Biochimica et Biophysica Acta 1844 (2014) 2306–2314

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Intrinsic disorder within the erythrocyte binding-like proteins from Plasmodium falciparum Manuel Blanc a,b,g, Theresa L. Coetzer c, Martin Blackledge d,e,f, Michael Haertlein b, Edward P. Mitchell a,g, V. Trevor Forsyth a,b, Malene Ringkjøbing Jensen d,e,f,⁎ a

Faculty of Natural Sciences and Institute for Science & Technology in Medicine, Keele University, Staffordshire ST5 5BG, UK Life Sciences Group, Institut Laue-Langevin, 71 avenue des Martyrs, 38000 Grenoble, France Wits Research Institute for Malaria (WRIM), Faculty of Health Sciences, University of the Witwatersrand, National Health Laboratory Service, Johannesburg, South Africa d Univ. Grenoble Alpes, IBS, F-38044 Grenoble, France e CNRS, IBS, F-38044 Grenoble, France f CEA, DSV, IBS, F-38044 Grenoble, France g ESRF, 71 avenue des Martyrs, CS 40220, 38043 Grenoble Cedex 9, France b c

a r t i c l e

i n f o

Article history: Received 7 August 2014 Received in revised form 18 September 2014 Accepted 26 September 2014 Available online 5 October 2014 Keywords: Malaria Erythrocyte binding-like protein Disorder prediction Nuclear magnetic resonance Chemical shift Residual dipolar coupling

a b s t r a c t The ability of the malaria parasite, Plasmodium falciparum, to proliferate within the human host depends on its invasion of erythrocytes. Erythrocyte binding-like (EBL) proteins play crucial roles in the attachment of merozoites to human erythrocytes by binding to specific receptors on the cell surface. In this study, we have carried out a bioinformatics analysis of the three EBL proteins EBA-140, EBA-175 and EBA-181 and show that they contain a large amount of intrinsic disorder in particular within the RIII–V domains. The functional role of these domains has so far not been identified, although antibodies raised against these regions were shown to inhibit parasite invasion. Here, we obtain a more complete structural and dynamic view of the EBL proteins by focusing on the biophysical characterization of a smaller construct of the RIII–V regions of EBA-181 (EBA-181945–1097). We show using a number of techniques that EBA-181945–1097 is intrinsically disordered, and we obtain a detailed structural and dynamic characterization of the protein at atomic resolution using nuclear magnetic resonance (NMR) spectroscopy. Our results show that EBA-181945–1097 is essentially a statistical coil with the presence of several turn motifs and does not possess transiently populated secondary structures as is common for many intrinsically disordered proteins that fold via specific, pre-formed molecular recognition elements. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Malaria remains a major global health problem. The World Health Organisation (WHO) estimates that there were 207 million cases of malaria in 2012 resulting in approximately 627,000 deaths, mainly in children under five living in sub-Saharan Africa (WHO World Malaria Report 2013). Malaria elimination is an ambitious long-term international goal, however, the lack of an effective vaccine and the development of resistance to anti-malaria drugs and insecticides, threaten the success of this programme. To combat malaria, a multi-disciplinary approach is imperative, which requires improved knowledge of the biology of the Plasmodium parasites and the Anopheles vectors. The genome sequence of the most virulent and deadly malaria parasite, Plasmodium falciparum, was published in 2002 and represents a major advance in our understanding of this pathogen [1]. The parasite transcriptome has also been extensively studied, however, the proteome ⁎ Corresponding author at: Institut de Biologie Structurale, 71, avenue des Martyrs, CS 10090, 38044 Grenoble Cedex 9, France. Tel.: + 33 4 57 42 86 68. E-mail address: [email protected] (M.R. Jensen).

http://dx.doi.org/10.1016/j.bbapap.2014.09.023 1570-9639/© 2014 Elsevier B.V. All rights reserved.

still presents a major challenge and about 60% of the approximately 5300 predicted proteins in P. falciparum have little similarity to proteins in other organisms and have no known function. Structural data on P. falciparum proteins is limited and represents another gap in our knowledge. The ability of P. falciparum to proliferate within the human host depends on its invasion of erythrocytes. Erythrocyte binding-like (EBL) proteins play crucial roles in the attachment of merozoites to human erythrocytes by binding to specific receptors on the cell surface [2–4]. The EBL family of proteins includes erythrocyte binding antigens EBA-140 [5] and EBA-175 [6–8] that target the glycophorin C and A receptors, respectively [9–13], as well as EBA-181 [14] for which the receptor remains unknown. Thus, the EBL proteins target different receptors on the cell surface consistent with multiple invasion pathways [15]. The EBL proteins are type 1 transmembrane proteins and share similar overall domain organizations (Fig. 1A) [16]. The proteins are divided into six regions, RI to RVI. RII is the receptor-binding domain containing two cysteine-rich domains (F1 and F2) that are homologs of the Duffy binding proteins of Plasmodium vivax and are therefore named Duffy binding-like (DBL) domains. The crystal structure of RII

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of EBA-175 was determined previously and shows that the two domains, F1 and F2, have very similar structures composed mainly of α-helices (Fig. 1A) [17]. RIV is also a cysteine-rich domain and the crystal structure of RIV from EBA-175 shows that this domain displays structural features similar to the KIX-binding domain of the co-activator CREB-binding protein [18]. Much less is known about the regions RIII–V. The functional role has so far not been identified, although antibodies raised against these regions of EBA-140 and EBA-175 were shown to inhibit parasite invasion suggesting a key role in the invasion process [19].

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Intrinsically disordered proteins (IDPs) have emerged in recent years as a new class of proteins that lack significant amounts of secondary and tertiary structures, but nevertheless play active roles in many biological processes [20–25]. Recent studies have highlighted the high abundance of IDPs within the P. falciparum proteome, and it was noted that low complexity regions are particularly prevalent [26,27]. The presence of low complexity regions hampers advances in structural studies as the number of homologous proteins are limited.

Fig. 1. Prediction of disorder within the erythrocyte binding-like proteins. (A) Domain organizations of three erythrocyte binding-like proteins EBA-175, EBA-140 and EBA-181. The crystal structures of regions RII and RVI of EBA-175 were solved previously and are shown in cartoon representations. The positions of the transmembrane regions are indicated in red. (B) Disorder prediction using the server IUPRED for EBA-175 (top), EBA-140 (middle) and EBA-181 (bottom). (C) Amino acid composition within the RIII–V regions of the EBL proteins EBA-140 (blue), EBA-175 (red) and EBA-181 (green) compared to the composition of the DisProt database release 6.02 (orange).

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In this study, we show that the EBL proteins EBA-140, EBA-175 and EBA-181 contain a large amount of intrinsic disorder in particular within the RIII–V domains. We focus on the biophysical characterization of a smaller construct encompassing residues 945–1097 within the RIII–V regions of EBA-181 (EBA-181945–1097). EBA-181945–1097 is particularly interesting as it has previously been shown by phage display technology and pull-down assays to interact with the 10 kDa domain of the erythrocyte membrane skeleton protein 4.1R [28,29]. We show using a number of biophysical techniques that EBA-181945–1097 is intrinsically disordered and we obtain a detailed structural and dynamic characterization of the protein using nuclear magnetic resonance (NMR) spectroscopy [30–35]. Our results show that EBA-181945–1097 is essentially a statistical coil with the presence of several turn motifs and does not possess transiently populated secondary structures as is common for many IDPs that fold via specific, pre-formed molecular recognition elements [36–40]. 2. Materials and methods 2.1. Expression and purification of EBA-181945–1097 The EBA-181 DNA sequence encompassing the amino acids 945 to 1097 was cloned into a pET15b vector thereby adding an N-terminal polyhistidine tag and a thrombin cleavage site. In vitro transposition was used to change the antibiotic resistance from ampicillin to kanamycin and the resulting plasmid was transformed into Escherichia coli Rosetta™ 2 (DE3) cells. The Ross medium [41] was used to express double labelled (13C/15N) EBA-181945–1097 protein. EBA-181945–1097 was expressed by inducing the bacterial cultures with 1 mM IPTG for 16 h at 30 °C. The EBA-181945–1097 protein was purified using immobilised metal affinity chromatography (IMAC, nickel resin). The histidine tag was cleaved using thrombin protease, followed by an IMAC step in order to remove the cleaved tag. Finally, the protein was further purified on a size exclusion chromatography column. 2.2. NMR spectroscopy The NMR spectral assignment was carried out using a 1.0 mM 13C, N labelled sample of EBA-181945–1097 in 50 mM phosphate buffer and 50 mM sodium chloride at pH 6.0 and 25 °C. The assignment was obtained using a series of BEST-type triple resonance experiments [42]: HNCO, intra-residue HN(CA)CO, HN(CO)CA, intra-residue HNCA, HNCOCACB and intra-residue HNCACB. The spectra were processed in NMRPipe [43], analyzed using Sparky [44] and automatic assignment of spin systems was achieved using MARS [45] followed by manual

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verification. Secondary chemical shifts were calculated using the random coil values from RefDB [46]. Site specific 15N R1, R2 (CPMG) and {1H}–15N heteronuclear nOes were measured using previously developed pulse sequences [47] at a 1 H frequency of 600 MHz and 25 °C. The decay of magnetization in the R1 experiment was sampled at the following time points: 0.6, 0.1, 1.5, 0, 0.4, 1.1, 0.8, 1.9, and 0.2 s with a repeat of the delay at 0.6 s for the purpose of error estimations. The decay of magnetization in the R2 experiment was sampled at: 0.07, 0.13, 0.01, 0.09, 0.25, 0.03, 0.21, 0.05, and 0.17 s with a repeat of the delay at 0.07 s. The heteronuclear nOes were obtained as the intensity ratio between two spectra acquired with and without initial proton saturation. Proton saturation was achieved using a 3 s WALTZ decoupling sequence that in the reference experiment was replaced by a delay of 3 s. The experiment was repeated twice and the values of the nOes were taken as the average between these two measurements. Residual dipolar couplings (RDCs) were measured in a liquid crystal composed of poly-ethylene glycol and 1-hexanol as described previously [48]. The alignment of the protein gave rise to a residual deuterium splitting of 18 Hz. 1H–15N RDCs (1DNH) were measured using a BESTtype HNCO experiment with coupling evolution in the 13C dimension [49]. 2.3. Structural ensemble selections on the basis of experimental NMR data Ensemble selections of EBA-181945–1097 were carried out as described previously [50,51]. Briefly, a large pool of statistical coil conformers (10,000 structures) was initially generated using Flexible-Meccano [52, 53]. Five representative ensembles of 200 conformers each were selected from the pool using ASTEROIDS [54,55] on the basis of the experimental chemical shifts using the following estimated errors on the experimental data: 1HN (0.04 ppm), 15N (0.2 ppm), 13Cα (0.1 ppm), 13C′ (0.1 ppm) and 13Cβ (0.1 ppm). The backbone dihedral angles were extracted from the five representative ensembles giving 1000 ϕ/ψ pairs for each residue. These dihedral angle distributions were used to generate a new pool of conformers of EBA-181945–1097 from which a new round of selections of five ensembles was carried out. This procedure was repeated five times (five iterations) until convergence with respect to the experimental data was achieved. The five ensembles obtained at the end of the fifth iteration were analyzed in terms of site-specific populations in four distinct regions of Ramachandran space defined as: αL {ϕ N 0°, −180° b ψ b 180°}; αR {ϕ b 0, −120° b ψ b 50°}; βP {−100° b ϕ b 0°, ψ N 50° or ψ b −120°}; βS {−180° b ϕ b −100°, ψ N 50° or ψ b −120°}. The populations of these quadrants are denoted p(αL), p(αR), p(βP) and p(βS), respectively.

Fig. 2. EBA-181945–1097 shows characteristics of a disordered protein in solution. (A) Determination of the molecular mass of EBA-181945–1097 from SEC combined with detection by MALLS and refractometry. The blue line shows the SEC elution profile as monitored by refractometry (left axis), while the red line shows the molecular mass calculated from light scattering and refractometry data across the elution peak (right axis). (B) SDS-PAGE of purified EBA-181945–1097. (C) Far-UV CD spectrum of EBA-181945–1097.

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This sub-division of Ramachandran space, that covers the entire ϕ/ψ space, facilitates the quantification of conformational sampling. It is, however, important to underline that this rather coarse division of Ramachandran space does not exclude a more thorough analysis of the formation of specific secondary structure motifs within the derived ϕ/ψ distributions obtained using ASTEROIDS. 3. Results 3.1. The RIII–V regions of the erythrocyte binding-like proteins are intrinsically disordered We carried out a disorder prediction on the three EBL proteins using the server IUPRED (Fig. 1B) [56]. A disorder score of 0 predicts that the protein is folded, while a score of 1 predicts a strong tendency to be

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intrinsically disordered. For all three proteins, the folded regions RII and RVI are easily identified by low disorder scores, while RIII–V are predicted to be intrinsically disordered. An analysis of the amino acid composition of RIII–V of the EBL proteins reveals that they are in general rich in charged amino acids such as lysines and glutamic and aspartic acids, as well as hydrophilic amino acids such as threonine and serine (Fig. 1C). On the other hand, the proteins are depleted in hydrophobic amino acids that are necessary for forming stable hydrophobic cores in folded proteins. Comparison with the amino acid composition of the DisProt database [57] reveals that the disordered regions of the EBL proteins are richer in asparagines, serines and glutamic acids, while lacking alanines that normally represent one of the most common amino acid types in IDPs (Fig. 1C). Alanine is expected to be under-represented due to the extreme AT nucleotide bias (N 80%) of the P. falciparum genome [1].

Fig. 3. 1H–15N HSQC spectrum of EBA-181945–1097 showing the limited signal dispersion in the 1H dimension characteristic of an intrinsically disordered protein. The labels indicate the spectral assignment of the NMR resonances.

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The functional role of RIII–V remains to be elucidated, however, they may function as interaction platforms for different cellular partners. We carried out a prediction of interaction sites using the server ANCHOR [58] indicating the presence of a large number of potential binding regions throughout RIII–V of all three EBL proteins (Fig. A.1). Interestingly, the sequence conservation within RIII–V is low (Figs. A.2–A.4) suggesting that the three proteins are not likely to bind the same cellular partners and therefore could exert different biological functions. 3.2. Structural and dynamic characterization of EBA-181945–1097 In order to obtain more insight into the conformational behaviour of RIII–V of EBA-181, we employed a divide-and-conquer approach and expressed and purified a smaller construct of EBA-181 encompassing residues 945–1097. Initially, we determined the oligomeric state of EBA-181945–1097 by size exclusion chromatography (SEC) combined with detection by multi-angle laser light scattering (MALLS) and refractometry (Fig. 2A). The data show that EBA-181945–1097 is monomeric in solution (molecular weight calculated from sequence is 17,075 Da). According to polyacrylamide gel electrophoresis (SDS-PAGE), EBA181945–1097 migrates corresponding to a molecular weight of approximately 50 kDa (Fig. 2B) indicating that EBA-181945–1097 is intrinsically disordered in solution, where a high content of charged residues leads to an aberrant migration pattern. The far-UV circular dichroism (CD)

spectrum of the protein displays a signature typical of random coil conformations with a minimum around 202 nm (Fig. 2C). Functionally disordered proteins often possess transiently populated secondary structures that serve as molecular recognition elements for other proteins [59–64]. In many cases, the molecular recognition elements undergo folding upon binding to their partner proteins into specific conformations in the complex, and it has been postulated that the pre-formation of secondary structure facilitates the binding process by lowering the entropic cost of folding from a completely unfolded chain [61,62]. In order to obtain information about transiently populated secondary structures in EBA-181945–1097, we characterized the protein at atomic resolution using NMR spectroscopy. The 1H– 15 N HSQC spectrum displays a limited dispersion of the NMR resonances in the 1H dimension confirming the intrinsically disordered nature of the protein (Fig. 3). The complete spectral assignment of the 1HN, 15 N, 13Cα, 13C′ and 13Cβ nuclei of the protein was carried out using a set of standard triple resonance experiments (Fig. 3, Table A.1). The secondary Cα chemical shifts show that the protein is intrinsically disordered without significantly populated secondary structure elements in agreement with the measurements from CD spectroscopy (Fig. 4A). We also characterized the backbone dynamics of the protein on the pico- to nano-second time scales through the measurements of 15N spin relaxation. The {1H}–15N heteronuclear nOes display a

Fig. 4. Characterization of the structure and dynamics of EBA-181945–1097. (A) Secondary Cα chemical shifts of EBA-181945–1097. (B) {1H}–15N heteronuclear nOes. (C) 15N R2 spin relaxation rates. (D) 15N R1 spin relaxation rates. All relaxation data were obtained at a 1H frequency of 600 MHz and 25 °C. Blue shading indicates regions of the protein for which a rigidification of the protein is observed corresponding to positive heteronuclear nOes.

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profile characteristic of a disordered protein with values close to zero at a 1H frequency of 600 MHz (Fig. 4B). The protein shows increased dynamics towards the C-terminus of the protein, while the N-terminus contains additional amino acids from the cloning site for which no spectral assignment was obtained. The region comprising residues 980–1025 shows on average larger heteronuclear nOes with two short stretches having positive values (residues 986–988 and 1016–1020) corresponding to a rigidification of these parts of the protein compared to the remainder of the chain. The 15N transverse and longitudinal spin relaxation rates follow a similar pattern (Fig. 4C, D), in particular, the 15 N R2 rates are enhanced in the regions also showing positive heteronuclear nOes. 3.3. Structural ensemble description of EBA-181945–1097 from experimental chemical shifts In order to obtain more details about the local conformational sampling of EBA-181945–1097 we carried out ensemble selections of the protein on the basis of the experimental chemical shifts. Thus, we generated a large pool of conformers (10,000 structures) of the protein using the statistical coil generator Flexible-Meccano [52,53] and we used the genetic algorithm ASTEROIDS [54,55] to select representative sub-ensembles of EBA-181945–1097 on the basis of the experimental chemical shifts as described previously [50,51,65]. Excellent agreement was obtained between the experimental chemical shifts and those back-calculated from the ASTEROIDS ensembles comprising 200 conformers (Fig. 5). We note

Fig. 5. Generation of representative structural ensembles of EBA-181945–1097 on the basis of experimental chemical shift values. Comparison of experimental secondary chemical shifts (red) and back-calculated values from a selected ASTEROIDS ensemble comprising 200 conformers (blue). From top to bottom: Cα, Cβ, C′, N and HN secondary chemical shifts.

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that by combining the statistical coil description with selection of subensembles using a genetic algorithm, we allow for a direct modulation of the potential energy landscape using the experimental data. Thus, the statistical coil library is used solely as a starting model for the conformational behaviour of the IDP, and we subsequently refine the conformational space using the experimental data. An explicit assumption in this approach is that the statistical coil library over-samples conformational space meaning that the residue-specific sampling of the IDP is contained within the amino acid specific coil distributions. An analysis of the site-specific conformational sampling in the selected ASTEROIDS ensembles reveals depletion (on the order of 15%) of the β-strand region of Ramachandran space compared to statistical coil sampling, as well as an increase in the poly-proline II (βP) populations in several regions of the protein. The most pronounced increase in βP sampling is observed in the vicinity of proline residues in the sequence for example around P945, P949, P1003, P1039, P1040 and P1044, where the increase reaches 20% additional sampling in the βP region compared to statistical coil populations (Fig. 6). In other regions, for example at the C-terminal residues 1085–1092, poly-proline II sampling is also enhanced, however, independently of the presence of proline residues. Further analysis of the conformational sampling of EBA-181945–1097 shows that the α-helical population is increased in four different regions of the protein: residues 979–982, 986–988, 1006–1009 and 1016–1019 coinciding with the regions displaying the largest heteronuclear nOes and transverse spin relaxation rates. These regions of the protein adopt turn motifs or short single turn α-helices with populations of around 15–20% leading to a rigidification of the protein on the pico- to nano-second time scales.

Fig. 6. Site-specific populations of EBA-181945–1097 in four distinct regions of Ramachandran space derived from ASTEROIDS ensembles selected on the basis of experimental chemical shift. Red bars indicate populations derived from the experimental chemical shifts, while blue lines correspond to statistical coil populations. Blue shading indicates regions of the protein for which elevated poly-proline II populations are observed, while yellow shading indicates regions for which α-helical sampling is increased.

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3.4. Validation of structural ensembles by experimental residual dipolar couplings As we increase the number of available ensemble descriptions of highly flexible systems, it becomes important to develop robust procedures that allow validation of these ensembles. As we have shown previously [51], chemical shifts and residual dipolar couplings (RDCs) are highly complementary structural parameters in IDPs, and the measurement of both of these parameters therefore offers a unique possibility to validate the derived structural ensembles. RDCs are in general powerful reporters on backbone conformational sampling in disordered proteins with a strong dependence on helical elements or turn motifs that give rise to a sign inversion of the RDCs compared to random coil conformations [66–69]. We measured 1DNH RDCs of EBA-181945–1097 in a liquid crystal composed of poly-ethylene glycol and 1-hexanol. The RDCs are negative as expected for a disordered protein that mainly samples statistical coil conformations, except for four short stretches comprising residues 987– 988, 998, 1006–1007 and 1016–1019 that display positive RDCs (Fig. 7). The experimental RDCs agree reasonably well with those predicted from a statistical coil ensemble generated using Flexible-Meccano with a reduced χ2 of 1.21 (Fig. 7A). Importantly, we observe a modest improvement in the reproduction of the experimental RDCs when using the conformational sampling of EBA-181945–1097 derived from the experimental chemical shifts (reduced χ2 of 1.08) (Fig. 7B). The biggest improvement in the reproduction of the RDCs is observed within the turn or short helical motifs identified by the experimental chemical shifts leading to positive back-calculated RDCs. In addition, some improvement is observed in the regions of the protein oversampling the poly-proline II region of Ramachandran space, i.e. towards the N- and C-termini of the protein as well as around P1039–P1044. The improvement in the reproduction of the RDCs compared to a standard statistical coil ensemble provides an independent verification of the validity of the ASTEROIDS ensembles and testifies to the predictive nature of these ensembles.

is their ability to interact with multiple partner proteins for example in so-called hub proteins that, by exploiting their structural plasticity, bind multiple proteins simultaneously. In this study, we carried out a bioinformatics analysis of the EBL family of proteins revealing that the RIII–V regions are intrinsically disordered with multiple predicted interaction sites. So far very few interaction partners have been identified and the role that these regions play in the invasion process remains largely unknown. Considering the variable length of RIII–V within the three EBL proteins, we suggest that these disordered regions direct the EBL proteins towards different cellular functions by discriminating between different partner proteins. This hypothesis is supported by the fact that the three proteins share very little sequence conservation within RIII–V (Figs. A.2–A.4), which provides a means of diversifying the invasion process across the EBL family. One can imagine that RIII–V bind proteins that are subsequently inactivated (sequestered) thereby facilitating invasion or that RIII–V interact with proteins that play a more direct role in the invasion process. Interactions with host erythrocyte proteins, such as protein 4.1R, could destabilize the membrane skeleton and may also impair host membrane repair pathways. In order to obtain more insight into the conformational sampling within the RIII-V regions of EBA-181, we carried out a structural and dynamic characterization of a shorter construct of EBA-181. The data show that EBA-181945–1097 is essentially a statistical coil with a number of turn or short α-helical motifs. Whether these motifs function as molecular recognition elements and thereby play a role in the biological function of the protein remains to be elucidated, however the motifs are present within regions of the protein predicted by ANCHOR to participate in protein–protein interactions (Fig. A.5). The spectral assignment obtained here of a shorter construct of the RIII–V regions of EBA-181 provides a new basis for mapping and identifying interactions with different partner proteins and that at amino acid resolution. This could provide valuable information on host–pathogen interactions during the invasion of erythrocytes.

4. Discussion and conclusions Acknowledgements In recent years, IDPs have emerged as a new class of proteins that defy the classical structure–function paradigm. They do not adopt welldefined, folded structures, but rather sample a large number of conformations in solution posing specific challenges for their structural and dynamic characterization. One of the most remarkable features of IDPs

MB, EPM and VTF acknowledge support from the ILL, the ESRF and Keele University for the provision of a PhD studentship to MB. Macromolecular labelling was carried out in ILL's Life Science Group using its Deuteration Laboratory platform, originally established with funding

Fig. 7. Validation of selected ASTEROIDS ensembles of EBA-181945–1097 using 1DNH residual dipolar couplings. (A) Comparison of experimental (red) and predicted (green) 1DNH RDCs from a statistical coil ensemble. (B) Comparison of experimental (red) and predicted (blue) 1DNH RDCs from the representative ensembles of EBA-181945–1097 derived on the basis of experimental chemical shifts only. Yellow shading and blue shading indicate regions for which the reproduction of RDCs is improved compared to a statistical coil ensemble prediction (shown in A).

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from the UK EPSRC (grant ref EP/C015452/1). This work used the platforms of the Grenoble Instruct centre (ISBG; UMS 3518 CNRS-CEAUJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). This work was carried out with financial support from the French Agence Nationale de la Recherche through ANR JCJC ProteinDisorder (to M.R.J.), ANR MALZ TAUSTRUCT and ANR ComplexDynamics (to MB). This work is based upon research supported by the National Research Foundation (NRF) of South Africa, grant numbers: CPR20100421000010585 and IFR2009021600003. Any opinion, findings and conclusions or recommendations expressed in this material are those of the authors and therefore the NRF do not accept any liability in regard thereto. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbapap.2014.09.023. References [1] M.J. Gardner, N. Hall, E. Fung, O. 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