Understanding Intramolecular Crosstalk in an Intrinsically Disordered

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Understanding the intramolecular crosstalk in an intrinsically disordered protein Francesca Troilo, Daniela Bonetti, Christophe Bignon, Sonia Longhi, and Stefano Gianni ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b01055 • Publication Date (Web): 04 Feb 2019 Downloaded from http://pubs.acs.org on February 7, 2019

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ACS Chemical Biology

Understanding the intramolecular crosstalk in an intrinsically disordered protein Francesca Troilo1, Daniela Bonetti1, Christophe Bignon2, Sonia Longhi2* and Stefano Gianni1,* 1Istituto

Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche “A. Rossi Fanelli” and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, 00185, Rome, Italy 2 Aix-Marseille Univ, CNRS, Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 7257, Marseille, France ABSTRACT: The interaction between NTAIL and XD from measles virus represents a paradigmatic example of molecular recognition between an intrinsically disordered protein and a folded partner. By binding to XD, a small portion of NTAIL (classically denoted as MoRE) undergoes a disorder to order transition, populating an α-helical structure, while the reminder of the protein remains disordered. Here we demonstrate an unexpected crosstalk between such a disordered region and the adjacent MoRE. This result was obtained by producing a series of truncation and site-directed variants of NTAIL while measuring the effects on the kinetics of folding and binding. We show that the disordered region of NTAIL exerts its inhibitory role by slowing down the folding step of the MoRE, thereby tuning the affinity of the interaction.

The relationship between structure and function represents the foundation of our current knowledge of proteins. In this context, understanding the role of disorder represents one of the major challenges of modern chemistry and biology 1-6. In fact, whilst a relevant fraction of the proteome lacks a well-defined three-dimensional structure, it displays important and diverse functions. This finding led to the view that intrinsic disorder is likely to confer a functional benefit to proteins, spanning from allowing interactions with multiple partners while maintaining specificity, to enabling easily tunable interactions with physiological partners 7,8. The C-terminal domain of the measles virus nucleoprotein (NTAIL, residues 401-525 of the nucleoprotein) is an intrinsically disordered domain 9, which is critical for transcription and replication of measles virus 10-13. Upon binding to the X domain of the viral phosphoprotein, residues 486-502 of NTAIL adopt an α-helical structure 14, while the reminder of the protein remains disordered 11,13,15,16. It was previously demonstrated that NTAIL and XD recognize each other following an induced fit scenario, whereby the molecular recognition element (MoRE) of NTAIL folds only after the initial recognition of the partner 17. Furthermore, we demonstrated how the kinetics of interaction between NTAIL and XD is particularly informative, allowing to analyze quantitatively both the folding and binding steps of the overall process by studying the observed rate constants as a function of reactant concentrations 18,19. We recently showed that the long N-terminal appendage (residues 400-485) of NTAIL modulates its affinity towards XD, such that its progressive truncation results in an increased binding strength between the two interacting domains 20. In this study, we resorted to analyze in detail the mechanism by which the disordered region of NTAIL affects the binding to XD. To analyze the mechanism by which the disordered region flanking the MoRE influences the binding and folding of the latter, we constructed different truncation variants of NTAIL (Figure 1). In particular, we produced, expressed and purified 9 different variants (named 401, 411, 421, 431, 441, 451, 461, 471 and 481), whose sequence was progressively deleted of 10 amino acids such that their N-terminal residue corresponds to residue 401, 411, 421, 431, 441, 451, 461, 471 and 481 respectively (Figure 1). In analogy to our previous studies on wild-type and site-directed variants of NTAIL 17-20, we performed temperature-jump binding experiments between each truncation variant and the pseudo-wild type XD Y480W.

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Figure 1. Truncation variants on NTAIL. Panel A: schematic representation of the truncation variant constructs cloned into the pETG20a plasmid. The variants were obtained by inserting a tobacco etch virus (TEV) protease cleavage site immediately upstream the codons encoding amino acids 401, 411, 421, 431, 441, 451, 461, 471 and 481 of NTAIL. After expression in E. coli and purification through IMAC, the variants were obtained by cleavage with TEV protease. Each variant is 10 amino acids shorter than the previous one (panel B).

The dependence of the observed rate constant of each truncation variant as a function of NTAIL concentration is reported in Figure 2. It is evident that, whilst 401, 411, 421, 431 and 441 variants display a trend similar to that observed with wild-type NTAIL, with a hyperbolic dependence of the observed rate constants, the variants 451, 461, 471 and 481 return a linear dependence. By analyzing the kinetics of binding in the presence of co-solvents stabilizing the formation of helical structure in the MoRE, we previously showed that the hyperbolic dependence of the observed rate constant arises from a change in rate limiting step, with folding becoming rate limiting at high reactant concentrations 17-20. Consequently, a qualitative comparison of the data reported in Figure 2 suggests that truncation of the N-terminal disordered region of NTAIL accelerates folding of the MoRE, with a clear change in the dependence arising upon deletion of the first 50 residues. This finding confirms and extends our earlier demonstration that truncation of these residues increases the binding strength between NTAIL and XD and, therefore, constitute an auto-inhibitory segment of the protein20.


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Figure 2. Kinetics of binding of NTAIL truncated variants to XD. Observed rate constants (kobs) were measured as a function of NTAIL concentration (typically ranging from 1 to 60 μM) by temperature-jump experiments, in the presence of XD Y480W at a constant concentration (5 μM) in buffer 10 mM sodium phosphate pH 7.0, 150 mM NaCl. In each panel, the data obtained for wild-type NTAIL are reported in grey, whereas those of each variant are reported in black.

The analysis of the kinetics of the truncation variants of NTAIL shows unambiguously that the disordered region flanking the MoRE influences its binding mechanism to XD. The dampening effect may arise either from steric hindrance brought by the disordered extension, or from the direct interaction between specific residues. To shed light on these two alternative hypotheses, we carried out site-directed mutagenesis in the region between the residues 451 and 482 and produced 19 site directed mutants. The dependence of the observed rate constant as a function of NTAIL concentration for each of these variants is reported in Figure 3. Surprisingly, whilst some of the variants display a hyperbolic kinetics similar to that of wild type NTAIL, it is evident that substitutions outside the MoRE have a clear effect on NTAIL binding to XD. In particular, it appears that substitutions at residues 451, 454, 459, 465, 466 and 467 increase the folding rate of the MoRE, suggesting that these residues play a direct role in the self-inhibition of folding.



Figure 3. Kinetics of binding of NTAIL and its site-directed variants to XD. Observed rate constants were measured as a function of NTAIL concentration (typically ranging from 1 to 50 μM) by temperature-jump experiments, in the presence of XD Y480W at a constant concentration (5 μM). The experiments were performed following the same experimental set up as that used for the different truncation variants (Figure 2). In each panel, the data obtained for wild-type NTAIL are reported in grey, whereas those of each variant are reported in black.

The understanding of protein functions often relies on experiments carried out on isolated domains. Yet, it is not rare to observe a different behavior when a domain is investigated in isolation as compared to the more complex scenario that may be found in the context of full-length proteins 21-24. This observation may be even more relevant for disordered segments that are endowed with an extreme allostery and may hence mediate the communication between contiguous domains. In the case of NTAIL, by systematically mutating the sequence both by truncation and by site-directed mutagenesis in regions contiguous to the MoRE, we could identify a hidden self-inhibitory interaction between residues 451, 454, 459, 465, 466 and 467 and the MoRE. Noteworthy, these interactions escaped identification in previous NMR studies 14-16, indicating that they are probably very dynamic and are, therefore, very elusive to an experimental characterization. Whilst a self-inhibitory role of disordered segments, or negative regulation, has been previously observed in other proteins 25-31, the results presented in this study are particularly interesting as they allow, for the first time, to assign a novel mechanism of inhibition exerted by the appendage. In fact, by comparing the kinetic behavior of the different variants, it is evident that inhibition relies on slowing down the folding step on the MoRE. In this context, the transient interactions between the MoRE and the fuzzy appendage appear to favor a disordered conformation, thereby tuning down the affinity between NTAIL and XD to match physiological requirements. Future work on other protein systems will further clarify the generality of these effects.

METHODS Constructs generation The constructs encoding N-terminally heaxahistidine tagged NTAIL truncation variants (from 401 to 481) with a tobacco etch virus (TEV) protease cleavage site (amino acidic sequence: ENLYFQGS) immediately upstream of the codons encoding amino acids 401, 411, 421, 431, 441, 451, 461, 471 and 481 of NTAIL were already available 20 and cloned into the pETG20A expression vector that drives the expression of the protein of interest fused to a Thioredoxin (Trx) solubility tag. The constructs encoding the site directed variants of NTAIL were obtained using the gene encoding wild-type NTAIL, inserted in the pET28 expression vector, as template to perform site-directed mutagenesis using the ACS Paragon Plus Environment

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QuickChange Lightning Site-Directed Mutagenesis kit (Agilent technologies) according to the manufacturer’s instructions. All substitutions were conservative and were confirmed by DNA sequencing. Protein expression and purification NTAIL site-directed variants were expressed in the Escherichia coli Rosetta T7 pLysS (Novagen) strain. Cultures were grown in Luria Bertani (LB) medium containing 100 μg/ml ampicillin and 34 μg/ml chloramphenicol at 37°C until the OD600 reached 0.6-0.8 and then protein expression was induced with 1 mM IPTG (isopropyl-β-Dthiogalactopyranoside). After induction, cells were grown at 25°C over night and then collected by centrifugation. Cells were resuspended in 5ml/gr of pellet of buffer A (50 mM sodium phosphate pH 7.2, 300 mM NaCl) supplemented with 0.1 mg/mL lysozyme, 10 μg/mL DNase I and protease inhibitor cocktail (Sigma) and disrupted by sonication. The lysate was clarified by centrifugation at 12,000 rpm for 40 minutes at 4 °C. The soluble fraction of the bacterial lysate was loaded onto a 5 ml His trap FF column (GE, Healthcare) preloaded with Ni2+ ions, previously equilibrated in buffer A. After a washing step with buffer A supplemented with 1 M NaCl, the proteins were eluted with buffer A containing 250 mM imidazole. The proteins were then loaded onto a Superdex 75 16/60 column (GE, Healthcare). Isocratic elution was carried out in SEC buffer (10 mM sodium phosphate pH 7.0, 150 mM NaCl). The NTAIL truncation variants were expressed in the same conditions described for the site-directed NTAIL variants except that Terrific Broth (TB) medium was used and cells were collected by centrifugation after 4 hours at 37°C. Cell lysis and the first step of purification through IMAC were carried out as described above. In this case, the buffer used to equilibrate the column was 50 mM Tris/HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM phenyl-methyl-sulphonyl-fluoride (PMSF). After sample injection, the column was washed with 50 mM Tris/HCl pH 8.0, 1 M NaCl, 20 mM Imidazole. Elution was carried out with 50 mM Tris/HCl pH 8.0, 300 mM NaCl, 250 mM Imidazole. Eluents from IMAC were desalted using a desalting column (GE, Healthcare) previously equilibrated with desalting buffer (20 mM Tris/HCl pH 8.0, 300 mM NaCl). Cleavage with TEV protease was carried out overnight at 4°C in desalting buffer using 1 mg of TEV protease per 20 mg of target protein. Samples were then loaded onto 2.5 ml Ni2+ IDA Agarose resin, previously equilibrated in desalting buffer, and incubated 15 minutes at 4°C. The non-retained fraction was recovered and then loaded onto a Superdex 75 16/60 column (GE Healthcare) and eluted in SEC buffer. The pseudo wild-type XD variant Y480W was expressed and purified as previously reported 17. Temperature-jump measurements Relaxation binding experiments were performed by using a Hi-Tech PTJ-64 capacitor-discharge temperaturejump apparatus (Hi-Tech, Salisbury, UK). The fluorescence change of N-acetyltryptophanamide (NATA) was used in control measurements. The experiments were carried out in pseudo first order conditions by mixing a constant concentration of pseudo wild-type Y480W XD (5 μM) with different NTAIL concentrations usually ranging from 2 uM to 50 uM. The buffer used was 10 mM sodium phosphate pH 7.0, 150 mM NaCl. The solutions, at equilibrium, were degassed and slowly pumped through the 0.5x2 mm quartz flow cell where a rapid discharge of 12 kV leads to a temperature jump of 9°C (from 16 to 25°C). The relaxation rate was then measured by following the change in the tryptophan fluorescence occurring in the reaction. Usually 10-20 traces were averaged for each sample. The excitation wavelength was 296 nm and the change in the fluorescence emission of the tryptophan was measured as a function of the time using a 320 nm cut-off filter. The same experiments were performed using the truncation variants and all the site-directed variants of NTAIL. In all cases, observed kinetics were consistent with single exponential time courses. Observed rate constants obtained were plotted as a function of NTAIL concentration and fitted to the following hyperbolic function, arising from an induced-fit model: 𝑘𝑜𝑏𝑠 =

[𝑁𝑇𝐴𝐼𝐿] ∙ 𝑘𝐹 [𝑁𝑇𝐴𝐼𝐿] + 𝐾′𝐷

+ 𝑘𝑈

where kF is the folding rate constant and kU is the unfolding rate constants of NTAIL and K’D is the dissociation constant of the initial complex between NTAIL unfolded and XD. Data were fitted to a hyperbolic function on the basis of the ACS Paragon Plus Environment


kinetic observations reported previously 18. Whilst some of the experimental points could not be recorded under the pseudo-first order assumption, it is important to note that, as the described in the text, the main conclusions of this work are based on the analysis of the data at high concentrations of NTAIL. AUTHOR INFORMATION Corresponding Author

Stefano Gianni: [email protected] Sonia Longhi: [email protected] Author Contributions

S.L. and S.G. designed research; F.T, D.B and C.B. performed research; F.T. and S.G. analyzed data; S.G. wrote the first draft of the manuscript and all the authors revised the manuscript. Funding Sources

No competing financial interests have been declared. Work partly supported by grants from the Italian Ministero dell’Istruzione dell’Università e della Ricerca (Progetto di Interesse ‘Invecchiamento’ to S.G.), Sapienza University of Rome (C26A155S48, B52F16003410005 and RP11715C34AEAC9B to S.G) and the Associazione Italiana per la Ricerca sul Cancro (Individual Grant - MFAG 2016, 18701 to S.G.). F.T. was supported by a fellowship from the Italo-French University. REFERENCES

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Understanding Intramolecular Crosstalk in an Intrinsically Disordered

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