Folding perspectives of an intrinsically disordered transactivation domain and its single mutation breaking the folding propensity

Journal Pre-proof Folding perspectives of an intrinsically disordered transactivation domain and its single mutation breaking the folding propensity

Nitin Sharma, Alexander V. Fonin, Olesya G. Shpironok, Sergey A. Silonov, Konstantin K. Turoverov, Vladimir N. Uversky, Irina M. Kuznetsova, Rajanish Giri PII:

S0141-8130(19)34479-4

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.11.111

Reference:

BIOMAC 13892

To appear in:

International Journal of Biological Macromolecules

Received date:

14 June 2019

Revised date:

1 November 2019

Accepted date:

12 November 2019

Please cite this article as: N. Sharma, A.V. Fonin, O.G. Shpironok, et al., Folding perspectives of an intrinsically disordered transactivation domain and its single mutation breaking the folding propensity, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2019.11.111

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© 2018 Published by Elsevier.

Journal Pre-proof

Folding perspectives of an intrinsically disordered transactivation domain and its single mutation breaking the folding propensity

Nitin Sharma,1,# Alexander V. Fonin,2,# Olesya G. Shpironok,2 Sergey A. Silonov,2 Konstantin K. Turoverov,2,3 Vladimir N. Uversky,4,5,* Irina M. Kuznetsova,2,* and Rajanish

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Giri1,6,*

School of Basic Sciences, Indian Institute of Technology Mandi, Himachal Pradesh 175005,

Laboratory of structural dynamics, stability and folding of proteins, Institute of Cytology,

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2

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India

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Russian Academy of Sciences, St. Petersburg, Russia

Peter the Great St. Petersburg Polytechnic University, Department of Biophysics,

Department of Molecular Medicine and USF Health Byrd Alzheimer's Research Institute,

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Polytechnicheskaya av. 29, St. Petersburg, Russia

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Morsani College of Medicine, University of South Florida, Tampa, FL, USA Laboratory of New Methods in Biology, Institute for Biological Instrumentation of the Russian

Academy of Sciences, Pushchino, Moscow region 142290, Russia 6

BioX Centre, Indian Institute of Technology Mandi, VPO Kamand, 175005, India

*Corresponding authors: VNU, E-mail: [email protected]; Phone: 1-813-9745816; Fax: 1-813-974-7357; IMK, E-mail: [email protected]; Phone: +7 812 2971957; Fax: +7 812 2970341; RG, E-mail: [email protected]; Phone: +91-1905-267134; Fax: 01905-267138 1

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ABSTRACT Transcriptional regulation is a critical facet of cellular development controlled by numerous transcription factors, among which are E-proteins (E2A, HEB, and E2-2) that play important roles in lymphopoiesis. For example, primary hematopoietic cells immortalisation is promoted by interaction of the conserved PCET motif consisting of the Leu-X-X-Leu-Leu (LXXLL) and

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Leu-Asp-Phe-Ser (LDFS) sequences of the transactivation domains (AD1) of E-proteins with the KIX domain of CBP/p300 transcriptional co-activators. Earlier, it was shown that the LXXLL

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motif is essential for the PCET-KIX interaction driven by the PCET helical transition. In this

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study, we analyzed the dehydration-driven gain of helicity in the conserved region (residues 11-

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28) of the AD1 domain of E-protein. Particularly, we showed that AD1 structure was

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dramatically affected by alcohols, but was insensitive to changes in pH or the presence of osmolytes sarcosine and taurine, or high polyethylene glycol (PEG) concentrations and DOPC

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Liposomes. These structure-forming effects of solvents were almost completely absent in the

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case of L21P AD1 mutant characterized by weakened interaction with KIX. This indicates that KIX interaction-induced AD1 ordering is driven by PCET motif dehydration. The L21P

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mutation-caused loss of molecular recognition function of AD1 is due to the mutation-induced disruption of the AD1 helical propensity.

Keywords: Intrinsically Disordered Proteins, transactivation domain, PCET Motif

1. INTRODUCTION Non-random chromosomal translocations are known to cause several malignancies. These translocations either result in production of novel chimeric factors, which stimulate uncontrolled 2

Journal Pre-proof cell growth and inhibit differentiation or lead to overexpression of endogenous genes [1]. One such translocation occurs between chromosome 1 and 19 (t (1; 19)), which fuses E2A gene (one of the E-protein-encoding genes) and the PBX1 gene encoding for the homeodomain protein PBX1, implicated in induction of acute lymphoblastic leukemia (ALL) [2]. In approximately 412% of pediatric ALL cases, t (1; 19) leads to the expression of oncogenic E2A-PBX1 fusion protein [3]. The E-proteins (E2A, HEB, and E2-2) are a family of class I basic helix-loop-helix

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(bHLH) mammalian transcription factors that play essential roles in lymphopoiesis [2]. Each

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member of this family contains a C-terminal domain (CTD) for protein dimerization and

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recognition of the E-box DNA sequence CANNTG and two N-terminal activation domains AD1

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and AD2 that function in recruitment of co-activators and co-repressors [4]. An important conserved region known as PCET motif of AD1 domain consists of two overlapping conserved

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protein recognition motifs Leu-xx-Leu-Leu (LXXLL) and Leu-Asp-Phe-Ser (LDFS) sequences.

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The LXXLL motif found in AD1 is an illustrative member of the highly conserved ϕ-x-x-ϕ-ϕ motif (where ϕ represents a bulky hydrophobic residue and x corresponds to any amino acid) that

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mediates interactions between transcription factors and transcriptional co-regulators [5]. On its

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turn, the conserved LDFS motif, which is found in the vertebrate class I HLH proteins and in a yeast HLH protein, stimulates transcription by direct recruitment of the SAGA histone acetyltransferase complex [6]. Previous reports have identified the role of PCET motif in interactions with transcriptional activators, where this conserved motif was shown to bind to a KIX domain of the members of the CBP/p300 transcriptional co-activator family [2]. This CBP/p300 family includes two closely related transcriptional co-activating proteins, CREBbinding protein (CBP or CREBBP) and E1A binding protein p300 (p300 or EP300) [7]. Both members of the CBP/p300 family are characterized by similar structures, containing five protein

3

Journal Pre-proof interaction domains, such as the nuclear receptor interaction domain (RID), the KIX domain (CREB and MYB interaction domain), the cysteine/histidine regions (TAZ1/CH1 and TAZ2/CH3), and the interferon response binding domain (IBiD) [7]. Studies have also identified this PCET motif as a target of the eTAFH domain of transcriptional corepressor ETO and leukemogenic fusion protein AML1-ETO [8,9]. These interactions prevent the binding of KIX domain to the same site and lead to the silencing of E-proteins.

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The molecular mechanism through which E2A-PBX1 plays a role in leukaemia induction is

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unclear. In literature, two potential mechanisms are consonant: recruitment of co-regulators to

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the target gene loci bound to E2A-PBX1 homeodomain or withdrawal of co-regulators away

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from the wild-type E-proteins. These mechanisms, in turn, reveal a critical role of the AD1

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domain of E2A-PBX1 in the recruitment of transcriptional co-regulators. Therefore, AD1 seems to be indispensable for E2A-PBX1 mediated oncogenesis [2].

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The role of the conserved region of AD1 domain of E-proteins (E2A and E2-2) in transcription

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activation of yeast and mammalian cells was demonstrated [10], where it was observed that the AD1 conserved putative helix region (PCET motif) is crucial for transactivation properties of E-

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proteins. The AD1 domain (residues 1-99) has reported being structured and characterised by the distinctive helical properties, but the synthetic peptide (residues 9-31) representing conserved region (PCET motif) was unstructured in aqueous solution and adopted a helical conformation with the increasing concentration of 2,2,2-trifluoroethanol (TFE) [10]. The analysis of the role of the interaction between E2A-PBX1 (1-483) and KIX domain of CBP/p300 in proliferation of primary hematopoietic cells revealed that this interaction with KIX domain requires helical AD1 and AD2 domains [11]. This study highlighted the functional cooperativity between the two domains and presented the importance of this interaction in the immortalisation of primary bone 4

Journal Pre-proof marrow cells retrovirally transduced with E2A-PBX1 [11]. As conserved PCET motif region of AD1 is unstructured, its interaction with KIX domain was elucidated to show that after interaction with KIX, AD1 conserved domain undergoes helical transition [12]. But the substitution of L20 in LXXLL motif has shown to impair PCET interaction with KIX and also inhibited cell immortalisation by E2A-PBX1 [12]. E2A binding requirements to the KIX domain was functionally mapped and compared with the c-Myb binding to KIX. This analysis revealed

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that the E2A interaction was less sensitive to the disruption of hydrophobic pocket and groove of

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KIX, suggesting different mode of KIX recognition by these activation domains and underscored

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the need of biophysical and structural characterisation of the E2A-KIX interaction further [12].

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The importance of LXXLL motif for the interactions of E-proteins with KIX and oncogenesis was also reported [2]. The LXXLL residues of PCET motif are essential for both binding KIX

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and bone marrow immortalisation by E2A-PBX1. However, despite the abundance of data about

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the function of both E-proteins and CBP/p300, how the interaction between E2A-PBX1 and CBP/p300 might contribute to leukaemia induction is still unclear. Therefore, there is a need for

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detailed structural studies to find the molecular mechanisms behind the abolishment of leukemia

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induction due to the single L20 mutations in the AD1 region of E2A-PBX1. Intrinsically disordered proteins (IDPs) and IDP regions (IDPRs) are unable to form stable structures, but still, exhibit important biological activities [13–20]. Their flexibility and structural instability are due to the specific amino acid sequence [17,21]. The penetrance of IDPs in viruses and the use of molecular recognition features by these viral IDPs have allowed them to fold upon interaction with the folding partners to perform various functions with small number of proteins [22,23]. The studies of IDPs involvement in pathogenic disease have proven the importance of the development of new disorder based therapeutic strategies [22–28]. Structured proteins can be

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Journal Pre-proof grouped according to their secondary and tertiary structures but IDPs and IDPRs are difficult to partition into structural groups [29–31]. The conserved PCET motif of the AD1 domain of Eproteins is considered to be IDPR, which folds upon binding to KIX domain of CBP. Binding of IDPs to their physiological partners, such as proteins and nucleic acids, is often coupled with structuration and molecular recognition features, a phenomenon known as “folding upon binding” or “coupled folding and binding” [29,32–35]. Coupled folding and binding is further

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divided into two most commonly discussed mechanisms, that is, conformational selection (i.e.,

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folding before binding) and induced fit (i.e., folding after binding) [36,37]. However, there are

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also more complex mechanisms, e.g., comprising conformational selection combined with

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induced fit. The cellular conditions of molecular crowding (where the concentration of biological macromolecules ranges from 80 to 400 mg/ml) occupy 5–40% of cellular volume [38–41]. This

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creates a unique crowded medium with restricted amounts of free water [40,42–46] are also an

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essential factor, which may play a role in structural transitions and functionality of IDPs [47,48]. It is known that intracellular environment contains high concentration of biological

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macromolecules that can be created not only by high-molecular substances (proteins, nucleic and

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ribonucleic acids), but also by low-molecular compounds. In particular, under extreme conditions, cells of a number of organisms contain high concentrations (up to 2.5 M) of osmolytes [49–52]. Osmolytes are small organic substances, the main function of which is to equalize the hydrostatic pressure between the inside of cell and the extracellular space. These low-molecular compounds accumulate in the cell in response to stress (osmotic, temperature, caused by changes in pH, salt concentration) and are able to increase the stability and functional activity of native proteins and promote folding of unfolded proteins. Therefore, osmolytes are often considered as ‘chemical chaperones’ [50,53–58].

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Journal Pre-proof Here, we have elucidated the structural properties of the conserved PCET motif (residues 11-28) of the E-protein HEB (reference from structure PDB: 2KWF) to evaluate the effects of pH, temperature, different solvents, osmolytes, and macromolecular crowding conditions. We have found the gain of helical content in the peptide corresponding to the wild type PCET motif in the presence of alcohols, such as ethanol, 2,2,2-trifluoroethanol (TFE), hexafluoroisopropanol (HFIP) and the osmolyte trimethylamine N-oxide (TMAO). Also, we have observed that the

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introduction of the proline (known as a helix breaker) at the 21st residue (L21P mutation) has

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disrupted the helical propensity of the peptide. Curiously, the previous analysis revealed that the

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L20A mutant of the analogous E2A-PCET peptide showed no detectable binding to KIX domain

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[2]. It is known that although proline serves as a helix breaker, alanine is known to display the highest propensity for helical structure among the amino acids. Therefore, these observations

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indicate that the mechanisms for disruption of KIX binding by L21P mutation in HEB PCET

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motif and by the L20A mutation in the E2A-PCET peptide are different. In other words, our analysis provided mechanistic foundation needed for better understanding of the roles of

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substitutions of the conserved leucine residue at 20th position of E-proteins in disrupting the

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transactivation and molecular recognition functions of E-proteins and in ultimate blocking of the leukaemia induction by E2A-PBX1.

2. MATERIAL AND METHODS Many chemicals were purchased from Sigma-Aldrich. The peptide comprising 19 amino acid residues (11-28) of the wild type PCET motif of AD1 domain of HEB (NH2GSGTDKELSDLLDFSAMFS-COOH) and mutant type (L21P) was chemically synthesized by ThermoFisher Scientific. The wild type peptide sequence was equivalent to the peptide used for obtaining the NMR solution structure of KIX domain bound to PCET (HEB) motif of E-protein 7

Journal Pre-proof activation domain 1 (AD1) with PDB: 2KWF. Lyophilised peptide was dissolved in buffer (10 mM potassium phosphate, 50 mM sodium sulphate, pH 7.0) at concentration of 1 mg/ml. Lipids were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Computational analysis: The intrinsic disorder predisposition of human E-protein HEB, also known as transcription factor 12 (TCF-12), class B basic helix-loop-helix protein 20 (bHLHb20), DNA-binding protein HTF4, E-box-binding protein, and transcription factor HTF-4 (UniProt ID:

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Q99081) was evaluated by a set of commonly used disorder predictors, such as PONDR® VLXT

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[59], PONDR® VSL2 [60], PONDR® VL3 [60,61]}, PONDR® FIT [62], IUPred_short, and

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IUPred_long [63–65]. Justification for the selection of these tools is provided in our previous

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publications (e.g., [66,67]). We also generated a mean per-residue intrinsic disorder profile for

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vitronectin by averaging the outputs of these six per-residue disorder predictors. The use of consensus or mean disorder propensity is motivated by empirical observations that this approach

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usually increases the predictive performance compared to the use of a single predictor [68–70]. The outputs of the evaluation of the per-residue disorder propensity by these tools are

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represented as the real numbers between 1 (ideal prediction of disorder) and 0 (ideal prediction

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of order). A threshold of ≥0.5 was used to identify disordered residues and regions in a query protein, whereas residues/regions characterized by the disorder scores ranging from 0.2 to 0.5 are classified as flexible. Liposome Preparation: Dioleoyl-phosphatidyl choline (DOPC) was used for preparation of neutral liposomes. Liposomes were prepared in 20mM sodium phosphate buffer pH 7.4 by 5 freeze thaw cycles in liq. N2 and water at 60oC. This is followed by the extrusion method using mini Avanti extruder with 100 nm polycarbonate filters by Avanti standard protocol [71]. The

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Journal Pre-proof size and uniformity of prepared liposomes was confirmed by Dynamic Light Scattering (DLS) using Zetasizer Nano ZS (Malvern Instruments Ltd., UK). CD spectroscopy: CD spectroscopy was used to assess the secondary structure composition of the AD1 domain of E-protein (HEB) in various solvents (such as water and aqueous mixtures of ethanol, TFE, and HFIP), osmolytes (such as sarcosine, taurine and TMAO), neutral liposomes such as DOPC, as well as in the presence of high concentration of crowding agent, PEG. Far-UV

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CD spectra were measured between 190 and 240 nm at a protein concentration of 20 µM using a

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JASCO-815 spectropolarimeter equipped with a thermostatically controlled cell holder.

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Measurements were conducted at 25°C using a quartz cuvette with the 1 path-length mm.

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Spectra were recorded at a scan speed of 50 nm/min and a bandwidth of 1 nm. The spectra were

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averaged over three scans to eliminate signal noise. The normalisation of data was performed by subtracting the baseline recorded for the buffer from values obtained for protein samples under

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similar conditions.

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Phase diagram analysis: The “phase diagram” method (also known as method of spectral diagrams, or parametric dependencies) was first suggested by Dr. Burstein in 1971 for the

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analysis of fluorescence data [72], and later, a generalized form was developed for any pair of extensive independent spectral parameters [73]. The indispensable property of this method of the phase diagrams is to develop the plot of I1 versus I2, where I1 and I2 are the spectral intensities measured on wavelengths 1 and 2 under distinct experimental conditions for a protein undergoing structural transformations. Spectral intensity can be described by any twocomponent system by a simple relationship: I()=1 I1() + 2 I2()

(1)

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Journal Pre-proof 1+2= 1, where I1() and I2() are the spectral intensities corresponding to the first and second components, whereas 1 and 2 are relative contents of these components in a system. Excluding

1 (1=1-2), the equation (1) could be rewritten as: I()=(1- 2) I1() + 2 I2() = I1() + 2(I2()-I1())

(2)

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Here, 2 may be determined from the spectral intensity measurements at two different

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wavelengths, 1 and 2. In fact:

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and

(4)

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I() = I1() + 2 (I2() - I1())

(3)

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I() = I1() + 2 (I2() - I1())

𝐼(𝜆2 )−𝐼1 (𝜆2 ) (𝜆2 ) 2 (𝜆2 )−𝐼

𝛼2 = 𝐼

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1

(5)

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The relationship between I() and I() may be determined by the substitution of 2 in (3) from

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(5):

𝐼(𝜆2 )−𝐼1 (𝜆2 ) (𝐼2 (𝜆1 ) 2 (𝜆2 )−𝐼1 (𝜆2 )

𝐼(𝜆1 ) = 𝐼1 (𝜆1 ) + 𝐼

𝐼 (𝜆 )−𝐼 (𝜆 )

− 𝐼1 (𝜆1 )) = 𝐼1 (𝜆1 ) − 𝐼2 (𝜆1 )−𝐼1 (𝜆1 ) 𝐼1 (𝜆2 ) + 2

𝐼2 (𝜆1 )−𝐼1 (𝜆1 ) 𝐼(𝜆2 ) 𝐼2 (𝜆2 )−𝐼1 (𝜆2 )

2

1

2

(6)

or: 𝐼(𝜆1 ) = 𝑎 + 𝑏𝐼(𝜆2 ),

(7)

where 𝐼 (𝜆 )−𝐼 (𝜆 )

𝑎 = 𝐼1 (𝜆1 ) − 𝐼2 (𝜆1 )−𝐼1 (𝜆1 ) 𝐼1 (𝜆2 ) and 𝑏 = 2

2

1

2

𝐼2 (𝜆1 )−𝐼1 (𝜆1 ) . 𝐼2 (𝜆2 )−𝐼1 (𝜆2 )

10

(8)

Journal Pre-proof In relation to protein unfolding experiments, the relationship (7) predicts that the dependence 𝐼(𝜆1 ) = 𝑓(𝐼(𝜆2 )) will be linear, if changes in protein environment result in the all-or-none transition between two different conformations. Contrarily, the non-linearity of this function imitates the sequential behavior of structural transformations. Furthermore, each linear portion of the 𝐼(𝜆1 ) = 𝑓(𝐼(𝜆2 )) dependence describes the individual all-or-none transition. In principle, 1

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and 2 are the arbitrary wavelengths of the spectrum [73]. Solution-state NMR Spectroscopy: All NMR experiments were performed on a Bruker

H] NOESY (Nuclear Overhauser Enhancement spectroscopy) experiments were carried out for

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1

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Advance III 500 MHz spectrometer equipped with the cryogenic triple-resonance probe. 2D [1H-

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protein resonance assignment and collected 2048 data points in f2 and 1024 in f1 on 1 mM

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samples of wild type and mutant E2A peptides in phosphate buffer, pH 7. Solvent suppression was achieved by pre-saturation of the water signal for samples in aqueous-D2O (5%). NOESY

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data were collected with a mixing time of 250 ms to derive 1H–1H distance constraints. A total of

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128 (NOESY) transients were acquired and all 2D spectra were recorded with a spectral width of 7,000 Hz. Bruker AG 3.5 and Topspin 2.1 software was used for acquisition, Fourier

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transformation, and processing of time-domain data. Computer Aided Resonance Assignment (CARA 1.9.2 alpha) software was used for backbone and side chain resonance analysis.

3. RESULTS To place the intrinsic disorder predisposition of the PCET motif within the context of the full length human HEB protein and to evaluate how this propensity is affected by the L21P mutation, we subjected the amino acid sequences of the wild type and mutant proteins to the

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Journal Pre-proof multiparametric computational analysis utilizing several commonly used per-residue disorder predictors. Results are shown in Figure 1. Figure 1A illustrates that the wild type human HEB is predicted as mostly disordered protein containing several relatively short ordered regions. The presence of such short regions of order embedded within the long IDPRs represents a hallmark feature of many IDPs and reflects their ability to adopt structure upon interaction with a binding partner. In fact, such segments are commonly classified as molecular recognition

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features (MoRFs), which are relatively short segments in IDPs that are capable of undergoing

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disorder to order transitions upon binding to their partners [29,74–80]. In its bound state, the

[29,74,75,80,81]. Because of specific sequence features, MoRFs are

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irregular structure

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ordered (or “frozen”) MoRF can contain elements of classic secondary structure or possess

predictable with rather high accuracy [29,77,78]. Using one of such tools, ANCHOR [78],

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human HEB was predicted to contain 20 MoRFs (residues 5-31, 41-52, 82-108, 114-143, 153-

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162, 171-196, 219-230, 246-256, 265-277, 291-298, 302-312, 315-353, 388-422, 424-490, 498-519, 546-554, 588-609, 614-632, 641-647, and 676-682). In other words, more than 60%

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of the residues of this protein might take part in disorder-based interaction with various

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partners. Figure 1B focuses on the first 50 residues of the wild type human HEB, a region containing the PCET motif. It is seen that similar to the full-length protein, the N-terminal tail of human HEB is mostly disordered. Importantly, the PCET motif (shown as a gray shaded are) is located within the first MoRF region, conforming the intrinsic propensity of this motif for binding to specific partners. Finally, Figure 1C represents the first 50 residues of the mutated HEB and clearly shows that the L21P mutation noticeably increases the intrinsic disorder propensity of PCET.

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Journal Pre-proof To elucidate the secondary structure of the PCET motif of the AD1 domain of HEB (a homologous sequence of E2A-PBX1) in solution, we carried out far-UV circular dichroism (CD) experiments at pH 7.0 and 25°C. The far-UV CD spectrum of the conserved region (residues 11-28) of AD1 domain of E-protein HEB reported in Figure 2A shows absence of ordered secondary structure, confirming that PCET motif of AD1 domain of E-proteins is an IDPR. Furthermore, Figure 2A shows that the L21P mutation has a minimal effect on the

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structure of this peptide.

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In order to determine which factors may cause the ordering of the PCET structure in its bound

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form, we have studied the structure of this peptide in various solvents. First, we investigated the

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spectral characteristics of PCET peptide in solutions with different pH. Results are summarized

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in Figure 2B illustrates that the structure of PCET motif was practically the same in solutions with acidic, neutral, and alkaline pH, indicating the independence of PCET conformation on

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environmental pH.

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Next, we looked at the effect of temperature on the secondary structure of the wild type and L21P mutant PCET peptides. Figure 3 summarises the results of these studies and shows that

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similar to many other IDPs [13], these peptides possess so-called “turned out” response to heat [82–84]. Figures 3A and 3B show that at low temperatures, both peptides possessed far-UV CD spectra typical of an unfolded peptide chain. As the temperature increased, the shape of spectra changed, reflecting the temperature-induced formation of transient structure content as documented in other IDPs representing the redistribution of statistical coils [85]. Note that these temperature-induced changes in secondary structure of these peptides were completely reversible. This temperature-induced changes of the wild type and L21P mutant PCET peptides is further illustrated by Figures 3C and 3D, which represent dependences of []222 and []198 of 13

Journal Pre-proof both peptides on temperature. Finally, Figures 3E and 3F shows that temperature-induced hydrodynamic changes in the structures of these peptides can be described in terms of response for temperature sensitivity generally correlated to the temperature induced backbone conformations in polypropylene II and -helix as reported by English and co-workers, since the corresponding phase diagrams represent mostly linear dependences [86]. The secondary structure analysis of CD spectra using K2D3 software had shown the inertness of both wild type and L21P

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peptide. The structure-forming effects of the elevated temperatures may be attributed to the

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stronger hydrophobic interaction at higher temperatures, which may be the major driving force

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for transient conformational changes.

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Membrane–protein interfaces are known to stabilize inter-residue hydrogen bond networks of

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proteins by the exclusion of the bulk solvent and reducing the effective dielectric constant [87]. Alcohols, by decreasing the dielectric constant around the peptide/protein, favor the formation

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of inter-residue hydrogen bonds, which stabilize secondary structure elements, such as α-

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helices [88]. Based on this analogy, alcohols such as TFE or HFIP are referred to as membrane-mimetic solvents for proteins. Based on these premises, we analyzed the effects of

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different alcohols (such as ethanol, TFE, and HFIP) on secondary structure of the wild type and L21P mutant of the PCET peptide. Results of these studies are shown in Figures 4-6 and clearly illustrate that the addition of any of the alcohols caused the noticeable increase in the ordered secondary structure content manifested by a decrease in the minimum at 198 nm accompanied by an increase in negative mean residual ellipticity around 222 nm. Importantly, these structural changes observed by CD were completely reversible and were independent of protein concentration (at least in the range of 0.1–2.5 mg/ml), indicating that the increase in structure of wild type PCET domain induced by alcohols represents an intramolecular process

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Journal Pre-proof and not self-association. Since pronounced helical structure was induced by all three alcohols in the wild type PCET peptide, one can conclude that this peptide possesses profound helical propensity that can be stabilized by alcohol-induced dehydration of protein causing the formation of intramolecular hydrogen bonds. The helical propensity of wild type HEB PCET peptide in aqueous TFE solutions has been quantified by the analysis of induced secondary structure elements from CD spectra using K2D3 software (as shown in supplementary Figure

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S1). The total helical propensity of PCET motif was found to be ~77% at a TFE concentration

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of 90% (Supplementary Table T1). This is in agreement with the previous studies on the

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modulation of secondary structure of peptides derived from globular proteins by alcohols,

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where it was shown that the helical peptides have a significant intrinsic tendency to adopt their

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native helical structure in aqueous alcohol solutions [89]. On the contrary, similar analysis conducted for the L21P mutant form in presence of ethanol

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and fluoroalcohols revealed that the increase in the concentration of all three alcohols had a minor effect on the structural properties of this peptide (see plots B, D, and F in Figures 4-6).

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The analysis of percent helicity of L21P from CD spectra using K2D3 has shown very minor

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changes at higher concentrations of TFE (as shown in Supplementary Figure S2). These observations indicate that the intrinsic helical propensity of the PCET peptide is dramatically distorted by the L21P mutation. We have also analyzed the conformational changes in WT peptide in the presence of neutral liposomes (DOPC) using far-UV CD spectroscopy. However, we have not found any significant effects of DOPC in the far-UV CD spectra of the peptide up to a concentration of 5 mM (see in Figure 7). At higher concentrations, DOPC led to the noticeable increase in the high-tension (HT) voltage, resulting in the dramatic increase in the spectral noise.

15

Journal Pre-proof Biological macromolecules are known to function in a cell under conditions of macromolecular crowding characterized by limited free volume. It is believed that this macromolecular crowding might limit the number of conformations that the polypeptide chain can adopt, which, in turn, can cause both the folding of the polypeptide chain into a more compact structure, and the misfolding of the proteins and their aggregation. The macromolecular crowding conditions in vitro are usually modeled using the highly concentrated solutions of inert polymers of different

of

molecular weights, such as polyethylene glycol (PEG), Dextran, Ficoll, etc. Due to the mutual

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impermeability of the dissolved macromolecules under such conditions, the steric interactions

-p

between the biological object under study and the solute molecules increase substantially. We

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investigated the effect of different concentrations of PEGs with different molecular weights on the secondary structure of PCET peptide. Results are summarised in Figure 8, which clearly

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shows that neither the concentration nor the molecular mass of PEG used in this study had a

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significant effect on the peptide structure. This indicates that the decrease in the accessible volume does not cause noticeable ordering of the PCET peptide.

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Next, we analyzed the effects of various osmolytes (sarcosine, taurine, and TMAO) on the

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structure of the PCET peptide and its L21P mutant. This analysis revealed that although structure of this domain was not affected by sarcosine and taurine, the increase in the TMAO concentration promoted a significant ordering of the wild type PCET (Figure 9). TMAO is known as one of the stronger osmolytes that can induce folding of various proteins [90]. It is believed that the effect of TMAO on the structure and stability of proteins is determined by the osmophobic effect; i.e., the preferential exclusion of water molecules from the near-surface layer of the protein [91–93]. On the contrary, none of the osmolytes had noticeable effects on the highly disordered structure of the L21P mutant (data not shown).

16

Journal Pre-proof The 2D NMR NOESY spectra of the wild type and mutant E2A peptides obtained in aqueous solution (5% D2O) did not indicate the presence of secondary structure. However, in the presence of 20% HFIP/water mixtures, the increase in the number and the distribution of new correlations indicated that significant secondary structure had been induced. The overlay of the 2D NMR 1H1

H NOESY spectra measured for the wild-type peptide in the aqueous and 20% HFIP solution

was used to qualitatively identify changes in the content of secondary structure elements in the

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peptide after the addition of helix-stabilizing alcohol (HFIP) as shown in Figure 10. Similarly,

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the overlay of the 2D NMR 1H-1H NOESY spectra measured for the mutant peptide L21P in the

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aqueous and 20% HFIP solutions was plotted as shown in Figure 11 to identify the induction of

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helical structure, which was not prominently visible in mutant peptide. The analysis of NH-NH connectivity regions (Figures 10C and 11A) has shown the decreased HFIP-induced helicity in

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the mutant PCET peptide as compared to the wild type peptide. Also, the low NH-H cross-

na

peaks (as shown in Figures 10C and 11C) have represented the decrease in intramolecular Hbonding in L21P peptide in comparison with the wild type PCET motif. These results correlate

ur

very well with the Circular Dichroism data obtained for these peptides in the presence of helix-

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inducing solvent conditions. Hence, the mutant (L20P) is capable of breaking the helix propensity of PCET motif of E proteins and thereby can decrease the efficiency of the E2A-KIX interactions.

4. DISCUSSION A highly conserved 17 residues N-terminal region (PCET motif) of transactivation domain AD1 is present in E-proteins. It consists of two overlapping protein recognition sites, the LXXLL

17

Journal Pre-proof (representative of the ϕxxϕϕ motif) and LDFS motifs, identified to be responsible for interaction with transcriptional activators. Previous reports have indicated the specific interactions between KIX domain (residues 589-685) of CBP/p300 and PCET motif (residues 11-28) of the AD1 domain of E-proteins are related to the regulation of B cell lymphopoiesis and are essential for leukemia induction by E2A-PBX1. In structural studies on the PCETHEB bound to KIX domain of CBP, a set of residues at the KIX/PCET interface important for interactions has been

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established. These included the hydrophobic residues L17, L20, and L21 from the LXXLL motif

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and the F23 from the overlapping LDFS site. The L21 residue has been found to be associated

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with the hydrophobic pocket on KIX surface formed by I611, I660, L664, and K667 residues.

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Analysis of the primary structure of the AD1-PCET motif indicates a high probability of the α-

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helical structure formation, which would suggest the presence of a high content of α-helical regions in the PCET structure [10]. However, computational analysis of the full-length HEB

na

protein by a set of commonly used disorder predictors suggested that this protein is expected to be mostly disordered. Furthermore, the PCET motif, being generally disordered, is located within

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the MoRF region, which supposes to fold into an ordered conformation at interaction with

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binding partner. In agreement with the results of these computational studies, the far-UV CD spectrum and the 2D NMR 1H-1H NOESY spectra of the peptide corresponding to the PCET motif in aqueous solutions have all characteristics of highly disordered polypeptide chain, and that there is practically no α-helical structure of this region from the transactivation domain of HEB peptide. These data are in accordance with the results of previous studies of the PCET motif using 1H-15N HSQC experiments, where this peptide was shown to display poor dispersion of the HN resonances, thereby indicating its intrinsically disordered nature [2]. Since PCET motif has a predominant α-helical structure in its complex with KIX domain, this observation suggests

18

Journal Pre-proof that PCET acts as a molecular recognition feature (MoRF), which is region of intrinsically disordered protein that is unstructured in the unbound form but can gain folded conformation at interaction with binding partners [29]. Analysis of the structure of the KIX complexed with AD1 (PDB ID: 2kwf) indicated that in its bound state, the PCET motif of the AD1 domain is characterized by low solvent accessibility. Therefore, based on all these observations, it can be hypothesized that the acquisition of the α-helical structure by PCET upon its binding to KIX is

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driven by the displacement of water from the close environment of the PCET motif.

ro

To test this idea, we investigated the effect of various solvent conditions on structural properties

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of the peptide corresponding to the wild PCET motif, as well as to the L21P mutant form of this

re

region. It is known that the replacement of leucine residue at the junction of the Leu-x-x-Leu-

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Leu and Leu-Asp-Phe-Ser motifs of PCET weakens the interaction of E2A-PBX1 with the KIX domain and affects induction of bone marrow leukemia in mice in vitro [12]. Our analyses

na

revealed that the environmental conditions causing dehydration of the polypeptide chain (such as alcohols and osmolyte TMAO) were able, in a cosolute concentration-dependent manner, to

ur

induce noticeable structural transformation of the wild type PCET peptide reflective of the

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pronounced α-helical structure formation. However, neither change in pH, or addition of sarcosine and taurine osmolytes, nor mimicking conditions of macromolecular crowding by inclusion to the solution of high concentrations of polyethylene glycol (PEG) of different molecular masses had comparable structure-forming effects on the wild type PCET peptide. On the other hand, the structure of the L21P PCET mutant was much less sensitive to changes in solutions, and this mutant peptide preserved mostly disordered structure in the presence of alcohols and TMAO.

19

Journal Pre-proof Therefore, our analyses revealed that among various solvent conditions, noticeable structural changes in the wild type PCET peptide were induced by ethanol, TFE, and HFIP, which are cosolvents that stabilize the α-helical structure in peptides and proteins with intrinsic helical propensity, and by the osmolyte TMAO that contributes to the dehydration of proteins. The observed transition of the mostly disordered peptide to a conformation with pronounced α-helical structure in alcohol solutions and TMAO suggested that it is the dehydration of this peptide that

of

contributes to the ordering of its structure. Also, the inertness of the L21P mutant of the PCET

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peptide to structural changes in presence of alcohols and TMAO indicated that this amino acid

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substitution was sufficient to dramatically diminish the intrinsic helical propensity of this region,

ACKNOWLEDGEMENTS

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probably due to the fact that proline can serve as a helix breaker.

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This work was supported by grants from the Department of Science and Technology (DST)

ur

India, Grant number: INT/RUS/RFBR/P-255 (to R.G.) and from Russian Foundation for Basic

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Research, Grant number: 17-54-45169 IND (to I.M.K.) and INT/RUS/RFBR/P-255 (R.G.). We thank Dr. Neel Sarovar Bhavesh, Group Leader at the International Center for Genetic Engineering and Biotechnology (ICGEB), for providing access to the NMR spectrometer at ICGEB New Delhi, which is procured and supported by the Department of Biotechnology (DBT), Government of India and ICGEB core funds.

AUTHOR’S CONTRIBUTION

20

Journal Pre-proof RG: conception and design. RG and IMK: study supervision. NS, AVF, and SAS: performed experiment with wild type peptide. VNU: conducted computational analyses. NS: performed experiments of L21P mutant of peptide. NS, AVF, VNU, IMK, and RG: data analysis and writing of the manuscript. OGS, SAS, and KKT: data analysis and review of manuscript.

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COMPETING INTERESTS

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The authors declare no competing interests and do not include any financial and non-financial

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interests.

S.E. Aspland, H.H. Bendall, C. Murre, The role of E2A-PBX1 in leukemogenesis,

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[1]

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FIGURE LEGENDS Figure 1. Intrinsic disorder propensity of human HEB protein and its PCET motif. A. Disorder propensity of the full-length wild type human HEB protein (UniProt ID: Q99081). B. Zoomed-in view of the disorder profile of the N-terminal region (first 50 residues) of the wild

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type human HEB protein. C. Zoomed-in view of the disorder profile of the N-terminal region

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(first 50 residues) of the L21P mutant form of human HEB protein. Disorder propensity was

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studied by PONDR® VLXT (black curves), PONDR® VL3 (red curves), PONDR® VSL2 (green curves), PONDR® FIT (pink curves), IUPred_short (yellow curves), and IUPred_long (blue

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curves). Access to the per-residue disorder predictors of PONDR® family and to the UniProt2A

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disorder predictor was provided by the DisProt database (http://original.disprot.org/) and UniProt

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platform (https://iupred2a.elte.hu/), respectively. Light pink shadow around PONDR® FIT curve shows error distribution. Mean disorder predisposition was calculated by averaging of all

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predictor-specific per-residue disorder profiles (bold, dashed, dark cyan curve). Light blue

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shadow around the mean disorder curve shows the distribution of standard deviations. Gray shaded area in each plot represents position of the PCET motif.

Figure 2. Far UV-CD spectra of the peptides and effect of pH. A. Far UV-CD spectra of the peptide corresponding to the PCET motif of the AD1 domain of HEB E-protein. Data for the wild type (WT) and L21P mutant are shown by black and red lines, respectively. Measurements were conducted in buffer containing 10 mM potassium phosphate and 50 mM sodium sulphate, pH 7.4 at 25oC. B. Far UV-CD spectra of the wild type PECT 34

Journal Pre-proof peptide measured in solutions with different pH.

Figure 3. Effects of temperature on far-UV-CD spectra of the peptides. Effects of temperature on the wild type PCET peptide (A, C, and E) and its L21P mutant form (B, C, and F). Plots A and B represent far UV-CD spectra measured at temperatures changing

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from 4 to 90oC at an interval of 5oC. Plots C and D show the temperature courses of the

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ellipticity at 198 nm (black) and 222 nm (red) with increasing temperature to 90oC. Plots E and F

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represent phase diagrams ([]222 vs. []198 dependences) to analyze the mechanism of changes in

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structural propensity of these peptides under increasing temperature.

Figure 4. Effects of ethanol on secondary structure of the wild type PCET peptide and its

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L21P mutant.

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Effects of ethanol on the wild type PCET peptide (A, C, and E) and its L21P mutant (B, C, and F). Plots A and B represent far UV-CD spectra measured at ethanol concentration ranging from

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0% to 90% at an interval of 5%. Plots C and D show the dependences of the ellipticity at 198 nm (black) and 222 nm (red) on increasing ethanol concentration to 90%. Plots E and F represent phase diagrams ([]222 vs. []198 dependences) to analyze the mechanism of changes in structural propensity of these peptides under increasing ethanol concentration.

Figure 5. Effects of TFE on secondary structure of the peptides.

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Journal Pre-proof Effects of TFE on the wild type PCET peptide (A, C, and E) and its L21P mutant form (B, C, and F). Plots A and B represent far UV-CD spectra measured at TFE concentration ranging from 0% to 90% at an interval of 5%. Plots C and D show the dependences of the ellipticity at 198 nm (black) and 222 nm (red) on increasing TFE concentration to 90%. Plots E and F represent phase diagrams ([]222 vs. []198 dependences) to analyze the mechanism of changes in structural

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propensity of these peptides under increasing TFE concentration.

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Figure 6. Effects of HFIP on secondary structure of the peptides. Effects of HFIP on the wild type PCET peptide (A, C, and E) and its L21P mutant form (B, C,

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and F). Plots A and B represent far UV-CD spectra measured at HFIP concentration ranging

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from 0% to 90% at an interval of 5%. Plots C and D show the dependences of the ellipticity at

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198 nm (black) and 222 nm (red) on increasing HFIP concentration to 90%. Plots E and F represent phase diagrams ([]222 vs. []198 dependences) to analyze the mechanism of changes in

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structural propensity of these peptides under increasing HFIP concentration.

Figure 7. Effect of neutral liposomes (DOPC) at a concentration of 1mM, 2.5mM and 5mM on secondary structure of WT (HEB) peptide.

Figure 8. Effect of molecular crowder on secondary structure of peptides. A. Far-UV CD spectra of the PCET peptide in solutions containing different concentrations of PEG with the molecular weight of 600 Da. B. Far-UV CD spectra of the PCET peptide in solutions containing different concentration of PEG with the molecular weight of 4,000 Da. C. 36

Journal Pre-proof Far-UV CD spectra of the PCET peptide in solutions containing different concentration of PEG with the molecular weight of 12,000 Da. D. Far-UV CD spectra of the PCET peptide in solutions containing PEG of different molecular weights at concentration of 200 mg/ml.

Figure 9. Effect of osmolytes on far-UV-CD spectra of the peptides.

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A. Far-UV CD spectra of the PCET peptide in the presence of different concentrations of

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sarcosine. B. Far-UV CD spectra of the PCET peptide in the presence of different concentrations of taurine. C. Far-UV CD spectra of the PCET peptide in the presence of different concentrations

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of TMAO. D. Dependences of the ellipticity at 222 nm in far UV CD spectra of the PCET

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peptide on TMAO concentration.

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Figure 10. 2D NMR 1H-1H NOESY spectra of the wild type PCET peptide.

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The figure is showing the effect of HFIP on secondary structure elements of PCET peptide. The overlay of wild type PCET peptide in aqueous condition (Black) and 20% HFIP aqueous mixture

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(Red) using qualitative analysis (Negative peaks are shown in green). A. Fingerprint NH-H region of 2D NMR 1H-1H NOESY spectrum acquired at 500 MHz B. Fingerprint NH-H( or ) region of 2D NMR 1H-1H NOESY spectrum C. Fingerprint NH-NH connectivity region of 2D NMR 1H-1H NOESY spectrum D. Fingerprint H-H connectivity region of 2D NMR 1H-1H NOESY spectrum E. Fingerprint of H- H region of 2D NMR 1H-1H NOESY spectrum of PCET wild-type peptide. F. Overlay of 2D NOESY wide range spectrum of PCET wild type peptide in water (black) and 20% HFIP (red) aqueous mixture.

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Journal Pre-proof Figure 11. 2D NMR 1H-1H NOESY spectra of the mutant PCET peptide. The effect of HFIP on secondary structure elements of mutant PCET peptide has been shown by the overlay of mutant PCET peptide in aqueous condition (Black) and 20% HFIP aqueous mixture (Red) using qualitative analysis. A. Connectivity of NH-H region of 2D NMR 1H-1H NOESY spectrum has been shown to differentiate the secondary structure elements B. Overlay of NH-H( or ) region of 2D NMR 1H-1H NOESY spectrum C. Overlay of NH-NH correlations

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of 2D NMR 1H-1H NOESY spectrum D. Overlay of H-H connectivity region of 2D NMR 1HH NOESY spectrum of mutant PCET peptide E. Fingerprint of H- H region of 2D NMR 1H-

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H NOESY spectrum of mutant peptide. F. Overlay of 2D NOESY wide range spectrum of

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1

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PCET mutant peptide in water (black) and 20% HFIP (red) aqueous mixture.

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Figure 11