Structural genomics applied to the rust fungus Melampsora larici-populina reveals two candidate effector proteins adopting cystine knot and NTF2-like protein folds

Rust fungi are plant pathogens that secrete an arsenal of effector proteins interfering with plant functions and promoting parasitic infection. Effectors are often species-specific, evolve rapidly, and display low sequence similarities with known proteins. How rust fungal effectors function in host cells remains elusive, and biochemical and structural approaches have been scarcely used to tackle this question. In this study, we produced recombinant proteins of eleven candidate effectors of the leaf rust fungus Melampsora larici-populina in Escherichia coli. We successfully purified and solved the three-dimensional structure of two proteins, MLP124266 and MLP124017, using NMR spectroscopy. Although both MLP124266 and MLP124017 show no sequence similarity with known proteins, they exhibit structural similarities to knottins, which are disulfide-rich small proteins characterized by intricate disulfide bridges, and to nuclear transport factor 2-like proteins, which are molecular containers involved in a wide range of functions, respectively. Interestingly, such structural folds have not been reported so far in pathogen effectors, indicating that MLP124266 and MLP124017 may bear novel functions related to pathogenicity. Our findings show that sequence-unrelated effectors can adopt folds similar to known proteins, and encourage the use of biochemical and structural approaches to functionally characterize effector candidates.

To infect their host, filamentous pathogens secrete effector proteins that interfere with plant physiology and immunity to promote parasitic growth 1 . Although progresses have been made in the past decade, how effectors act in host cells remains a central question in molecular plant pathology. Effectors of filamentous pathogens are secreted and either stay in the apoplast or penetrate inside the cell through specialized infection structures such as haustoria 2 . Effectors are detected by the host plant by two layers of immune receptors at the cell surface or inside the cell, which trigger plant defence response 3 .
To evade recognition by the host immune system, pathogen effector genes evolve rapidly, notably through the diversification of the amino acid sequence of the encoded proteins 4 . Such diversification impairs the identification of amino acid motifs or sequences similar to known proteins, which could give insights on effector functions inside the host cell 5 . Several superfamilies of effector proteins, such as the fungal MAX or the oomycete WY-domain families, have members showing similar fold but divergent primary sequences 6,7 , the fold being conserved probably due to the strong link between protein structure and function 8 . Research efforts have been set in this direction and applied in order to determine the structure of several effector proteins 6,[9][10][11] .
Successful production and purification of three candidate effectors in E. coli. To investigate the structural properties of the 11 selected candidate effectors ( Fig. 1), we first aimed at obtaining the recombinant proteins. To this end, the sequence encoding mature proteins (i.e. without signal peptide) were cloned into pET-26b (for Mlp124111, Mlp124478, Mlp124530, Mlp124561, Mlp37347, Mlp109567, Mlp107772, Mlp124202, Mlp124266, and Mlp124499) or pET-28a (for Mlp124017) expression vectors, in order to incorporate a C-terminal 6-histidine tag (Table S1). Small-scale expression assays achieved into E. coli Rosetta2 (DE3) pLysS strain indicated that nine out of the eleven proteins accumulated using a standard induction protocol (i.e.  22 and/or Germain et al. 24 . b Immunolocalization performed on infected poplar leaves by Hacquard et al. 16 . addition of 100 µM IPTG in mid-exponential growth phase and further growing for 3 to 4 hours at 37 °C). Among those, five (MLP124111, MLP124561, MLP37347, MLP107772 and MLP124202) accumulated in the insoluble protein fraction, and one (MLP109567) was not expressed (Fig. 2), despite the use of other E. coli expression strains (SoluBL21 (DE3), Origami2 (DE3) pLysS, Rosetta-Gami2 (DE3) pLysS) and modification of the culture conditions (induction time, temperature, and osmolarity). Among the five remaining soluble proteins we choose MLP124017, MLP124266, and MLP124499, the most stable along the purification procedure, for further analyses. We thus purified the His-tagged recombinant proteins in native conditions using a two-step protocol including immobilized-metal affinity chromatography (IMAC), then size exclusion chromatography (Fig. 3). The purified proteins, yielding respectively 50 mg/L (cell culture), 0.5 mg/L, and 0.5 mg/L for MLP124017, MLP124266 and MLP124499, respectively, eluted in size exclusion chromatography as a single peak corresponding to an estimated apparent molecular mass compatible with a monomeric organization data not shown.
MLP124266 is a thermostable protein that exhibits a cystine knot. From a previous study, we reported that the Mlp124266 and Mlp124499 genes are strongly expressed and induced during poplar leaf colonization by M. larici-populina, and belong to large multigene families of 13 and 31 members, respectively, in M. larici-populina 16 . Mlp124266 and Mlp124499 encode mature proteins of 69 and 50 amino acids, respectively (Fig. S1). MLP124266 has an N-terminal part enriched in charged residues and a C-terminal region that possesses six conserved cysteines predicted to form a cystine knot structure (Fig. S1A). This typical protein organization is shared by all members of the family as well as by alleles of M. lini AvrP4 33,34 . In MLP124499, several acidic residues are found in the N-terminal part whereas the C-terminal part contains three conserved cysteines (Fig. S1B).
Prediction programs indicate that all members of both protein families exhibit highly conserved N-terminal signal peptides for protein secretion. Following the production and the purification of both MLP124266 and MLP124499, we undertook a structural characterization of each recombinant protein using a NMR spectroscopy approach. Standard homonuclear 2D experiments and 15 N-edited TOCSY-HSQC and NOESY-HSQC experiments carried out on MLP124499 allowed the assignment of 1 H and 15 N resonances except for the four N-terminal residues (Fig. S2A). However, several minor peaks were observed, especially for Ala 14 -Glu 16 , Gly 25 -Gln 26 , Glu 30 , Asp 49 residues, suggesting the presence of multiple forms or conformations. Changing the temperature and the ionic strength, or adding dithiothreitol failed to improve the quality of the MLP124499 NMR spectra. Consequently, very few experimental restraints could be derived and structure calculations led to very ill-defined models.
For MLP124266, the assignment of 1 H, 15 N and 13 C resonances has been obtained for all residues except for the five N-terminal amino acids (Fig. S2B) and its 3D structure could further be modelled by the NMR derived constraints (Table S2;

Priority candidate effectors
SSPs induced during infecƟon, expressed in haustoria, with no known funcƟon, and specific to Pucciniales

High priority candidate effectors
SSPs target a specific plant cell compartment or protein complex, or homologous to known effectors Protein expression in E. coli SDS-PAGE assays detect the accumulaƟon of recombinant proteins in E. coli

Proteins in soluble fracƟon
Cell fracƟonaƟon assays detect recombinant proteins in the soluble protein fracƟon

Proteins purified to homogeneity
Tandem affinity/size exclusion chromatography purifies recombinant proteins to homogeneity NMR-resolved protein structure NMR spectroscopy determines protein threedimensional structure   IF  TF  SF  IF  TF  SF  IF  TF  SF  IF  MM  TF  SF  IF  TF  www.nature.com/scientificreports www.nature.com/scientificreports/ the models, respectively. Backbone dynamic properties of MLP124266 have been investigated by 15 N relaxation measurements. Heteronuclear 1 H- 15 N NOE values showed a contrasted profile with low values for N-terminal residues (indicative of a flexible structure) and high values for C-terminal residues (indicative of a rigid structure). Indeed, amino acids Asp 6 to Gly 38 and Cys 39 to Leu 69 presented heteronuclear NOE averaged values of 0.26 ± 0.05 and 0.66 ± 0.10, respectively. Several secondary structure prediction softwares predict a helix between residues 11 and 18 (data not shown) and a Consurf analysis shows that the proportion of conserved amino acids in the N-terminal region is much higher than in the C-terminal part (Fig. S4). Interestingly 6 out of the 8 cysteines are gathered in the C-terminal region between Cys 39 and Cys 69 , following a spacing (Cys-X 2-7 -Cys-X 3-10 -Cys-X 0-7 -Cys-X 1-17 -Cys-X 4-19 -Cys) typical of cystine knot structures, (i.e., three intricate disulfide bridges that confer very high stability to proteins; Fig. 4) 35 . Hence, it is likely that rigidity originates from the structure formed by these cysteines that are highly conserved in the protein family, as indicated by the Consurf analysis (Figs. S1 and S4). Thus, we sought to determine whether these disulfides are formed and whether they influence the stability and/or the oligomerization state of the protein by covalent bonds. A single peak corresponding to the theoretical mass of MLP124266 monomer was obtained by mass spectrometry (data not shown). The titration of free thiol groups in an untreated recombinant MLP124266 gave an averaged value of 1 mole SH per mole of protein.
Considering the presence of 8 cysteines in the protein, these results are consistent with the existence of three intramolecular disulfide bridges (Fig. S3). The thermostability of recombinant protein was estimated by heating the protein for 10 min at 95 °C. The observation that the protein remained in solution (i.e. no precipitation was observed) indicates that it is thermosoluble. In order to investigate the role of the disulfides for such property, we should compare the results obtained with an oxidized and a reduced protein. However, as assessed by thiol titration experiments, we failed to obtain a complete reduction of these disulfides despite extensive incubation of the protein at high temperatures, in denaturing and reducing environments. Altogether, these results indicate that recombinant MLP124266 is properly folded by E. coli, and that the disulfide bridges, which are partially resistant to reduction, confer a high rigidity and stability to the protein.
MLP124017 is part of the nuclear transport factor 2-like protein superfamily. MLP124017 is a small-secreted protein (167 amino acids with its signal peptide; 150 amino acids in its mature form, with a molecular mass of 18 kDa) of unknown function, highly expressed during infection of poplar leaves by M. larici-populina 16 . MLP124017 shares sequence similarity with neither other M. larici-populina nor other rust fungal proteins. In a previous study, we demonstrated the nucleocytoplasmic localization of MLP124107 in N. benthamiana and its interaction with poplar TOPLESS-related 4 protein 22 . To further investigate MLP124017 structure and to get insights into its function, we first attempted to solve its 3D structure by crystallization coupled to X-ray diffraction. We were unable to obtain exploitable diffracting crystals despite the use of different versions (untagged or N-or C-terminal His-tagged) of MLP124017 protein and therefore switched to NMR. The recombinant 15 N and 13 C-labelled MLP124017 protein was used for structure determination by two-and three-dimensional NMR experiments (Table S3). The assigned 1 H, 15 N-HSQC spectra were well dispersed but the peaks for residues from the N-terminal 1-14 and 86-95 segments were missing (Fig. S5). From preliminary structures, the production of a truncated recombinant protein for the first eight N-terminal residues that could mask residues 86-95 did not improve the data. The solution structure of MLP124017 was determined based on 1727 NOE-derived distance restraints, 214 dihedral angle restraints and 102 hydrogen bond restraints. All proline residues have been determined to be in a trans-conformation according to the 13 54 and Pro 146 respectively. The best conformers with the lowest energies, which exhibited no obvious NOE violations and no dihedral violations >5° were selected for final analysis. The Ramachandran plot produced shows that 99.6% of the residues are in favoured regions (Table S4). MLP124017 structure is composed of a α + β barrel with seven β-strands forming one mixed β-sheet, four β-hairpins, four β-bulges, and four α-helices (Fig. 5A). Residues 1-14 and 150-151 having missing assignments are not defined in the final models. This arrangement of secondary structure produces a cone-shaped fold for the protein, which generates a distinctive hydrophobic cavity (Fig. 5B, Fig. 5C). www.nature.com/scientificreports www.nature.com/scientificreports/ To identify potential structural homologs of MLP124017, we performed structural similarity searches using the Dali server 36 . Queries identified SBAL_0622 (PDB code 3BLZ) and SPO1084 (PDB code 3FKA) as the closest structural homologs with the highest Z-score of 6.0 and 6.3, respectively, and a RMSD of 4.1 and 3.5 Å, respectively (Fig. S6). These two proteins, which are from the bacteria Shewanella baltica and Ruegeria pomeroyi, have no known function, but share a common Nuclear Transport Factor 2-like (NTF2) fold. The NTF2 superfamily comprises a large group of proteins that share a common fold and that are widespread in both prokaryotes and eukaryotes 37 . Taken together these results show that although MLP124017 do not share sequence similarities or domain with other proteins in sequence databases, its structure is similar to proteins of the NTF2 folding superfamily.

Discussion
In this study, we have set up a small-throughput effectoromics pipeline based on recombinant protein production and structural characterization to get insights on 11 candidate effectors of the poplar leaf rust fungus M. larici-populina.
Although the production of recombinant proteins in E. coli is a valuable approach to perform biophysical and biochemical analyses of candidate effectors 5,25 , we have faced issues for the production of soluble small-cysteine rich proteins in this system. Indeed, among the eleven candidate effectors screened for expression, only three were found in the soluble protein fraction and stable enough to tolerate the purification procedure. Although we tested different E. coli strains and protein expression induction protocols, the other candidate effectors were either not expressed or expressed as inclusion bodies. It is possible to purify recombinant proteins from inclusion bodies by using denaturing extraction conditions and further refolding proteins 38 . However, this approach is not recommended for structural analysis as the refolding of the proteins in vitro may alter folding. Another limit of prokaryote systems to produce eukaryote proteins is the lack of post-translational modifications such as methylations. www.nature.com/scientificreports www.nature.com/scientificreports/ An alternative to this is to use the yeast Pichia pastoris, which has proven useful for several fungal effectors such as Leptospheria maculans AvrLm4-7 or Cladosporium fulvum Avr2 and Avr4 39,40 . We have tried this system to produce the candidate effector (MLP107772), but without success (data not shown). Nevertheless, this system may be useful and deserves to be considered as an alternative to assay other rust effectors for which we were not able to obtain production in E. coli.
Out of the three effectors successfully purified as recombinant proteins, two were structurally characterized by NMR spectroscopy. MLP124266 is a homolog of the M. lini AvrP4 effector protein 41 , and we showed that it exhibits a cystine knot (or knottin) structural motif commonly encountered in small disulfide-rich proteins. MLP124017 is an orphan protein in M. larici-populina with no known ortholog in Pucciniales. MLP124017 physically associates with the poplar TOPLESS-related protein 4 (TRP4) 22 , and we showed that it exhibits a fold similar to two bacterial proteins that belong to the Nuclear-Transport factor 2-like protein superfamily.
We showed that MLP124266 possesses two distinct regions with contrasted structural properties. The C-terminal part is rigidified by a cystine knot motif whereas the N-terminal part is globally flexible. The knottin folded proteins display a variety of functions such as venoms and spider toxins 42,43 but also antimicrobial properties such as the cyclotides 44 . Some are also found to interact with protease inhibitors found in plants, insects and plant parasites 45 . The three disulfide bridges within the C-terminal part of MLP124266 confer its rigidity and probably contribute to the high protein stability 35 . MLP124266 presents a β-sheet structure typical of knottins, but interestingly it also has an additional helix between β2 and β3 strands. To our knowledge, the presence of such a helix in knottins has been reported in cyclotides only, and more precisely in bracelet cyclotides containing six or seven residues in the loop between Cys(III) and Cys(IV) 46 . This loop often contains an alanine, which favours the formation of the helix as well as a highly conserved glycine allowing its connection to the cystine knot 47 . Interestingly, the loop in MLP124266 has such residues, i.e. Ala 52 and Gly 54 Ala and Gly, but consists of four residues only. In Viola odorata cycloviolacin O2 (cO2), the additional helix is located in a hydrophobic loop that interacts with the membrane-mimicking micelles 48 . Therefore, it might help disrupting membranes and thus contribute to the cytotoxicity activity of cO2 and play a role in plant defence. In MLP124266, the helical turn is not particularly hydrophobic (Fig. S4B) and may not have these properties. To our knowledge, MLP124266 is the first fungal protein to present a knottin-like structure 49 . It would be interesting to collect structural data from other potential fungal knottins to find out whether the additional helix is always present and to clarify its role.
The intrinsic disorder of the N-terminus of MLP124266 also deserve to be pointed out. This region, approximately extending up to residue 35 and thus representing half of the primary sequence, globally presents a high flexibility, as demonstrated by the NMR dynamic results. Nevertheless, a few residues exhibit a propensity to form helical structures, which may support a biochemical role that remains to be elucidated. Interestingly, other effectors possess a predicted disordered N-terminal region 29 . For instance, M. lini effectors AvrL567 and AvrM have predicted disordered N-terminal regions that are susceptible to protease degradation 11,50 . Flexible folds are known to be adaptable linkers that favour the ability to bind partners 51 . As the N-terminus of many cytoplasmic effectors is anticipated to mediate protein entry into host cells 2 , it is tempting to speculate that this flexible part may bind a target important for cell entry. The structure of MLP124017 solved by NMR spectroscopy showed a fold similar to members of the NTF2-like superfamily. The NTF2-like superfamily is a group of protein domains sharing a common fold, but showing no sequence similarity. MLP124017 is structurally similar to two bacterial proteins, despite the lack of sequence similarity. The structures of these two bacterial proteins consist of a β-sheet surrounding a binding pocket and α-helices acting as a lid 52 . The NTF2 family regroups catalytic and non-catalytic proteins that contain cone-like structured proteins with a cavity that often acts as a molecular container involved in a wide range of cellular functions 37 . Interestingly, the cone-shaped structure of MLP124017 is widespread across both prokaryotes and eukaryotes. The first proteins of the NTF2 family were reported to play a role in the transport of molecules from the cytoplasm to the nucleus. Arabidopsis NTF2 protein is required to import nuclear proteins via the recognition of a nuclear localization signal (NLS). This protein also plays a role in the nuclear import of the small-GTPase Ran-GDP that is a central protein in various signal transduction pathways (e.g. mitotic spindle formation, nuclear envelope assembly, or responses to biotic stresses) [53][54][55][56] . In bacteria, some NTF2-like proteins play a role in bacterial conjugation as part of the type IV secretion system 57 , whereas non-catalytic NTF2-like domains act as immunity proteins 58 . In fungi, Saccharomyces cerevisiae NTF2 mutant is defective for nuclear import 59 . Although NTF2 folded proteins are widespread across kingdoms, very few is known about their role. A recent study presented that the silencing of NTF2 in wheat decreased the resistance against avirulent isolates of the wheat stripe rust fungus P. striiformis f. sp. tritici 60 . Since MLP124017 has been shown to interact with TOPLESS and TOPLESS-related proteins 22 , it is tempting to speculate that the cavity formed by the β-sheet could be involved in the association with these plant partners.
Although MLP124266 and MLP124017 show no primary sequence similarity to known proteins, they adopt a three dimensional fold similar to knottins and NTF2 family members, respectively. Thus, knowing the structure of both candidate effectors allowed us to classify them as members of large superfamilies of proteins. The concept of structural families whose members share no, or very limited, primary sequence homology emerges in effector biology 61 . This concept promises to revolution the way we predict and categorize effector proteins 6,7 . For instance, members of the MAX effector family share a common β-sandwich fold, but show no primary sequence similarity 6 . Similarly, members of the WY superfamily of RXLR effectors in oomycetes share a common three-to four-helix bundle 29 . Such features are now used to search and categorize fungal and oomycete effector proteins into structural superfamilies 1,6 . To our knowledge, MLP124266 and MLP124017 are the first effector proteins to adopt knottins and NTF2 folds. Whether other effector proteins adopt similar folds remains to be identified to determine if knottins and NTF2 folds define structural superfamilies in fungi.

Experimental Procedures
Sequence analyses and names. Alignment  Cloning of selected effector encoding sequences. Open reading frames coding for the mature forms (i.e. devoid of the sequence encoding N-terminal secretion peptide) of MLP124266 and MLP124499 of M. larici-populina isolate 98AG31 were ordered as synthetic genes cloned in pBSK(+) vectors (Genecust). Coding sequence of the mature forms of the nine other candidate effectors (Mlp124111, Mlp124478, Mlp124530, Mlp124561, Mlp37347, Mlp109567, Mlp124017, Mlp107772, and Mlp124202) were amplified by polymerase chain reaction (PCR) using cDNAs from leaves of the poplar hybrid Beaupré infected by M. larici-populina (isolate 98AG31) and further cloned into pICSL01005 vector as described previously 22 . The sequences encoding the mature form of each effector were subsequently cloned by PCR in either pET26b or pET28a vector between NdeI and XhoI (or NotI) or NcoI and XhoI restriction sites, respectively, using primers shown in Table S1.

Expression and purification of recombinant proteins in Escherichia coli. Expression of recombinants
proteins was performed at 37 °C using the E. coli SoluBL21 (DE3) pRARE2 (Amsbio Abington, UK), Rosetta2 (DE3) pLysS, Origami2 (DE3) pLysS or RosettaGami2 (DE3) pLysS strains (Novagen) containing the adequate pET expression vector coding for the selected candidate effector (Table S1) in LB medium supplemented with kanamycin (50 μg/ ml) and chloramphenicol (34 μg/ml). When the cell culture reached an OD 600nm of 0.7, protein expression was induced with 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) and cells were grown for a further 4 h. To improve the solubility of some recombinant candidate effectors, other protocols were used as follows. First, we added 0.5% (v/v) of ethanol in the medium when culture reached an OD 600nm of 0.7. The cells were cooled to 4 °C for 3 h, recombinant protein expression induced with 0.1 mM IPTG and cells further grown for 18 h at 20 °C. We also tested a combination of an osmotic and a thermal shock 62 . When the culture reached an OD 600nm of 0.5-0.6, 500 mM NaCl and 2 mM of betaine were added to the culture medium and the culture incubated at 47 °C for 1 hour under stirring. Cells were then cooled to 20 °C and the expression of recombinant proteins induced with 0.1 mM IPTG. After induction, cells were harvested by centrifugation, suspended in a 30 mM Tris-HCl pH 8.0 and 200 mM NaCl lysis buffer, and stored at −20 °C. Cell lysis was completed by sonication (three times for 1 min with intervals of 1 min). The cell extract was then centrifuged at 35 000 g for 25 min at 4 °C to remove cellular debris and aggregated proteins. After the addition of 10 mM imidazole, soluble fraction containing C-terminal His-tagged recombinant proteins were then purified by gravity-flow chromatography on a nickel nitrilotriacetate (Ni-NTA) agarose resin (Qiagen). After a washing step with lysis buffer supplemented with 20 mM imidazole, the proteins were eluted using lysis buffer containing 250 mM imidazole. www.nature.com/scientificreports www.nature.com/scientificreports/ of interest were pooled, concentrated by ultrafiltration then injected onto a gel filtration HiLoad 16/600 Superdex 75 prep grade (GE Healthcare) column connected to an ÄKTA Purifier TM (GE Healthcare) and eluted with lysis buffer. The fractions containing recombinant MLP124017 were pooled, concentrated, and stored at −20 °C as such, whereas for MLP124266 and MLP124499 fractions were pooled, dialyzed against 30 mM Tris-HCl, 1 mM EDTA (TE) pH 8.0 buffer, and stored at 4 °C until further use.
Protein sample preparation for NMR spectroscopy. Uniformly labelled 15  Nuclear magnetic resonance spectroscopy. For MLP124017, spectra were acquired on 800 and 700 MHz Avance Bruker spectrometers equipped with triple-resonance ( 1 H, 15 N, 13 C) z-gradient cryo-probe at 298 K. Experiments were recorded using the TOPSPIN pulse sequence library (v. 2.1) (Table S2). All spectra are referenced to the internal reference DSS for the 1 H dimension and indirectly referenced for the 15 N and 13 C dimensions 63 . Sequential assignment was performed using 3D 15 N-NOESY-HSQC, 15 N-TOCSY-HSQC, HNCO, HNCACO, HNCA, HNCOCACB, and HNCACB. Side chain 1 H assignments were carried out using combined analysis with 3D 15 N-NOESY-HSQC, 15 N-TOCSY-HSQC, and 2D NOESY and TOCSY with D 2 O samples. A series of three HSQC spectra was performed after lyophilisation and dilution of the first sample in D 2 O to determine amide protons in slow exchange (Table S2).
For MLP124266 and MLP1124499, NMR spectra were acquired on a Bruker DRX 600 MHz spectrometer equipped with a TCI cryoprobe. Structure calculation. For MLP124017 structure calculation, NOE peaks identified in 3D 15 N-NOESY-HSQC and 2D NOESY experiments were automatically assigned during structure calculations performed by the program CYANA 2.1 67 . The 15 N, H N , 13 C' , 13 Cα, Hα, and 13 Cβ chemical shifts were converted into ϕ/Ψ dihedral angle constraints using TALOS + (v. 1.2) 68 . Hydrogen bond constraints were determined according to 1 H/ 2 H exchange experiments of backbone amide protons (H N ). Each hydrogen bond was forced using following constraints: 1.8-2.0 Å for H N ,O distance and 2.7-3.0 Å for N H ,O distance. Final structure calculations were performed with CYANA (v. 2.1) using all distance and angle restraints (Table S3). 600 structures were calculated with CYANA 2.1, of which the 20 conformers with the lowest target function were refined by CNS (v. 1.2) using the refinement in water of RECOORD 69 and validated using PROCHECK 70 .
NMR assignment and structure coordinates have been deposited in the Biological Magnetic Resonance Data Bank (BMRB code 34423 and 34298) and in the RCSB Protein Data Bank (PDB code 6SGO and 6H0I), respectively.

Data availability
The data that support the findings of this study are openly available in the Biological Magnetic Resonance Data Bank (http://www.bmrb.wisc.edu/) and in the RCSB Protein Data Bank (http://www.rcsb.org/).