¹; HeV N_TAILΔ3Δ4: 12 mg mL⁻¹; NiV N_TAILΔ1: 12.2 mg mL⁻¹; NiV N_TAILΔ4: 17 mg mL⁻¹; NiV N_TAILΔ1Δ2: 14.5 mg mL⁻¹; NiV N_TAILΔ3Δ4: 17.7 mg mL⁻¹.

Table 1 Stokes radii (R^obs_S, Å) as obtained by SEC-MALLS and expected values for the various conformational states. R^NF_S: R_S expected for a natively folded form; R^IDP_S: R_S expected for an IDP based on the simple power-law model or (values in parentheses) from the sequence-based model; R^PMG_S: R_S expected for a premolten globule; R^U_S: R_S expected for a fully unfolded form; MM^Obs: molecular mass as determined by SEC-MALLS. MM^theo: expected molecular mass. CI¹: Compaction index calculated using the reference R_S values for unfolded and natively folded proteins as defined by Uversky.⁶¹ CI²: Compaction index calculated using the references R_S values for unfolded and natively folded proteins as defined by Marsh and Forman-Kay⁶⁰

	MM^Obs (kDa)	MM^theo (kDa)	R ^ObsS	R ^NFS	R ^IDPS	R ^PMGS	R ^US	R ^ObsS/R^PMGS	R ^ObsS/R^US	CI^1	CI^2
a Values were derived by previously reported mass spectrometry studies.^29 b Values were derived by previously reported SEC studies.^29
HeV
NTAIL	15.3^a	15.2	28.0^b ± 3	19.4	30.8 (26.6)	28.0	34.0	1.001	0.826	0.410	0.475
NTAILΔ1	15.5	14.5	30.5 ± 3	19.1	29.9 (25.9)	27.4	33.0	1.112	0.922	0.184	0.247
NTAILΔ1Δ2	9.8	10	22.8 ± 3	16.7	24.6 (21.2)	23.6	27.2	0.965	0.837	0.421	0.485
NTAILΔ4	15.3	15.7	31.4 ± 3	19.7	31.3 (26.4)	28.4	34.5	1.107	0.909	0.211	0.285
NTAILΔ3Δ4	11.1	10.3	21.3 ± 3	16.8	25.3 (21.0)	23.9	27.7	0.891	0.770	0.590	0.683
NiV
NTAIL	14.9^a	14.9	28.0^b ± 3	19.4	30.8 (25.7)	27.7	33.6	1.009	0.834	0.391	0.475
NTAILΔ1	14.0	14.3	31.2 ± 3	19.0	29.9 (25.0)	27.2	32.8	1.145	0.952	0.115	0.197
NTAILΔ1Δ2	10.0	9.8	25.3 ± 3	16.6	24.6 (20.2)	23.5	27.0	1.078	0.936	0.166	0.230
NTAILΔ4	15.5	15.5	27.0 ± 3	19.6	31.3 (25.5)	28.1	34.2	0.959	0.790	0.492	0.569
NTAILΔ3Δ4	10.5	10.1	21.9 ± 3	16.8	25.3 (20.7)	23.8	27.5	0.921	0.797	0.521	0.625

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On the other hand, the finding that both HeV and NiV N_TAILΔ1 proteins are the least compact proteins is not in agreement with the in silico analyses, although these latter predict Henipavirus N_TAILΔ1 proteins to be less compact than the parental, full-length forms (see Fig. 2).

The compaction trend of HeV N_TAILΔ1Δ2 and HeV N_TAILΔ4 is inversed with respect to that of the corresponding NiV proteins: indeed, while HeV N_TAILΔ1Δ2 has a compaction slightly higher than that of the parental full-length form and HeV N_TAILΔ4 is less compact, the opposite scenario is observed in the case of the NiV proteins (see Table 1). This indicates that while in the case of HeV N_TAIL, removal of Box4 leads to a more extended form, in the case of NiV N_TAIL it is the removal of Box1 plus Box2 which leads to a decrease in compaction. This suggests the occurrence of differences in these boxes and/or in the remainder of the polypeptide chain in terms of local structural propensities and/or abilities to form tertiary contacts in the HeV and NiV N_TAIL proteins.

Altogether, these data support a major role in both HeV and NiV N_TAIL proteins for Box1 as a determinant of protein compaction. It is conceivable that Box1 could exert this effect by establishing long-range tertiary contacts and/or by adopting a transiently populated regular conformation that would result in a PMG-like form. The role of Box2 and Box4 is more dubious, given the poor agreement between the hydrodynamic and the in silico analyses and the inconsistencies between the two viruses. By contrast, experimental and computational data for both viruses consistently indicate that removal of Box3 plus Box4 results in a more compact form. The possibility that the shortening of polypeptide chain length might be a determinant of protein compaction is ruled out by the observation that the NiV N_TAILΔ1Δ2 protein, which has an even slightly shorter chain (see Table S2, ESI‡), adopts a more extended conformation with respect to both HeV and NiV N_TAILΔ3Δ4 proteins.

Spectroscopic properties and folding propensities of N_TAIL deletion proteins

In order to further investigate the local structural propensities of the various boxes, we carried out far-UV circular dichroism (CD) studies. As expected for proteins that are truncated forms of an IDP where putative transiently structured regions have been removed, the far-UV circular dichroism spectra of the N_TAIL deletion proteins at neutral pH are all typical of predominantly unstructured proteins, as seen by their large negative ellipticity at 198 nm and very low ellipticity at 190 nm (Fig. 4). These results indicate that the HeV and NiV N_TAILΔ3Δ4 proteins, although predicted to have a borderline order and found to be more compact in hydrodynamic studies, yet adopt a mostly disordered conformation, lacking highly populated, stable regular secondary structure elements.

Fig. 4 Far-UV CD spectra of HeV (A) and NiV (B) N_TAIL proteins in 10 mM sodium phosphate pH 7 in the absence or in the presence of either 20% TFE or 3 M TMAO. The protein concentration was 0.1 mg mL⁻¹. Spectra were recorded at 20 °C. Due to high background noise at lower wavelength (beyond 210 nm), the spectra in the presence of TMAO are shown to the point up to which the dyna voltage was in the permissible range. Data are representative of one out of two independent acquisitions.

Trifluoroethanol (TFE) and trimethylamine N-oxide (TMAO) are secondary structure stabilizers that can be used as probes of hidden structural propensities of peptides and proteins. TMAO and other osmolytes may fold unstructured proteins due to the osmophobic effect, a solvophobic thermodynamic force, arising from the unfavorable interaction between the osmolyte and the peptide backbone.^63–66 Although both TMAO and TFE act on the peptide backbone, the molecular mechanisms underlying their effects are different. Both solutes act by mimicking the hydrophobic environment experienced by proteins in protein–protein interactions, and can thus be used as probes to unveil regions having a propensity to undergo induced folding.^67–70 It has long been known that TFE increases the propensity of amino acids to form an α-helix, presumably by strengthening peptide hydrogen bonds in TFE/H₂O mixtures and through favorable interactions of hydrophobic amino acid side chains with TFE.^71,72 Peptide hydrogen bonds in helices are believed to be stabilized indirectly by weakening the hydrogen bonding of water molecules to the peptide backbone in the coil form.⁷² As a result of weakening the hydrophobic interactions within the protein interior, TFE might promote helical structure in most peptides and proteins, even though this helical structure may be non-native.^73–75 In contrast, TMAO increases the driving forces for protein folding due to its solvophobic effect on the backbone, forcing thermodynamically unstable proteins to fold without altering the rules for folding to a native-like conformation.⁶⁴ Furthermore, in opposition to TFE solutions, the propensities of hydrophobic groups to interact with solvent are essentially the same in water as they are in TMAO solutions.^76,77 Thus, due to the weakening of hydrophobic interactions, the dominant effect of TFE on proteins is protein denaturation accompanied by the preferential formation of α-helices as a result of the strengthening of peptide hydrogen bonds. Contrarily to that, TMAO promotes folding of unfolded proteins by providing an additional force for folding that has no preference for any particular secondary structure.⁶⁶ It should be pointed out however, that even if TFE is known to stabilize α-helices more than β-strands, gain of α-helicity in the presence of TFE is not a general rule: for instance, (i) the acidic activator domain of GCN4 forms little or no α-helix in TFE concentrations as high as 30% and folds mostly as β-sheets in 50% TFE,⁷⁸ (ii) the intrinsically disordered dehydrin Rab18 has an α-helical content as low as 2% in the presence of 90% TFE⁷⁹ and (iii) the intrinsically disordered rat seminal vesicle protein IV exclusively undergoes β transitions in the presence of TFE (P. Palladino, S. Vilasi, R. Ragone and F. Rossi, personal communication). Furthermore, in the case of measles virus N_TAIL, we have recently shown that TFE promotes α-helical folding of the 488–502 region only, with the downstream region only becoming slightly less mobile while retaining an extended conformation in the presence of 20% TFE.⁸⁰ These observations attest the ability of TFE to show β propensities.

In order to assess the folding potential of the truncated N_TAIL proteins, and taking into account the fact that both HeV and NiV N_TAIL possess a predicted MoRE of the dubious β state, namely Box2,²⁹ we recorded the CD spectra of N_TAIL proteins in the presence of either TFE or TMAO, this latter being used with the specific purpose of unveiling possible β propensities. The TFE concentration in these studies was set to 20%, this choice being dictated by previous CD studies focused on full-length Henipavirus N_TAIL proteins showing that most unstructured-to-structured transitions take place at this concentration.²⁹

TMAO experiments were initially carried out with full-length proteins and the concentration of the osmolyte was gradually increased from 0 to 5 M (with 1 M steps). At TMAO concentrations as high as 4 and 5 M, protein precipitation occurred and the spectra were extremely noisy thus precluding meaningful analyses. Because of this observation and also taking into account the fact that previous studies carried out by others showed that 3 M TMAO was found to promote most dramatic structural transitions in various IDPs (for examples see ref. 81–83), we carried out subsequent experiments at 3 M TMAO, a concentration where significant structural transitions were observed and no protein precipitation occurred. Under these conditions, the spectra show a poor signal-to-noise ratio due to the strong absorption of TMAO in the far-UV region. This high background noise prevented meaningful analyses of the spectra beyond 210 nm, as the dyna voltage above this wavelength was found to fall in the non-permissible range.

All the N_TAIL proteins, including the full-length forms (see Fig. S1, ESI‡ and ref. 29), show a loss of unordered structure upon addition of 20% TFE, as indicated by the characteristic increase in ellipticity at 190 and 198 nm (Fig. 4 and Fig. S1, ESI‡). Likewise, and in spite of the noisy nature of the spectra in the presence of TMAO, this latter was found to trigger a disorder-to-order transition in all N_TAIL proteins, as judged from the drop in the ellipticity in the 210–240 nm region (Fig. 4 and Fig. S1, ESI‡). For both HeV and NiV N_TAIL proteins, and irrespective of whether TFE or TMAO was added, the disorder-to-order transition is more pronounced for N_TAILΔ1 and N_TAILΔ1Δ2 than for N_TAILΔ4 and N_TAILΔ3Δ4, suggesting a role for Box3 and Box4 in the gain of regular secondary structure. Interestingly, NiV N_TAILΔ1Δ2 exhibits a lower ability to undergo a structural transition in the presence of the osmolytes as compared to the HeV N_TAILΔ1Δ2 protein (Fig. 4). This observation suggests the existence of subtle differences in the inherent folding propensities of Box1 and Box2 between the HeV and the NiV N_TAIL proteins, with these differences having been already unveiled by hydrodynamic studies.

In order to achieve a quantitative assessment of the TFE and TMAO impact, the CD spectra were deconvoluted (see Experimental), thereby allowing the percentage of the various secondary structures to be inferred (Fig. 5). Although deconvolution approaches notoriously lead to estimations that cannot be taken as fully reliable, i.e. they often significantly deviate from the actual content in secondary structure as observed in experimentally determined structures (for examples see ref. 48 and 84), the estimated α-helical content is nevertheless very useful for comparative purposes. Note that the spectra recorded in the presence of TMAO could not be deconvoluted, as the wavelength range in which the dyna voltage was in the permissible range (namely, 260–210 nm) was too small to allow deconvolution by any of the programs implemented in the DICHROWEB server.

Fig. 5 Secondary structure content of HeV (A) and NiV (B) N_TAIL proteins in 0 and 20% TFE. The percentages of α-helices, β-strands, turns and unordered regions were calculated using CDSSTR. Values for full-length HeV and NiV N_TAIL proteins were derived from spectra shown in Fig. S1 (ESI‡).

As shown in Fig. 5, spectral deconvolution pointed out that for both viruses, N_TAILΔ3Δ4 is the N_TAIL protein with the lowest α-helical content, followed by the N_TAILΔ1Δ2 proteins. In both viruses, N_TAILΔ1 and N_TAILΔ4 were found to have an α-helical content similar to that of the full-length form, arguing for a poor, if any, ability of Box1 and Box4 to adopt an α-helical conformation in water. On the other hand, data support a role for Box3 and, to a minor extent, for Box2 in sampling, at least transiently, α-helical conformations. Consistent with the hypothesis that Box1 poorly contributes to the overall α-helical content of both HeV and NiV N_TAIL proteins, the addition of 20% TFE to both HeV and NiV N_TAILΔ1 proteins pointed out a gain in α-helicity similar to that of the full-length form (see Fig. 5). Conversely, and in agreement with a possible involvement of Box3 and Box2 in α-helical folding, addition of TFE to Henipavirus N_TAILΔ3Δ4 and N_TAILΔ1Δ2 proteins led to a dramatic or significant drop, respectively, in the ability to undergo α-helical folding as compared to the full-length form (see Fig. 5). In particular, for both viruses, N_TAILΔ3Δ4 is the N_TAIL protein with the lowest ability to undergo α-helical folding, as judged from the finding that the HeV and NiV N_TAILΔ3Δ4 proteins have the lowest α-helical content in 20% TFE. Surprisingly however, in both viruses, N_TAILΔ4 does not gain α-helicity in the presence of TFE (see Fig. 5). This behavior, which suggests a role for Box4 in α-helical folding, was quite unexpected based on the observation that this latter form has an α-helical content similar to that of the parental, full-length form in the absence of osmolytes. Notably, for all N_TAIL proteins, the addition of 20% TFE triggers a decrease in the β content, arguing for the lack of strong β propensities within the HeV and NiV N_TAIL proteins (see Fig. 5). This observation, together with the finding that Box2 has an α-helical propensity, suggests that Box2 likely adopts an α-helical conformation, thus shedding light on the hitherto dubious state of this Box.²⁹

Taking into account however the fact that TFE may favor (non-native) α-helical folding, we also sought at performing a quantitative analysis of spectra recorded in the presence of TMAO. To this endeavor, since deconvolution was not feasible for spectra recorded in the presence of TMAO, we derived the α-helical content of the various N_TAIL proteins from the ellipticity at 222 nm as described in ref. 85 (see Fig. S2, ESI‡). In order to allow meaningful comparisons with spectra recorded in the absence of any osmolyte, as well as with spectra recorded in TFE, we also used this method to infer the α-helical content of the spectra of the N_TAIL proteins alone and in the presence of 20% TFE (see Fig. S2, ESI‡). The absolute values of the α-helical content derived by this analysis turned out to be different from those provided by the deconvolution (cf.Fig. 5 and Fig. S2, ESI‡). Comparison of data obtained in the presence of TFE with data obtained in the presence of TMAO revealed that this latter is less efficient than TFE in promoting α-helical folding, as judged from the lower α-helical content of all N_TAIL proteins in the presence of TMAO as compared to TFE. This finding is in agreement with previous reports (for examples see ref. 81 and 86). It should be pointed out however that the lower folding potential of 3 M TMAO with respect to 20% TFE may also arise from the possibility that this TMAO concentration does not correspond to the condition where the midpoint transition takes place.

In spite of the numerical differences between the two calculations, an overall similar trend, with a few exceptions, was observed. In agreement with the results obtained by deconvoluting the spectra, the data shown in Fig. S2 (ESI‡) show that (i) the N_TAILΔ3Δ4 proteins were found to possess the lowest α-helical content in the absence of osmolytes and to have the lowest ability to undergo α-helical folding in the presence of TFE or TMAO, (ii) the N_TAILΔ1Δ2 proteins have a borderline (HeV) or significantly reduced (NiV) ability to undergo α-helical folding in both TFE and TMAO as compared to the parental full-length forms, and (iii) the N_TAILΔ4 proteins were found to possess an α-helical content similar (NiV) or slightly higher (HeV) than that of the parental proteins. In contrast with the data obtained by deconvoluting the spectra, the data shown in Fig. S2 (ESI‡) support the ability of both HeV and NiV N_TAILΔ4 proteins to undergo α-helical folding in the presence of osmolytes, consistent with the lack of strong α-helical propensities within Box4. Another notable difference between the data obtained from the deconvolution of the whole CD spectra or from the sole ellipticity at 222 nm concerns N_TAILΔ1. Based on the ellipticity at 222 nm, both HeV and NiV N_TAILΔ1 proteins were found to possess an even higher ability to undergo α-helical folding than the parental, full-length proteins, although their α-helical content in the absence of osmolytes is comparable to that of the full-length protein (see Fig. S2, ESI‡). It should be pointed out however that the values of α-helical content obtained upon deconvoluting the spectra recorded over the whole wavelength range are likely to be more reliable than the data inferred from a single ellipticity value, with this being especially true for highly noisy spectra such as those recorded in the presence of TMAO.

Having in mind the reported ability of TMAO to unveil possible β propensities, we also attempted at obtaining insights about the β content from the spectra recorded in the presence of TMAO. To this end, we checked those spectra for the presence of a possible minimum at 218 nm, with a pronounced negative peak at this wavelength being a hallmark of β structure.⁸⁷ None of the N_TAIL spectra in the presence of TMAO displays such a minimum, with perhaps the only exception of HeV and NiV N_TAILΔ4 proteins that have a spectral shape reminiscent of that of a partly β folded protein (see Fig. 4). Since however this spectral signature was not found in the spectra of the parental, full-length proteins in the presence of TMAO (see Fig. S1, ESI‡), we concluded that the present data do not support any pronounced β propensity within Henipavirus N_TAIL.

Altogether, the present data support a role for Box3, and to a minor extent, for Box2 in α-helical folding of N_TAIL. By contrast, Box1 was found to be devoid of α-helical propensities and not to be involved in α-helical folding. The observation that Box2 has an α, rather than a β, inherent structural propensity provides insights into this MoRE, the structural state of which has been uncertain so far.²⁹ The finding that Box3 plays a major role in the osmolyte-induced α-helical transition provides support to our previous hypothesis that Box3 could be the region implicated in the P_XD-induced α-helical folding of Henipavirus N_TAIL.²¹ Data are also consistent with the lack of pronounced α-helical propensity within Box4, thus providing additional support to our previous computational analyses that predicted the occurrence of an irregular MoRE (I-MoRE) within residues 523–532. We can speculate that the removal of Box4 could reduce the ability of N_TAIL to undergo α-helical folding through a mechanism that is independent from the inherent folding ability of this box and that rather reflects a possible stabilization of the α-helices within Box2 and Box3 through long-range tertiary contacts.

Combining computational and spectroscopic analyses

Although the CD spectra of all N_TAIL proteins are typical of predominantly unfolded proteins, the observed ellipticity values at 200 and 222 nm (Θ₂₀₀ and Θ₂₂₂) of the N_TAIL proteins are consistent with the existence of some residual secondary structure, as observed in IDPs adopting a PMG conformation (Fig. 6A). Indeed, Uversky noticed that IDPs can be subdivided in PMG-like and random coil-like (RC-like) forms as a function of their Θ₂₀₀ and Θ₂₂₂ values.³⁴ Strikingly, except for NiV N_TAILΔ3Δ4 that falls in a position close to the RC-like region of the plot, the other N_TAIL proteins are all located in the twilight zone between RC-like and PMG-like proteins (Fig. 6A). This suggests that while these latter are endowed with some transiently populated structure, the NiV N_TAILΔ3Δ4 protein would possess less residual regular secondary structure.

Fig. 6 (A) 222–200 ellipticity plot modified from Uversky.³⁴ The mean residue ellipticity at 222 nm (Θ₂₂₂) of a set of well-characterized unfolded, random coil-like (RC-like) or premolten globule-like (PMG-like) proteins (from ref. 34) has been plotted against the mean residue ellipticity at 200 nm (Θ₂₀₀). The position in the plot of Henipavirus N_TAIL proteins is highlighted. (B) Plot of the ratio between the ellipticity at 222 nm and the ellipticity at 200 nm (Θ₂₂₂/Θ₂₀₀) of the same set of well-characterized RC-like or PMG-like proteins shown in (A). The position in the plot of Henipavirus N_TAIL proteins is highlighted. The dashed line corresponds to the boundary between RC-like and PMG-like proteins. (C) CH-ellipticity plot of Henipavirus N_TAIL proteins. For each N_TAIL protein, the distance from the boundary in the CH plot, referred to as CH distance, has been plotted as a function of the distance from the boundary in the Θ₂₂₂/Θ₂₀₀ plot shown in (B).

We then plotted the ratio between the Θ₂₂₂ and Θ₂₀₀ of the N_TAIL proteins and of a set of well-characterized RC-like and PMG-like proteins³⁴ (Fig. 6B). This ratio has the advantage over the individual Θ₂₂₂ and Θ₂₀₀ of being less affected by errors in estimations of protein concentrations, and of providing a unified parameter reflecting the content in both unordered and α-helical structure. We then arbitrarily set a threshold of the Θ₂₂₂/Θ₂₀₀ (see the dashed line in Fig. 6B) that allows RC-like IDPs to be discriminated from IDPs adopting a PMG-like conformation.

We then generated a plot in which the distance of each N_TAIL protein from this threshold was plotted as a function of its CH-distance (Fig. 6C). This analysis is intended to combine, and hence extend, the two methods previously introduced by Uversky.^34,58 Indeed, it combines a computational and a spectroscopic analysis and is in principle meant to allow RC-like forms to be distinguished from PMG-like forms among proteins predicted to be intrinsically disordered by the hydropathy/charge method. In this plot, increasingly negative CH distances designate proteins with increasing disorder, while increasingly positive θ₂₂₂/θ₂₀₀ distances designate IDPs becoming progressively more collapsed, as a consequence of an increased content in regular secondary structure. Thus, the left bottom quadrant is expected to correspond to IDPs adopting a RC-like conformation, while the right bottom quadrant is supposed to designate IDPs adopting a PMG conformation.

Surprisingly however, in the case of the N_TAIL proteins, this analysis revealed that the HeV and NiV N_TAILΔ3Δ4 proteins, which are predicted to be the most ordered by the charge/hydropathy method and which additionally were also shown to be the most compact forms by the hydrodynamic analyses (see Table 1), are the N_TAIL proteins exhibiting the lowest distance from the θ₂₂₂/θ₂₀₀ boundary (see Fig. 6B) and hence expected to adopt the most extended conformation (see Fig. 6C). Thus, this analysis would wrongly predict the HeV and NiV N_TAILΔ3Δ4 proteins to be more extended than the parental, full-length forms by virtue of their increased content in unordered structure and decreased content in α-helical structure. CD studies allowed the lower α-helical content of the HeV and NiV N_TAILΔ3Δ4 proteins to be unambiguously accounted for by the lack of Box3, with the MoRE located within Box3 having been shown to be the one with the most pronounced α-helical propensity (Fig. 5 and Fig. S2, ESI‡).

The present data suggest that the amount of regular secondary structure is not seemingly a major determinant of protein compaction, with IDPs depleted in MoREs being nevertheless able to adopt a collapsed state. The bottom line of these observations is that the distance from the θ₂₂₂/θ₂₀₀ boundary, while being informative in terms of the relative content in unordered and α-helical structure, is not a reliable indicator of the extent of compaction of the Henipavirus N_TAIL proteins. Whether this is a unique property of the Henipavirus N_TAIL proteins or rather a general rule of IDPs remains to be established.

Binding affinities between P_XD and N_TAIL deletion proteins

In order to precisely assess the role of the various boxes in binding to P_XD, we investigated the binding abilities of the N_TAIL deletion proteins using isothermal titration calorimetry (ITC), an approach that gives access to the stoichiometry, the equilibrium association constant, and the variation in enthalpy and entropy.⁸⁸ Purified N_TAIL proteins were loaded into the calorimeter sample cell, and titrated with the corresponding P_XD, achieving a molar ratio of 2 (2.5 for the NiV N_TAILΔ1Δ2/P_XD pair) at the end of the titration (Fig. 7). The data, following integration and correction for the heats of dilution, were fit with a standard model allowing for a set of independent and equivalent binding sites (Fig. 7). The equilibrium dissociation constant (K_D) was derived from the K_A, as the reciprocal of this latter. The estimates for the model parameters (see Table 2) confirm a 1 : 1 stoichiometry, as already reported for the parental, full-length N_TAIL proteins.²¹ Results revealed that the binding reactions are both enthalpy and entropy-driven, with the only exception of the NiV N_TAILΔ1Δ2–P_XD and NiV N_TAILΔ4–P_XD pairs for which an unfavorable (−4.50 cal mol⁻¹ deg⁻¹) and small favorable (0.615 cal mol⁻¹ deg⁻¹) entropic contributions, respectively, were found. Interestingly, this was also the case of the binding reaction of the full-length NiV N_TAIL protein, that was found to have a similarly unfavorable (−4.84 cal mol⁻¹ deg⁻¹) entropic contribution.²¹ For both viruses, the measured K_D for the N_TAILΔ1, N_TAILΔ1Δ2 and N_TAILΔ4 proteins are close to the values found for the parental, full-length proteins (2.1 μM for the NiV pair and 8.7 μM for the HeV pair) (cf.Tables 2 and 3), arguing for the lack of involvement of Box1, Box2 and Box4 in P_XD binding. Conversely, under similar experimental conditions, no binding was observed for the N_TAILΔ3Δ4 proteins, thereby precluding estimation of the K_D (Fig. 7). Note that experiments with 1.5 mM N_TAILΔ3Δ4 and 15 mM P_XD, which expectedly might have corresponded to an experimental condition suitable for obtaining interpretable binding curves, were not feasible because of limitations in protein solubility. A rough estimation of the K_D between N_TAILΔ3Δ4 and P_XD that would reflect as low as 1–10% of complex formation, which could indeed escape detection, can however be derived from the following equation:According to eqn (1), the corresponding calculated equilibrium dissociation constants would be approximately 13 mM and 148 mM, assuming, respectively, 1% or 10% of complex formation. A K_D in this range would support a fortuitous encounter at best, and would thus not be physiologically relevant.

Fig. 7 ITC studies of homologous complex formation between P_XD and HeV (A) or NiV (B) N_TAIL deletion proteins. Data are representative of at least two independent experiments where the initial concentrations of N_TAIL proteins in the microcalorimeter cell and of the homologous P_XD in the microsyringe were slightly tuned. Panels (A) and (B) show the data obtained using 150 μM N_TAIL and 1.5 mM P_XD. Graphs shown in the bottom of each panel correspond to integrated and corrected ITC data fit to a single set of sites model (all sites identical and equivalent). The filled squares represent the experimental data, whereas the solid line corresponds to the model. The derived equilibrium dissociation constant (K_D) as well as the stoichiometric number are shown.

Table 2 Equilibrium dissociation constants and binding parameters for the binding reactions between N_TAIL truncated proteins and the homologous P_XD as derived from ITC studies. Data are representative of two independent trials

	Stoichiometry, n	K D (μM)	Binding enthalpy, ΔH (cal mol^−1)	ΔS (cal mol^−1 deg^−1)
HeV NTAILΔ1	0.92 ± 0.015	5.18 ± 0.85	−2771 ± 69.87	14.7
HeV NTAILΔ1Δ2	0.93 ± 0.010	3.98 ± 0.48	−4082 ± 61.41	10.8
HeV NTAILΔ4	0.72 ± 0.015	7.14 ± 1.10	−4222 ± 119.9	9.14
NiV NTAILΔ1	1.31 ± 0.036	9.00 ± 2.10	−2305 ± 95.52	15.2
NiV NTAILΔ1Δ2	0.75 ± 0.016	7.24 ± 1.09	−8184 ± 241.6	−4.5
NiV NTAILΔ4	0.91 ± 0.02	9.00 ± 1.63	−6586 ± 238.4	0.615

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Table 3 Equilibrium dissociation constants and binding parameters for the binding reactions between homologous and heterologous Henipavirus N_TAIL–P_XD complexes as derived from ITC studies. Data are representative of two independent trials

	Stoichiometry, n	K D (μM)	Binding enthalpy, ΔH (cal mol^−1)	Binding entropy, ΔS (cal mol^−1 deg^−1)
a Data were taken from ref. 21.
HeV NTAIL–HeV PXD^a	1.37 ± 0.01	8.7 ± 0.55	−5584 ± 62	4.09
NiV NTAIL–HeV PXD	1.02 ± 0.007	7.6 ± 0.46	−6001 ± 56.38	2.92
NiV NTAIL–NiV PXD^a	0.93 ± 0.07	2.1 ± 0.24	−9034 ± 108.9	−4.84
HeV NTAIL–NiV PXD	1.12 ± 0.21	6.9 ± 1.32	−2494 ± 81.24	15.1

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The present results support a role for Box3 as a major determinant of P_XD binding. These findings validate the previously proposed involvement of Box3 in complex formation, and provide additional support to our structural models of the Henipavirus N_TAIL–P_XD complexes.²¹

Having shown that Box3 is the N_TAIL region indispensable for binding to P_XD, we sought at assessing whether the Box3 regions could be functionally interchanged between the two viruses. We therefore carried out ITC studies in which the parental full-length N_TAIL proteins were loaded in the cell and then titrated with the heterologous P_XD protein (Fig. S3, ESI‡ and Table 3). As shown in Table 3, replacing HeV N_TAIL by NiV N_TAIL in the binding reaction with HeV P_XD led to binding parameters quite comparable (i.e. within the error bars) to those observed for the homologous pair. Substituting NiV N_TAIL with HeV N_TAIL in the binding reaction to NiV P_XD yielded a slight, though significant, increase in the K_D with a concomitant variation in the enthalpic and entropic contributions: while the enthalpic contribution is reduced for the heterologous pair, the entropic penalty is suppressed and the ΔS becomes favorable, possibly reflecting a less pronounced disorder-to-order transition. In support of the significance of the interaction, it should be noted that no interaction was found between Henipavirus N_TAIL and measles virus P_XD (Habchi et al., unpublished).

These data suggest that the HeV and NiV Box3 regions can be functionally replaced by each other in binding to P_XD, consistent with previous studies showing that Henipavirus N–P can form heterologous complexes.²³ The HeV P_XD scaffold seems to be more tolerant (i.e. equally able to accommodate the homologous and the heterologous Box3 region) than the NiV P_XD. We can speculate that this difference could reflect the higher buried surface area (and hence the higher surface complementarity) of the NiV N_TAIL–P_XD complex as compared to the HeV one.²¹

P_XD-induced folding of N_TAIL deletion proteins

In order to be able to draw definitive conclusions about the role of Box3 not only in osmolyte-induced folding but also in P_XD-induced folding, we carried out far-UV CD studies where the various N_TAIL deletion proteins were mixed with the homologous P_XD. The experimental design was the same already used to document P_XD-induced α-helical folding of parental, full-length N_TAIL proteins.²¹ Briefly, after recording the CD spectra of individual P_XD and N_TAIL proteins, for each N_TAIL protein we also recorded the CD spectrum of a mixture containing a two-fold molar excess of the homologous P_XD. Under these experimental conditions, the N_TAIL and P_XD concentrations (350 μM and 700 μM, respectively) were well above the estimated K_D (and hence expected to lead to 100% complex formation) of the various N_TAIL–P_XD pairs, with the only exception of the N_TAILΔ3Δ4–P_XD pair for which the K_D could not be determined. In the case of the N_TAILΔ1, N_TAILΔ1Δ2 and N_TAILΔ4 proteins from both viruses, the observed CD spectra of the mixtures differed from the corresponding theoretical average curves calculated from the individual N_TAIL and P_XD spectra (Fig. 8). Since the theoretical average curves correspond to the spectra that would be expected if no structural variations occur, deviations from these curves indicate structural transitions. The experimentally observed spectra of the mixtures support an α-helical transition, as judged from the more pronounced minima at 208 and 222 nm, and by the higher ellipticity at 190 nm of the experimentally observed spectra compared to the corresponding theoretical average curves (Fig. 8). By contrast, P_XD was found to be unable to trigger such an α-helical transition in both HeV and NiV N_TAILΔ3Δ4 proteins, as judged from the good superimposition of the spectra of N_TAIL + P_XD mixtures with the corresponding average spectra (Fig. 8). These results indicate that Box3 is critical for N_TAIL to undergo P_XD-induced α-helical folding, since a form devoid of this box fails to fold onto P_XD.

Fig. 8 Far-UV CD studies of HeV (A) and NiV (B) N_TAIL proteins either alone (black line) or in the presence of a two-fold molar excess of the homologous P_XD protein (full circles). The CD spectra of P_XD proteins alone (grey line) are also shown, as are the theoretical average curves (empty circles) calculated by assuming that no structural variations occur (see Experimental). Spectra were recorded at 20 °C in 10 mM Tris/HCl pH 7.5, NaCl 200 mM. Protein concentrations were 10 mg mL⁻¹. In N_TAIL/P_XD mixtures the N_TAIL concentration was 350 μM, while that of P_XD was 700 μM. The path length was 0.01 mm. Data are representative of at least two independent experiments.

Conclusions

In the present work, we carried out an in-depth biochemical characterization of Henipavirus N_TAIL deletion proteins that bear different combinations of four putative MoREs.

The first interesting observation that we made concerns the anomalous electrophoretic behavior of the N_TAIL proteins, which could not be exclusively accounted for by their content in acidic residues, suggesting that other additional parameters can be responsible for the abnormal electrophoretic migration of IDPs in SDS-PAGE. A possible hint in this regard is provided by the behavior of the NiV N_TAILΔ1Δ2 protein. Indeed, this protein was found to display the most aberrant electrophoretic mobility. Besides, its electrophoretic behavior is more anomalous than that of the cognate HeV N_TAIL protein. While the two proteins have the same content in acidic residues, the NiV N_TAILΔ1Δ2 protein was found to adopt a more extended conformation than the corresponding HeV protein. This observation suggests that the degree of protein extension in solution could be a possible additional parameter affecting the electrophoretic mobility of IDPs. In further support of this hypothesis, analysis of the N_TAIL proteins by native polyacrylamide gel electrophoresis (see Fig. S4, ESI‡) clearly shows that the NiV N_TAILΔ1Δ2 protein migrates much more slowly than the HeV N_TAILΔ1Δ2 protein, consistent with the lower compaction of the NiV N_TAILΔ1Δ2 protein as compared to the HeV one. In the same vein, the NiV N_TAILΔ4 protein migrates much faster than the HeV N_TAILΔ4 protein, reflecting the higher extent of compaction of the NiV protein as compared to the HeV one.

By combining computational, hydrodynamic and spectroscopic analyses, we could unveil regions playing a major role in protein compaction, as well as regions sampling transiently populated α-helices (Fig. 9A). In particular, the spectroscopic studies herein described revealed the structural state of Box2. The present results also support the occurrence within Henipavirus N_TAIL of two I-MoREs (e.g. Box1 and Box4) and of two α-MoREs (e.g. Box2 and Box3), thus arguing for the lack of a transiently populated β structure within Box2 (Fig. 9A). Among the two α-MoREs, Box3 was found to be a major determinant of α-helical folding and ITC studies showed that this region is strictly required for binding to P_XD (Fig. 9A). These results confirm the previously postulated role of Box3 in binding to P_XD and provide direct experimental support for the proposed Henipavirus N_TAIL–P_XD complexes.²¹ In addition, ITC studies in which the full-length N_TAIL proteins were titrated with the heterologous P_XD, showed that the two Box3 regions are functionally interchangeable between the two viruses, consistent with previous immunoprecipitation studies showing that Henipavirus N–P can form heterologous complexes.²³ The HeV P_XD was found to be seemingly more tolerant than the NiV P_XD (i.e. equally able to accommodate the homologous and the heterologous Box3 region), with this property possibly reflecting a higher surface complementarity in the case of the NiV N_TAIL–P_XD complex as compared to the HeV one.²¹

Fig. 9 (A) Sequence alignment between the HeV and NiV N_TAIL proteins. Hydrophobic residues (L, I, V, F, W and M) are shown in green, acidic residues in red and basic residues in blue. Asterisks and colons below the alignment denote identity and similarity, respectively, while dots indicate weakly similar residues. Lack of symbol below the alignment denotes strongly different residues. The front numbers correspond to the amino acid position in the N sequence. Dots above the alignment indicate intervals of 10 residues. The four boxes are shown, as is the location of the transiently populated α-helices, as unveiled by CD studies. (B) Schematic diagram summarizing the data obtained by computational and hydrodynamic analyses. The red and green arrows highlight the discrepancies found between the two viruses in terms of compaction properties of N_TAILΔ1Δ2 and N_TAILΔ4 proteins.

Strikingly, and in agreement with previous studies,⁶⁰ the content in regular secondary structure was found not to be a major determinant of protein compaction, with N_TAIL proteins depleted in MoREs being nevertheless able to adopt a collapsed state (Fig. 9B). Indeed, removal of Box3 plus Box4, where Box3 has a strong inherent α-helical propensity as judged from CD studies, led to a more compact form (see Fig. 9B). This finding suggests that the region downstream Box3 (i.e. residues 493–532) is endowed with a large conformational freedom that confers to the full-length protein an overall extended character. Thus, the occurrence of a transiently populated α-helix within Box3 would not impart strong compaction constraints to N_TAIL and would be unable to compensate for the high flexibility of the downstream region. Lack of a strict relationship between the content in regular secondary structure and protein compaction is further supported by hydrodynamic and spectroscopic studies focused on N_TAILΔ1, which showed that Box1 plays a role as a major determinant of compaction, with this property being quite independent from its ability to adopt a regular secondary structure: indeed CD studies unveiled that removal of Box1 does not lead to any significant reduction in the α-helical content nor does it affect the α-helical folding abilities of N_TAILΔ1 as compared to the full-length form (see Fig. 9). Therefore, in the case of the N_TAIL proteins, the PMG state likely mainly arises from the occurrence of either long-range or short-range tertiary contacts rather than from local constraints imposed by transiently populated regular secondary structure elements. The occurrence of long-range tertiary contacts within IDPs has been already well documented (for examples see ref. 89–97), suggesting that long-range contacts may serve as major determinants of protein compaction not only in Henipavirus N_TAIL proteins but in IDPs in general. The present findings are expected to stimulate future studies in this field and hence to shed light on the conformational behavior of IDPs and on how this latter is encoded by the amino acid sequence.

We also devised a method that combines computational and spectroscopic analyses, which was meant to allow RC-like IDPs to be discriminated from PMG-like IDPs. This method, which is a combination of two methods previously introduced by Uversky,^34,58 revealed once again that the content in unordered and α-helical structure is a poor predictive criterion for the prediction of protein compaction. These findings somehow challenge the well-established notion that PMG-like IDPs have more residual secondary structures than RC-like IDPs.³⁴

Given the poor agreement between the hydrodynamic and the in silico analyses and the inconsistencies between the two viruses in the case of the N_TAILΔ1Δ2 and N_TAILΔ4 proteins (see Fig. 9B), no definitive conclusions could be drawn as to the role of Box2 and Box4 in determining protein compaction. We reasoned that the observed discrepancies might be accounted for by differences in the N_TAIL sequence leading to a different pattern in the tertiary contacts (whether long- or short-range) in the two viruses. In order to get insights into this issue, we compared the amino acid sequences of the HeV and NiV N_TAIL proteins in view of highlighting possible differences. As shown in Fig. 9A, the N_TAIL sequence is highly conserved in the two viruses (76% identity and 90% similarity). As expected for disordered regions not predicted to be involved in partner recognition, the regions connecting Box1 to Box2 and Box3 to Box4 are the most variable ones. The local sequence variability between Box3 and Box4 might account for conformational differences between HeV and NiV N_TAILΔ1Δ2. On the other hand, the conformational differences between the HeV and NiV N_TAILΔ4 proteins are more difficult to explain. Indeed, the possibility that the differences occurring in the region between Box1 and Box2 could be responsible for the differences between HeV and NiV N_TAILΔ4 can be ruled out, based on the conformational similarity between HeV and NiV N_TAILΔ3Δ4. Likewise, since the variable region between Box3 and Box4 does not seemingly lead to any significant difference in the conformational behavior of the HeV and NiV N_TAILΔ1 proteins, it is difficult to explain how differences in this region could be responsible for the observed conformational differences between HeV and NiV N_TAILΔ4. More subtle parameters are certainly at play, which remain however elusive so far.

As for Box3, while residues 474–487 are fully conserved in the two viruses, the last five residues are not (Fig. 9A). Given the functional equivalence of Box3 between the two viruses, this observation may imply that either residues 488–493 are not as much involved in P_XD binding as the upstream residues, or that the partner can equally accommodate the IKESTA and AKEAAS stretches.

In conclusion, the present studies provide the first detailed mapping of the P_XD binding region within N_TAIL and provide the first quantitative estimation of the ability of Henipavirus N_TAIL and P_XD to form heterologous complexes. These studies designate specific Henipavirus N_TAIL regions as targets for future site-directed mutagenesis studies and/or for the design of inhibitors capable to block the N_TAIL–P_XD interaction.

Experimental

Bioinformatics analyses

Sequences for this study were obtained from the VaZyMolO data base.⁹⁸ Sequence accession numbers for N_TAIL are VaZy82 (HeV) and VaZy1 (NiV). Disorder predictions were carried out using the MeDor metaserver for disorder prediction⁵⁵ and the PONDR server (http://www.disorder.wsu.edu/) using the default integrated predictor VL-XT.^56,57 Access to PONDR was provided by Molecular Kinetics (P.O. Box 2475 CS, Pullman, WA 99165-2475; E-mail: mkinetics@turbonet.com) under license from the WSU Research Foundation. PONDR is copyright ©1999 by the WSU Research Foundation, all rights reserved.

The charge–hydropathy (CH) plot was generated as described in ref. 34. The CH plot is divided into two regions by a line, which corresponds to the equation H = (R + 1.151)/2.785, where R is the mean net charge and H is the mean hydrophobicity. In the left part of the diagram (where H < (R + 1.151)/2.785), a protein is predicted as disordered, whereas it is predicted as ordered in the right part.³⁴H_Boundary was computed according to ref. 34: H_Boundary = (R + 1.15)/2.785. The mean net charge (R) of a protein is defined as the absolute value of the difference between the number of positively and negatively charged residues at pH 7 divided by the total number of amino acid residues. It was calculated using the program ProtParam at the EXPASY server (http://www.expasy.ch/tools). The mean hydrophobicity (H) is the sum of normalized hydrophobicities of individual residues divided by the total number of amino acid residues minus 4 residues (to take into account fringe effects in the calculation of hydrophobicity). Individual hydrophobicities were determined using the Protscale program at the EXPASY server (http://www.expasy.ch/tools), using the options “Hphob/Kyte & Doolittle”, a window size of 5, and normalizing the scale from 0 to 1.

The net charge per residue (NCR) of the various N_TAIL truncated proteins was calculated as described in ref. 59 as the difference between the fraction of positively charged residues (f+) and the fraction of negatively charged residues (f−).

The pairwise sequence alignment between the HeV and NiV N_TAIL proteins was obtained using ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index.html).⁹⁹ Sequence similarity and identity were calculated using the Emboss program¹⁰⁰ (http://www.ebi.ac.uk/Tools/emboss/align/index.html).

Construction of expression plasmids

The Henipavirus pDest14/N_TAILHN constructs, encoding residues 400–532 of either HeV or NiV N with a hexahistidine tag fused to their N-termini, and the pDest14/P_XD constructs, encoding residues 660–709 (NiV) or 657–707 (HeV) of P with a hexahistidine tag fused to their C-termini, have already been described.^21,29

All N_TAIL constructs were obtained by PCR using Phusion (Finnzymes) polymerase and synthetic N genes (GenScript) optimized for the expression in E. coli, as templates. Primers (Operon) were designed to introduce an AttB1 and AttB2 sites at the 5′ and 3′ ends, respectively, and to amplify the desired part of the N_TAIL ORF. After digestion with DpnI (New England Biolabs) to remove the methylated DNA template, and purification (Nucleospin Extract II, Macherey-Nagel), the PCR products were cloned using the Gateway® recombination system (Invitrogen) into the pDest17OI expression vector, a modified pDest17® (Invitrogen) to which LacO and LacI were inserted to allow a better control of protein expression.¹⁰¹ The resulting constructs encode N-terminally histidine tagged proteins in which the native sequence is preceded by a vector-encoded MSYYHHHHHHLESTSLYKKAGS amino acid stretch. The pDest17OI/N_TAILΔ1 and pDest17OI/N_TAILΔ1Δ2 gene constructs encode residues 423–532 and 465–532 of Henipavirus N proteins, respectively. The pDest17OI/N_TAILΔ4 and pDest17OI/N_TAILΔ3Δ4 gene constructs encode residues 400–522 and 400–472 of Henipavirus N proteins, respectively.

Selection and amplification of DNA constructs were carried out using CaCl₂-competent E. coli TAM1 cells (Active Motif). The sequence of the coding region of all expression plasmids was verified by sequencing (GATC Biotech) and found to conform to expectations.

Bacterial expression and purification of Henipavirus P_XD and N_TAIL constructs

The E. coli Rosetta [DE3] pLysS strain (Novagen) was used for the expression of all the Henipavirus PXD and N_TAIL constructs. This strain, which is optimized for the expression of recombinant proteins, also carries the lysozyme gene thus allowing a tight regulation of the expression of the recombinant gene, as well as a facilitated lysis. Cultures were grown overnight to saturation in 2YT medium containing 100 μg mL⁻¹ ampicillin and 34 μg mL⁻¹ chloramphenicol. An aliquot of the overnight culture was diluted 1/25 in 2YT medium and grown at 37 °C. When the optical density at 600 nm (OD₆₀₀) reached 0.6–0.8, isopropyl β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM, and the cells were grown at 37 °C for 3 additional hours. The induced cells were harvested, washed and collected by centrifugation (5000 g, 10 min). The resulting pellets were frozen at −20 °C.

The N_TAIL truncated proteins were purified using the protocol already described for Henipavirus wt N_TAIL proteins²⁹ with minor modifications. Briefly, cellular pellets were resuspended in 5 volumes (v/w) buffer A (10 mM Tris/HCl pH 8, 300 mM NaCl, 10 mM Imidazole, 1 mM phenyl-methyl-sulfonyl-fluoride – PMSF) supplemented with lysozyme 0.1 mg mL⁻¹, DNAse I 10 μg mL⁻¹, 20 mM MgSO₄ and protease inhibitor cocktail (Roche, one tablet per 50 mL of bacterial lysate). After a 20 min incubation with gentle agitation, the cells were disrupted by sonication (using a 750 W sonicator and 4 cycles of 30 s each at 45% power output). The lysate was clarified by centrifugation at 30 000 g for 30 min. Starting from a 1 L culture, the clarified supernatant was incubated for 1 h with gentle shaking with 4 mL Chelating Sepharose Fast Flow Resin preloaded with Ni²⁺ ions (GE, Healthcare), previously equilibrated in buffer A. The resin was washed with buffer A supplemented with 20 mM imidazole, and the recombinant protein was eluted in buffer A supplemented with 250 mM imidazole. Eluates were analyzed by SDS-PAGE for the presence of the desired protein product. The fractions containing the recombinant protein were combined, and then loaded onto a Superdex 200 HR 16/60 column (GE, Healthcare). The elution buffer was 50 mM Tris/HCl pH 7.5, supplemented with either 300 mM (N_TAILΔ1 and N_TAILΔ4) or 500 mM (N_TAILΔ1Δ2 and N_TAILΔ3Δ4) NaCl. Purification of Henipavirus P_XD and full-length N_TAIL samples was carried out as described.²¹

The proteins were concentrated using Centricon Plus-20 (molecular cutoff of 3000 Da for P_XD, and of 5000 Da for N_TAIL) (Millipore). All proteins were stored at −20 °C. Dialysis was used to exchange the buffer and adjust it to the ensuing analyses. All purification steps, except for gel filtrations, were carried out at 4 °C.

Protein concentrations of N_TAIL truncated forms were derived using the theoretical absorption coefficients at 280 nm, as obtained using the program ProtParam at the EXPASY server (http://www.expasy.ch/tools). In the case of P_XD samples, protein concentrations were estimated using the BCA protein assay reagent (Pierce), since estimations based on the theoretical absorption coefficients at 280 nm turned out to be not fully reliable.²¹

The purified N_TAIL proteins were also analyzed by native polyacrylamide gel electrophoresis. In those experiments, migration was carried out in the absence of SDS. In addition, the sample was not boiled before being subjected to electrophoresis and the loading buffer was devoid of both SDS and β-mercaptoethanol. Proteins were thus separated as a function of their inherent charge and compaction.

Mass spectrometry (MALDI-TOF)

The identity of all purified Henipavirus N_TAIL proteins was confirmed by mass spectral analysis of tryptic fragments. The latter was obtained by digesting (0.25 μg trypsin) 1 μg of purified recombinant protein obtained after separation onto SDS-PAGE. The tryptic peptides were analyzed using an Autoflex II TOF/TOF. Spectra were acquired in the linear mode. Samples (0.7 μL containing 15 pmol) were mixed with an equal volume of sinapinic acid matrix solution, spotted on the target, then dried at room temperature for 10 min. The tryptic fragments were analyzed in the Autoflex matrix-assisted laser desorption ionization/time of flight (MALDI-TOF)¹ (Bruker Daltonics, Bremen, Germany). Peptide fingerprints were obtained and compared with in silico protein digest (Biotools, Bruker Daltonics, Germany). The mass standards were either autolytic tryptic peptides or peptide standards (Bruker Daltonics).

Analytical SEC combined with on-line multi-angle laser light scattering (MALLS) and refractometry (RI) and calculations of hydrodynamic radii

Analytical SEC was carried out on a high pressure liquid chromatography (HPLC) system (Alliance 2695, Waters) using silica-based columns (Shodex). A 15 mL KW-802.5 column was used for the characterization of N_TAIL truncated proteins. Proteins were eluted with a 50 mM Tris/HCl pH 7.3, 200 mM NaCl buffer at a flow of 0.5 mL min⁻¹. Separation was performed at room temperature. Typically, 30 μL of a protein solution at 12–23 mg mL⁻¹ in 50 mM Tris/HCl pH 7.5, supplemented with either 300 mM (N_TAILΔ1 and N_TAILΔ4) or 500 mM (N_TAILΔ1Δ2 and N_TAILΔ3Δ4) NaCl, were injected. Detection was performed using a triple-angle light-scattering detector (MiniDAWN™ TREOS, Wyatt Technology), a quasi-elastic light-scattering instrument (Dynapro™, Wyatt Technology) and a differential refractometer (Optilab® rEX, Wyatt Technology). Molecular mass and R_S determination was performed by the ASTRA V software (Wyatt Technology) using a dn/dc value of 0.185 mL g⁻¹. The column was calibrated with proteins of known Stokes radii and molecular masses.

The theoretical Stokes radii (R_S, in Å) expected for a natively folded (R^NF_S), fully unfolded random coil state in urea (R^U_S) and natively unfolded PMG (R^PMG_S) protein with an expected molecular mass (MMtheo) (in Daltons) were calculated according to ref. 61:

log(R^NF_S) = 0.369 × log(MMtheo) − 0.254 (2)

log(R^U_S) = 0.521 × log(MMtheo) − 0.649 (3)

log(R^PMG_S) = 0.403 × log(MMtheo) − 0.239 (4)

The hydrodynamic radius of a natively unfolded protein with N residues was also calculated according to ref. 60 using either the simple power-law model:

R^IDP_S = R₀N^ν (5)

or the sequence-based model:⁶⁰

R^IDP_Sseq = (AP_Pro + B) × (C|Q| + D)S_his × R₀N^ν (6)

where A = 1.24, P_Pro = fractional proline content, B = 0.904, C = 0.00759, |Q| = absolute value of the difference between the total number of negatively charged and positively charged residues, D = 0.963, S_his = 0.901 (correction factor for histidine tag), R₀ = 2.49 and ν = 0.509.

The compaction index (CI) is expressed as:

CI = (R^U_S − R^obs_S)/(R^U_S − R^NF_S) (7)

according to ref. 62.

Circular dichroism (CD)

CD spectra were recorded at 20 °C on a Jasco 810 dichrograph, equipped with a Peltier thermoregulation system. Spectra of N_TAIL truncated proteins in the presence of 20% TFE (Sigma-Aldrich) or 3 M TMAO were recorded using 1 mm thick quartz cells. When recording the CD spectra of N_TAIL + P_XD mixtures, 0.01 mm thick quartz cells were used. In the case of spectra recorded in the presence of TFE or TMAO, the buffer was 10 mM sodium phosphate pH 7. In experiments where the ability of N_TAIL proteins to undergo P_XD-induced folding was assessed, the buffer was 10 mM Tris/HCl pH 7.5, NaCl 200 mM. CD spectra were measured between 190 and 260 nm, with a scanning speed of 20 nm min⁻¹ and a data pitch of 0.2 nm. Spectra were averaged from three scans. Moreover, for each protein sample, at least two independent acquisitions were carried out so as to estimate the experimental error arising from sample preparation. The contribution of buffer was subtracted from experimental spectra. Spectra were smoothed using the “means-movement” smoothing procedure implemented in the SpectraManager package. Structural variations in N_TAIL proteins were measured as a function of changes in the initial CD spectrum upon addition of either 20% TFE or 3 M TMAO or a two-fold molar excess of P_XD to N_TAIL proteins.

Mean ellipticity values per residue ([Θ]) were calculated as [Θ] = 3300 m ΔA/(lcn), where l (path length) in cm, n = number of residues, m = molecular mass in daltons and c = protein concentration expressed in mg mL⁻¹. Number of residues (n) are 132 for N_TAILΔ1 proteins, 90 for N_TAILΔ1,2 proteins, 145 for N_TAILΔ4 proteins, 95 for N_TAILΔ3,4 proteins, 58 for HeV P_XD and 57 for NiV P_XD. Mass (m) values for HeV N_TAIL proteins are 14 519 Da (N_TAILΔ1), 10 012 Da (N_TAILΔ1Δ2), 15 775 Da (N_TAILΔ4), and 10 319 Da (N_TAILΔ3Δ4). Mass values for NiV N_TAIL proteins are 14 286 Da (N_TAILΔ1), 9862 Da (N_TAILΔ1Δ2), 15 482 Da (N_TAILΔ4), and 10 176 Da (N_TAILΔ3Δ4). Mass values for P_XD proteins are 6871 Da for HeV and 6733 Da for NiV. Protein concentrations of 0.1 mg mL⁻¹ were used when recording spectra of N_TAIL proteins either in the absence or in the presence of 20% TFE or 3 M TMAO.

When 0.01 mm thick quartz cells were used, protein concentrations as high as 10 mg mL⁻¹ could be used. Use of high protein concentrations ensured achievement of saturation in induced folding experiments in the presence of the P_XD homologous partner. In the case of protein mixtures (i.e. N_TAIL + P_XD), mean ellipticity values per residue ([Θ]) were calculated as [Θ] = 3300 ΔA/{[(C₁n₁/m₁) + (C₂n₂/m₂)]l}, where l (path length) = 0.001 cm, n₁ or n₂ = number of residues, m₁ or m₂ = molecular mass in daltons and C₁ or C₂ = protein concentration expressed in mg mL⁻¹ for each of the two proteins in the mixture. The theoretical average ellipticity values per residue ([Θ]_Ave), assuming that neither unstructured-to-structured transitions nor secondary structure rearrangements occur, were calculated as follows: [Θ]_Ave = [([Θ]₁n₁) + ([Θ]₂n₂R)]/(n₁ + n₂R), where [Θ]₁ and [Θ]₂ correspond to the measured mean ellipticity values per residue, n₁ and n₂ to the number of residues for each of the two proteins, and R to the excess molar ratio of protein 2.

The experimental data in the 190–260 nm range were analyzed using the DICHROWEB website (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml) which was supported by grants to the BBSRC Centre for Protein and Membrane Structure and Dynamics.^102,103 The CDSSTR deconvolution method was used to estimate the content in secondary structure using the reference protein set 7. For the spectra recorded from TMAO-containing mixtures, which could be recorded in the 210–260 nm range only, the α-helical content was derived from the ellipticity at 222 nm as described in ref. 85.

Isothermal titration calorimetry (ITC)

ITC experiments were carried out on a ITC200 isothermal titration calorimeter (Microcal, Northampton, USA) at 20 °C. In these studies, the concentration of N_TAIL proteins was initially adjusted to 150–180 μM in the microcalorimeter cell (0.2 mL). The corresponding P_XD protein (stock solution at 1.5–1.8 mM) was added from a computer-controlled 40 μL microsyringe via a total of 19 injections of 2 μL each at intervals of 180 s. Whatever the binding reaction being studied, the pair of proteins used in each binding assay were dialyzed against a 10 mM Tris/HCl pH 7.5 buffer, supplemented with 300 mM NaCl, to minimize undesirable buffer-related effects. The dialysis buffer was used in all preliminary equilibration and washing steps.

Heat dilution of the ligand was taken into account from peaks measured after full saturation of the protein sample contained in the microcalorimeter cell by the ligand. A theoretical titration curve was fitted to the experimental data using the ORIGIN software (Microcal). This software uses the relationship between the heat generated by each injection and ΔH° (enthalpy change in cal mole⁻¹), K_A (association binding constant in M⁻¹), n (number of binding sites per monomer), total protein concentration and free and total ligand concentrations. The variation in the entropy (ΔS in cal mol⁻¹ deg⁻¹) of each binding reaction was inferred from the variation in the free energy (ΔG), where this latter was calculated from the following relation: ΔG = −RTLn 1/K_A.

Funding

This work was carried out with the financial support of the Agence Nationale de la Recherche, specific program “Physico-Chimie du Vivant”, ANR-08-PCVI-0020-01, and with the partial support of the CNRS. DB is supported by a joint doctoral fellowship from the Direction Générale de l'Armement (DGA) and the CNRS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Acknowledgements

We wish to thank Nicholas Armstrong and the mass spectrometry platform of the IFR48 of Marseille for mass spectrometry analyses. We also thank Catarina Rodrigues for help with SEC-MALLS measurements. We are very grateful to Vladimir Uversky for kindly providing us with the ellipticity values of a set of well-characterized premolten globule-like and random coil-like proteins.

References

1. R. Stone, Science, 2011, 331, 1128–1131.
2. B. T. Eaton, J. S. Mackenzie and L. F. Wang, Henipaviruses, in Fields Virology, ed. B. N. Fields, D. M. Knipe and P. M. Howley, 5th edn, Lippincott-Raven, Philadelphia, 2007, pp. 1587–1600.
3. J. F. Drexler, V. M. Corman, F. Gloza-Rausch, A. Seebens, A. Annan, A. Ipsen, T. Kruppa, M. A. Muller, E. K. Kalko, Y. Adu-Sarkodie, S. Oppong and C. Drosten, PLoS One, 2009, 4, e6367.
4. M. Enserink, Science, 2004, 303, 1121.
5. D. Butler, Nature, 2004, 429, 7.
6. V. P. Hsu, M. J. Hossain, U. D. Parashar, M. M. Ali, T. G. Ksiazek, I. Kuzmin, M. Niezgoda, C. Rupprecht, J. Bresee and R. F. Breiman, Emerging Infect. Dis., 2004, 10, 2082–2087.
7. N. Homaira, M. Rahman, M. J. Hossain, J. H. Epstein, R. Sultana, M. S. Khan, G. Podder, K. Nahar, B. Ahmed, E. S. Gurley, P. Daszak, W. I. Lipkin, P. E. Rollin, J. A. Comer, T. G. Ksiazek and S. P. Luby, Epidemiol. Infect., 2010, 138, 1630–1636.
8. J. J. Sejvar, J. Hossain, S. K. Saha, E. S. Gurley, S. Banu, J. D. Hamadani, M. A. Faiz, F. M. Siddiqui, Q. D. Mohammad, A. H. Mollah, R. Uddin, R. Alam, R. Rahman, C. T. Tan, W. Bellini, P. Rota, R. F. Breiman and S. P. Luby, Ann. Neurol., 2007, 62, 235–242.
9. L. F. Wang, M. Yu, E. Hansson, L. I. Pritchard, B. Shiell, W. P. Michalski and B. T. Eaton, J. Virol., 2000, 74, 9972–9979.
10. B. T. Eaton, C. C. Broder, D. Middleton and L. F. Wang, Nat. Rev. Microbiol., 2006, 4, 23–35.
11. D. Karlin, S. Longhi and B. Canard, Virology, 2002, 302, 420–432.
12. S. Longhi, V. Receveur-Brechot, D. Karlin, K. Johansson, H. Darbon, D. Bhella, R. Yeo, S. Finet and B. Canard, J. Biol. Chem., 2003, 278, 18638–18648.
13. G. Schoehn, M. Mavrakis, A. Albertini, R. Wade, A. Hoenger and R. W. Ruigrok, J. Mol. Biol., 2004, 339, 301–312.
14. D. Bhella, Measles virus nucleocapsid structure, conformational flexibility and the rule of six, in Measles virus nucleoprotein, ed. S. Longhi, Nova Publishers Inc., Hauppage, NY, 2007.
15. D. Bhella, A. Ralph, L. B. Murphy and R. P. Yeo, J. Gen. Virol., 2002, 83, 1831–1839.
16. D. Bhella, A. Ralph and R. P. Yeo, J. Mol. Biol., 2004, 340, 319–331.
17. M. Ringkjøbing Jensen, G. Communie, E. D. Ribeiro Jr., N. Martinez, A. Desfosses, L. Salmon, L. Mollica, F. Gabel, M. Jamin, S. Longhi, R. W. Ruigrok and M. Blackledge, Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 9839–9844.
18. W. S. Tan, S. T. Ong, M. Eshaghi, S. S. Foo and K. Yusoff, J. Med. Virol., 2004, 73, 105–112.
19. M. Eshaghi, W. S. Tan, S. T. Ong and K. Yusoff, J. Clin. Microbiol., 2005, 43, 3172–3177.
20. S. T. Ong, K. Yusoff, C. L. Kho, J. O. Abdullah and W. S. Tan, J. Gen. Virol., 2009, 90, 392–397.
21. J. Habchi, S. Blangy, L. Mamelli, M. Ringkjobing Jensen, M. Blackledge, H. Darbon, M. Oglesbee, Y. Shu and S. Longhi, J. Biol. Chem., 2011, 286, 13583–13602.
22. K. Halpin, B. Bankamp, B. H. Harcourt, W. J. Bellini and P. A. Rota, J. Gen. Virol., 2004, 85, 701–707.
23. Y. P. Chan, C. L. Koh, S. K. Lam and L. F. Wang, J. Gen. Virol., 2004, 85, 1675–1684.
24. M. Omi-Furutani, M. Yoneda, K. Fujita, F. Ikeda and C. Kai, J. Virol., 2010, 84, 9793–9799.
25. Z. Lou, Y. Xu, K. Xiang, N. Su, L. Qin, X. Li, G. F. Gao, M. Bartlam and Z. Rao, FEBS J., 2006, 273, 4538–4547.
26. T. A. Bowden, M. Crispin, D. J. Harvey, A. R. Aricescu, J. M. Grimes, E. Y. Jones and D. I. Stuart, J. Virol., 2008, 82, 11628–11636.
27. T. A. Bowden, A. R. Aricescu, R. J. Gilbert, J. M. Grimes, E. Y. Jones and D. I. Stuart, Nat. Struct. Mol. Biol., 2008, 15, 567–572.
28. T. A. Bowden, M. Crispin, D. J. Harvey, E. Y. Jones and D. I. Stuart, J. Virol., 2010, 84, 6208–6217.
29. J. Habchi, L. Mamelli, H. Darbon and S. Longhi, PLoS One, 2010, 5, e11684.
30. J. Habchi and S. Longhi, Mol. BioSyst., 2011 10.1039/c1mb05204g.
31. J. Habchi, L. Mamelli and S. Longhi, Structural disorder within the nucleoprotein and phosphoprotein from measles, Nipah and Hendra viruses, in Flexible viruses: structural disorder in viral proteins, ed. V. N. Uversky and S. Longhi, John Wiley and Sons, Hoboken, New Yersey, 2011.
32. A. K. Dunker, J. D. Lawson, C. J. Brown, R. M. Williams, P. Romero, J. S. Oh, C. J. Oldfield, A. M. Campen, C. M. Ratliff, K. W. Hipps, J. Ausio, M. S. Nissen, R. Reeves, C. Kang, C. R. Kissinger, R. W. Bailey, M. D. Griswold, W. Chiu, E. C. Garner and Z. Obradovic, J. Mol. Graphics Modell., 2001, 19, 26–59.
33. A. K. Dunker and Z. Obradovic, Nat. Biotechnol., 2001, 19, 805–806.
34. V. N. Uversky, Protein Sci., 2002, 11, 739–756.
35. V. N. Uversky, Cell. Mol. Life Sci., 2003, 60, 1852–1871.
36. H. J. Dyson and P. E. Wright, Nat. Rev. Mol. Cell Biol., 2005, 6, 197–208.
37. P. Radivojac, L. M. Iakoucheva, C. J. Oldfield, Z. Obradovic, V. N. Uversky and A. K. Dunker, Biophys. J., 2007, 92, 1439–1456.
38. A. K. Dunker, C. J. Oldfield, J. Meng, P. Romero, J. Y. Yang, J. W. Chen, V. Vacic, Z. Obradovic and V. N. Uversky, BMC Genomics, 2008, 9(Suppl 2), S1.
39. A. K. Dunker, I. Silman, V. N. Uversky and J. L. Sussman, Curr. Opin. Struct. Biol., 2008, 18, 756–764.
40. V. N. Uversky, J. Biomed. Biotechnol., 2010, 2010, 568068.
41. V. N. Uversky and A. K. Dunker, Biochim. Biophys. Acta, 2010, 1804, 1231–1264.
42. K. K. Turoverov, I. M. Kuznetsova and V. N. Uversky, Prog. Biophys. Mol. Biol., 2010, 102, 73–84.
43. T. Chouard, Nature, 2011, 471, 151–153.
44. C. J. Oldfield, Y. Cheng, M. S. Cortese, P. Romero, V. N. Uversky and A. K. Dunker, Biochemistry, 2005, 44, 12454–12470.
45. A. Mohan, C. J. Oldfield, P. Radivojac, V. Vacic, M. S. Cortese, A. K. Dunker and V. N. Uversky, J. Mol. Biol., 2006, 362, 1043–1059.
46. V. Vacic, C. J. Oldfield, A. Mohan, P. Radivojac, M. S. Cortese, V. N. Uversky and A. K. Dunker, J. Proteome Res., 2007, 6, 2351–2366.
47. M. Fuxreiter, P. Tompa and I. Simon, Bioinformatics, 2007, 23, 950–956.
48. K. Johansson, J. M. Bourhis, V. Campanacci, C. Cambillau, B. Canard and S. Longhi, J. Biol. Chem., 2003, 278, 44567–44573.
49. R. L. Kingston, D. J. Hamel, L. S. Gay, F. W. Dahlquist and B. W. Matthews, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 8301–8306.
50. D. Karlin, S. Longhi, V. Receveur and B. Canard, Virology, 2002, 296, 251–262.
51. J. M. Bourhis, V. Receveur-Bréchot, M. Oglesbee, X. Zhang, M. Buccellato, H. Darbon, B. Canard, S. Finet and S. Longhi, Protein Sci., 2005, 14, 1975–1992.
52. I. Sambi, P. Gatti-Lafranconi, S. Longhi and M. Lotti, FEBS J., 2010, 277, 4438–4451.
53. P. Tompa, Trends Biochem. Sci., 2002, 27, 527–533.
54. L. M. Iakoucheva, A. L. Kimzey, C. D. Masselon, R. D. Smith, A. K. Dunker and E. J. Ackerman, Protein Sci., 2001, 10, 1353–1362.
55. P. Lieutaud, B. Canard and S. Longhi, BMC Genomics, 2008, 9, S25.
56. X. Li, P. Romero, M. Rani, A. K. Dunker and Z. Obradovic, Genome Inform. Ser., 1999, 10, 30–40.
57. P. Romero, Z. Obradovic, X. Li, E. C. Garner, C. J. Brown and A. K. Dunker, Proteins, 2001, 42, 38–48.
58. V. N. Uversky, J. R. Gillespie and A. L. Fink, Proteins, 2000, 41, 415–427.
59. A. H. Mao, S. L. Crick, A. Vitalis, C. L. Chicoine and R. V. Pappu, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 8183–8188.
60. J. A. Marsh and J. D. Forman-Kay, Biophys. J., 2010, 98, 2383–2390.
61. V. N. Uversky, Eur. J. Biochem., 2002, 269, 2–12.
62. S. Brocca, L. Testa, F. Sobott, M. Samalikova, A. Natalello, E. Papaleo, M. Lotti, L. De Gioia, S. M. Doglia, L. Alberghina and R. Grandori, Biophys. J., 2011, 100, 2243–2252.
63. I. Baskakov, A. Wang and D. W. Bolen, Biophys. J., 1998, 74, 2666–2673.
64. I. Baskakov and D. W. Bolen, J. Biol. Chem., 1998, 273, 4831–4834.
65. I. V. Baskakov, R. Kumar, G. Srinivasan, Y. S. Ji, D. W. Bolen and E. B. Thompson, J. Biol. Chem., 1999, 274, 10693–10696.
66. D. W. Bolen and I. V. Baskakov, J. Mol. Biol., 2001, 310, 955–963.
67. G. Tell, L. Perrone, D. Fabbro, L. Pellizzari, C. Pucillo, M. De Felice, R. Acquaviva, S. Formisano and G. Damante, Biochem. J., 1998, 329, 395–403.
68. Q. X. Hua, W. H. Jia, B. P. Bullock, J. F. Habener and M. A. Weiss, Biochemistry, 1998, 37, 5858–5866.
69. K. Dahlman-Wright and I. J. McEwan, Biochemistry, 1996, 35, 1323–1327.
70. K. Watt, T. J. Jess, S. M. Kelly, N. C. Price and I. J. McEwan, Biochemistry, 2005, 44, 734–743.
71. P. Luo and R. L. Baldwin, Biochemistry, 1997, 36, 8413–8421.
72. A. Cammers-Goodwin, T. J. Allen, S. L. Oslick, K. F. McClure, J. H. Lee and D. S. Kemp, J. Am. Chem. Soc., 1996, 118, 3082–3090.
73. M. Buck, H. Schwalbe and C. M. Dobson, Biochemistry, 1995, 34, 13219–13232.
74. P. Fan, C. Bracken and J. Baum, Biochemistry, 1993, 32, 1573–1582.
75. Y. Luo and R. L. Baldwin, J. Mol. Biol., 1998, 279, 49–57.
76. V. N. Uversky, V. P. Kutyshenko, N. Protasova, V. V. Rogov, K. S. Vassilenko and A. T. Gudkov, Protein Sci., 1996, 5, 1844–1851.
77. A. Wang and D. W. Bolen, Biochemistry, 1997, 36, 9101–9108.
78. M. Van Hoy, K. K. Leuther, T. Kodadek and S. A. Johnston, Cell, 1993, 72, 587–594.
79. J. M. Mouillon, P. Gustafsson and P. Harryson, Plant Physiol., 2006, 141, 638–650.
80. V. Belle, S. Rouger, S. Costanzo, E. Liquiere, J. Strancar, B. Guigliarelli, A. Fournel and S. Longhi, Proteins: Struct., Funct., Bioinf., 2008, 73, 973–988.
81. I. V. Baskakov, R. Kumar, G. Srinivasan, Y. S. Ji, D. W. Bolen and E. B. Thompson, J. Biol. Chem., 1999, 274, 10693–10696.
82. R. Kumar, J. M. Serrette, S. H. Khan, A. L. Miller and E. B. Thompson, Arch. Biochem. Biophys., 2007, 465, 452–460.
83. L. B. Dyksterhuis, E. A. Carter, S. M. Mithieux and A. S. Weiss, Arch. Biochem. Biophys., 2009, 487, 79–84.
84. M. Couturier, M. Buccellato, S. Costanzo, J. M. Bourhis, Y. Shu, M. Nicaise, M. Desmadril, C. Flaudrops, S. Longhi and M. Oglesbee, J. Mol. Recognit., 2010, 23, 301–315.
85. J. K. Myers, C. N. Pace and J. M. Scholtz, Biochemistry, 1997, 36, 10923–10929.
86. M. Foucault, K. Mayol, V. Receveur-Brechot, M. C. Bussat, C. Klinguer-Hamour, B. Verrier, A. Beck, R. Haser, P. Gouet and C. Guillon, Proteins, 2009, 78, 1441–1456.
87. S. M. Kelly and N. C. Price, Curr. Protein Pept. Sci., 2000, 1, 349–384.
88. M. W. Freyer and E. A. Lewis, Methods Cell Biol., 2008, 84, 79–113.
89. P. Bernado, C. W. Bertoncini, C. Griesinger, M. Zweckstetter and M. Blackledge, J. Am. Chem. Soc., 2005, 127, 17968–17969.
90. M. M. Dedmon, K. Lindorff-Larsen, J. Christodoulou, M. Vendruscolo and C. M. Dobson, J. Am. Chem. Soc., 2005, 127, 476–477.
91. C. W. Bertoncini, Y. S. Jung, C. O. Fernandez, W. Hoyer, C. Griesinger, T. M. Jovin and M. Zweckstetter, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 1430–1435.
92. C. W. Bertoncini, C. O. Fernandez, C. Griesinger, T. M. Jovin and M. Zweckstetter, J. Biol. Chem., 2005, 280, 30649–30652.
93. P. Vise, B. Baral, A. Stancik, D. F. Lowry and G. W. Daughdrill, Proteins, 2007, 67, 526–530.
94. D. F. Lowry, A. C. Hausrath and G. W. Daughdrill, Proteins, 2008, 73, 918–928.
95. D. F. Lowry, A. Stancik, R. M. Shrestha and G. W. Daughdrill, Proteins, 2008, 71, 587–598.
96. M. K. Cho, G. Nodet, H. Y. Kim, M. R. Jensen, P. Bernado, C. O. Fernandez, S. Becker, M. Blackledge and M. Zweckstetter, Protein Sci., 2009, 18, 1840–1846.
97. L. Salmon, G. Nodet, V. Ozenne, G. Yin, M. R. Jensen, M. Zweckstetter and M. Blackledge, J. Am. Chem. Soc., 2010, 132, 8407–8418.
98. F. Ferron, C. Rancurel, S. Longhi, C. Cambillau, B. Henrissat and B. Canard, J. Gen. Virol., 2005, 86, 743–749.
99. M. A. Larkin, G. Blackshields, N. P. Brown, R. Chenna, P. A. McGettigan, H. McWilliam, F. Valentin, I. M. Wallace, A. Wilm, R. Lopez, J. D. Thompson, T. J. Gibson and D. G. Higgins, Bioinformatics, 2007, 23, 2947–2948.
100. P. Rice, I. Longden and A. Bleasby, Trends Genet., 2000, 16, 276–277.
101. R. Vincentelli, S. Canaan, V. Campanacci, C. Valencia, D. Maurin, F. Frassinetti, L. Scappucini-Calvo, Y. Bourne, C. Cambillau and C. Bignon, Protein Sci., 2004, 13, 2782–2792.
102. L. Whitmore and B. A. Wallace, Nucleic Acids Res., 2004, 32, W668–W673.
103. L. Whitmore and B. A. Wallace, Biopolymers, 2008, 89, 392–400.

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Footnotes

† Published as part of a Molecular BioSystems themed issue on Intrinsically Disordered Proteins; Guest Editor: M. Madan Babu.

‡ Electronic supplementary information (ESI) available: Fig. S1–S4 and Tables S1 and S2. See DOI: 10.1039/c1mb05401e

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