# Structural characterization of stem cell factors Oct4, Sox2, Nanog and Esrrb disordered domains, and a method to detect phospho-dependent binding partners

Chafiaa Bouguechtouli, Rania Ghouil, Ania Alik, Florent Dingli, Damarys Loew, et al.

Loew, Francois-Xavier Theillet

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Chafiaa Bouguechtouli, Rania Ghouil, Ania Alik, Florent Dingli, Damarys Loew, et al. Structural characterization of stem cell factors Oct4, Sox2, Nanog and Esrrb disordered domains, and a method to detect phospho-dependent binding partners. Comptes Rendus. Chimie, 2024, 26 (S3), pp.1 - 19. 10.5802/crchim.272. hal-04705165

HAL Id: hal-04705165

https://hal.science/hal-04705165v1

Submitted on 22 Sep 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_3.png" /></figure>

INSTITUT DE FRANCE
Académie des sciences

# Comptes Rendus
Chimie

Chafiaa Bouguechtouli, Rania Ghouil, Ania Alik, Florent Dingli, Damarys Loew and Francois-Xavier Theillet

**Structural characterization of stem cell factors Oct4, Sox2, Nanog and Esrrb disordered domains, and a method to detect phospho-dependent binding partners**

Published online: 19 April 2024

**Part of Special Issue:** Breaking Barriers in Chemical Biology – Toulouse 2022

**Guest editors:** Marie Lopez (CNRS-Univ. Montpellier-ENSCM, IBMM, Montpellier, France), Elisabetta Mileo (Aix-Marseille Univ, CNRS, BIP, IMM, Marseille, France), Eric Defrancq (Univ. Grenoble-Alpes-CNRS, DCM, Grenoble, France), Agnes Delmas (CNRS, CBM, Orléans, France), Boris Vauzeilles (CNRS-Univ. Paris-Saclay, ICSN, Gif-sur-Yvette, France), Dominique Guianvarch (CNRS-Univ. Paris-Saclay, ICMMO, Orsay, France) and Christophe Biot (CNRS-Univ. Lille, UGSF, Lille, France)

https://doi.org/10.5802/crchim.272

<figure><img src="image_4.png" /></figure>

This article is licensed under the
CREATIVE COMMONS ATTRIBUTION 4.0 INTERNATIONAL LICENSE.
http://creativecommons.org/licenses/by/4.0/

<figure><img src="image_5.png" /></figure>

The Comptes Rendus. Chimie are a member of the
*Mersenne Center for open scientific publishing*
www.centre-mersenne.org — e-ISSN : 1878-1543
cell differentiation [4–9]. Consistently, their misreg-
ulation correlates with cancer malignancy and stem-
ness [10–14].

Comprehensive structural descriptions of OSNE
are still missing to the best of our knowledge. The
folded DNA-binding domains (DBDs) of OSNE have
been structurally characterized in complex with
their DNA target sequences [15–19] together with
the ligand-binding domain (LBD) of Esrrb [20]. Re-
cent studies have depicted even splendid struc-
tures of Oct4's and Sox2's DBDs bound to nucle-
osomes, hence deciphering their "pioneer factor"
abilities [21–29]. Another structure of Sox2 bound
to the importin Impα3 has also been published,
showing how its two Nuclear Localization Sequences
(NLSs) flanking the DBD are involved in Sox2 nuclear
import [30]. The other segments of OSNE have been
predicted to be intrinsically disordered regions of
proteins (IDRs) [31], i.e., they should have no stable
tertiary fold when isolated [32–36].

These IDRs appear to have important roles in
binding partners involved in epigenetic reprogram-
ming, chromatin reorganization, and in recruiting
transcription or repression machineries [5,37–41].
These functions are poorly understood, and, to the
best of our knowledge, no experimental characteri-
zation of the structural behavior of these regions in
N- and C-terminal of DBDs has been released yet.
Recent studies have shown that C-terminal regions
of Oct4 and Sox2 are important for their reprogram-
ming capacities [42,43], notably by contributing to
the engagement in molecular phase-separated con-
densates with the Mediator complex [44]. More gen-
erally, the activating or repressive activities of IDRs of
transcription factors (TFs) have been scarcely stud-
ied at the structural level: these segments are thought
to contain hydrophobic patches flanked by acidic
amino acids, which favors DNA-binding specificity,
phase separation, and low-specificity interactions,
notably with the Mediator subunit Med15 [44–53];
more specific interactions have been described in
some cases [45,54,55].

Post-translational modifications (PTMs) add a
layer of complexity by often regulating IDRs' interac-
tions [32,33,56,57] and notably TFs' activity [58–61].
PTMs are classical carriers of cell signaling by
regulating the stability and the interactions of
proteins. An increasing number of PTMs have
been described on OSNE's IDRs in the recent years

[5,37,38,62–77], notably phosphorylation by cyclin-
dependent kinases (CDKs) [66,78–87] or by mitogen-
activated protein kinases (MAPKs) [83,88,89], or
their complementary Ser/Thr O-GlcNAcylation by
OGT [65,90–96].

In order to prompt future studies on this topic,
we describe here a feasibility study (i) for pro-
ducing well-characterized samples made of post-
translationally modified IDRs of PluriTFs and (ii) to
use these as baits in pull-down assays for detecting
PTMs' related binding partners. Hence, we charac-
terized some of the phosphorylation reactions of Es-
rrb and Sox2 by p38α/β, Erk2 and Cdk1/2. Then, we
showed that biotinylated chimera of Sox2 and Esrrb
coupled to an AviTag peptide could be attached to
streptavidin-coated beads. Finally, we loaded trun-
cated segments of the C-terminal IDR of Sox2 (phos-
phorylated or not) on these beads, and exposed
them to extracts of mouse ESCs (mESCs) in pull-
down assays, which we analyzed using quantitative
mass spectrometry-based proteomics. Among the
quantified (phospho-)Sox2 binders, we verified the
phospho-dependent interaction between the pro-
line cis-trans isomerase Pin1 and Sox2 using NMR
spectroscopy.

## 2. Material and methods

2.1. *Production of recombinant fragments of Oct4, Sox2, Nanog and Esrrb*

We used human protein sequences, unless speci-
fied. Codon-optimized (for expression in *Escherichia
coli*) genes coding for human Oct4(aa1-145) and
Oct4(aa286-380) were synthesized in the context of
larger genes coding for Tev-Oct4(aa1-145)-Tev-GB1
and Tev-Oct4(aa286-380)-Tev-GB1 by Genscript and
cloned into pET-41a(+) vector between SacII and
HindIII restriction sites, hence permitting the
expression of GST-His6-Tev1-Oct4(aa1-145)-Tev2-
GB1 and GST-His6-Tev1-Oct4(aa286-380)-Tev2-
GB1; Tev1 and Tev2 are the heptapeptide ENLYFQG
cleavage site of the TEV protease, Tev2 is separated
by GAGGAGG from GB1 (T2Q variant of the im-
munoglobulin binding domain B1 of the protein G
from group G Streptococcus [97,98]). The C-terminal
GB1 tag was added to avoid any C-terminal prote-
olysis of the IDR of interest during the expression
and the first purification steps; we did not test con-
structs without this supplementary folded domain,
whose necessity for the stability of the IDR is thus
not proven.

The same rationale (cDNA synthesis, cloning,
vectors, chimera constructs) was used for producing
Nanog(aa154-305), Nanog(aa154-215), Nanog(aa154-
272), Nanog(aa154-305_C185A-C227A-C243A-C251A),
Nanog(aa154-272_C185A-C227A-C243A-C251A), and
a very similar rationale (chimera constructs missing
the C-terminal Tev2-GB1) for Sox2(aa1-42), Sox2
(aa115-317_C265A), Sox2(aa115-187), Sox2(aa115-
236), Sox2(aa115-282_C265A), Esrrb(aa1-102_C12A-
C72A-C91A), Esrrb(aa1-102_C12A-C91A), Nanog
(aa1-85) (this latter was cloned in the MfeI/HindIII
restriction sites from pET-41a(+)).

The recombinant production and the purification
of the protein constructs followed the procedures de-
scribed previously [99], using the soluble fraction of
bacterial lysates, except for the constructs contain-
ing the Sox2 C-terminal fragments. These latter con-
structs were recovered from the insoluble fractions
of the lysates and resolubilized in 8 M urea; these
were submitted to a His-tag purification in urea, and
the last size-exclusion chromatography (SEC) had to
be carried out in 2 M urea, which avoided clogging
of the column and permitted obtaining regular elu-
tion peak widths (these were otherwise extremely
broad, up to 100 mL for the longest Sox2(aa115-
317_C265A) construct). The samples were concen-
trated and stored at -20 °C, and thawed just before
the NMR experiments. The Sox2 samples containing
2 M urea were submitted to 2-3 cycles of concentra-
tion/dilution in Hepes at 20 mM, NaCl at 75 mM to
generate samples in urea at 0.25 or 0.125 M.

All purification steps were carefully carried out at
4 °C; protein eluates from every purification step
were immediately supplemented with protease
inhibitors (EDTA-free cOmplete, Roche) (together
with DTT at 10 mM for cysteine-containing protein
constructs), before being submitted to a concentra-
tion preparing the next purification step.

Chimera constructs of Sox2's and Esrrb's IDR frag-
ments containing a 15-mer peptide AviTag GLN-
DIFEAQKIEWHE were produced using procedures
similar to those described earlier for OSNE con-
structs. The construct Sox2(aa234-317)-AviTag-His6
was soluble and did not require to be purified in
urea.

More details about the production of OSNE pep-
tides are given in the Supplementary Material.

2.2. *Production of the biotin ligase BirA and specific biotinylation of the AviTag–peptide chimera*

The biotin ligase BirA was produced using recom-
binant production in *E. coli* BL21(DE3)Star trans-
formed with a pET21-a(+) plasmid containing a gene
coding for BirA cloned at EcoRI and HindIII restric-
tion sites. pET21a-BirA was a gift from Alice Ting
(Addgene plasmid #20857) [100]. The expression was
carried out overnight at 20 °C in a Luria–Bertani cul-
ture medium. The construct contained a His6 tag in
C-terminal and was purified using a two-step purifi-
cation procedure including a His-trap followed by a
SEC. Details about the production of BirA are given
in the Supplementary Material.

The biotinylation was executed using a rationale
inspired by a published protocol [101], at room tem-
perature during 90 min, in samples containing the
AviTag-chimera of interest at 100 µM and BirA at
0.7 µM in a buffer containing ATP at 2 mM, biotin
at 600 µM, MgCl₂ at 5 mM, DTT at 1 mM, HEPES
at 50 mM, NaCl at 150 mM, protease inhibitors (fi-
nal concentration 1×, EDTA-free cOmplete, Roche),
at pH 7.0. To remove some possible proteolyzed
peptides and BirA, the biotinylated constructs were
purified using a SEC in a column (Superdex 16/60
75 pg, Cytiva) preequilibrated with a buffer con-
taining phosphate at 20 mM, NaCl at 150 mM at
pH 7.4 (buffer called thereafter Phosphate Buffer
Saline, PBS). The eluted fractions of interest were
concentrated and stored at –20 °C.

2.3. *Assignment of NMR signals from OSNE*
*fragments and structural propensities*

The assignment strategy was the same as in previous
reports from our laboratory [99]. The ¹⁵N relaxation
data were recorded and analyzed according to the
methods described in previous reports [102]. Details
are given in the Supplementary Material.

Disorder prediction was calculated using the
ODINPred website (https://st-protein.chem.au.dk/
odinpred) [103]. Experimental secondary structure
propensities of unmodified OSNE peptides were
obtained using the neighbor-corrected structural
propensity calculator ncSCP [104,105] (http://www.
protein-nmr.org/, https://st-protein02.chem.au.dk/
ncSPC/) from the experimentally determined, DSS-
referenced Cα and Cβ chemical shifts as input, with a correction for Gly-Pro motifs (−0.77 ppm instead of −2.0 ppm) [106]. We also used the δ2D method to get requested verifications of the experimental secondary structure propensities [107]. Some signals were too weak in 3D spectra from Sox2(aa115–317_C265A) recorded at 950 MHz, and their chemical shifts were not defined. In these cases, chemical shifts from 3D spectra of Sox2(115–236) or His6–AviTag–Sox2(aa234–317_C265A) were used to complete the lists of chemical shifts used to calculate the chemical shift propensities shown in Figure 2.

$^1$H$_N$/$^{15}$N/$^{13}$Ca/$^{13}$Cb/$^{13}$CO NMR assignments of OSNE peptides, together with the corresponding experimental details, have been deposited in the Biological Magnetic Resonance Data Bank (BMRB) with accession numbers 51534 (Sox2 aa1–42), 51717 (Esrrb aa1–102), 51756 (Oct4 aa1–145), 51758 (Oct4 aa286–360), 51782 (His6–AviTag–Sox2 aa234–317_C265A), and 51780 (Nanog aa1–85).

## 2.4. NMR monitoring of phosphorylation reactions and production of phosphorylated peptides

We performed the phosphorylation kinetics presented in Figure 4a using commercial recombinant kinases GST–p38β at 10 µg/mL (Sigma-Aldrich, ref. B4437), GST–Erk2 at 20 µg/mL (Sigma-Aldrich, ref. E1283), GST–Cdk1/CyclinA2 at 20 µg/mL (Sigma-Aldrich, stock ref. C0244), and GST–Cdk2/CyclinA2 at 20 µg/mL (Sigma-Aldrich, ref. C0495). Then, we used kinases produced in-house in *E. coli*, using plasmids containing optimized genes coding for p38α(aa1–360, full-length) and Erk2(aa8–360); these were produced, activated, and purified in house as described previously [99]; in-house p38α was used at 40 µg/mL for the experiments shown in Figure 4. Indeed, the limited activities and high costs of commercial kinases motivated us to develop in-house capacities in kinase production. p38α was the most accessible to produce among the MAPKs and CDKs; we produced it and activated it using recombinant MKK6.

Phosphorylation reactions were carried out using $^{15}$N-labeled IDRs at 50 µM, in Hepes 20 mM, NaCl 50 mM, DTT or TCEP at 4 mM, ATP 1.5 mM, MgCl$_2$ at 5 mM, protease inhibitors (Roche), 7.5% D$_2$O, pH 6.8 at 25 °C in 100 µL using 3 mm diameter Shigemi tubes. We monitored the phosphorylation

kinetics by recording time series of $^1$H–$^{15}$N SOFAST-HMQC spectra at 600 or 700 MHz, and by quantifying the NMR signal intensities of the disappearing unphospho- and appearing phospho-residues. We applied the methods that we described in earlier publications [108–111]. More details are given in the Supplementary Material.

## 2.5. Pull-down assays

The mESCs extracts were obtained from mESCs cultured in the conditions previously described [112]. Homogeneous extracts were obtained using DNA shearing by sonication in the presence of benzonase, as described by Gingras and colleagues [113].

The pull-down assays were executed using 25 µL of streptavidin-coated magnetic beads (Magbeads streptavidine, Genscript) loaded with 1 nmol of the biotinylated bait-peptides of interest. These were incubated for one hour at room temperature with mESCs extracts, washed in PBS and eluted using a 2× Laemmli buffer. Details are given in the Supplementary Material.

## 2.6. Mass spectrometry-based proteomics analysis of pull-down assays

The pull-down samples were treated on-beads by trypsin/LysC (Promega). The resulting peptides were loaded and separated on a C18 column for online liquid chromatography performed with an RSLCnano system (Ultimate 3000, Thermo Scientific) coupled to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific). Maximum allowed mass deviation was set to 10 ppm for monoisotopic precursor ions and 0.6 Da for MS/MS peaks. The resulting files were further processed using myProMS v3.9.3 (https://github.com/bioinfo-pf-curie/myproms; Poullet et al. [114]). False-discovery rate (FDR) was calculated using Percolator [115] and was set to 1% at the peptide level for the whole study. Label-free quantification was performed using extracted-ion chromatograms (XICs) of peptides, computed with MassChroQ [116] v.2.2.1. The complete details are given in the Supplementary Material.
## 2.7. Recombinant production of Pin1 and NMR analysis of its interaction with Sox2 or phospho-Sox2

The plasmid containing the gene coding for the Pin1-WW domain was a kind gift from Isabelle Landrieu. The production was executed according to the previously published protocol [117]. NMR analysis of the binding with phospho-Sox2(aa115-240) was performed at 283 K and pH 7.0 with the GST-Pin1-WW construct and ¹⁵N-labeled Sox2(aa115-240) mixed in stoichiometric proportions, either at 50 or 10 µM for non-phospho- and phospho-Sox2, respectively. The details on the NMR acquisition, processing, and analysis are given in the Supplementary Information.

# 3. Results

## 3.1. Structural characterization of the N- and C-terminal regions of Oct4

We produced and purified protein constructs containing the fragments of human Oct4(aa1-145) and Oct4(aa286-360), which were both predicted to be mostly disordered (Supplementary material 3.2.3). The 2D ¹H-¹⁵N HSQC NMR spectra showed cross-peaks in the region where random coil peptides resonances are usually found (Figure 1). We assigned the backbone NMR signals of ¹Hₙ, ¹⁵N, ¹³Cα, ¹³Cβ, ¹³CO for both segments, which permitted to calculate experimentally derived secondary structure propensities (Figures 1c, f, Supplementary Figure S4). We did not find any sign of a stable secondary structure, the highest α-helical propensities reaching about 25% in short stretches of about 5 consecutive amino acids. Hence, we verified experimentally that these N- and C-terminal fragments of human Oct4 are IDRs. We noticed that one motif RTWLSF (aa33-38) generates cross-peaks out of the random coil area in the 2D ¹H-¹⁵N HSQC, and its chemical shifts reveal the strongest α-helical propensity (about 25% according to two distinct software products, see Figures 1c, f, Supplementary Figure S4). This might typically correspond to an interaction site: IDRs' binding motifs often adopt secondary structure in complexes, whose formation is energetically favored by local conformational preferences for the bound structure in the free state [57, 106, 118].

We can highlight the fact that Oct4's IDRs contain a high density of prolines, which are not directly

detectable in the present ¹Hₙ-detected experiments, even though most of the ¹³Cα, ¹³Cβ, and ¹³CO resonances were characterized via HNCAB and HNCO experiments. We have shown previously that the ¹³C-detected experiments ¹³Cα, ¹³CO permitted observing all these Pro residues in Oct4(aa1-145) [99], whose chemical shifts were those of random coil peptides.

## 3.2. Structural characterization of the N- and C-terminal regions of Sox2

We produced and purified peptide fragments of human Sox2, namely Sox2(aa1-42), Sox2(aa115-187), Sox2(aa115-236), Sox2(aa115-282), Sox2(aa234-317_C265A), and Sox2(aa115-317_C265A). We also produced and purified chimera peptides His6-AviTag-Sox2(aa115-240) and His6-AviTag-Sox2(aa234-317_C265A). These were all predicted to be disordered (Supplementary material 3.3.3).

We had solubility issues with all of them but Sox2(aa1-42), Sox2(aa234-317_C265A), and His6-AviTag-Sox2(aa234-317_C265A). We had to recover these troublesome peptides from the insoluble fraction of the bacterial extract after overexpression at 37 °C. We even had to carry out our final SEC purification step in a buffer containing urea at 2 M (at 4 °C) for Sox2(aa115-236), Sox2(aa115-282), His6-AviTag-Sox2(aa115-240), and Sox2(aa115-317_C265A). The assignments of these latter constructs were achieved in 0.25-0.5 M urea, after executing 2 to 3 concentration-dilution steps. Aggregates were forming during the acquisition, which made the assignment rather painful. This behavior correlated with liquid-liquid phase separation (LLPS) propensities (Supplementary Figure S5), which we observed a few months before such a behavior was reported by Young and collaborators [44]. The assignment of Sox2(aa115-317_C265A) was possible only at 950 MHz with the help of the previously assigned smaller fragments Sox2(aa115-236) and His6-AviTag-Sox2(aa234-317_C265A). Some stretches of amino acids were particularly difficult to observe in 3D spectra, e.g., the region aa160-185, because of an apparent fast T2 relaxation. We may investigate these phenomena in later reports.

We observed cross-peaks in the 2D ¹H-¹⁵N HSQC NMR spectra that were all resonating in the spectral region of random coil peptides' resonances
<figure><img src="image_6.png" /><figcaption>**Figure 1.** (a,d) 2D ¹H–¹⁵N HSQC spectra of the N- and C-terminal IDRs of human Oct4, the labels indicating the assignments; the grey areas show the spectral regions where random coil amino acids resonate usually; (b,e) primary structures of Oct4; dark and light colors indicate the folded and disordered domains, respectively; blue and red sticks indicate the positions of Glycines and Prolines, respectively; (c,f) secondary structure propensities calculated from the experimental chemical shifts of the peptide backbone Cα and Cβ, using the ncSPC algorithm [104,105].</figcaption></figure>

(Figure 2). The spectra of the short fragments of the C-terminal region of Sox2 were exactly overlapping with those of Sox2(aa115–317_C265A) (Supplementary Figure S1). This shows that all these fragments have very similar local conformational behaviors,

a phenomenon regularly observed with IDRs. We assigned the backbone NMR signals of ¹Hₙ, ¹⁵N, ¹³Cα, ¹³Cβ, ¹³CO for Sox2(aa1–42), Sox2(aa115–236), His6–AviTag–Sox2(aa234–317_C265A) and partially for Sox2(aa115–317_C265A). We aggregated the lists
of chemical shifts of the C-terminal fragments and used them to calculate the experimental secondary structure propensities (Figures 2c, f and Supplementary Figure S4). This confirmed the absence of any stable secondary structure elements in Sox2 N- and C-terminal region. The C-terminal region is poorly soluble below 0.25 M urea; this should not affect a stable fold, so we can affirm that these regions of Sox2 are experimentally proven IDRs.

### 3.3. *Structural characterization of the N-terminal region of Nanog*

We produced and purified the N-terminal peptide fragment of human Nanog(aa1-85). All our attempts to purify C-terminal regions of Nanog failed, even after alanine-mutation of cysteines in Nanog(aa154-305), Nanog(aa154-272), and Nanog(aa154-215). We managed to resolubilize our construct GST-His6-Tev-Nanog(aa154-305) from the insoluble fraction of the bacterial extract, to partially purify it and cleave it using the TEV protease. However, the resulting Nanog(aa154-305) peptide was barely soluble in a detergent (NP-40 at 2% v/v), and not in high-salt buffers, or not even in the presence of urea at 4 M. The 10 tryptophane residues are probably playing a role in this behavior, in the context of a primary structure containing not enough hydrophobic amino acids favoring stable folds.

The cross-peaks of Nanog(aa1-85) in the 2D ¹H-¹⁵N HSQC NMR spectrum were all in the spectral region of random coil peptides' resonances (Figure 3). The assignment of the backbone NMR signals of ¹HN, ¹⁵N, ¹³Cα, ¹³Cβ, ¹³CO permitted calculating experimental secondary structure propensities, which were low through the whole peptide (Figure 3b, Supplementary Figure S4). Thus, we confirmed that this Nanog N-terminal is an IDR.

### 3.4. *Structural characterization of the N-terminal region of Esrrb*

We produced and purified the N-terminal fragment of human Esrrb(aa1-102), which was predicted to be disordered (Supplementary material 3.5.3), in the alanine-mutated versions Esrrb(aa1-102_C12A-C72A-C92A) and Esrrb(aa1-102_C12A-C92A). This was a strategic choice to attenuate the formation of disulfide bonds; the wild-type N-terminal

fragments might however be workable too. Mutating cysteines permitted working in more comfortable conditions and maintaining our construct monomeric for longer periods of time in the next phosphorylation and biotinylation experiments. Cysteines are indeed highly solvent-accessible in IDRs and they are consequently difficult to keep in their thiol, non-disulfide forms, even in the presence of fresh DTT or TCEP at neutral pH. We also produced chimera constructs Esrrb(aa1-102_C12A-C72A-C92A)-AviTag-His6 and Esrrb(aa1-102_C12A-C72A)-AviTag-His6.

All the 2D ¹H-¹⁵N HSQC NMR spectra revealed cross-peaks in the spectral region of random coil peptides (Figure 3). These spectra are overlapping to a large extent, confirming the weak influence of the mutations of cysteines: the mutation Cys72Ala has almost no consequences on the chemical shifts, below 0.05 ppm even for the neighboring amino acids (Supplementary Figure S2a,b); the mutation Cys91Ala has more impact, with chemical shifts perturbations of about 0.1 ppm for the next 5 amino acids (Supplementary Figure S2c,d), which is at least partially due to the fact that the Ala substitution favors an increase in local α-helicity (about 25%, see Supplementary Figure S2e,f). This N-terminal fragment of human Esrrb is thus an IDR, according to the calculated secondary structure propensities (Figure 3f, Supplementary Figures S2e,f, S4).

### 3.5. *Phosphorylation of Esrrb and Sox2 by p38α, Erk2, Cdk1/2 as monitored by NMR spectroscopy*

We reported recently the site-specific phosphorylation kinetics of Oct4 by p38α using ¹³C-direct NMR detection [99]. Here, we used more standard ¹H-detected/¹⁵N-filtered experiments to rapidly characterize the site preferences of MAPKs and CDKs on Esrrb and Sox2, which we thought to use as baits for performing phospho-dependent pull-down assays (see below).

To start with, we used commercial aliquots of MAPKs, namely p38β and Erk2, and CDKs, namely Cdk1/CyclinA2 and Cdk2/CyclinA2, on Esrrb(aa1-102). We observed the progressive phosphorylation of its three Ser-Pro motifs (at Ser22, Ser34 and Ser58)
<figure><img src="image_7.png" /><figcaption>**Figure 2.** (a,d) 2D $^{1}$H–$^{15}$N HSQC spectra of the N- and C-terminal IDRs of human Sox2, the labels indicating the assignments; the grey areas show the spectral regions where random coil amino acids resonate usually; (b,e) primary structures of human Sox2; dark and light colors indicate the folded and disordered domains, respectively; (c,f) secondary structure propensities calculated from the experimental chemical shifts of the peptide backbone Cα and Cβ, using the ncSPC algorithm [104,105].</figcaption></figure>

in agreement with the consensus motifs of these ki-
nases [119,120]. The kinetics reveal a classical dis-
tributive mechanism, where phosphorylation sites
are processed independently according to their re-
spective kcat and KM. Ser22 is the preferred target in
all cases, while Ser34 is the least processed by CDKs,
if at all: the commercial CDKs are poorly active in
our hands, which we have verified with a number of
other targets for years in the laboratory; this makes
it difficult to distinguish between sites that are only

mildly disfavored or those that are more stringently
ignored by CDKs in NMR-monitored assays.

Then, we used a potent home-made activated
p38α on His6-AviTag-Sox2(aa115-240) and His6-
AviTag-Sox2(aa234-317). It phosphorylated all the
Ser/Thr-Pro motifs of these two peptides, and also
T306 in a PGT306AI context, which shows a favorable
proline in position -2 [121], and a less common S212
in a MTS212SQ context.

Hence, we were able to generate AviTag-IDR
<figure><img src="image_8.png" /><figcaption>**Figure 3.** (a,d) 2D $^{1}$H–$^{15}$N HSQC spectra of the N-terminal IDRs of human Nanog and Esrrb, the labels indicating the assignments; the grey areas show the spectral regions where random coil amino acids resonate usually; (b,e) primary structures of human Nanog and Esrrb; dark and light colors indicate the folded and disordered domains, respectively; orange and black sticks indicate the positions of cysteines and tryptophanes, respectively; (c,f) secondary structure propensities calculated from the experimental chemical shifts of the peptide backbone Cα and Cβ, using the ncSPC algorithm [104,105].</figcaption></figure>

chimera in well-defined phosphorylated states. To
produce phosphorylated $^{14}$N-AviTag-IDR dedicated
to pull-down assays, we executed the same protocol
on $^{14}$N-peptides, while NMR-monitoring in parallel
“identical” but $^{15}$N-labeled peptides. This allowed us

to produce a well-defined phosphorylation state of
the $^{14}$N-AviTag-IDR for the next experiments.
<figure><img src="image_9.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_12.png" /></figure>

**Figure 4.** (a) Overlay of 2D ¹H–¹⁵N HSQC spectra of Esrrb(aa1–102_C12A-C72A-C91A) before (black) and after (grey and phosphosites colored in red/purple/blue) phosphorylation by p38β; (b) residue specific time courses of the phosphorylation of Esrrb(aa1–102) executed by commercial kinases p38β, Erk2, Cdk2 or Cdk1, as measured in time series of 2D ¹H–¹⁵N SOFAST-HMQC spectra recorded during the reaction; (c) overlay of 2D ¹H–¹⁵N HSQC spectra of AviTag-Sox2(aa115–240) before (black) and after (red) phosphorylation by p38α; the inset at the top-right shows the residue specific phosphorylation build-up curves, as measured in time series of 2D ¹H–¹⁵N SOFAST-HMQC spectra; (d) same as (c) with AviTag-Sox2(aa234–317_C265A).
### 3.6. *Structural characterization of the AviTag-peptide chimera and its biotinylated versions*

We aimed at detecting new partners of OSNE using pull-down assays. We thought to use chimera containing GST at the N-terminus, which appeared as a convenient approach: vectors integrating a GST-coding DNA sequence for overexpression in *E. coli* are available and of common use; glutathione-coated beads are also accessible and permit efficient and specific binding of GST-containing chimera peptides. However, we were unsatisfied by the performances of the method: GST binding to glutathione-coated beads is slow at low temperature (necessary to avoid IDR proteolysis); moreover, it appeared that GST-IDRs chimera are hampered by the IDRs' "molecular cloud" and are even weaker and slower to bind to the beads. Our attempts to bind GST-IDRs to the beads were thus resulting in poor yields, which were not very reproducible. In the context of our aims, i.e., to establish a method allowing quantitative detection of IDRs' binding partners, this unsatisfying lack of reproducibility was only foreboding supplementary variable parameters.

Thus, we decided to switch to another strategy: the use of the specifically biotinylated 15-mer peptide tag called AviTag [101]. This is efficiently and specifically biotinylated by biotin ligase BirA (Figure 5d), which permits high-affinity binding to streptavidin-coated beads. We designed AviTag-IDR chimera, with the AviTag in N-terminal position for Sox2's IDR constructs, and in C-terminal for Esrrb's IDR constructs. We characterized the AviTag and its impact on the IDRs of interest using NMR: the AviTag is unfolded and it does not affect the Sox2 and Esrrb fragments, according to the observed negligible chemical shift perturbations (Figure 5)—the GST Tag provoked also only very weak chemical shift changes on Esrrb(aa1-102) (Supplementary Figure S3). The biotinylated AviTag peptide appears to be slightly less mobile than the common IDPs on the ps-ns timescale, according to the heteronuclear ¹⁵N-{¹H} NOEs (Figure 5b).

We observed that the biotinylation of the AviTag provokes weak but distinguishable chemical shift perturbations for the close neighbors of the biotinylated lysine, but had no effect on the peptides of interest (Figure 5c). It generated also the appearance of

a HN-ester NMR signal, similar to that of acetylated lysine [108,122]. Hence, we could quantify and monitor the reaction advancement using NMR, and determine the incubation time that was necessary and sufficient to obtain a complete biotinylation of our chimera AviTag-IDRs (see Section 2.2). This was one among many optimization steps permitting the production of sufficient quantities of intact IDRs for the pull-down assays.

Next, we tested the binding of the biotinylated AviTag-IDRs on streptavidin-coated beads. This produced very satisfying results, i.e., stoichiometric, specific binding in one hour with no leakage (Supplementary Figure S6). This approach was thus selected for the pull-down assays.

### 3.7. *Use of AviTag-Sox2 for pull-down assays from mESCs extracts*

We prepared the four peptides AviTag-Sox2(aa115-240) and AviTag-Sox2(aa234-317) in their non-phosphorylated and phosphorylated versions, using p38α to execute the phosphorylation reactions (Figure 6a). These peptides were also biotinylated, and later bound to streptavidin-coated beads, which we used as baits for pull-down assays in extracts from mESCs (Figure 6b). Importantly, an additional SEC was carried out between every step to discard proteolyzed peptides, the enzymes (kinases of BirA) and their contaminants. We performed the pull-down assays with the four samples in parallel with the same cell extract, in duplicate, and then analyzed the bound fractions using quantitative LC-MS/MS analysis (see Supplementary material 1.6 for full description). Hence, we could detect and evaluate the relative quantities of proteins retained by the four AviTag-peptides (Figure 6). On paper, this presents the important advantage of removing false-positive binders, which can interact unspecifically with the streptavidin-coated beads.

We present here results that should be interpreted carefully: we produced only duplicates for every condition, using one single cell extract. To deliver trustful information, the common standards in the field recommend 3 to 5 replicates. Here, we considered only proteins with at least 2 distinct peptides across the 2 replicates (Figure 6c). We observed a two-fold change or more (FC > 2) of some TFs pulled out by AviTag-Sox2(aa115-240), among which the PluriTFs Oct4 and Klf5 are significantly enriched
<figure><img src="image_13.png" /><figcaption>**Figure 5.** (a) 2D ¹H–¹⁵N HSQC spectrum of ¹⁵N-His6-AviTag-Sox2(aa234–317_C265A), the blue labels indicated the assigned signals from the AviTag residues; (b) secondary structure propensities calculated from the experimental chemical shifts of the peptide backbone Cα and Cβ, using the ncSPC algorithm [104,105]; the residue specific ¹⁵N–{¹H} NOEs, ¹⁵N-R1 (grey) and ¹⁵N-R2 (black) measured at 600 MHz are shown below (the profiles show values averaged over three consecutive residues); (c) overlay of 2D ¹H–¹⁵N HSQC spectra of the Esrrb(aa1–102_C12A-C72A-C91A)–AviTag–His6 before (black) et after (magenta) biotinylation by BirA; the NMR signals from the residues neighboring the biotinylation site are indicated, which permit the quantification of the biotinylated population; (d) scheme of the reaction of AviTag biotinylation executed by the ATP-dependent BirA.</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>**Figure 6.** Differential interactomics of Sox2 constructs upon p38α phosphorylation. (a) We have produced unmodified/phospho-Sox2 truncations carrying N-ter biotinylation on the AviTag, and later attached on streptavidin-magnetic beads. (b) We have generated mESCs nuclear extracts for pull-down assays using our Sox2 constructs as baits. (c) The volcano plots of the log2 ratios, showing a quantitative analysis of the proteins present at the end of the pull-down assays; the dashed line indicates the threshold of *p*-value < 0.05; we highlighted interesting partners in: **blue**: transcription factors associating with Sox2(aa115-240); **magenta**: phospho-dependent partners of Sox2(aa115-240) and or Sox2(aa234-317). Pull-downs have been performed in duplicates, using 15 million cells per sample (extract protein conc.: 5 mg/mL), and 1 nmol of bait protein. Experimental conditions may be improved (higher number of replicates, cells, washing conditions, ...).</figcaption></figure>

(>4 peptides, *p*-value < 0.02). Also, we found out that pSox2(aa115-240) was pulling out the three isoforms of CK1 (>3 peptides, *p*-values < 10⁻⁵) and the proline isomerase Pin1 (>2 peptides, *p*-values

< 2 × 10⁻⁴). To confirm the value of the method and of the detected interactions, one should at least test them using an orthogonal approach, e.g., NMR spectroscopy of purified proteins.
## 3.8. NMR characterization of the interaction between Pin1 and phospho-Sox2

We decided to test the interaction between pSox2(aa115-240) and Pin1. We recorded ¹H-¹⁵N NMR spectra of ¹⁵N-labeled Sox2(aa115-240) or pSox2(aa115-240) alone or in the presence of the Pin1-WW domain (natural abundance peptide, i.e., 0.6% ¹⁵N, 99.4% ¹⁴N, hence "NMR-invisible" in ¹⁵N-filtered experiments). We observed localized losses in signal intensities for the residues neighboring the three pSox2(aa115-240) phosphosites when mixed with Pin1 (Figure 7b); in contrast, no significant differences showed up in the spectra obtained with non-phosphorylated Sox2(aa115-240) in the absence or presence of Pin1-WW (Figure 7a). Hence, these signal losses are the typical signs of a position-specific interaction between an IDR and a folded protein in the intermediate or slow NMR timescale (µs-s), which corresponds to submicromolar affinities for this type of molecules. Interactions with this range of affinity can therefore be detected using the presented pull-down assay approach.

# 4. Discussion

The structural biochemistry analysis reported here can be applied to a large list of transcription factors (TFs). These are essential actors of cell signaling: they are key elements for inducing or maintaining pluripotency or differentiation, for cell proliferation or cell cycle arrest, by activating or repressing gene transcription [61,123]. About 90% of the ~1600 TFs are predicted to contain large disordered segments (>30 consecutive amino acids), which is particularly true for PluriTFs [31,124]. A correlation exists actually between TFs and predicted IDRs in all kingdoms of life [45,125,126]. The IDRs of these TFs recruit transcription co-factors or the transcription machinery, which is still not very well characterized in detail [44-55].

Indeed, the fine understanding of TFs' interactions via their IDRs appears to be hampered by the nature of these interactions, which are characterized by weak affinities and multivalency. Moreover, possible redundancy between TFs can emerge from coacervation. Post-translational modifications (PTMs), which can switch on or off IDRs' interactions, are a supplementary source of confusion when searching for binding partners. Among the difficulties, we

should also mention the basic biochemistry issues: IDRs are difficult to produce and manipulate in vitro, because they are prone to degradation or aggregation. Here, we have tried to demonstrate the feasibility and interest of some biochemical and spectroscopic approaches to better characterize IDRs of TFs, their phosphorylation and the associated binding partners.

Using a residue specific NMR analysis, we have shown experimentally that the pluripotency TFs OSNE contained IDRs. None of these regions do show any strong secondary structure propensity, which often reveals functional binding sites. Faint ~20% helicities were detected by two independent algorithms (ncIDP and δ2D [104,105,107]) on 5-6 amino acid stretches, which are indicated on Figures 1 and 2 for Oct4 and Sox2. Altogether, the analyzed peptides show local conformational behaviors close to that of a random coil, without any obvious structural elements except the Oct4 stretch between amino acids 33 and 38. Finally, we shall highlight the high propensity for liquid-liquid phase separation of the fragments of Sox2 containing the region aa115-236.

Like other TFs, pluripotency TFs OSNE are post-translationally modified (see Section 1), notably by CDKs and MAPKs [66,78-88]. These two classes of kinases are fundamental actors in all aspects of eukaryotic cellular life, and understanding their activity and regulation in pluripotency or differentiation is of high significance. Interesting questions are still pending: what is the phosphorylation status of OSNE's IDRs in pluripotent cells, what is the impact on their interaction networks, and how does it affect pluripotency or differentiation? The inhibition of MAPK Erk signaling is necessary to maintain pluripotency in the standard culture conditions of ESCs and iPSCs [66,82-84], while these cells show a high CDK activity [8,86,87]; these two kinases family have the same core consensus sites (Ser/Thr-Pro) motifs, which are abundant in OSNE's IDRs and whose phosphorylation has apparently consequences for initiating differentiation [73, 78,80,83,85]. We have shown that we could produce well-defined phosphorylation status of these peptides, using recombinant kinases and NMR analysis, which makes it possible to study their interactions in vitro. We have also demonstrated our capacity to use these peptides as baits in pull-down assays for detecting potential new binding partners.
<figure><img src="image_15.png" /><figcaption>**Figure 7.** (a) Overlay of 2D ¹H–¹⁵N HSQC spectra of ¹⁵N-Sox2(aa115–240) alone at 50 µM (black) or mixed with Pin1 in isotopic natural abundance and in stoichiometric amounts (grey); inset up-right: residue specific NMR signal intensity ratios as measured in the two HSQC spectra. (b) Overlay of 2D ¹H–¹⁵N HSQC spectra of ¹⁵N-phosphoSox2(aa115–240) alone at 10 µM (red) or mixed with the Pin1–WW domain in stoichiometric amounts (grey); insets, up-right: residue specific NMR signal intensity ratios as measured in the two HSQC spectra (in the absence/presence of Pin1–WW domain).</figcaption></figure>

However, the detected interactions between
phospho-Sox2(aa115-240)-pS212-pS220-pT232 and
Pin1 or CK1 correspond to widespread, degenerate
interactions, whose biological significance may be
questionable [127-129]. This is one of the major
drawbacks in the field of IDRs' studies: they partic-
ipate in multiple, degenerate and transient interac-
tions of weak affinities, which can be easily released
during the washes of our pull-down assays. In this
regard, the "proximity labeling" approaches (BioID,
APEX and their derivates) appeared recently to be
quite adapted to transient interactions: these meth-
ods, developed in the last ten years, use chimera
constructs containing enzymes that transfer chemi-
cal groups to their intracellular neighbors, which can
be later identified by mass spectrometry [130-133].
IDRs are very flexible, solvent-exposed and establish
a lot of poorly specific transient interactions. This
was raising concerns about the possible production
of many false positives if one used proximity labeling
methods to detect IDRs' binding partners. This has
been partially confirmed by a recent study, but this
bias appears to be limited [134]. Interactomes of 109
TFs have actually been described using Bio-ID and
affinity purification MS, showing the complemen-
tarity between proximity labeling and the pull-down

approach proposed here [135]. Yeast two-hybrid,
which can detect ~20 µM affinity interactions, and
novel phage display approaches will also help in this
task [136-139].

Another difficulty in studying IDRs of TFs is their
propensity to coacervate [44–55]. Here, we have
tried to use Sox2 as a bait in pull-down assays,
a protein that has been later recognized to favor
liquid–liquid phase separation [44,140]. We met
this difficulty during the production steps, which
forced us to purify most of the Sox2 constructs in
urea at 2 M. We could straightforwardly observe
liquid–liquid phase separation of Sox2(aa115–317) at
4 µM using differential interference contrast (DIC)
microscopy in the presence of Ficoll-70 (Supple-
mentary Figure S5), but also progressive aggrega-
tion and low solubility thresholds while working with
our purified samples. These are clear limiting fac-
tors for sample production and NMR characteriza-
tion, which will hamper a number of other studies
on IDRs of TFs. This might also affect the results
of pull-down assays: we noticed an enrichment in
TFs in the samples obtained from pull-downs using
Sox2(aa115–240) as a bait, which has a much higher
coacervation propensity than Sox2(aa234–317). Is
it possible to generate local surface liquid–liquid
phase separation on the surface of streptavidin-coated beads? This might be at the same time a blessing and a curse for future studies, by helping the formation of biologically significant assemblies, or by favoring unspecific, non-native macromolecules interactions.

A final bottleneck in the studies of these IDRs is the capacity to produce post-translationally modified samples. The commercial enzymes are not very well adapted to our NMR studies, because of the required quantities. Here, we have used in-house production of kinase p38α. Since we carried out the present work, we have developed our capacities in producing activated Erk2, Cdk2/CyclinA1 and Cdk1/CyclinB1. These will be part of our future studies. TFs are indeed quite adapted to NMR investigations: they are 300 to 500 residues long and contain large IDRs (~100 amino acids) separating small folded domains (also ~100 amino acids) [31,45]. Their structural characterization would permit understanding a number of cell-signaling mechanisms at the atomic scale, and possibly identifying new therapeutic targets, even though this class of proteins is notoriously difficult to inhibit [60,141,142].

## 5. Conclusion

We have applied NMR techniques to carry out a primary analysis of the pluripotency transcription factors Oct4, Sox2, Nanog and Esrrb, in particular of their intrinsically disordered regions. We have shown experimentally that they did not adopt a stable fold when isolated, and that we were able to conduct a residue-specific analysis. This relies on the delicate production and purification of these peptides, which are prone to proteolysis and aggregation; producing them in a well-defined PTM status was an even more arduous challenge. We have demonstrated the feasibility of these tasks using recombinant kinases and NMR analysis. We have also evaluated the usefulness of such protein constructs as baits in pull-down assays to detect new binding partners of IDRs. These characterizations and the associated methods provide firm basis for future investigations on transcription factors. The proposed experimental scheme is thus a promising methodology that still needs to be developed and to prove its merits in revealing novel and significant interactions.

## Declaration of interests

The authors do not work for, advise, own shares in, or receive funds from any organization that could benefit from this article, and have declared no affiliations other than their research organizations.

## Funding

This work was supported by the CNRS and the CEA-Saclay, by the French Infrastructure for Integrated Structural Biology (https://frisbi.eu/, grant number ANR-10-INSB-05-01, Acronym FRISBI) and by the French National Research Agency (ANR; research grants ANR-14-ACHN-0015 and ANR-20-CE92-0013). Financial support from the IR INFRANALYTICS FR2054 for conducting the research is also gratefully acknowledged. This work was also supported by grants from the "Région Ile-de-France" and "Fondation pour la Recherche Médicale" (DL and LSMP).

## Acknowledgements

We thank Thaleia Papadopoulou, Amandine Moliex and Navarro Pablo for providing mESCs extracts and for fruitful discussions. We thank Nadia Izadi-Pruneyre for providing the bench space necessary to carry out the pull-down experiments with fresh mESCs extracts. We thank Romain LeBars and the microscopy facility of I2BC Imagerie-Gif for the preliminary tests on liquid-liquid phase separation. We thank the IR-RMN staff (now part of Infranalytics), notably Nelly Morellet, François Giraud and Ewen Lescop, for their reactivity, their support, and their long-standing, efficient care of the 950 MHz spectrometer. We thank Marie Sorin, Baptiste Nguyen and Benjamin Bacri, who contributed to this study during their Master1-Master2 internships.

## Supplementary data

Supporting information for this article is available on the journal's website under https://doi.org/10.5802/crchim.272 or from the author.

## References

[1] H. Inoue, N. Nagata, H. Kurokawa, S. Yamanaka, *EMBO J.*, 2014, **33**, 409-417.
[2] Y. Shi, H. Inoue, J. C. Wu, S. Yamanaka, *Nat. Rev. Drug Discov.*, 2017, **16**, 115-130.

[3] R. G. Rowe, G. Q. Daley, *Nat. Rev. Genet.*, 2019, **20**, 377-388.

[4] W. Deng, E. C. Jacobson, A. J. Collier, K. Plath, *Curr. Opin. Genet. Dev.*, 2021, **70**, 89-96.

[5] T. W. Theunissen, R. Jaenisch, *Stem Cell*, 2014, **14**, 720-734.

[6] M. Li, J. C. I. Belmonte, *Nat. Rev. Genet.*, 2017, **18**, 180-191.

[7] C. Chronis, P. Fiziev, B. Papp, S. Butz, G. Bonora, S. Sabri, J. Ernst, K. Plath, *Cell*, 2017, **168**, 442-459, e20.

[8] N. Festuccia, I. Gonzalez, N. Owens, P. Navarro, *Development*, 2017, **144**, 3633-3645.

[9] I. Gonzalez, A. Molliex, P. Navarro, *Curr. Opin. Cell Biol.*, 2021, **69**, 41-47.

[10] P. Mu, Z. Zhang, M. Benelli, W. R. Karthaus, E. Hoover, C.-C. Chen, J. Wongvipat, S.-Y. Ku, D. Gao, Z. Cao, N. Shah, E. J. Adams, W. Abida, P. A. Watson, D. Prandi, C.-H. Huang, E. de Stanchina, S. W. Lowe, L. Ellis, H. Beltran, M. A. Rubin, D. W. Goodrich, F. Demichelis, C. L. Sawyers, *Science*, 2017, **355**, 84-88.

[11] A. C. Hepburn, R. E. Steele, R. Veeratterapillay, L. Wilson, E. E. Kounatidou, A. Barnard, P. Berry, J. R. Cassidy, M. Moad, A. El-Sherif, L. Gaughan, I. G. Mills, C. N. Robson, R. Heer, *Oncogene*, 2019, **38**, 4412-4424.

[12] S. Mirzaei, M. D. A. Paskeh, M. Entezari, S. reza Mirmazloomi, A. Hassanpoor, M. Aboutalebi, S. Rezaei, E. S. Hejazi, A. Kakavand, H. Heidari, S. Salimimoghadam, A. Taheriazam, M. Hashemi, S. Samarghandian, *Biomed. Pharmacother.*, 2022, **156**, article no. 113860.

[13] E.-H. Ervin, R. French, C.-H. Chang, S. Pauklin, *Semin. Cancer Biol.*, 2022, **87**, 48-83.

[14] A. Chaudhary, S. S. Raza, R. Haque, *Semin. Cancer Biol.*, 2023, **88**, 123-137.

[15] D. Esch, J. Vahokoski, M. R. Groves, V. Pogenberg, V. Cojocaru, H. vom Bruch, D. Han, H. C. A. Drexler, M. J. Araúzov-Bravo, C. K. L. Ng, R. Jauch, M. Wilmanns, H. R. Schöler, *Nat. Cell Biol.*, 2013, **15**, 295-301.

[16] A. Reményi, K. Lins, L. J. Nissen, R. Reinbold, H. R. Schöler, M. Wilmanns, *Genes Dev.*, 2003, **17**, 2048-2059.

[17] M. D. Gearhart, S. M. A. Holmbeck, R. M. Evans, H. J. Dyson, P. E. Wright, *J. Mol. Biol.*, 2003, **327**, 819-832.

[18] R. Jauch, C. K. L. Ng, K. S. Saikatendu, R. C. Stevens, P. R. Kolatkar, *J. Mol. Biol.*, 2008, **376**, 758-770.

[19] Y. Hayashi, L. Caboni, D. Das, F. Yumoto, T. Clayton, M. C. Deller, P. Nguyen, C. L. Farr, H.-J. Chiu, M. D. Miller, M.-A. Elsliger, A. M. Deacon, A. Godzik, S. A. Lesley, K. Tomoda, B. R. Conklin, I. A. Wilson, S. Yamanaka, R. J. Fletterick, *Proc. Natl. Acad. Sci. USA*, 2015, **112**, 4666-4671.

[20] B. Yao, S. Zhang, Y. Wei, S. Tian, Z. Lu, L. Jin, Y. He, W. Xie, Y. Li, *J. Mol. Biol.*, 2020, **432**, 5460-5472.

[21] S. O. Dodonova, F. Zhu, C. Dienemann, J. Taipale, P. Cramer, *Nature*, 2020, **580**, 669-672.

[22] A. K. Michael, R. S. Grand, L. Isbel, S. Cavadini, Z. Kozicka, G. Kempf, R. D. Bunker, A. D. Schenk, A. Graff-Meyer, G. R. Pathare, J. Weiss, S. Matsumoto, L. Burger, D. Schübeler, N. H. Thomä, *Science*, 2020, **368**, 1460-1465.

[23] K. Echigoya, M. Koyama, L. Negishi, Y. Takizawa, Y. Mizukami, H. Shimabayashi, A. Kuroda, H. Kurumizaka, *Sci. Rep.*, 2020, **10**, article no. 11832.

[24] G. A. Roberts, B. Ozkan, I. Gachulincová, M. R. O'Dwyer, E. Hall-Ponsele, M. Saxena, P. J. Robinson, A. Soufi, *Nat. Cell Biol.*, 2021, **23**, 834-845.

[25] E. Morgunova, J. Taipale, *Curr. Opin. Struct. Biol.*, 2021, **71**, 171-179.

[26] W. Kagawa, H. Kurumizaka, *Curr. Opin. Struct. Biol.*, 2021, **71**, 59-64.

[27] E. Luzete-Monteiro, K. S. Zaret, *Curr. Opin. Struct. Biol.*, 2022, **75**, article no. 102425.

[28] B. D. Sunkel, B. Z. Stanton, *IScience*, 2021, **24**, article no. 103132.

[29] F. C. M. Gadea, E. N. Nikolova, *J. Mol. Biol.*, 2023, **435**, article no. 167916.

[30] B. Jagga, M. Edwards, M. Pagin, K. M. Wagstaff, D. Aragão, N. Roman, J. D. Nanson, S. R. Raidal, N. Dominado, M. Stewart, D. A. Jans, G. R. Hime, S. K. Nicolis, C. F. Basler, J. K. Forwood, *Nat. Commun.*, 2021, **12**, article no. 28.

[31] B. Xue, C. J. Oldfield, Y.-Y. Van, A. K. Dunker, V. N. Uversky, *Mol. BioSyst.*, 2012, **8**, 134-150.

[32] M. M. Babu, *Biochem. Soc. Trans.*, 2016, **44**, 1185-1200.

[33] P. E. Wright, H. J. Dyson, *Nat. Struct. Mol. Biol.*, 2015, **16**, 18-29.

[34] D. Piovesan, M. Necci, N. Escobedo, A. M. Monzon, A. Hatos, I. Mičetić, F. Quaglia, L. Paladin, P. Ramasamy, Z. Dosztanyi, W. F. Vranken, N. E. Davey, G. Parisi, M. Fuxreiter, S. C. E. Tosatto, *Nucleic Acids Res.*, 2021, **49**, D361-D367.

[35] P. Kulkarni, S. Bhattacharya, S. Achuthan, A. Behal, M. K. Jolly, S. Kotnala, A. Mohanty, G. Rangarajan, R. Salgia, V. Uversky, *Chem. Rev.*, 2022, **122**, 6614-6633.

[36] D. Piovesan, A. Del Conte, D. Clementel, A. M. Monzon, M. Bevilacqua, M. C. Aspromonte, J. A. Iserte, F. E. Orti, C. Marino-Buslje, S. C. E. Tosatto, *Nucleic Acids Res.*, 2023, **51**, D438-D444.

[37] Y. Buganim, D. A. Faddah, R. Jaenisch, *Nat. Struct. Mol. Biol.*, 2013, **14**, 427-439.

[38] A. Rizzino, *Stem Cells*, 2013, **31**, 1033-1039.

[39] S. Jerabek, F. Merino, H. R. Schöler, V. Cojocaru, *Biochim. Biophys. Acta Gene Regul. Mech.*, 2014, **1839**, 138-154.

[40] A. Saunders, F. Faiola, J. Wang, *Stem Cells*, 2013, **31**, 1227-1236.

[41] S. E. Bondos, A. K. Dunker, V. N. Uversky, *Cell Commun. Signal*, 2022, **20**, article no. 20.

[42] K.-P. Kim, Y. Wu, J. Yoon, K. Adachi, G. Wu, S. Velychko, C. M. MacCarthy, B. Shin, A. Röpke, M. J. Arauzo-Bravo, M. Stehling, D. W. Han, Y. Gao, J. Kim, S. Gao, H. R. Schöler, *Sci. Adv.*, 2020, **6**, article no. eaaz7364.

[43] I. Aksoy, R. Jauch, V. Eras, W. A. Chng, J. Chen, U. Divakar, C. K. L. Ng, P. R. Kolatkar, L. W. Stanton, *Stem Cells*, 2013, **31**, 2632-2646.

[44] A. Boija, I. A. Klein, B. R. Sabari, A. Dall'Agnese, E. L. Coffey, A. V. Zamudio, C. H. Li, K. Shrinivas, J. C. Manteiga, N. M. Hannett, B. J. Abraham, L. K. Afeyan, Y. E. Guo, J. K. Rimel, C. B. Fant, J. Schuijers, T. I. Lee, D. J. Taatjes, R. A. Young, *Cell*, 2018, **175**, 1842-1855.

[45] L. Staby, C. O'Shea, M. Willemoës, F. Theisen, B. B. Kragelund, K. Skriver, *Biochem. J.*, 2017, **474**, 2509-2532.

[46] C. N. Ravarani, T. Y. Erkina, G. De Baets, D. C. Dudman, A. M. Erkine, M. M. Babu, *Mol. Syst. Biol.*, 2018, **14**, article no. e8190-14.
[47] S. Brodsky, T. Jana, K. Mittelman, M. Chapal, D. K. Kumar, M. Carmi, N. Barkai, *Mol. Cell*, 2020, **79**, 459-471, e4.

[48] A. Erijman, L. Kozlowski, S. Sohrabi-Jahromi, J. Fishburn, L. Warfield, J. Schreiber, W. S. Noble, J. Söding, S. Hahn, *Mol. Cell*, 2020, **78**, 890-902, e6.

[49] G. Næs, J. O. Storesund, P. Udayakumar, M. Ledsaak, O. S. Gabrielsen, *FEBS Open Bio*, 2020, **10**, 2329-2342.

[50] A. L. Sanborn, B. T. Yeh, J. T. Feigerle, C. V. Hao, R. J. Townshend, E. Lieberman Aiden, R. O. Dror, R. D. Kornberg, *ELife*, 2021, **10**, article no. e68068.

[51] L. M. Tuttle, D. Pacheco, L. Warfield, D. B. Wilburn, S. Hahn, R. E. Klevit, *Nat. Commun.*, 2021, **12**, article no. 2220.

[52] L. F. Soto, Z. Li, C. S. Santoso, A. Berenson, I. Ho, V. X. Shen, S. Yuan, J. I. Fuxman Bass, *Mol. Cell*, 2022, **82**, 514-526.

[53] M. V. Staller, E. Ramirez, S. R. Kotha, A. S. Holehouse, R. V. Pappu, B. A. Cohen, *Cell Syst.*, 2022, **13**, 334-345, e5.

[54] B. Bourgeois, T. Gui, D. Hoogeboom, H. G. Hocking, G. Richter, E. Spreitzer, M. Viertler, K. Richter, T. Madl, B. M. T. Burgering, *Cell Rep.*, 2021, **36**, article no. 109446.

[55] K. Teilum, J. G. Olsen, B. B. Kragelund, *Biochem. J.*, 2021, **478**, 2035-2050.

[56] F.-X. Theillet, A. Binolfi, T. Frembgen-Kesner, K. Hingorani, M. Sarkar, C. Kyne, C. Li, P. B. Crowley, L. Gierasch, G. J. Pielak, A. H. Elcock, A. Gershenson, P. Selenko, *Chem. Rev.*, 2014, **114**, 6661-6714.

[57] N. E. Davey, *Curr. Opin. Struct. Biol.*, 2019, **56**, 155-163.

[58] T. M. Filtz, W. K. Vogel, M. Leid, *Trends Pharmacol. Sci.*, 2014, **35**, 76-85.

[59] D. Han, M. Huang, T. Wang, Z. Li, Y. Chen, C. Liu, Z. Lei, X. Chu, *Cell Death Dis*, 2019, **10**, article no. 290.

[60] M. Qian, F. Yan, T. Yuan, B. Yang, Q. He, H. Zhu, *Drug Discov. Today*, 2020, **25**, 1502-1512.

[61] P. Weidemüller, M. Kholmatov, E. Petsalaki, J. B. Zaugg, *Proteomics*, 2021, **21**, article no. 2000034.

[62] N. Cai, M. Li, J. Qu, G.-H. Liu, J. C. Izpisua Belmonte, *J. Mol. Cell Biol.*, 2012, **4**, 262-265.

[63] L. Fang, L. Zhang, W. Wei, X. Jin, P. Wang, Y. Tong, J. Li, J. X. Du, J. Wong, *Mol. Cell*, 2014, **55**, 537-551.

[64] D. S. Yoon, Y. Choi, Y. Jang, M. Lee, W. J. Choi, S.-H. Kim, J. W. Lee, *Stem Cells*, 2014, **32**, 3219-3231.

[65] H. Jang, T. W. Kim, S. Yoon, S.-Y. Choi, T.-W. Kang, S.-Y. Kim, Y.-W. Kwon, E.-J. Cho, H.-D. Youn, *Stem Cell*, 2012, **11**, 62-74.

[66] J. Brumbaugh, Z. Hou, J. D. Russell, S. E. Howden, P. Yu, A. R. Ledvina, J. J. Coon, J. A. Thomson, *Proc. Natl. Acad. Sci. USA*, 2012, **109**, 7162-7168.

[67] S. Dan, B. Kang, X. Duan, Y.-J. Wang, *Biochem. Biophys. Res. Commun.*, 2015, **456**, 714-720.

[68] Y. Cho, H. G. Kang, S.-J. Kim, S. Lee, S. Jee, S. G. Ahn, M. J. Kang, J. S. Song, J.-Y. Chung, E. C. Yi, K.-H. Chun, *Cell Death Differ.*, 2018, **25**, 1781-1795.

[69] C. A. C. Williams, A. Soufi, S. M. Pollard, *Semin. Cancer Biol.*, 2019, **67**, 30-38.

[70] X. Abulaiti, H. Zhang, A. Wang, N. Li, Y. Li, C. Wang, X. Du, L. Li, *Stem Cell Rep.*, 2017, **9**, 1630-1641.

[71] D. K. Kim, B. Song, S. Han, H. Jang, S.-H. Bae, H. Y. Kim, S.-H. Lee, S. Lee, J. K. Kim, H.-S. Kim, K.-M. Hong, B. I. Lee, H.-D. Youn, S.-Y. Kim, S. W. Kang, H. Jang, *Cancers*, 2020, **12**, article no. 2601.

[72] N. P. Mullin, J. Varghese, D. Colby, J. M. Richardson, G. M. Findlay, I. Chambers, *FEBS Lett.*, 2021, **595**, 14-25.

[73] K. T. G. Rigbolt, T. A. Prokhorova, V. Akimov, J. Henningsen, P. T. Johansen, I. Kratchmarova, M. Kassem, M. Mann, J. V. Olsen, B. Blagoev, *Sci. Signal*, 2011, **4**, article no. rs3.

[74] Y. Kamachi, H. Kondoh, *Development*, 2013, **140**, 4129-4144.

[75] J. Shin, T. W. Kim, H. Kim, H. J. Kim, M. Y. Suh, S. Lee, H.-T. Lee, S. Kwak, S.-E. Lee, J.-H. Lee, H. Jang, E.-J. Cho, H.-D. Youn, *Elife*, 2016, **5**, article no. e10877.

[76] P. N. Malak, B. Dannenmann, A. Hirth, O. C. Rothfuss, K. Schulze-Osthoff, *Cell Cycle*, 2015, **14**, 3748-3754.

[77] T. Schaefer, C. Lengerke, *Oncogene*, 2020, **39**, 278-292.

[78] J. Ouyang, W. Yu, J. Liu, N. Zhang, L. Florens, J. Chen, H. Liu, M. Washburn, D. Pei, T. Xie, *J. Biol. Chem.*, 2015, **290**, 22782-22794.

[79] S. Lim, A. Bhinge, S. Bragado Alonso, I. Aksoy, J. Aprea, C. F. Cheok, F. Calegari, L. W. Stanton, P. Kaldis, *Mol. Cell Biol.*, 2017, **37**, article no. e00201-17-24.

[80] H. J. Kim, J. Shin, S. Lee, T. W. Kim, H. Jang, M. Y. Suh, J.-H. Kim, I.-Y. Hwang, D. S. Hwang, E.-J. Cho, H.-D. Youn, *Nucleic Acids Res.*, 2018, **46**, 6544-6560.

[81] M. Moretto-Zita, H. Jin, Z. Shen, T. Zhao, S. P. Briggs, Y. Xu, *Proc. Natl. Acad. Sci. USA*, 2010, **107**, 13312-13317.

[82] S.-H. Kim, M.-O. Kim, Y.-Y. Cho, K. Yao, D. J. Kim, C.-H. Jeong, D. H. Yu, K. B. Bae, E.-J. Cho, S. K. Jung, M. H. Lee, H. Chen, J. Y. Kim, A. M. Bode, Z. Dong, *Stem Cell Res.*, 2014, **13**, 1-11.

[83] J. Brumbaugh, J. D. Russell, P. Yu, M. S. Westphall, J. J. Coon, J. A. Thomson, *Stem Cell Rep.*, 2014, **2**, 18-25.

[84] A. Saunders, D. Li, F. Faiola, X. Huang, M. Fidalgo, D. Guallar, J. Ding, F. Yang, Y. Xu, H. Zhou, J. Wang, *Stem Cell Rep.*, 2017, **8**, 1115-1123.

[85] L. Liu, W. Michowski, H. Inuzuka, K. Shimizu, N. T. Ni-hira, J. M. Chick, N. Li, Y. Geng, A. Y. Meng, A. Ordureau, A. Kolodziejczyk, K. L. Ligon, R. T. Bronson, K. Polyak, J. W. Harper, S. P. Gygi, W. Wei, P. Sicinski, *Nat. Cell Biol.*, 2017, **19**, 177-188.

[86] L. Liu, W. Michowski, A. Kolodziejczyk, P. Sicinski, *Nat. Cell Biol.*, 2019, **21**, 1060-1067.

[87] S. Jirawatnotai, S. Dalton, M. Wattanapanitch, *Semin. Cell Dev. Biol.*, 2020, **107**, 63-71.

[88] R. Spelat, F. Ferro, F. Curcio, *J. Biol. Chem.*, 2012, **287**, 38279-38288.

[89] K. B. Bae, D. H. Yu, K. Y. Lee, K. Yao, J. Ryu, D. Y. Lim, T. A. Zykova, M.-O. Kim, A. M. Bode, Z. Dong, *Stem Cell Rep.*, 2017, **9**, 2050-2064.

[90] Y. Hao, X. Fan, Y. Shi, C. Zhang, D. Sun, K. Qin, W. Qin, W. Zhou, X. Chen, *Nat. Commun.*, 2019, **10**, 1-13.

[91] N. S. Sharma, V. K. Gupta, P. Dauer, K. Kesh, R. Hadad, B. Giri, A. Chandra, V. Dudeja, C. Slawson, S. Banerjee, S. M. Vickers, A. Saluja, S. Banerjee, *Theranostics*, 2019, **9**, 3410-3424.

[92] D. K. Kim, J.-S. Lee, E. Y. Lee, H. Jang, S. Han, H. Y. Kim, I.-Y. Hwang, J.-W. Choi, H. M. Shin, H. J. You, H.-D. Youn, H. Jang, *Exp. Mol. Med.*, 2021, **53**, 1759-1768.

[93] S. Constable, J.-M. Lim, K. Vaidyanathan, L. Wells, *Glycobiology*, 2017, **27**, 927-937.

[94] T. Miura, S. Nishihara, *Trends Glycosci. Glycotechnol.*, 2019, **31**, E69-E75.

[95] L. Ciraku, E. M. Esquea, M. J. Reginato, *Cell. Signal*, 2022, **90**, article no. 110201.
[96] J. Ma, C. Hou, C. Wu, *Chem. Rev.*, 2022, **122**, 15822-15864.

[97] A. M. Gronenborn, D. R. Filipula, N. Z. Essig, A. Achari, M. Whitlow, P. T. Wingfield, G. M. Clore, *Nature*, 1991, **253**, 657-661.

[98] C. K. Smith, J. M. Withka, L. Regan, *Biochemistry*, 1994, **33**, 5510-5517.

[99] A. Alik, C. Bouguechtouli, M. Julien, W. Bermel, R. Ghouil, S. Zinn-Justin, F.-X. Theillet, *Angew. Chem. Int. Ed.*, 2020, **59**, 10411-10415.

[100] M. Howarth, K. Takao, Y. Hayashi, A. Y. Ting, *Proc. Natl. Acad. Sci. USA*, 2005, **102**, 7583-7588.

[101] M. Fairhead, M. Howarth, *Methods Mol. Biol.*, 2015, **1266**, 171-184.

[102] F.-X. Theillet, A. Binolfi, B. Bekei, A. Martorana, H. M. Rose, M. Stuiver, S. Verzini, D. Lorenz, M. van Rossum, D. Goldfarb, P. Selenko, *Nature*, 2016, **530**, 45-50.

[103] R. Dass, F. A. A. Mulder, J. T. Nielsen, *Sci. Rep.*, 2020, **10**, article no. 14780.

[104] K. Tamiola, F. A. A. Mulder, *Biochem. Soc. Trans.*, 2012, **40**, 1014-1020.

[105] J. T. Nielsen, F. A. A. Mulder, *J. Biomol. NMR*, 2018, **70**, 141-165.

[106] W. Borcherds, F.-X. Theillet, A. Katzer, A. Finzel, K. M. Mishall, A. T. Powell, H. Wu, W. Manieri, C. Dieterich, P. Selenko, A. Loewer, G. W. Daughdrill, *Nat. Chem. Biol.*, 2014, **10**, 1000-1002.

[107] C. Camilloni, A. De Simone, W. F. Vranken, M. Vendruscolo, *Biochemistry*, 2012, **51**, 2224-2231.

[108] F.-X. Theillet, C. Smet-Nocca, S. Liokatis, R. Thongwichian, J. Kosten, M.-K. Yoon, R. W. Kriwacki, I. Landrieu, G. Lippens, P. Selenko, *J. Biomol. NMR*, 2012, **54**, 217-236.

[109] F.-X. Theillet, H. M. Rose, S. Liokatis, A. Binolfi, R. Thongwichian, M. Stuiver, P. Selenko, *Nat. Protoc.*, 2013, **8**, 1416-1432.

[110] A. Mylona, F.-X. Theillet, C. Foster, T. M. Cheng, F. Miralles, P. A. Bates, P. Selenko, R. Treisman, *Science*, 2016, **354**, 233-237.

[111] M. Julien, C. Bouguechtouli, A. Alik, R. Ghouil, S. Zinn-Justin, F.-X. Theillet, in *Intrinsically Disordered Proteins: Methods and Protocols* (B. B. Kragelund, K. Skriver, eds.), Springer US, New York, 2020, 793-817.

[112] N. Festuccia, N. Owens, A. Chervova, A. Dubois, P. Navarro, *Development*, 2021, **148**, article no. dev199604.

[113] J.-P. Lambert, M. Tucholska, T. Pawson, A.-C. Gingras, *J. Proteomics*, 2014, **100**, 55-59.

[114] P. Poullet, S. Carpentier, E. Barillot, *Proteomics*, 2007, **7**, 2553-2556.

[115] M. The, M. J. MacCoss, W. S. Noble, L. Käll, *J. Am. Soc. Mass Spectrom*, 2016, **27**, 1719-1727.

[116] B. Valot, O. Langella, E. Nano, M. Zivy, *Proteomics*, 2011, **11**, 3572-3577.

[117] C. Smet-Nocca, H. Launay, J.-M. Wieruszeski, G. Lippens, I. Landrieu, *J. Biomol. NMR*, 2013, **55**, 323-337.

[118] S. Elkjær, A. D. Due, L. F. Christensen, F. F. Theisen, L. Staby, B. B. Kragelund, K. Skriver, *Commun. Biol.*, 2023, **6**, article no. 63.

[119] W. Peti, R. Page, *Protein Sci.*, 2013, **22**, 1698-1710.

[120] D. J. Wood, J. A. Endicott, *Open Biol.*, 2018, **8**, article no. 180112.

[121] D. L. Sheridan, Y. Kong, S. A. Parker, K. N. Dalby, B. E. Turk, *J. Biol. Chem.*, 2008, **283**, 19511-19520.

[122] S. Liokatis, A. Stützer, S. J. Elsässer, F.-X. Theillet, R. Klingberg, B. van Rossum, D. Schwarzer, C. D. Allis, W. Fischle, P. Selenko, *Nat. Struct. Mol. Biol.*, 2012, **19**, 819-823.

[123] S. A. Lambert, A. Jolma, L. F. Campitelli, P. K. Das, Y. Yin, M. Albu, X. Chen, J. Taipale, T. R. Hughes, M. T. Weirauch, *Cell*, 2018, **172**, 650-665.

[124] J. Liu, N. B. Perumal, C. J. Oldfield, E. W. Su, V. N. Uversky, A. K. Dunker, *Biochemistry*, 2006, **45**, 6873-6888.

[125] I. Yruela, C. J. Oldfield, K. J. Niklas, A. K. Dunker, *Genome Biol. Evol.*, 2017, **9**, 1248-1265.

[126] K. Cermakova, H. C. Hodges, *Trends Biochem. Sci.*, 2023, **48**, 477-490.

[127] X. Z. Zhou, K. P. Lu, *Nat. Struct. Mol. Biol.*, 2016, **16**, 463-478.

[128] Y. Chen, Y. Wu, H. Yang, X. Li, M. Jie, C. Hu, Y. Wu, S. Yang, Y. Yang, *Cell Death Dis.*, 2018, **9**, article no. 883.

[129] J. Gebel, M. Tuppi, A. Chaikuad, K. Hötte, M. Schröder, L. Schulz, F. Lohr, N. Gutfreund, F. Finke, E. Henrich, J. Mezhyrova, R. Lehnert, F. Pampaloni, G. Hummer, E. H. K. Stelzer, S. Knapp, V. Dötsch, *Nat. Chem. Biol.*, 2020, **16**, 1078-1086.

[130] W. Qin, K. F. Cho, P. E. Cavanagh, A. Y. Ting, *Nat. Methods*, 2021, **18**, 133-143.

[131] J.-P. Lambert, M. Tucholska, C. Go, J. D. R. Knight, A.-C. Gingras, *J. Proteomics*, 2015, **118**, 81-94.

[132] A.-C. Gingras, K. T. Abe, B. Raught, *Curr. Opin. Chem. Biol.*, 2019, **48**, 44-54.

[133] X. Liu, K. Salokas, R. G. Weldatsadik, L. Gawriyski, M. Varjosalo, *Nat. Protoc.*, 2020, **15**, 3182-3211.

[134] D.-P. Minde, M. Ramakrishna, K. S. Lilley, *Commun. Biol.*, 2020, **3**, article no. 38.

[135] H. Göös, M. Kinnunen, K. Salokas, Z. Tan, X. Liu, L. Yadav, Q. Zhang, G.-H. Wei, M. Varjosalo, *Nat. Commun.*, 2022, **13**, article no. 766.

[136] C. P. Wigington, J. Roy, N. P. Damle, V. K. Yadav, C. Blikstad, E. Resch, C. J. Wong, D. R. Mackay, J. T. Wang, I. Krystkowiak, D. A. Bradburn, E. Tsekitsidou, S. H. Hong, M. A. Kaderali, S.-L. Xu, T. Stearns, A.-C. Gingras, K. S. Ullman, Y. Ivarsson, N. E. Davey, M. S. Cyert, *Mol. Cell*, 2020, **79**, 342-358, e12.

[137] Y. Ueki, T. Kruse, M. B. Weisser, G. N. Sundell, M. S. Y. Larsen, B. L. Mendez, N. P. Jenkins, D. H. Garvanska, L. Cressey, G. Zhang, N. Davey, G. Montoya, Y. Ivarsson, A. N. Kettenbach, J. Nilsson, *Mol. Cell*, 2019, **76**, 953-964, e6.

[138] C. Benz, M. Ali, I. Krystkowiak, L. Simonetti, A. Sayadi, F. Mihalic, J. Kliche, E. Andersson, P. Jemth, N. E. Davey, Y. Ivarsson, *Mol. Syst. Biol.*, 2022, **18**, article no. e10584.

[139] N. E. Davey, L. Simonetti, Y. Ivarsson, *Trends Biochem. Sci.*, 2022, **47**, 547-548.

[140] G. Krainer, T. J. Welsh, J. A. Joseph, J. R. Espinosa, S. Wittmann, E. de Csilléry, A. Sridhar, Z. Toprakcioglu, G. Gudiškytė, M. A. Czekalska, W. E. Arter, J. Guillén-Boixet, T. M. Franzmann, S. Qamar, P. S. George-Hyslop, A. A. Hyman, R. Collepardo-Guevara, S. Alberti, T. P. J. Knowles, *Nat. Commun.*, 2021, **12**, article no. 1085.

[141] K. Tsafou, P. B. Tiwari, J. D. Forman-Kay, S. J. Metallo, J. A. Toretsky, *J. Mol. Biol.*, 2018, **430**, 2321-2341.

[142] A. Chen, A. N. Koehler, *Trends Mol. Med.*, 2020, **26**, 508-518.
Research article

# Structural characterization of stem cell factors Oct4, Sox2, Nanog and Esrrb disordered domains, and a method to detect phospho-dependent binding partners

Chafiaa Bouguechtouli<sup>a</sup>, Rania Ghoulil<sup>®,a</sup>, Ania Alik<sup>a</sup>, Florent Dingli<sup>®,b</sup>, Damarys Loew<sup>®,b</sup> and Francois-Xavier Theillet<sup>®,*,a</sup>

<sup>a</sup> Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France

<sup>b</sup> Institut Curie, PSL Research University, Centre de Recherche, CurieCoreTech Spectrométrie de Masse Protéomique, Paris cedex 05, France

*Current addresses:* Structural Motility, Institut Curie, Paris Université Sciences et Lettres, Sorbonne Université, CNRS UMR144, 75005 Paris, France (C. Bouguechtouli), Université de Paris, Institut Cochin, CNRS UMR8104, INSERM U1016, Paris, France (A. Alik)

*E-mail:* francois-xavier.theillet@cnrs.fr (F.-X. Theillet)

## 1. Experimental procedures

### 1.1. OSNE peptides production

The TEV (Tobacco Etch Virus) protease was produced in-house recombinantly in *E. coli* BL21(DE3)Star, from a construct containing a hexahistidine tag (His6).

All peptides were produced in *E. coli* (strain BL21(DE3)Star) transformed with the plasmids presented in the main text. Cells were grown in M9 medium containing $^{15}$NH$_4^+$ (0.5 g/L) and $^{13}$C-glucose (2 g/L) as sole sources of nitrogen and carbon for producing samples used for NMR assignment, and natural abundance $^{12}$C-glucose (2 g/L) otherwise. Media were supplemented with kanamycine at 50 µg/mL, and the expression was induced at an optical density $OD_{600}$ = 0.8 by supplementing the medium with ITPG at 1 mM at 37 °C. Cells were harvested by

centrifugation (5 min at 5000 g) 4 h later and cell pellets were stored at -20 °C. Cells were lysed using sonication in Tris 20 mM, NaCl 150 mM, at pH 7.4 (buffer called “Tris Buffer Saline”, TBS) in presence of benzonase (E1014 Sigma-Aldrich), lysozyme, protease inhibitors 1x (EDTA-free cOmplete, Roche) and 10 mM DTT.

Soluble and insoluble fractions were separated by 15 min of centrifugation at 15,000 g. Oct4-, Nanog- and Esrrb-peptides were purified from the soluble fractions. The lysates were loaded on a His-Trap FF column (5 mL, Cytiva) and eluted using a gradient of imidazole (in TBS). The eluted fractions were concentrated, submitted to TEV treatment for 1 h in TBS+imidazole supplemented with 10 mM DTT, and then diluted in TBS and re-loaded on the His-Trap column. Fractions containing the peptide of interest were submitted to a size-exclusion chromatography (SEC) in a column (Superdex 16/60 75 pg, Cytiva) previously equilibrated with Hepes at 10 mM or 20 mM, or phosphate at 20 mM, and NaCl 50 mM or 150 mM, at pH 6.8 (low-salt samples for NMR assignments,

<footnote>* Corresponding author.</footnote>
high-salt samples for phosphorylation kinetics or
pull-down assays). For the cysteine-containing pep-
tides, the eluted fractions of interest were immedi-
ately supplemented with DTT or TCEP at 2 mM, con-
centrated and stored at –20 °C. Fresh DTT or TCEP
was supplemented further after thawing before the
NMR experiments.

The constructs containing Sox2(aa1-42),
Sox2(aa115-187) and Sox2(aa234-317)-AviTag-His6
were also purified from the soluble fraction, as ex-
plained above. The other Sox2-constructs were re-
covered from the insoluble fractions of the lysates,
and resolubilized in TBS supplemented with 8 M
urea, loaded on a His-Trap FF column (5 mL, Cytiva)
and eluted using a gradient of imidazole; the eluates
were then supplemented with β-mercaptoethanol
at 50 mM and incubated at room temperature for
15 min, before being dialyzed in TBS supplemented
with DTT at 1 mM, in order to refold the GST domain.
The samples were then submitted to TEV cleavage
in 0.5 M urea, and a second His-Trap purification
was carried out in TBS supplemented with urea at
2 M. The fractions of interest were concentrated and
submitted to a SEC in Hepes at 10 mM or phosphate
at 20 mM, and NaCl 50 mM or 150 mM, urea at 2 M,
at pH 6.8. The samples were concentrated and stored
at –20 °C. Before the NMR experiments, they were
thawed and submitted to 2–3 cycles of concentra-
tion/dilution in Hepes at 20 mM, NaCl 75 mM to
generate samples in urea at 0.25 or 0.125 M. We paid
attention to avoid precipitation during the concen-
tration steps, because these peptides had a limited
solubility, about 100–150 µM.

We achieved some liquid-liquid phase separation
assays, using DIC microscopy at room temperature in
Ficoll-70 at 100 mg/mL. These were carried out with
Sox2 peptides previously centrifuged during 10 min
at 15,000 g to remove the aggregates: for example,
coacervates were observed at 4 µM of Sox2(aa115-
317_C265A), and some aggregates were rapidly form-
ing under the microscope at 20 µM.

## 1.2. *Production of BirA and biotinylation of AviTag-peptide chimera*

Bacteria transformed with pET21a-BirA were precul-
tured at 37 °C overnight in a Luria-Bertani (LB)
culture medium supplemented with ampicillin at
100 µg/mL. Then, these were cultured in a larger

volume of LB supplemented with ampicillin at
50 µg/mL at 37 °C, and at 30 °C when they reached
an optical density (OD) of 0.4. At an OD = 0.8, the
culture was transferred to 20 °C and the protein ex-
pression was induced by supplementing the medium
with IPTG at 0.5 mM. The incubation was carried out
overnight, the bacteria were harvested by centrifuga-
tion at 4500 g for 5 min and the pellets were stored at
-20 °C.

The purification was carried out at 4 °C. Cells
were lysed using sonication in TBS at pH 7.5 in pres-
ence of 0.5 µL of benzonase (E1014 Sigma-Aldrich),
lysozyme, PMSF at 1 mM (Sigma-Aldrich) and DTT
at 10 mM. The soluble and insoluble fractions were
separated by 15 min centrifugation at 15,000 g. The
lysate (supernatant, soluble fraction) was loaded on
a His-Trap column (His-Trap FF 5 mL, Cytiva) and
eluted in TBS using a gradient of imidazole. The
eluted fractions of interest were concentrated in
presence of DTT at 10 mM, and later submitted to a
SEC in a column (Superdex 16/60 75 pg, Cytiva) previ-
ously equilibrated with TBS at pH 7.5, supplemented
with 10% v/v glycerol. The fractions of interest were
concentrated in presence of DTT at 2 mM. Final
concentrations of BirA were about 100 µM. The ob-
tained sample was aliquoted, flash-frozen and stored
at -80 °C.

The primary sequence of the expressed construct
is:

MKDNTVPLKLIALANGEFHSGEQLGETLGMSRAAINKHIQTLRDGW
VDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYL
LDRIGELKSGDACIAEYQQAGRGRGRKWFSPFGANLYSMFWRLEQ
GPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPNDLYLQDRKLAGIL
VELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGWITLQEAGINLDR
NTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGD
KEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEKKLAAALEH
HHHHH*

## 1.3. Assignment of NMR signals from OSNE fragments, and structural propensities

Almost all NMR spectra were recorded on a 700 MHz Bruker Avance Neo spectrometer or a 600 MHz Bruker Avance II, equipped with cryogenically cooled triple resonance ¹H[¹³C/¹⁵N] probes optimized for ¹H-detection, a TCI and a TXI, respectively. Assignment spectra of Sox2 aa115-317 were recorded on a 950 MHz Bruker Avance III
spectrometer, equipped with a cryogenically cooled triple resonance ¹H[¹³C/¹⁵N] probe (TCI). All spectra were processed in Topspin 3 or Topspin 4. 3D spectra analysis was carried out using CccpNmr 2.4.2. DSS at 100 µM and 7.5% D₂O were added in all samples.

NMR assignments of backbone amide resonances of uniformly-labeled peptides (¹³C/¹⁵N) was achieved using BEST-HNCO, -HN(CA)CO, -HNCACB,[⁴] and (H)N(CA)NH 3D experiments, in HEPES at 10 mM, NaCl at 50 mM, DTT or TCEP at 2 to 5 mM, at pH 6.8 and 283 K, and at peptide concentrations ranging from 150 to 900 µM in 5 mm diameter Shigemi tubes.

Assignments of Oct4_aa286-360, Sox2_aa1-42, Nanog_aa1-85, Esrrb_aa1-102 were carried out at 700 MHz; those of Oct4_aa1-145, Sox2_aa115-236, His6-AviTag-Sox2_aa234-317_C265A at 600 MHz; those of Sox2_aa115-317_C265A at 950 MHz.

### Oct4_aa286-360: interscan delay: 0.5 s

B-HNCO and were carried out with 1024 (¹H) × 96 (¹³C) × 80 (¹⁵N) complex points and sweep widths of 12.98 ppm (¹H), 8 ppm (¹³C) and 22 ppm (¹⁵N),

B-HN(CA)CO were carried out with 1024 (¹H) × 96 (¹³C) × 64 (¹⁵N) complex points and sweep widths of 12.98 ppm (¹H), 8 ppm (¹³C) and 22 ppm (¹⁵N),

B-HNCACB 1024 (¹H) × 128 (¹³C) × 64 (¹⁵N) complex points and sweep widths of 12.98 ppm (¹H), 60 ppm (¹³C) and 22 ppm (¹⁵N),

B-(H)N(CA)NH with 1024 (¹H) × 64 (¹⁵N) × 64 (¹⁵N) complex points and sweep widths of 12.98 ppm (¹H), and 22 ppm (¹⁵N).

### Sox2_aa1-42: interscan delay: 0.5 s

B-HNCO and was carried out with 2048 (¹H) × 88 (¹³C) × 88 (¹⁵N) complex points and sweep widths of 12.98 ppm (¹H), 8 ppm (¹³C) and 26 ppm (¹⁵N),

HN(CA)CO was carried out with 2048 (¹H) × 72 (¹³C) × 72 (¹⁵N) complex points and sweep widths of 12.98 ppm (¹H), 8 ppm (¹³C) and 26 ppm (¹⁵N),

B-HNCACB 2048 (¹H) × 80 (¹³C) × 80 (¹⁵N) complex points and sweep widths of 12.98 ppm (¹H), 60 ppm (¹³C) and 26 ppm (¹⁵N),

B-(H)N(CA)NH with 2048 (¹H) × 64 (¹⁵N) × 64 (¹⁵N) complex points and sweep widths of 12.98 ppm (¹H), and 26 ppm (¹⁵N).

### Esrrb_aa1-102_3Cys->3Ala: interscan delay: 0.5 s

B-HNCO and was carried out with 2048 (¹H) × 92 (¹³C) × 92 (¹⁵N) complex points and sweep widths of 12.98 ppm (¹H), 8 ppm (¹³C) and 26 ppm (¹⁵N),

HN(CA)CO was carried out with 2048 (¹H) × 922 (¹³C) × 72 (¹⁵N) complex points and sweep widths of 12.98 ppm (¹H), 8 ppm (¹³C) and 26 ppm (¹⁵N),

B-HNCACB 2048 (¹H) × 128 (¹³C) × 72 (¹⁵N) complex points and sweep widths of 12.98 ppm (¹H), 60 ppm (¹³C) and 26 ppm (¹⁵N),

B-(H)N(CA)NH with 2048 (¹H) × 72 (¹⁵N) × 72 (¹⁵N) complex points and sweep widths of 12.98 ppm (¹H), and 26 ppm (¹⁵N).

### Nanog_aa1-85: interscan delay: 0.5 s

B-HNCO was carried out with 1024 (¹H) × 96 (¹³C) × 92 (¹⁵N) complex points and sweep widths of 12.98 ppm (¹H), 8 ppm (¹³C) and 26 ppm (¹⁵N),

B-HN(CA)CO: 1024 (¹H) × 88 (¹³C) × 72 (¹⁵N) complex points and sweep widths of 12.98 ppm (¹H), 8 ppm (¹³C) and 26 ppm (¹⁵N),

B-HNCACB: 1024 (¹H) × 96 (¹³C) × 72 (¹⁵N) complex points and sweep widths of 12.98 ppm (¹H), 60 ppm (¹³C) and 22 ppm (¹⁵N),

B-(H)N(CA)NH: 1024 (¹H) × 72 (¹⁵N) × 72 (¹⁵N) complex points and sweep widths of 12.98 ppm (¹H), and 26 ppm (¹⁵N).

### Oct4_aa1-145: interscan delay: between 0.12 and 0.2 s

B-HNCO was carried out with 1536 (¹H) × 92 (¹³C) × 72 (¹⁵N) complex points and sweep widths of 16.6 ppm (¹H), 10 ppm (¹³C) and 24 ppm (¹⁵N),

B-HN(CA)CO: 1536 (¹H) × 92 (¹³C) × 64 (¹⁵N) complex points and sweep widths of 16.6 ppm (¹H), 10 ppm (¹³C) and 24 ppm (¹⁵N),

B-HNCACB: 1536 (¹H) × 128 (¹³C) × 64 (¹⁵N) complex points and sweep widths of 16.6 ppm (¹H), 65 ppm (¹³C) and 24 ppm (¹⁵N),

B-(H)N(CA)NH: 1536 (¹H) × 64 (¹⁵N) × 64 (¹⁵N) complex points and sweep widths of 16.6 ppm (¹H), and 24 ppm (¹⁵N).

### Sox2_aa115-236 (together with Sox2_aa1-187): interscan delay: between 0.12 and 0.2 s

B-HNCO was carried out with 1536 (¹H) × 92 (¹³C) × 64 (¹⁵N) complex points and sweep widths of 16.6 ppm (¹H), 10 ppm (¹³C) and 25 ppm (¹⁵N),
B-HN(CA)CO: 1536 ($^{1}$H) × 64 ($^{13}$C) × 64 ($^{15}$N) complex points and sweep widths of 16.6 ppm ($^{1}$H), 10 ppm ($^{13}$C) and 26 ppm ($^{15}$N),

B-HNCACB: 1536 ($^{1}$H) × 84 ($^{13}$C) × 64 ($^{15}$N) complex points and sweep widths of 16.6 ppm ($^{1}$H), 65 ppm ($^{13}$C) and 26 ppm ($^{15}$N),

B-(H)N(CA)NH: 1536 ($^{1}$H) × 32 ($^{15}$N) × 32 ($^{15}$N) complex points and sweep widths of 16.6 ppm ($^{1}$H), and 24 ppm ($^{15}$N).

His6-AviTag-Sox2_aa234-317: interscan delay: between 0.12 and 0.2 s.

B-HNCO was carried out with 1536 ($^{1}$H) × 92 ($^{13}$C) × 72 ($^{15}$N) complex points and sweep widths of 16.6 ppm ($^{1}$H), 11 ppm ($^{13}$C) and 24 ppm ($^{15}$N),

B-HN(CA)CO: 1536 ($^{1}$H) × 92 ($^{13}$C) × 64 ($^{15}$N) complex points and sweep widths of 16.6 ppm ($^{1}$H), 11 ppm ($^{13}$C) and 24 ppm ($^{15}$N),

B-HNCACB: 1536 ($^{1}$H) × 128 ($^{13}$C) × 64 ($^{15}$N) complex points and sweep widths of 16.6 ppm ($^{1}$H), 65 ppm ($^{13}$C) and 24 ppm ($^{15}$N),

B-(H)N(CA)NH: 1536 ($^{1}$H) × 64($^{15}$N) × 64 ($^{15}$N) complex points and sweep widths of 16.6 ppm ($^{1}$H), and 24 ppm ($^{15}$N).

Sox2_aa115-317_C265A: d1 = 0.2 s.

B-HNCO: 2126 ($^{1}$H) × 128 ($^{13}$C) × 128 ($^{15}$N) complex points and sweep widths of 14 ppm ($^{1}$H), 7 ppm ($^{13}$C) and 22 ppm ($^{15}$N).

B-HN(CA)CO: 2126 ($^{1}$H) × 92 ($^{13}$C) × 92 ($^{15}$N) complex points and sweep widths of 14 ppm ($^{1}$H), 7 ppm ($^{13}$C) and 22 ppm ($^{15}$N). Non-uniform sampling at 35%.

B-HNCACB: 2126 ($^{1}$H) × 128 ($^{13}$C) × 128 ($^{15}$N) complex points and sweep widths of 14 ppm ($^{1}$H), 60 ppm ($^{13}$C) and 22 ppm ($^{15}$N). Non-uniform sampling at 35%.

B-(H)N(CA)NH: 18218($^{1}$H) × 92 ($^{15}$N) × 92 ($^{15}$N) complex points and sweep widths of 14 ppm ($^{1}$H), and 22 ppm ($^{15}$N). Non-uniform sampling at 35%.

Spectra were processed with linear prediction of 16 or 32 complex points in both $^{13}$C, and $^{15}$N dimensions, cosine apodization in $^{1}$H and $^{15}$N dimensions, no apodization in $^{13}$C dimension, and zero filling to 2048, 512 and 256 complex points in $^{1}$H, $^{13}$C, and $^{15}$N dimensions, respectively. Assignment 2D $^{1}$H-$^{15}$N HSQC spectra were recorded using at least 1536 ($^{1}$H) × 256 ($^{15}$N) complex points and sweep widths of 16.23 ppm ($^{1}$H) and 30 ppm ($^{15}$N), and processed

with zero filling to 4 K and 1 K in the proton and nitrogen dimensions, respectively.

### 1.4. NMR monitoring of phosphorylation reactions and production of phosphorylated peptides

Phosphorylation reactions were carried out using $^{15}$N-labeled IDRs at 50 µM, in Hepes 20 mM, NaCl 50 mM, DTT or TCEP at 4 mM, ATP 1.5 mM, MgCl₂ at 5 mM, protease inhibitors (Roche), 7.5% D₂O, pH6.8 at 25 °C in 100 µL using 3 mm diameter Shigemi tubes. We monitored the phosphorylation kinetics by recording time series of $^{1}$H-$^{15}$N SOFAST-HMQC spectra on a 600 MHz Bruker Avance II or a 700 MHz Bruker Avance Neo spectrometer, both equipped with cryogenically cooled triple resonance $^{1}$H[$^{13}$C/$^{15}$N] probes optimized for $^{1}$H detection.

The kinase was spiked in the IDR sample on ice just before filling the NMR tube, which was immediately placed in the spectrometer. About 2 min were necessary to reach a temperature equilibrium. The automatic shimming procedure was then executed, and short 1D $^{1}$H and $^{1}$H($^{15}$N-filtered)-SOFAST-HMQC spectra were recorded before the 2D spectra.

We recorded 2D spectra during the phosphorylation reactions as follows: 2D $^{1}$H-$^{15}$N SOFAST-HMQC experiments were recorded using 2048 ($^{1}$H) × 96 ($^{15}$N) complex points and sweep widths of 16.6 ppm ($^{1}$H) and 26 ppm ($^{15}$N), 128 scans and interscan delays of 0.04 s; hence, the acquisition of one spectrum took 30 min. All spectra were processed zero filling to 2 K and 1 K in the direct and indirect dimensions, respectively. No apodization was applied for $^{1}$H-$^{15}$N SOFAST-HMQC spectra.

After processing spectra in Topspin3, we measured peak intensities in NMRFAM-SPARKY [1]. Peaks were centered in every spectrum to follow peak shifting because of pH drifts. Progress curves were plotted and fitted in Kaleidagraph 4.5. In the case of the phosphorylation reactions that were not complete, we used decay curves to normalize phosphorylation build-up curves. At the opposite, for the phosphorylation reactions reaching ~100%, we used the phospho-peaks intensities for normalizing the build-up curves. Detailed descriptions of the methods can be found in previous reports and published protocols [2–5].
## 1.5. Pull-down assays for interactomic analysis

Mouse Embryonic Stem Cells (mESCs) were harvested using a classical trypsin treatment (2 × 150 cm² cell culture dishes, 70% confluency). The production of nuclear extracts was inspired by the procedures previously published by Gingras and colleagues [6]. Trypsin was blocked, and cell pellets were washed three times in PBS, before being resuspended in a first gentle lysis buffer containing HEPES at 10 mM, KCl at 10 mM, EDTA at 0.5 mM, DTT at 1 mM, PMSF at 0.5 mM, 1% v/v NP40. After 10 min on ice, the cells were centrifuged 10 min at 15,000 g. The supernatant containing the cytosolic fraction was discarded, and the pellets containing the nuclei were resuspended in a second lysis buffer containing HEPES at 20 mM, KCl at 250 mM, EDTA at 0.5 mM, DTT at 1 mM, PMSF at 0.5 mM, phosphatase inhibitors (2 × PhosSTOP, Roche), 5% v/v glycerol, supplemented with 2 µL of benzonase (>250 units/µL, E1014 Sigma-Aldrich), before being sonicated on ice using a microtip sonicator and 3 pulses of 10 seconds. The lysis of nuclei was verified visually under the microscope using a cell counting chamber. The extract concentration used for the pull-downs was about 5 mg/mL, as measured by Bradford protein assay.

The pull-down assays were executed using 25 µL of streptavidin-coated magnetic beads (Magbeads streptavidine, Genscript), i.e. 50 µL of resuspended beads in the storing buffer. After every step described below, the tubes were placed on a magnetic rack to collect the beads, while the supernatant was removed with a pipette. The fresh beads were washed 3 times during 5 min in 500 µL of a PBS buffer. 1 nmol of biotinylated (using BirA, see above) AviTag-chimera peptides (either AviTag-Sox2(aa115-240), AviTag-Sox2(aa234-317_C265A), phospho-AviTag-Sox2(aa115-240), or phospho-AviTag-Sox2(aa234-317_C265A)) were diluted in 500 µL of PBS and incubated with the beads during one hour at room temperature under rotary agitation. The supernatant was then removed, the beads were washed 3 times during 5 min in 500 µL of a PBS buffer.

The mESCs extract (200 µL, generated from 15 million cells) was mixed with the beads, and then incubated during one hour at room temperature under rotary agitation. The supernatant was removed and the beads were washed 3 times during 5 min in 500 µL of a buffer containing HEPES at 20 mM, KCl

at 250 mM, EDTA at 0.5 mM, DTT at 1 mM, PMSF at 0.5 mM + PhosSTOP 1×.

## 1.6. Mass spectrometry-based proteomics analysis of pull-down assays

### 1.6.1. Sample preparation

The beads were resuspended in 100 µL of 25 mM NH₄HCO₃ and digested by adding 0.2 µg of trypsin/LysC (Promega) for 1 h at 37 °C. Samples were then loaded into custom-made C18 StageTips packed by stacking three AttractSPE® disk (#SPE-Disks-Bio-C18-100.47.20 Affinisep) into a 200 µL micropipette tip for desalting. Peptides were eluted using a ratio of 40:60 CH₃CN:H₂O + 0.1% formic acid and vacuum concentrated to dryness with a SpeedVac apparatus. Peptides were reconstituted in 10 µL of injection buffer in 0.3% trifluoroacetic acid (TFA) before liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

### 1.6.2. LC-MS/MS analysis

Online chromatography was performed with an RSLCnano system (Ultimate 3000, Thermo Scientific) coupled to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific). Peptides were trapped on a C18 column (75 µm inner diameter × 2 cm; nanoViper Acclaim PepMap™ 100, Thermo Scientific) with buffer A (2/98 MeCN/H₂O in 0.1% formic acid) at a flow rate of 4.0 µL/min over 4 min. Separation was performed on a 50 cm × 75 µm C18 column (nanoViper Acclaim PepMap™ RSLC, 2 µm, 100 Å, Thermo Scientific) regulated to a temperature of 55 °C with a linear gradient of 5% to 25% buffer B (100% MeCN in 0.1% formic acid) at a flow rate of 300 nL/min over 100 min. Peptides were ionized by a nanospray ionization (NSI) ion source at 2.2 kV. Full-scan MS in the Orbitrap was set at a scan range of 400–1500 with a resolution at 120,000 and ions from each full scan were fragmented in higher-energy collisional dissociation mode (HCD) and analyzed in the linear ion trap in rapid mode. The fragmentation was set to top speed mode in data-dependent analysis (DDA). We selected ions with charge state from 2+ to 7+ for screening. Normalized collision energy (NCE) was set to 30, AGC target to 20,000 and the dynamic exclusion to 30 s.
### 1.6.3. Data analysis

For identification, the datasets were searched against the *Mus Musculus* (UP000000589) UniProt database using Sequest HT through proteome discoverer (version 2.2). Enzyme specificity was set to trypsin and a maximum of two miss cleavages sites were allowed. Oxidized methionine, phosphorylation of serines, threonines and tyrosines, carbamidomethylation of cysteines and N-terminal acetylation were set as variable modifications. Maximum allowed mass deviation was set to 10 ppm for monoisotopic precursor ions and 0.6 Da for MS/MS peaks. The resulting files were further processed using myProMS v3.9.3 (https://github.com/bioinfo-pf-curie/myproms; Poullet et al. [7]). False-discovery rate (FDR) was calculated using Percolator [8] and was set to 1% at the peptide level for the whole study. Label-free quantification was performed using peptide extracted ion chromatograms (XICs), computed with MassChroQ [9] v.2.2.1. For protein quantification, XICs from proteotypic peptides shared between compared conditions (TopN matching) were used, missed cleavages and peptide modifications were not allowed. Median and scale normalization at peptide level was applied on the total signal to correct the XICs for each biological replicate (N = 2). To estimate the significance of the change in protein abundance, a linear model (adjusted on peptides and biological replicates) was performed, and p-values were adjusted using the Benjamini-Hochberg FDR procedure.

The mass spectrometry proteomics raw data have been deposited to the ProteomeXchange Consortium

via the PRIDE partner repository dataset [10]: identifier PXD 040573 (reviewer_pxd040573@ebi.ac.uk and Password: sVM686zJ).

### 1.7. Recombinant production of Pin1 and NMR analysis of its interaction with Sox2 or phospho-Sox2

The plasmid containing the gene coding for the Pin1-WW domain was a kind gift from Isabelle Landrieu. The production was executed according to the previously published protocol [11]. The NMR analysis of binding with phosphoSox2(aa115-240) were performed with the GST-Pin1-WW construct and ¹⁵N-labeled Sox2(aa115-240) mixed in stoichio-metric proportions, either at 50 or 10 µM for non-phospho and phosphoSox2, respectively. The solution contained Hepes at 20 mM, NaCl at 50 mM, urea at 0.25 mM (left-overs from Sox2(aa115-240) stock, stored at 2 M urea for solubility, see above), 5% D₂O and DSS at 0.1 mM, at pH = 7.0. The 2D ¹H-¹⁵N SOFAST-HMQC spectra were recorded at 283 K, using a 600 MHz Bruker Avance II equipped with a cryogenically cooled triple resonance ¹H[¹³C/¹⁵N] probe and a 5 mm diameter Shigemi tube.

The experiments were recorded using 1536 (¹H) × 128 (¹⁵N) complex points and sweep widths of 16.0 ppm (¹H) and 264 ppm (¹⁵N), 64 or 128 scans and interscan delays of 0.04 s. The spectra were processed with zero filling to 2 K and 1 K in the direct and indirect dimensions, respectively. Cosine apodization was applied in both dimensions. After processing spectra in Topspin3, we measured peak intensities in NMRFAM-SPARKY [1], and plotted the intensity ratios in Kaleidagraph 4.5.
## 2. Supplementary figures

<figure><img src="image_16.png" /><figcaption>**Supplementary Figure S1.** Overlays of 2D ¹H–¹⁵N HSQC spectra of Sox2(aa115-317_C265A) and Sox2(aa115-187), Sox2(aa115-236) and Sox2(aa115-282_C265A). These spectra have been recorded in a buffer containing urea at 0.25 M, except for Sox2(aa115-187), at 283 K and 700 MHz.</figcaption></figure>
<figure><img src="image_17.png" /><figcaption>**Supplementary Figure S2.** Caption continued on next page.</figcaption></figure>
**Supplementary Figure S2. (cont.)** (a) Overlay of 2D ¹H–¹⁵N HSQC spectra of Esrrb(aa1-102_C12A-C72A-C91A) (black) and Esrrb(aa1-102_C12A-C91A) (green); (b) chemical shift perturbations between the two constructs in (a) using $\Delta\delta = [((\Delta\delta_{1H}^2 + (\Delta\delta_{15N}/5)^2)/2)^{1/2}$; (c) overlay of 2D ¹H–¹⁵N HSQC spectra of Esrrb(aa1-102_C12A-C72A-C91A) (black) and Esrrb(aa1-102_C12A-C72A) (orange); (d) chemical shift perturbations between the two constructs in (a); (e) 2D ¹H–¹⁵N HSQC spectrum of the N-terminal IDRs of human Esrrb(aa1-102_C12A-C72A-C91A), the labels indicating the assignments; (f) primary structure of human Esrrb, and Secondary structure propensities of Esrrb(aa1-102_C12A-C72A-C91A) and Esrrb(aa1-102_C12A-C72A) calculated from the experimental chemical shifts of the peptide backbone Cα and Cβ, using the ncSPC algorithm [12,13].

<figure><img src="image_18.png" /></figure>

**Supplementary Figure S3.** (a) Overlay of 2D ¹H–¹⁵N HSQC spectra of GST-His6-Tev-Esrrb(aa1-102_C12A-C72A-C91A) (black), Esrrb(aa1-102_C12A-C72A-C91A) (red) and GST-His6-Tev (blue); (b) chemical shift perturbations between the GST-His6-Tev-Esrrb(aa1-102_C12A-C72A-C91A) and Esrrb(aa1-102_C12A-C72A-C91A) after TEV cleavage, using $\Delta\delta = [((\Delta\delta_{1H}^2 + (\Delta\delta_{15N}/5)^2)/2)^{1/2}$.
<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

**Supplementary Figure S4.** Secondary structure propensities calculated from the experimental chemical shifts of the peptide backbone HN, NH, CO, Cα and Cβ, using the δ2D algorithm [14].
<figure><img src="image_23.png" /><figcaption>**Supplementary Figure S5.** Liquid-liquid phase separation of Sox2_aa115-317 at 4 µM in PBS supplemented with Ficoll70 at 100 g/L, observed under differential interference contrast (DIC) (TCS SP8-X inversed FALCON, Leica, 63× PLAN oil immersion implemented with DIC, Numerical Aperture: 1.4, Leica).</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>**Supplementary Figure S6.** (a–c) SDS-PAGE analysis of binding assays: 1 nmol of biotinylated AviTag-IDRs (Input: Inp.) were incubated with 25 µL of streptavidin-coated magnetic beads; the 3 washing steps showed the stability of the binding; the elutions were performed with a Laemmli buffer, which provokes also the release of the streptavidine, whose band is shown in the last lane and unfortunately overlaps with the AviTag-IDRs constructs here. (d) We show here one of our tests with a batch of AviTag-Sox2(aa115-317_C265A), which was partially proteolyzed; this is to show that one of the peptides that do not contain the AviTag is removed by the first wash.</figcaption></figure>
### 3.2.3. Disorder prediction

https://st-protein.chem.au.dk/odinpred

https://www.nature.com/articles/s41598-020-71716-1

<figure><img src="image_25.png" /></figure>

### 3.2.4. Coding DNA sequences, produced protein constructs

#### Oct4-aal-145

**Synthesized sequence:**

Ccgcggtgagaacctgtacttccaaggcatggcggtcacctggcgagcgattttgcgtttagcccggccgggtggtggtggtgacggtccgggtggccgggaaccgggttgggtgatccgcgtacctggctgagcttccaaggtccggcggtggccgggtattggtccgggtgtggcccggtagcgaggtttggggtattccgccgtggccggccgtacgaatttttgcggtggcatggcggtattgcggtccgcaagtgggcgttggtctggttccgcaaggtggcctggaaaccagccagccggagggtaagcgggcgtgggtggtgagagcaacagcgatggtgcgagcccggaaccgtgcaccggtaccccggtgcggttaagctggagaaggaaaaactggagcagaaccggaggaaagccaagatatcaaggcgctgcagaaagaaaaacctgtactttcaaggtggcgcgggtggcgcgggtggccagtataaaactgattctgaacggcaagaccctgaaaggtgaaaccaccaccgaagcggtggatgcggcgaccgctgagaaggttttcaaaacagtacgcgaacgacaacggcgtggatggcgagtggacctatgacgatgcgaccaagacctttaccgttaccgaaggtggctaaaaagctt

**Translates into:**

AGENLYFQGMAGHLASDFAFSPPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPGVGPGSEVWGIPPCPPPYEFCGGMAYCGPQVGVGLV
PQGGLETSQPEGEAGVGVESNSDGASEPECTVTPGAVKLEKEKLEQNPEESQDIKALQKENLYFQGGAGGAGGQYKLILNGKTLKGETTTEAVD
AATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGG*

**After TEV-cleavage (leaving 1 Gly in N-ter, and ENLYFQ in C-ter):**

<table><tr><td>G</td><td>MAGHLASDFA</td><td>FSPPPGGGGD</td><td>GPGGPEPGWV</td><td>DPRTWLSFQG</td><td>PPGGPGIGPG</td><td>VGPGSEVWGI</td></tr><tr><td>PPCPPPYEFC</td><td>GGMAYCGPQV</td><td>GVGLVPQGGL</td><td>ETSQPEGEAG</td><td>VGVESNSDGA</td><td>SPEPCTVTPG</td><td></td></tr><tr><td>AVKLEKEKLE</td><td>QNPEESQDIK</td><td>ALQKENLYFQ</td><td></td><td></td><td></td><td></td></tr></table>
Oct4-aa286-360

Synthesized sequence:

Cgcgggtgagaacctgtactttcaggcaagcgtagcagcagcgactatgcgcaacgtgaggatttcgaagcggcgggtagcccgtttagcgg
tggccgggtgagcttcccgctggccgggtccgcactttggtaccccggtttatggcagcccgcacttcaccgcgctgtatagcagcgttccg
ttcccgagggtgaagcgtttccgccgggtgagcgttaccacctgggcagcccgatgcacagcaacgaaaatctgtactttcaggtggcgcgg
gtggcgcgggtggccaatataagctgatcctgaacggcaagaccctgaaaggcgaaaccaccaccgaagcggtggatgCGGCGaccgctgagaa
ggtttttaaacagtacgcgaacgacaacggtgtggatggcgagtggacctatgacgatgcgaccaaaaccttcaccgttaccgaaggtggctaa
aagctt

Translates into:

AGENLYFQGKRSSSDYAQREDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFPEGEAFPPVSVTTLGSPMHSNENLYFQGGAG
GAGGQYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGG*

After TEV-cleavage (leaving 1 Gly in N-ter, and ENLYFQ in C-ter):

290 300
G KRSSS DYAQREDFEA

310 320 330 340 350 360
AGSPFSGGPV SFPLAPGPHF GTPGYGSPHF TALYSSVPFP EGEAFPPVSV TTLGSPMHSN ENLYFQG
### 3.3. Sox2

#### 3.3.1. Sequence alignment: mammals

S: Serine

P: Proline

SP: phosphorylation motif for MAPK and Cdk

FILVYW: hydrophobic

DE: Asp/Glu

KR: Lys/Arg

T: Thr

XXX: DND-BD in crystal (1GT0)

<table><tr><td>sox2-human</td><td>MNMMETELKPPGPQQT</td><td>GGGGG</td><td>---</td><td>N</td><td>TAAAGGNQKN</td><td>SPDRVKRP</td><td>PMNAFMVWSR</td><td>53</td></tr><tr><td>sox2-mus</td><td>MNMMETELKPPGPQQA</td><td>GGGGGGGG</td><td>---</td><td>N</td><td>TAAATAGGNQKN</td><td>SPDRVKRP</td><td>PMNAFMVWSR</td><td>55</td></tr><tr><td>sox2-Bos</td><td>MNMMETELKPPGPQQT</td><td>GGGGGGGG</td><td>---</td><td>N</td><td>TAAAGGNQKN</td><td>SPDRVKRP</td><td>PMNAFMVWSR</td><td>56</td></tr><tr><td>sox2-Canis</td><td>MNMMETELKPPGPQQT</td><td>GGGGGGGGGGGG</td><td>---</td><td>N</td><td>TAAAGGNQKN</td><td>SPDRVKRP</td><td>PMNAFMVWSR</td><td>60</td></tr><tr><td>sox2-Capra</td><td>MNMMETELQPGQJHN</td><td>GGGGGGGG</td><td>---</td><td>N</td><td>TAAAGGNQKN</td><td>SPDRVKRP</td><td>PMNAFMVWSR</td><td>56</td></tr><tr><td>sox2-Balaenoptera</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>---</td><td>AAAAGGNQKN</td><td>SPDRGKR</td><td>PMNAFMVWSR</td><td>27</td></tr></table>

***:********* **************

<table><tr><td>sox2-human</td><td>GQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYRPR</td><td>113</td></tr><tr><td>sox2-mus</td><td>GQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYRPR</td><td>115</td></tr><tr><td>sox2-Bos</td><td>GQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYRPR</td><td>116</td></tr><tr><td>sox2-Canis</td><td>GQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYRPR</td><td>120</td></tr><tr><td>sox2-Capra</td><td>GQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYRPR</td><td>116</td></tr><tr><td>sox2-Balaenoptera</td><td>GQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYRPR</td><td>87</td></tr></table>

************************:*************************

<table><tr><td>sox2-human</td><td>RKTKTMKKDKT</td><td>TPGGGLAFGGNSMA</td><td>GGVGVGAGAGAG</td><td>NQRMD</td><td>SAHMNGNS</td><td>SM</td><td>173</td></tr><tr><td>sox2-mus</td><td>RKTKTMKKDKT</td><td>TPGGGLAFGGNSMA</td><td>GGVGVGAGAGAG</td><td>NQRMD</td><td>SAHMNGNS</td><td>SM</td><td>175</td></tr><tr><td>sox2-Bos</td><td>RKTKTMKKDKT</td><td>TPGGGLAFGGNSMA</td><td>GGVGVGAGAGAG</td><td>NQRMD</td><td>SAHMNGNS</td><td>SM</td><td>176</td></tr><tr><td>sox2-Canis</td><td>RKTKTMKKDKT</td><td>TPGGGLAFGGNSMA</td><td>GGVGVGAGAGAG</td><td>NQRMD</td><td>SAHMNGNS</td><td>SM</td><td>180</td></tr><tr><td>sox2-Capra</td><td>RKTKTMKKDKT</td><td>TPGGGLAFGGNIMA</td><td>GGVGVGAGAG</td><td>NQRMD</td><td>SAHMNGNS</td><td>SM</td><td>176</td></tr><tr><td>sox2-Balaenoptera</td><td>RKTKTMKKDKT</td><td>RRAGGLAFGGNSMA</td><td>GGVGVGAGAG</td><td>NQRMD</td><td>SAHMNGNS</td><td>SM</td><td>147</td></tr></table>

*************************, *************************

<table><tr><td>sox2-human</td><td>MQDQGLQFQHEG</td><td>NAHGAAQMQ</td><td>PMHRYDVSA</td><td>QALNMTS</td><td>SQTMMNGSP</td><td>TPSM</td><td>SQGQCTP</td><td>233</td></tr><tr><td>sox2-mus</td><td>MQDQGLQFQHEG</td><td>NAHGAAQMQ</td><td>PMHRYDVSA</td><td>QALNMTS</td><td>SQTMMNGSP</td><td>TPSM</td><td>SQGQCTP</td><td>235</td></tr><tr><td>sox2-Bos</td><td>MQDQGLQFQHEG</td><td>NAHGAAQMQ</td><td>PMHRYDVSA</td><td>QALNMTS</td><td>SQTMMNGSP</td><td>TPSM</td><td>SQGQCTP</td><td>236</td></tr><tr><td>sox2-Canis</td><td>MQDQGLQFQHEG</td><td>NAHGAAQMQ</td><td>PMHRYDVSA</td><td>QALNMTS</td><td>SQTMMNGSP</td><td>TPSM</td><td>SQGQCTP</td><td>240</td></tr><tr><td>sox2-Capra</td><td>MQDQGLQFQHEG</td><td>NAHGAAQMQ</td><td>PMHRYDVSA</td><td>QALNMTS</td><td>SQTMMNGSP</td><td>TPSM</td><td>SQGQCTP</td><td>236</td></tr><tr><td>sox2-Balaenoptera</td><td>MQDQGLQFQHEG</td><td>NAHGAAQMQ</td><td>PMHRYDVSA</td><td>QALNMTS</td><td>SQTMMNGSP</td><td>TPSM</td><td>SQGQCTP</td><td>207</td></tr></table>

*************************, *************************

<table><tr><td>sox2-human</td><td>GMAIGSGMG</td><td>YVK</td><td>EASSSP</td><td>YTSCH</td><td>HARPCQAG</td><td>DRMD</td><td>MYLPGA</td><td>EPEAAP</td><td>SLRH</td><td>293</td></tr><tr><td>sox2-mus</td><td>GMAIGSGMG</td><td>YVK</td><td>EASSSP</td><td>YTSCH</td><td>HARPCQAG</td><td>DRMD</td><td>MYLPGA</td><td>EPEAAP</td><td>SLRH</td><td>295</td></tr><tr><td>sox2-Bos</td><td>GMAIGSGMG</td><td>YVK</td><td>EASSSP</td><td>YTSCH</td><td>HARPCQAG</td><td>DRMD</td><td>MYLPGA</td><td>EPEAAP</td><td>SLRH</td><td>296</td></tr><tr><td>sox2-Canis</td><td>GMAIGSGMG</td><td>YVK</td><td>EASSSP</td><td>YTSCH</td><td>HARPCQAG</td><td>DRMD</td><td>MYLPGA</td><td>EPEAAP</td><td>SLRH</td><td>300</td></tr><tr><td>sox2-Capra</td><td>GMAIGSGMG</td><td>YVK</td><td>EASSSP</td><td>YTSCH</td><td>HARPCQAG</td><td>DRMD</td><td>MYLPGA</td><td>EPEAAP</td><td>SLRH</td><td>296</td></tr><tr><td>sox2-Balaenoptera</td><td>GMAIGSGMG</td><td>YVK</td><td>EASSSP</td><td>YTSCH</td><td>HARPCQAG</td><td>DRMD</td><td>MYLPGA</td><td>EPEAAP</td><td>SLRH</td><td>267</td></tr></table>

*************************, *************************

<table><tr><td>sox2-human</td><td>MSQHWQGP</td><td>PGTA</td><td>NGT</td><td>PLSHM</td><td>317</td></tr><tr><td>sox2-mus</td><td>MAQHWQGP</td><td>PGTA</td><td>NGT</td><td>PLSHM</td><td>319</td></tr><tr><td>sox2-Bos</td><td>MSQHWQGP</td><td>PGTA</td><td>NGT</td><td>PLSHM</td><td>320</td></tr><tr><td>sox2-Canis</td><td>MSQHWQGP</td><td>PGTA</td><td>NGT</td><td>PLSHM</td><td>324</td></tr><tr><td>sox2-Capra</td><td>MSQHWQGP</td><td>PGTA</td><td>NGT</td><td>PLSHM</td><td>320</td></tr><tr><td>sox2-Balaenoptera</td><td>MSQHWQGP</td><td>PGTA</td><td>NGT</td><td>PLSHM</td><td>291</td></tr></table>

*:********* ********* *********

**>sox2-human**

MYNMMETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPMPNAFMVWSRGQRRKMAQENPKMHNSE
ISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYRPRKTKTLMKKDKTYLPGGLLAPGGNSMA
SGVGVGAGLGAGVNRQMDSYAHMGWSNGSYSMMQDQLGYPHPGLNAHGAAQMOPMHRYDVSALQYNSM
TSSQTYMNGSPTYSMSYSQQGTPGMALGSMGVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMISMYLPG
AEVPEPAPSRLHMSQHYQSGPVPGTAINGTLPLSHM
Translates into :

AGENLYFQGMYNNMNETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVK*

After TEV-cleavage (leaving 1 Gly in N-ter):

10 20 30 40
G MYNMMETEL KPPGPQQTSG GGGGNSTAAA AGGNQKNSPD RVK

Sox2-aa115-317_C265A

Synthesized sequence :

cgcgggtgagaacctgtacttccaggcaagaccaaaaccctgatgaagaaagacaagtataccctgccgggtggcctgctggcccggtggc
aacagcatggcgagcggtgtgggcgttgtgctgggcctgggtcggggcgtgaaccagcgtatggacagctacgcgcacatgaacggttggagca
acggcagctacagcatgatgcaggatcaactgggttatccgcaacatccgggtctgaacgcgcatggtgcggcgcagatgcaaccgatgcaccg
ttacgatgttagcgcgctgcagtatacacagcatgaccagcagccaaacctatatgaacggcagcccgacctacagcatgagctatagccaacaa
ggtaccccggttatggcgcctgggtagcatgggcagcgtggttaaaagcgaagcgagcagcagcccgccgggtggttaccagcagccacagcc
gtgccgccggcgaagcggtgacctgcgtgatatgatcagcatgtacctgccgggtgcggaagtgcggaaaccggctgcgccgagccgtctgca
catgagccagcactatcaaagcggtccggttccgggcaccgcgattaacggtaccctgccgctgagccacatgtaaaagctt

Translates into :

AGENLYFQGKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMNGWSNGSYSMMQDQLGYPQHPGLNAHGAAQMQPMHR
YDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPAQAGDLRDMISMYLPGAEVPEPAAPSRLH
MSQHYQSGPVPGTAINGTLPLSHM*

After TEV-cleavage (leaving 1 Gly in N-ter):

120
G KTKTLM

130 140 150 160 170 180
KKDKYTLPGG LLAPGGNSMA SGVGVGAGLG AGVNQRMDSY AHMNGWSNGS YSMMQDQLGY

190 200 210 220 230 240
PQHPGLNAHG AAQMOPMHRY DVSALQYNSM TSSQTYMNGS PTYSMSYSQQ GTPGMALGSM

250 260 270 280 290 300
GSVVKSEASS SPPVVTSSSH SRAPAQAGDL RDMISMYLPG AEVPEPAAPS RLHMSQHYQS

310
GPVPGTAING TLPLSHM

Sox2-aa115-187

Coding sequence - mutation from Sox2-aa115-317_C265A :

Cgcgggtgagaacctgtacttccaggcaagaccaaaaccctgatgaagaaagacaagtataccctgccgggtggcctgctggcccggtggc
aacagcatggcgagcggtgtgggcgttgtgctgggcctgggtgcgggcgtgaaccagcgtatggacagctacgcgcacatgaacggttggagc
aacggcagctacagcatgatgcaggatcaactgggttatccgcaacatccgggtctgaaactaaaagctt

Translates into :

AGENLYFQGKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMNGWSNGSYSMMQDQLGYPQHPGLN*
After TEV-cleavage (*leaving 1 Gly in N-ter*):

120
G KTKTLM

130 140 150 160 170 180
KKDKYTLPGG LLAPGGNSMA SGVGVGAGLG AGVNQRMDSY AHMNGWSNGS YSMMQDQLGY

190
PQHPGLN

## Sox2-aa115-236

Coding sequence - mutation from Sox2-aa115-317_C265A :

ccgCGGGTGagaACCTGTACTTCCAGGGCAAGACCAAAACCCTGATGAAGAAAGACAAGTATACCCTGCCGGGTGGCCTGCTGGCGCCGGGTGG
CAACAGCATGGCGAGCGGTGTGGGCGTTGGTGCGGGCCTGGGTGCGGGCGTGAACCAGCGTATGGACAGCTACGCGCACATGAACGGTTGGAGC
AACGGCAGCTACAGCATGATGCAGGATCAACTGGGTTATCCGCAACATCCGGGTCTGAACGCGCATGGTGCGGCGCAGATGCAACCGATGCACC
GTTACGATGTTAGCGCGCTGCAGTATAACAGCATGACCAGCAGCCAAACCTATATGAACGGCAGCCCGACCTACAGCATGAGCTATAGCCAACA
AGGTACCCCGGGTATGGCGTAAAAAGCTT

Translates into :

AGENLYFQGKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMNGWSNGSYSMMQDQLGYPQHPGLNAHGAAQMQPMHR
YDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMA*

After TEV-cleavage (*leaving 1 Gly in N-ter*):

120
G KTKTLM

130 140 150 160 170 180
KKDKYTLPGG LLAPGGNSMA SGVGVGAGLG AGVNQRMDSY AHMNGWSNGS YSMMQDQLGY

190 200 210 220 230 240
PQHPGLNAHG AAQMOPMHRY DVSALQYNSM TSSQTYMNGS PTYSMSYSQQ GTPGMA

## Sox2-aa115-282_C265A

Coding sequence - mutation from Sox2-aa115-317_C265A :

ccgCGGGTGagaACCTGTACTTCCAGGGCAAGACCAAAACCCTGATGAAGAAAGACAAGTATACCCTGCCGGGTGGCCTGCTGGCGCCGGGTGG
CAACAGCATGGCGAGCGGTGTGGGCGTTGGTGCGGGCCTGGGTGCGGGCGTGAACCAGCGTATGGACAGCTACGCGCACATGAACGGTTGGAGC
AACGGCAGCTACAGCATGATGCAGGATCAACTGGGTTATCCGCAACATCCGGGTCTGAACGCGCATGGTGCGGCGCAGATGCAACCGATGCACC
GTTACGATGTTAGCGCGCTGCAGTATAACAGCATGACCAGCAGCCAAACCTATATGAACGGCAGCCCGACCTACAGCATGAGCTATAGCCAACA
AGGTACCCCGGGTATGGCGCTGGGTAGCATGGGCAGCGTGGTTAAAAGCGAAGCGAGCAGCAGCCCGCCGGTGGTTACCAGCAGCCACAGC
CGTGCGCCGGCGCAAGCGGGTGACCTGCGTGAATATGATCAGCATGTACCTGCCGGGTGCGGAATAAAAGCTT

Translates into :

AGENLYFQGKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMNGWSNGSYSMMQDQLGYPQHPGLNAHGAAQMQPMHR
YDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPAQAGDLRDMISMYLPGAE*
After TEV-cleavage (leaving 1 Gly in N-ter):

120
G KTKTLM

130 140 150 160 170 180
KKDKYTLPGG LLAPGGNSMA SGVGVGAGLG AGVNQRMDSY AHMNGWSNGS YSMMQDQLGY

190 200 210 220 230 240
PQHPGLNAHG AAQMQPMHRY DVSALQYNSM TSSQTYMNGS PTYSMSYSQQ GTPGMALGSM

250 260 270 280
GSVVKSEASS SPPVVTSSSH SRAPAQAGDL RDMISMYLPG AE

AviTag-Sox2-115-317_C265A

Coding sequence - mutation from Sox2-aa115-317_C265A :

Ggtaccggcctgaacgacatttttgaagcgcagaagatcgagtggcacgaggcgcgggcaagaccaagaccctgatgaagaaggacaagtata
ccctgccgggtggcctgctggcgccgggtgcaacagcatggcgagcggtgtgggcgttggtgcgggcctgggtgcgggcgtgaaccagcgtat
ggacagctacgcgcacatgaacggttgagcaacggcagctacagcatgatgcaggatcaactgggttatccgcaacatccgggtctgaacgcg
catggtgcggcgcagatgcaaccgatgcaccgttacgacgttagcgcgctgcagtataacagcatgaccagcagccaaacctatatgaacggta
gccgcacctacagcatgagctatagccaacagggcaccccggttatggcctgggtagcatgggcagcgtggttaaaagcaggcgagcagcag
ccgcgccgtggttaccagcagcagccacagccgtgcgccggcgcaggcgggtgacctgcgtatgatgatcagcatgtacctgccgggtgcggaa
gtgccggaaccggcggcgcgccagccgtctgcacatgagccaacactatcagagcggtccggttccgggcaccgcgattaacggcaccctgccgc
tgagccatatgtaaaagctt

Translates into :

GTGLNDIFEAQKIEWHEGAGKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMNGWSNGSYSMMQDQLGYPQHPGLNA
HGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPAQAGDLRDMISMYLPGAE
VPEPAAPSRLHMSQHYQSGPVPGTAINGTLPLSHM*

Expressed peptide:

MAHHHHHHVGTGLNDIFEAQKIEWHEGAGKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMNGWSNGSYSMMQDQLG
YPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPAQAGDLRDM
ISMYLPGAEVPEPAAPSRLHMSQHYQSGPVPGTAINGTLPLSHM*

120
MAHHHHHHVGTGLNDIFEAQKIEWHEGAGG KTKTLM

130 140 150 160 170 180
KKDKYTLPGG LLAPGGNSMA SGVGVGAGLG AGVNQRMDSY AHMNGWSNGS YSMMQDQLGY

190 200 210 220 230 240
PQHPGLNAHG AAQMQPMHRY DVSALQYNSM TSSQTYMNGS PTYSMSYSQQ GTPGMALGSM

250 260 270 280 290 300
GSVVKSEASS SPPVVTSSSH SRAPAQAGDL RDMISMYLPG AEVPEPAAPS RLHMSQHYQS

310
GPVPGTAING TLPLSHM
## AviTag-Sox2-115-240

### Coding sequence - mutation from Sox2-aa115-317_C265A :

Ggtaccggcctgaacgacatttttgaagcgcagaagatcgagtggcacgaggcgcgggcaagaccaagaccctgatgaagaaggacaagtata ccctgccgggtggcctgctggccgggtggcaacagcatggcgagcgggtgtgggcgttggtgccgggcctgggtcgggcgtgaaccagcgtat ggacagctacgcgcacatgaacggttggagcaacggcagctacagcatgatgcaggatcaactgggttatccgcaacatccgggtctgaacgcg catggtgcggcgcagatgcaaccgatgcaccgttacgacgttagcgcgctgcagtataacagcatgaccagcagccaaacctatatgaacggta gccgcacctacagcatgagctatagccaacagggcaccccggtatggcgctgggtagcatgtaaaagctt

### Translates into :

GTGLNDIFEAQKIEWHEGAGKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMNGWSNGSYSMMQDQLGYPQHPGLNA HGAAQMOPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSM*

### Expressed peptide :

MAHHHHHHVGTGLNDIFEAQKIEWHEGAGKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMNGWSNGSYSMMQDQLG YPQHPGLNAHGAAQMOPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSM*

<table><thead><tr><th colspan="7">120</th></tr><tr><th colspan="7">MAHHHHHHVGTGLNDIFEAQKIEWHEGAGG KTKTLM</th></tr></thead><tbody><tr><td>130</td><td>140</td><td>150</td><td>160</td><td>170</td><td>180</td></tr><tr><td>KKDKYTLPGG</td><td>LLAPGGNSMA</td><td>SGVGVGAGLG</td><td>AGVNQRMDSY</td><td>AHMNGWSNGS</td><td>YSMMQDQLGY</td></tr><tr><td>190</td><td>200</td><td>210</td><td>220</td><td>230</td><td>240</td></tr><tr><td>PQHPGLNAHG</td><td>AAQMOPMHRY</td><td>DVSALQYNSM</td><td>TSSQTYMNGS</td><td>PTYSMSYSQQ</td><td>GTPGMALGSM</td></tr></tbody></table>

## AviTag-Sox2-234-317_C265A

### Coding sequence - mutation from Sox2-aa115-317_C265A :

ggtaccggcctgaacgacatttttgaagcgcagaagatcgagtggcacgaggcgcgggtatggcgctgggtagcatgggcagcgtggttaaaa gcgaggcgagcagcagccgccgggtggttaccagcagcagccacagccgtgcgccggcgcaggcggtgacctgcgtgatatgatcagcatgta ccctgccgggtgcggaagtgccggaaccggcggcgcgagccgtctgcacatgagccaacactatcagagcggtccgggttccgggcaccgcgatt aacggcacctgccgctgagccatatgtaaaaagctt

### Translates into :

GTGLNDIFEAQKIEWHEGAGMALGSMGSVVKSEASSSPPVVTSSSHSRAPAQAGDLRDMISMYLPGAEVPEPAAPSRLHMSQHYQSGPVPGTAI NGTLPLSHM*

### Expressed peptide :

MAHHHHHHVGTGLNDIFEAQKIEWHEGAGMALGSMGSVVKSEASSSPPVVTSSSHSRAPAQAGDLRDMISMYLPGAEVPEPAAPSRLHMSQHYQ SGPVPGTAINGTLPLSHM*

<table><thead><tr><th colspan="7">140</th></tr><tr><th colspan="7">MAHHHHHHVGTGLNDIFEAQKIEWHEGAG MALGSM</th></tr></thead><tbody><tr><td>250</td><td>260</td><td>270</td><td>280</td><td>290</td><td>300</td></tr><tr><td>GSVVKSEASS</td><td>SPPVVTSSSH</td><td>SRAPAQAGDL</td><td>RDMISMYLPG</td><td>AEVPEPAAPS</td><td>RLHMSQHYQS</td></tr><tr><td>310</td><td colspan="5"></td></tr><tr><td>GPVPGTAING</td><td>TLPLSHM</td><td colspan="4"></td></tr></tbody></table>
## 3.4. Nanog

### 3.4.1. Sequence alignment: mammals

S: Serine

P : Proline

SP: phosphorylation motif for MAPKand Cdk

FILVYW : hydrophobic

DE : Asp/Glu

KR : Lys/Arg

T : Thr

XXX: DND-BD in crystal (4RBO)

<table><tr><td>Nanog-human</td><td>MSVDPAPQPQLE-TEASE-KESSPMPVILGPEENMPSLQMESSAEMPHETVSPLFS-SM</td><td>58</td></tr><tr><td>nanog-Mus</td><td>MAJGLPGPHBESFEAAGNSGNASSMPAVEHP-ENYSCHOGATEMLCTEAAASPRPT-GE</td><td>58</td></tr><tr><td>NANOG-Bos</td><td>MAJGPAPOQL-GPEAAGNSRESSPMP----EESWVQTQADTLDTDTSP-PS-SM</td><td>53</td></tr><tr><td>Nanog-Canis</td><td>--------MPA-GFQAPNSDRFSPMPVEVAPGRGNPABPMSSAETBHAETSP-PS-SM</td><td>49</td></tr><tr><td>Nanog-Capra</td><td>MAJDPAPQQL-GPEAAGNSRESSPMP----EESWVQTQADTLDTDTSP-PS-SM</td><td>53</td></tr><tr><td>NANOG-Balaenoptera</td><td>MSVDPAPPOS-R-GPEAAGNSRESSPMPETYGPEENMVSPLQMESSVTHDMETSP-LFGSM</td><td>59</td></tr></table>

<table><tr><td>Nanog-human</td><td>DLLQSPDSSTSPFGK-QOPTSAEK-SVAKKEDKVPVKKQKTRTFSSTQCVINDRQR</td><td>116</td></tr><tr><td>nanog-Mus</td><td>DLPQGSPDSSTBPKQKSSPEADKGPEEEE-NKVLRARKQKMRTVSQACALKDRQK</td><td>117</td></tr><tr><td>NANOG-Bos</td><td>DLLTQSPDSSTBPRKPSPEVEE-TEK-EETVPKKKQKRTVTSQTCVINDRQR</td><td>111</td></tr><tr><td>Nanog-Canis</td><td>DLLTQSPDSSTBPRKLPPTGEE-RTARKEDATQGKKQKMRTVTSQTCVINDRQR</td><td>108</td></tr><tr><td>Nanog-Capra</td><td>DLLTHDNPSDSTBPRKPSPEAE-TEK-EEKVPKKKQKIRTVTSQTCVINDRQR</td><td>111</td></tr><tr><td>NANOG-Balaenoptera</td><td>DLLTQSPDSSTBPRKLLATAADK-TEKKEEKLLKKQKTRTFSSTQCVINDRQR</td><td>118</td></tr></table>

<table><tr><td>Nanog-human</td><td>QKVLSQQMQELSNTILNSMKQKTWEQNORMKSKRQKNNBKNKSGNTQKA-APTVP</td><td>175</td></tr><tr><td>nanog-Mus</td><td>QKVLSQQMQELSNTILNSMKQKTWEQNORMKCKRQKNNQLKTSGNLQKGSAPVEVP</td><td>177</td></tr><tr><td>NANOG-Bos</td><td>QKVLSQQMQELSNTILNSMKQKTWEQNORMKCKRQKNNBRNGNGMPQGP-AMAEP</td><td>170</td></tr><tr><td>Nanog-Canis</td><td>QKVLSQQMQELSNTILNSMKQKTWEQNORMKSKRQKSNBKEENSNTQNSSATTVEA</td><td>168</td></tr><tr><td>Nanog-Capra</td><td>QKVLSQQMQELSNTILNSMKQKTWEQNORMKCKRQKNNPRNNDVQDP-ATAEP</td><td>170</td></tr><tr><td>NANOG-Balaenoptera</td><td>QKVLSQQMQELSNTILNSMKQKTWEQNORMKCKRQKNNPRNNTVQGP-ATTEVP</td><td>177</td></tr></table>

<table><tr><td>Nanog-human</td><td>SZLQHQLGLVNTGPNEMG------------NOTWNNTPLNOTQNQLOGNHANTOT</td><td>225</td></tr><tr><td>nanog-Mus</td><td>HCLPQGYLVNAGGSLEMGSQTWTNPTVSSQTWTNPTVNNQTWTNPTVSSQATAQ</td><td>237</td></tr><tr><td>NANOG-Bos</td><td>GFYSHQHGLVNSPGNEMG------------NOTWNNPTVSNQWSNSQGNHANQA</td><td>219</td></tr><tr><td>Nanog-Canis</td><td>GFYPCRQGYLLNPGNEV---------SSQAWNNPNPSQTQNSNSQSHANQT</td><td>217</td></tr><tr><td>Nanog-Capra</td><td>SFYSHQHGLVNSPRNMEMG------------NOTWNNPTVSNQWSNSQGNHANQA</td><td>219</td></tr><tr><td>NANOG-Balaenoptera</td><td>GFYSHQHGLVNSGNLEMWG------------NOTWNNPTVSNQWSNSQGNHPNWNQT</td><td>226</td></tr></table>

<table><tr><td>Nanog-human</td><td>WQTQWNQAISNP-TNNGEEQDQMQUQENSPADLEAAEAEAGEGNVIQQTTF</td><td>284</td></tr><tr><td>nanog-Mus</td><td>W---NGQPNAAPHNNGEDFQPQVQQQNFSAQLEEVNLEATRESH-AHF</td><td>285</td></tr><tr><td>NANOG-Bos</td><td>WFQAANQQPNQNN-NNNEEFPQGTQQQNSPVCDLEATGTAGENVIQQTVKF</td><td>278</td></tr><tr><td>Nanog-Canis</td><td>WFQAANQQNNDE-LHNEEESQPFTQQNS-MGDELETAGESHGVQQSTKVF</td><td>275</td></tr><tr><td>Nanog-Capra</td><td>WFQAANQQNNQCNNNEEFPQGTQQQNSPVCDLEATGTAGENVIQQAVKF</td><td>278</td></tr><tr><td>NANOG-Balaenoptera</td><td>WFQAANQQTSNNO-NNNEEFPQQTQQQNSPVCDLEATLETAGESNIIQQTAKV</td><td>285</td></tr></table>

<table><tr><td>Nanog-human</td><td>STP-QTMDLFINNMMNQPEDV</td><td>305</td></tr><tr><td>nanog-Mus</td><td>STP-QALELFINVAVTE-FGE</td><td>305</td></tr><tr><td>NANOG-Bos</td><td>NSQQQTDLFENMFINQPEDV</td><td>300</td></tr><tr><td>Nanog-Canis</td><td>STP-QMDFFENJXNQPEDV</td><td>296</td></tr><tr><td>Nanog-Capra</td><td>SSQQQTDLFENMFINQPEDV</td><td>307</td></tr><tr><td>NANOG-Balaenoptera</td><td>NSQQQMDLFINNKNQPEDV</td><td>307</td></tr></table>

>Nanog-human

MSVDPACPQSLPCFEASDCKESSPMPVICGPEENYPSLQMSSAEMPHETVSPLPSSMDLLIQDSPDSST
SPKGKQPTSAEKSVAKKEDKVPVKKQKTRTVFSSSTQLCVLNDFRQKQYLSLQQMQELSNIILNLSYKQVK
TWFQNORMKSKRWQKNNWPKNSGNVQTQKASAAPTYPSLYSSYHQGCLVNPPTGNLPMWSNQTWNNSTWSNQT
QNIQSWNSHWSNTQTWCTQSWNNQAWNSPFYNYNGEESLQSCMQFQPNSPASDLEAAEAEGLNVIQQT
TRYFSTPQTMDLFLNYSMNMQPEDV
### 3.4.2. Sequence alignment: vertebrates

### 3.4.3. Disorder prediction

<figure><img src="image_26.png" /></figure>

### 3.4.4. Coding DNA sequences, produced protein constructs

#### Nanog-aa1-85

Name Genscript : TEV-NanogNter

Coding sequence (mutated from Nanog-aa1-85-Tev):

Caattggtgaaaatctgtacttccagggcatgtccgtcgatccggcgtgtccgcagagcctgccgtgctttgaagcgagcgactgtaaagaatc
gagcccgatgccgtcatttgccggccggaagaaaaactatccgtctctgcagatgagctctgcagaaatgccgcatacggaaaccgtgagcccg
ctgccgagttccatggatctgctgatccaggatagtccggactcatcgacgtcccccgaaaggtaaacaaccgaccagcgcggaaaaatctgtgg
cctaaaagctt

Translates into :

IGENLYFQGMSVDPACPQSLPCFEASDCKESSPMPVICGPEENYPSLQMSSAEMPHTETVSPLPSSMDLLIQDSPDSSTSPKGKQPTSAEKSV
*

Before TEV-cleavage:

GST-His-Tev_NanogCter_aa1-85

MSPILGYWKIKGLVQPTRLLEYLEEKYEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISM
LEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVLYMDPMCLDAFPKLVCFKKRIEAIPQ
IDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGSTSGGHHHHHHSAGLVPRGSTAIGENLYFQGMSVDPACPQSLPCFEASDCKESSPMPVIC
GPEENYPSLQMSSAEMPHTETVSPLPSSMDLLIQDSPDSSTSPKGKQPTSAEKSVA*

Number of amino acids: 338

Molecular weight: 38016.43

Theoretical pI: 5.54

Total number of negatively charged residues (Asp + Glu): 47

Total number of positively charged residues (Arg + Lys): 35

Ext. coefficient 46340

Abs 0.1% (=1 g/l) 1.219, assuming all pairs of Cys residues form cystines

Ext. coefficient 45840

Abs 0.1% (=1 g/l) 1.206, assuming all Cys residues are reduced
After Tev-cleavage (*leaving 1 Gly in N-ter*):

GMSVDPACPQSLPCFEASDCKESSPMPVICGPEENYPSLQMSSAEMPHTETVSPLPSSMDLLIQDSPDSSTSPKGKQPTSAEKSAVA

10 20 30 40 50 60
G MSVDPACPQS LPCFEASDCK ESSPMPVICG PEENYPSLQM SSAEMPHTET VSPLPSSMDL

70 80
LIQDSPDSST SPKGKQPTSA EKSVA

## Nanog-aa154-305-Tev

Synthesized sequence :

Cgcgggtgagaacctgtacttccaaggcatggcggtcacctggcgagcgattttgcgtttagcccggccgggtggtggtggtgacggtccgggtggccggaaaccgggtgggtggatccgcgtacctggctgagcttccaaggtccggccgggtggccgggtattggtccgggtgtggcccggttagcgaggtttgggggtattccgccgtggccggccgtacgaattttgcggtggcatggcgtattgcggtccgcaagtgggcgttggtctggttccgcaaggtggcctggaaaccagccagccggagggtgaagcgggcgtgggtggttgagagcaacagcgatggtgcgagcccggaaaccgtgcac cgtgacccgggtgcggttaagctggagaaggaaaaactggagcagaacccggaggaaagccaagatatcaaggcgtgcagaaagaaaaacctg tacctttcaaggtggcgcgggtggcgcgggtggccagtataactgattctgaacggcaagacctgaaaggtgaaaccaccaccgaagcggtgg atgcggcgaccgctgagaaggttttcaaaacagtacgcgaacgacaacggcgtggatggcgagtggacctatgacgatgcgaccaagacctttac cgttaccgaaggtggctaaaagctt

Translates into :

ENLYFQGKQNNWPKNSGNVQTQKASAPTYPSLYSSYHQGCLVNPTGNLPMWSNQTWNNSTWSNQTQNIQSWSNHSWNTQTWCTQSWNNQAWNSPFYNCGEESLQSCMQFQPNSPASDLEAALEAAGEGLNVIIQQTTRYFSTPQTMDLFLNYSMNMQPEDVENLYFQGGAGGAGGQYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGG*

Before TEV-cleavage:

GST-His-Tev_NanogCter_aa154-305_Tev-GB1 from pET41a+

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPPYYIDGDVLTQSMSAIIRYIADKHNMLGGCPKERAEISM
LEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVLYMDPMCLDAFPKLVCFKKRIEAIPQ
IDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGSTSGGHHHHHHSAGLPRGSTAIGMKETAENLYFQGKQNNWPKNSGVTQKASAPTYPSL
YSSYHQGCLVNPTGNLPMWSNQTWNNSTWSNQTQNIQSWSNHSWNTQTWCTQSWNNQAWNSPFYNYCGEESLQSCMQFQPNSPASDLEAALEAAG
EGLNVIIQQTTRYFSTPQTMDLFLNYSMNMQPEDVENLYFQGGAGGAGGQYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD
ATKTFTVTEGG

After Tev-cleavage (*leaving 1 Gly in N-ter and ENLYFQ in C-ter):

GKQNNWPKNSGNVQTQKASAPTYPSLYSSYHQGCLVNPTGNLPMWSNQTWNNSTWSNQTQNIQSWSNHSWNTQTWCTQSWNNQAWNSPFYNYCGEE
SLQSCMQFQPNSPASDLEAALEAAGEGLNVIIQQTTRYFSTPQTMDLFLNYSMNMQPEDVENLYFQ

160 170 180
G QKNNWPK NSNGVTQKAS APTYPSLYSS

190 200 210 220 230 240
YHQGCLVNPT GNLPMWSNQT WNNSTWSNQT QNIQSWSNHS WNTQTWCTQS WNNQAWNSPF

250 260 270 280 290 300
YNCGEESLQS CMQFQPNSPA SDLEAALEAA GEGLNVIIQQT TRYFSTPQTM DLFLNYSMNM

QPEDV ENLYFQ
## Nanog-aa154-305

### Coding sequence (mutated from Nanog-aa154-305-Tev):

ccgCGGGTgaaaACCTGTACTTCCAGGGTCAAAAGAACAACTGGCCGAAAAACAGCAACGGTGTGACCCAAAAAGGCGAGCGCGCCGACCTATCCGAGCCTGTACAGCAGCTATCACCAGGGTTGCCTGGTTAACCCGACCGGCAACCTGCCGATGTGGAGCAACCAAAACCTGGAACAACAGCACCTGGAGCAACCAGACCCAAAACATCCAGAGCTGGAGCAACCACAGCTGGAACACCAGACCTGGTCACCCAAAGCTGGAACAACCAGGCGTGGAAACAGCCCGTTCTACAACTGCGCGAGGAAAGCCTGCAAAGCTGATCGAGTTTCAACCGAACAGCCCGCGAGCGACCTGGAGCGCGCTGGAAAGCGGCGGGTGAAGGCCTGAACGTGATCCAGCAAACCACCGTTACTTCAGCACCCCGCAAAACCATGGACCTGTTTCTGAACTATAGCATGAACATGCAGCCGGAGGATGTTTAAAAAGCTT

### Translates into:

ENLYFQGQKNNWPKNSNGVTQKASAPTYPSLYSSYHQGCLVNPTGNLPMWSNQTWNNSTWSNQTQNIQSWSNHSWNTQTWCTQSWNNQAWNSPFYNCGEEESLQSCMQFQPNSPASDLEAALEAAGEGLNVIIQQTTRYFSTPQTMDLFLNYSMNMQPEDVENLYFQGGAGGAGGQYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGG*

### After Tev-cleavage (leaving 1 Gly in N-ter):

GQKNNWPKNSNGVTQKASAPTYPSLYSSYHQGCLVNPTGNLPMWSNQTWNNSTWSNQTQNIQSWSNHSWNTQTWCTQSWNNQAWNSPFYNYCGEE SLQSCMQFQPNSPASDLEAALEAAGEGLNVIIQQTTRYFSTPQTMDLFLNYSMNMQPEDV*

<table><thead><tr><th></th><th>160</th><th>170</th><th>180</th></tr></thead><tbody><tr><td>G QKNNWPK</td><td>NSNGVTQKAS</td><td>APTYPSLYSS</td></tr><tr><td>190</td><td>200</td><td>210</td></tr><tr><td>YHQGCLVNPT</td><td>GNLPMWSNQT</td><td>WNNSTWSNQT</td></tr><tr><td>250</td><td>260</td><td>270</td></tr><tr><td>YNCGEESLQS</td><td>CMQFQPNSPA</td><td>SDLEAALEAA</td></tr></tbody></table><table><thead><tr><th></th><th>220</th><th>230</th><th>240</th></tr></thead><tbody><tr><td></td><td>WNTQTWCTQS</td><td></td><td>WNNQAWNSPF</td></tr><tr><td></td><td>280</td><td>290</td><td>300</td></tr><tr><td></td><td>GEGLNVIIQQT</td><td>TRYFSTPQTM</td><td>DLFLNYSMNM</td></tr></tbody></table>

QPEDV

## Nanog-aa154-215-Tev

### Coding sequence (mutated from Nanog-aa154-305-Tev):

CcgCGGGTgaaaACCTGTACTTCCAGGGTCAAAAGAACAACTGGCCGAAAAACAGCAACGGTGTGACCCAAAAAGGCGAGCGCGCCGACCTATCCGAGCCTGTACAGCAGCTATCACCAGGGTTGCCTGGTTAACCCGACCGGCAACCTGCCGATGTGGAGCAACCAAAACCTGGAACAACAGCACCTGGAGCAACCAGACCCAAAACATCCAGAGCGAAAAACCTGTACTTTCAAGGTGGCGCGGGTGGCGCGGGTGGCCAGTATAAGCTGATTCTGAACGGCAAGACCTGAAAGGCGAAACCACCACGGAGCGGTGGATCGGCGACCGCTGAGAAGGTTTTCAAACAGTACGCGAACGACAACGGTGTGGATGGCGATGGACCTATGACGATCGACCAAAACCTTTACCGTTACCGAGGGTGGCTAAAAGCTT

### Translates into:

AGENLYFQGQKNNWPKNSNGVTQKASAPTYPSLYSSYHQGCLVNPTGNLPMWSNQTWNNSTWSNQTQNIQSENLYFQGGAGGAGGQYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGG*

### After Tev-cleavage (leaving 1 Gly in N-ter and ENLYFQ in C-ter):

GQKNNWPKNSNGVTQKASAPTYPSLYSSYHQGCLVNPTGNLPMWSNQTWNNSTWSNQTQNIQSENLYFQ

<table><thead><tr><th></th><th>160</th><th>170</th><th>180</th></tr></thead><tbody><tr><td>G QKNNWPK</td><td>NSNGVTQKAS</td><td>APTYPSLYSS</td></tr><tr><td>190</td><td>200</td><td>210</td></tr><tr><td>YHQGCLVNPT</td><td>GNLPMWSNQT</td><td>WNNSTWSNQT</td></tr></tbody></table><table><thead><tr><th></th><th>QNIQS</th><th>ENLYFQ</th></tr></thead></table>
## Nanog-aa154-272-Tev

### Coding sequence (mutated from Nanog-aa154-305-Tev):

Ccgcggtgaaaacctgtacttccaggttcaaaagaacaaactggccgaaaaacagcaacggtgtgacccaaaaaggcgagcgcgccgacctatcc
gagcctgtacagcagctatcaccaggttgccctggttaacccgaccggcaacctgccgatgtggagcaaccaaacctggaacaacagcacctgg
agcaaccagacccaaaaacatccagagctggagcaaccacagctggaacaccagacctggtgcacccaaagctggaacaaccaggctggaaca
gccgttctacaaactgcggcgaggaaagcctgcaaagctgcatgcagtttcaaccgaacagccggcgagcgacctggaggcggcgctggaagc
ggcgggtgaagaaaaacctgtactttcaaggtggcggcggtggcggcggtggccagtataagctgattctgaacggcaagaccctgaaaggcgaa
accaccaccgaggcggtggatgcggcaccgctgagaaggttttcaaacagtacgcgaacgacaacggtgtggatggcgaatggacctatgacg
atgcaccaaaacctttaccgttaccgaggtggctaaaagctt

### Translates into:

AGENLYFQGKNNWPKNNSNGVTQKASAPTYPSLYSSYHQGCLVNPTGNLPMWSNQTWNNSTWSNQTQNIQSWSNHSWNTQTWCTQSWNNQAWNS
PFYNCGEESLQSCMQFQPNSPASDLEAALEAAAGEENLYFQGGAGGAGGQYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDD
ATKTFTVTEGG*

### After Tev-cleavage (leaving 1 Gly in N-ter and ENLYFQ in C-ter):

GKNNWPKNNSNGVTQKASAPTYPSLYSSYHQGCLVNPTGNLPMWSNQTWNNSTWSNQTQNIQSWSNHSWNTQTWCTQSWNNQAWNSPFYNYCGEE
SLQSCMQFQPNSPASDLEAALEAAAGEENLYFQ

160 170 180
G QKNNWPK NSNGVTQKAS APTYPSLYSS
190 200 210 220 230 240
YHQGCLVNPT GNLPMWSNQT WNNSTWSNQT QNIQSWSNHS WNTQTWCTQS WNNQAWNSPF
250 260 270
YNCGEESLQS CMQFQPNSPA SDLEAALEAA GE ENLYFQG

## Nanog-aa154-305_4C4A

### Coding sequence (mutated from Nanog-aa154-305-Tev):

Ccgcggtgagaatctgtatttccaaggcaaaacaaactggccgaagaacagcaatggtgtgacccaaaaagcgagcgcgccgacctatccgag
cctgtacagcagctatcaccaaggtgcgctggtgaacccgaccggtaacctgccgatgtggagcaaccaaacctggaacaacagcacctggagc
aaccagacccaaaaacatccagagctggagcaaccacagctggaacaccagacctgggcgacccaaagctggaacaaccaggcgtggaacagcc
cgttctacaacgcgggcgaggaaagcctgcagagcgcgatgcagtttcaaccgaacagccggcgagcgacctggaggcggcgctggaagcggc
gggtgaaggcctgaacgttattcagcaaccaccgcgtatttccagcaccggcaaccacatggacctgtttctgaattatagcatgaatatgcag
ccggaggatgtgtaaaagctt

### Translates into:

AGENLYFQGKNNWPKNNSNGVTQKASAPTYPSLYSSYHQGALVNPTGNLPMWSNQTWNNSTWSNQTQNIQSWSNHSWNTQTWATQSWNNQAWNSPF
FYNAGEESLQSAMQFQPNSPASDLEAALEAAAGEGLNVIQQTTRYFSTPQTMDLFLNYSMNMQPEDV*
## After Tev-cleavage (leaving 1 Gly in N-ter)

GKNNWPKNSNGVTQKASAPTYPSLYSSYHQGALVNPTGNLPMWSNQTWNNSTWSNQTQNIQSWSNHSWNTQTWATQSWNNQAWNSPFYNAGEES
LQSAMQFQPNSPASDLEAALEAAAGEGLNVIQQTTRYFSTPQTMDLFLNYSMNMQPEDV

160 170 180
G QKNNWPK NSNGVTQKAS APTYPSLYSS
190 200 210 220 230 240
YHQGALVNPT GNLPMWSNQT WNNSTWSNQT QNIQSWSNHS WNTQTWATQS WNNQAWNSPF
250 260 270 280 290 300
YNAGEESLQS AMQFQPNSPA SDLEAALEAA GEGLNVIQQT TRYFSTPQTM DLFLNYSMNM

QPEDV

## Nanog-aa154-272_4C4A

### Coding sequence (mutated from Nanog-aa154-305_4C4A):

Ccgcggtgagaatctgtatttccaaggcaaaaaacaactggccgaagaacagcaatggtgtgacccaaaaagcgagcgcgccgacctatccgag
cctgtacagcagctatcaccaaggtgcgctggtgaacccgaccggtaacctgccgatgtggagcaaccaaacctggaacaacagcacctggagc
aaccagacccaaaaacatccagagctggagcaaccacagctggaacacccagacctgggcgacccaaagctggaacaaccaggcgtggaacagcc
cgttctacaacgcgggcgaggaaagcctgcagagcgcgatgcagtttcaaccgaacagccggcgagcgacctggaggcggcgctggaagcggc
gggtgaataaaagctt

### Translates into:

AGENLYFQGKNNWPKNSNGVTQKASAPTYPSLYSSYHQGALVNPTGNLPMWSNQTWNNSTWSNQTQNIQSWSNHSWNTQTWATQSWNNQAWNSPF
FYNAGEESLQSAMQFQPNSPASDLEAALEAAAGE

### After Tev-cleavage (leaving 1 Gly in N-ter):

GKNNWPKNSNGVTQKASAPTYPSLYSSYHQGALVNPTGNLPMWSNQTWNNSTWSNQTQNIQSWSNHSWNTQTWATQSWNNQAWNSPFYNAGEES
LQSAMQFQPNSPASDLEAALEAAAGE

160 170 180
G QKNNWPK NSNGVTQKAS APTYPSLYSS
190 200 210 220 230 240
YHQGALVNPT GNLPMWSNQT WNNSTWSNQT QNIQSWSNHS WNTQTWATQS WNNQAWNSPF
250 260 270
YNAGEESLQS AMQFQPNSPA SDLEAALEAA GE
## 3.5. Esrrb

### 3.5.1. Sequence alignment: mammals

S: Serine

P : Proline

SP : phosphorylation motif for MAPKand Cdk

FILVYW : hydrophobic

DE : Asp/Glu

KR : Lys/Arg

T : Thr

P : Cys

<table><tr><td>human</td><td>-----------------------</td><td>M</td><td>S</td><td>E</td><td>D</td><td>R</td><td>H</td><td>G</td><td>S</td><td>S</td><td>G</td><td>G</td><td>I</td><td>K</td><td>T</td><td>E</td><td>P</td><td>S</td><td>S</td><td>S</td><td>G</td><td>D</td><td>A</td><td>H</td><td>H</td><td>S</td><td>P</td><td>S</td><td>G</td><td>S</td></tr><tr><td>mouse</td><td>-----------------------</td><td>M</td><td>S</td><td>E</td><td>D</td><td>R</td><td>H</td><td>G</td><td>S</td><td>S</td><td>G</td><td>G</td><td>I</td><td>K</td><td>T</td><td>E</td><td>P</td><td>S</td><td>S</td><td>S</td><td>S</td><td>G</td><td>D</td><td>A</td><td>H</td><td>H</td><td>S</td><td>P</td><td>S</td><td>G</td><td>S</td></tr><tr><td>capra</td><td>-----------------------</td><td>M</td><td>D</td><td>V</td><td>S</td><td>E</td><td>L</td><td>C</td><td>V</td><td>P</td><td>D</td><td>P</td><td>L</td><td>G</td><td>Y</td><td>H</td><td>N</td><td>Q</td><td>L</td><td>L</td><td>N</td><td>R</td><td>M</td><td>A</td><td>D</td><td>R</td><td>H</td><td>S</td><td>S</td><td>S</td><td>G</td><td>G</td><td>I</td><td>K</td><td>T</td><td>E</td><td>P</td><td>S</td><td>S</td><td>S</td></tr><tr><td>bos</td><td>-----------------------</td><td>M</td><td>D</td><td>V</td><td>S</td><td>E</td><td>L</td><td>C</td><td>V</td><td>P</td><td>D</td><td>P</td><td>L</td><td>G</td><td>Y</td><td>H</td><td>N</td><td>Q</td><td>L</td><td>L</td><td>N</td><td>R</td><td>M</td><td>A</td><td>D</td><td>R</td><td>H</td><td>S</td><td>S</td><td>S</td><td>G</td><td>G</td><td>I</td><td>K</td><td>T</td><td>E</td><td>P</td><td>S</td><td>S</td><td>S</td></tr><tr><td>balaenoptera</td><td>-----------------------</td><td>M</td><td>D</td><td>V</td><td>S</td><td>E</td><td>L</td><td>C</td><td>I</td><td>P</td><td>D</td><td>P</td><td>L</td><td>G</td><td>Y</td><td>H</td><td>N</td><td>Q</td><td>L</td><td>L</td><td>N</td><td>R</td><td>M</td><td>A</td><td>D</td><td>R</td><td>H</td><td>V</td><td>S</td><td>S</td><td>S</td><td>G</td><td>G</td><td>I</td><td>K</td><td>T</td><td>E</td><td>P</td><td>S</td><td>S</td><td>S</td></tr></table>

********:***, . : ***************************, . : ***************************

<table><tr><td>human</td><td>D</td><td>A</td><td>G</td><td>G</td><td>G</td><td>L</td><td>A</td><td>S</td><td>T</td><td>H</td><td>A</td><td>N</td><td>G</td><td>L</td><td>S</td><td>P</td><td>P</td><td>M</td><td>A</td><td>G</td><td>A</td><td>G</td><td>L</td><td>G</td><td>N</td><td>D</td><td>R</td><td>K</td><td>G</td><td>Y</td><td>E</td><td>D</td><td>T</td><td>A</td><td>G</td><td>I</td><td>M</td><td>E</td><td>D</td><td>A</td><td>K</td><td>K</td><td>E</td><td>Y</td><td>M</td><td>L</td><td>A</td><td>P</td></tr><tr><td>mouse</td><td>D</td><td>A</td><td>G</td><td>G</td><td>G</td><td>L</td><td>A</td><td>S</td><td>T</td><td>H</td><td>A</td><td>N</td><td>G</td><td>L</td><td>S</td><td>P</td><td>P</td><td>M</td><td>A</td><td>G</td><td>A</td><td>G</td><td>L</td><td>G</td><td>N</td><td>D</td><td>R</td><td>K</td><td>G</td><td>Y</td><td>E</td><td>D</td><td>T</td><td>A</td><td>G</td><td>I</td><td>M</td><td>E</td><td>D</td><td>A</td><td>K</td><td>K</td><td>E</td><td>Y</td><td>M</td><td>L</td><td>A</td><td>P</td></tr><tr><td>capra</td><td>D</td><td>A</td><td>G</td><td>G</td><td>G</td><td>L</td><td>A</td><td>G</td><td>A</td><td>H</td><td>A</td><td>N</td><td>G</td><td>L</td><td>S</td><td>P</td><td>P</td><td>M</td><td>A</td><td>G</td><td>A</td><td>G</td><td>L</td><td>G</td><td>T</td><td>R</td><td>K</td><td>G</td><td>Y</td><td>E</td><td>D</td><td>A</td><td>G</td><td>G</td><td>L</td><td>M</td><td>E</td><td>D</td><td>A</td><td>K</td><td>K</td><td>E</td><td>Y</td><td>M</td><td>L</td><td>A</td><td>P</td></tr><tr><td>bos</td><td>D</td><td>A</td><td>G</td><td>G</td><td>G</td><td>L</td><td>A</td><td>G</td><td>A</td><td>H</td><td>A</td><td>N</td><td>G</td><td>L</td><td>S</td><td>P</td><td>P</td><td>M</td><td>A</td><td>G</td><td>A</td><td>G</td><td>L</td><td>G</td><td>T</td><td>R</td><td>K</td><td>G</td><td>Y</td><td>E</td><td>D</td><td>A</td><td>G</td><td>G</td><td>L</td><td>M</td><td>E</td><td>D</td><td>A</td><td>K</td><td>K</td><td>E</td><td>Y</td><td>M</td><td>L</td><td>A</td><td>P</td></tr><tr><td>balaenoptera</td><td>D</td><td>A</td><td>G</td><td>G</td><td>G</td><td>L</td><td>A</td><td>G</td><td>A</td><td>H</td><td>A</td><td>N</td><td>G</td><td>L</td><td>S</td><td>P</td><td>P</td><td>M</td><td>A</td><td>G</td><td>A</td><td>G</td><td>L</td><td>G</td><td>T</td><td>R</td><td>K</td><td>G</td><td>Y</td><td>E</td><td>D</td><td>A</td><td>G</td><td>G</td><td>L</td><td>M</td><td>E</td><td>D</td><td>A</td><td>K</td><td>K</td><td>E</td><td>Y</td><td>M</td><td>L</td><td>A</td><td>P</td></tr></table>

********:***, . : ***************************, . : ***************************

<table><tr><td>human</td><td>KRL</td></tr><tr><td>mouse</td><td>KRL</td></tr><tr><td>capra</td><td>KRL</td></tr><tr><td>bos</td><td>KRL</td></tr><tr><td>balaenoptera</td><td>KRL</td></tr></table>

***

>sp|095718|ERR2_HUMAN Steroid hormone receptor ERR2 OS=Homo sapiens GN=ESRRB PE=1 SV=2

MSSDDRHLGSSCGSFIKTEPSSPSSGIDALSHHSPSGSSDASGGFGLALGTHANGLDSPP

MFAGAGLGGTPCRKSYEDCASGIMEDSAIKCEYMLNAIPKRLCLVCGDIASGYHYGVASC

EACKAFFKRTIQGNIEYSCPATNECEITKRRRKSCQACRFMKCLKVGMLEKEGVRLDRVRG

GRQKYKRRLDSESSPYLSLQISPPAKKPLTKIVSYLLVAEPDKLYAMPPPGMEGDIKAL

TTLCDLADRELVVIIGWAKHIPGFSSLSLGDQMSLLQSAWMEILILGIVRSLPYDDKLV

YAEDYIMDEEHSRLAGLLELYRAILQLVRRYKLLKVEKEEFVTLKALALANSDSMYIEDL

EAVQKLQDLLHEALQDYELSQRHEEPWRTGKLLLLTLPLLRQTAAKAVQHFYSVKLQGKVP

MHKLFLEMLEAKVGQEQLRGSPKDERMSSHDGKCPFQSAAFTSRDQNSPGIPNPRPSSP TPLNERGRQISPSTRTPGGQGKHLWLTM
3.5.2. Sequence alignment: vertebrates

: cysteines in N-ter

XX: DNA-BD ordered in NMR structure 1L01 (construct: human aa96-194_C163A)

XX: folded: aa235-432 in Ligand-Binding Domain crystal structures 6LIT, 6LN4 (construct: human aa204-433_Y215H, mutation for solubility/stability)

<table><tbody><tr><td>mouse</td><td>MSSERHLGSS</td><td>GSFIKTEPSSPSSGIDALSHSPSGSS</td><td>39</td></tr><tr><td>human</td><td>MSSDDRHLGSS</td><td>GSFIKTEPSSPSSGIDALSHSPSGSS</td><td>39</td></tr><tr><td>goat</td><td>MDVSELCPDPLGYHNQLLNMSADDRHLSS</td><td>GSFIKTEPSSPSSGIDALSHSPSGSS</td><td>60</td></tr><tr><td>whale</td><td>MDVSELCPDPLGYHNQLLNMSADDRHLVSS</td><td>GSFIKTEPSSPSSGIDALSHSPSGSS</td><td>60</td></tr><tr><td>chicken</td><td>MDISELCISDPLGYHNQLLGRMATEERHLSS</td><td>GSFIKTEPSSPSSGIDAISHSPSGSS</td><td>60</td></tr><tr><td>fish</td><td>MAADERHLPSS</td><td>GSYIKTEPSSPSSVIDTVSHSPSGNS</td><td>39</td></tr><tr><td>mouse</td><td>DASGGFGIALSTHANGLDSSPPMAGAGLGGNP</td><td>RKSYEDTSGIMEDSAIKTEYMLNAIP</td><td>99</td></tr><tr><td>human</td><td>DASGGFGALGTHANGLDSSPPMAGAGLGGTP</td><td>RKSYEDTSGIMEDSAIKTEYMLNAIP</td><td>99</td></tr><tr><td>goat</td><td>DASGGFGALGAHANGLDSSPPMAGAGLGGTP</td><td>RKGYEDAGGLMEDSAIKTEYMLNAIP</td><td>120</td></tr><tr><td>whale</td><td>DASGGFGALGAHANGLDSSPPMAGAGLGGTP</td><td>RKGYEDAGGLMEDSAIKTEYMLNAIP</td><td>120</td></tr><tr><td>chicken</td><td>DASGGYIAMGGHPNGLDSSPPMFNGTGIGGGS</td><td>RKRYDDASAIMEDSPTEYMLNAIP</td><td>120</td></tr><tr><td>fish</td><td>DASGGYVSTMNSHNSLDPSPMFTPSGLGAGT</td><td>RKRYDDASTIMEDSSRTEYMLNSLP</td><td>99</td></tr><tr><td>mouse</td><td>KRLCLVCGDIASGYHYGVASCEACKAFKFRTIQGNIEYNCPATNECEITKRRRKSCQACR</td><td></td><td>159</td></tr><tr><td>human</td><td>KRLCLVCGDIASGYHYGVASCEACKAFKFRTIQGNIEYSCPATNECEITKRRRKSCQACR</td><td></td><td>159</td></tr><tr><td>goat</td><td>KRLCLVCGDIASGYHYGVASCEACKAFKFRTIQGNIEYSCPATNECEITKRRRKSCQACR</td><td></td><td>180</td></tr><tr><td>whale</td><td>KRLCLVCGDIASGYHYGVASCEACKAFKFRTIQGNIEYSCPATNECEITKRRRKSCQACR</td><td></td><td>180</td></tr><tr><td>chicken</td><td>KRLCLVCGDIASGYHYGVASCEACKAFKFRTIQGNIEYSCPATNECEITKRRRKSCQACR</td><td></td><td>180</td></tr><tr><td>fish</td><td>KRLCLVCGDIASGYHYGVASCEACKAFKFRTIQGNIEYSCPATNECEITKRRRKSCQACR</td><td></td><td>159</td></tr><tr><td>mouse</td><td>FMKCLKVGMKEGVRLDRVRGGQRKYKRRLDSENSPYLNLISPPAKKPLTKIVSNLLGV</td><td></td><td>219</td></tr><tr><td>human</td><td>FMKCLKVGMKEGVRLDRVRGGQRKYKRRLDSESSPYLSLQISPPAKKPLTKIVSYLLVA</td><td></td><td>219</td></tr><tr><td>goat</td><td>FMKCLKVGMKEGVRLDRVRGGQRKYKRRLDSESSPYLSLQISPPAKKPLTKIVSYLLVA</td><td></td><td>240</td></tr><tr><td>whale</td><td>FMKCLKVGMKEGVRLDRVRGGQRKYKRRLDSESSPYLSLQISPPAKKPLTKIVSYLLVA</td><td></td><td>240</td></tr><tr><td>chicken</td><td>FMKCLKVGMKEGVRLDRVRGGQRKYKRRLDSESSTYLSLQIPPPAKKPLTKIVSHLLVA</td><td></td><td>240</td></tr><tr><td>fish</td><td>FMKCLKVGMKEGVRLDRVRGGQRKYKRRLDSENNPYLGTLPPPTKKPLTKIVSHLLVA</td><td></td><td>219</td></tr><tr><td>mouse</td><td>EQDKLYAMPPNDIPEGDIKALTTLCELADRELVFLINWAKHPGFPSLTLGDQMSLLQSA</td><td></td><td>279</td></tr><tr><td>human</td><td>EPDKLYAMPPGMPEGDIKALTTLCDLADRELVVIIGWAKHPIGFSSLSLGDQMSLLQSA</td><td></td><td>279</td></tr><tr><td>goat</td><td>EPDKLYAMPPGMPEGDIKALTTLCDLADRELVVIIGWAKHPGFSNLSLGDQMSLLQSA</td><td></td><td>300</td></tr><tr><td>whale</td><td>EPDKLYAMPPGMPEGDIKALTTLCDLADRELVVIIGWAKHPGFSNLSLGDQMSLLQSA</td><td></td><td>300</td></tr><tr><td>chicken</td><td>EPEKIYAMPDPMTESDIKALTTLCDLADRELVVIIGWAKHPGFSNLSLGDQMSLLQSA</td><td></td><td>300</td></tr><tr><td>fish</td><td>EPEKIYAMPDPMTESDIKALTTLCDLADRELVVIIGWAKHPGFSTLSLGDQMSLLQSA</td><td></td><td>279</td></tr><tr><td>mouse</td><td>WMEILILGIVYRSLPYDDKLAYEADYIMDEEHSRLVGLLLDYRAILQLVRRYYKKLKVEKE</td><td></td><td>339</td></tr><tr><td>human</td><td>WMEILILGIVYRSLPYDDKLVYAEDYIMDEEHSRLAGLLELYRAILQLVRRYYKKLKVEKE</td><td></td><td>339</td></tr><tr><td>goat</td><td>WMEILILGIVYRSLPYDDKLVYAEDYIMDEEHSRLAGLLELYRAILQLVRRYYKKLKVEKE</td><td></td><td>360</td></tr><tr><td>whale</td><td>WMEILILGIVYRSLPYDDKLVYAEDYIMDEEHSRLAGLLELYRAILQLVRRYYKKLKVEKE</td><td></td><td>360</td></tr><tr><td>chicken</td><td>WMEILILGIVYRSLPYDKLVAEYIMDEEHSRLTGLELYLAILQLVRRYYKKLKVEKE</td><td></td><td>360</td></tr><tr><td>fish</td><td>WMEILILSVFRSLPYDELVYAEDYIMDEEHSRLTGLLDYVSILQLVRKYKKLKVEKE</td><td></td><td>339</td></tr><tr><td>mouse</td><td>EFMILKALALANSDMYIENLEAVQKLQDLLHEALQDYELSQRHEEPRRAGKLLLLTLPLL</td><td></td><td>399</td></tr><tr><td>human</td><td>EFVTLKALALANSDMYIEDLEAVQKLQDLLHEALQDYELSQRHEEPWRTGKLLLLTLPLL</td><td></td><td>399</td></tr><tr><td>goat</td><td>EFVTLKALALANSDMYIEDLEAVQKLQDLLHEALQDYELSQRHEEPRTGKLLLLTLPLL</td><td></td><td>420</td></tr><tr><td>whale</td><td>EFVTLKALALANSDMYIEDLEAVQKLQDLLHEALQDYELSQRHEEPRTGKLLLLTLPLL</td><td></td><td>420</td></tr><tr><td>chicken</td><td>EFVTLKALALANSDMSHIEDMDAVQKLQDLLHEALQDYELSQRNEEPRAGKLLLLTLPLL</td><td></td><td>420</td></tr><tr><td>fish</td><td>EFVTLKAIALANSDMSHIEDMEAVQKLQDALHEALQDFECSQHQEDPRRAGKLIMTLPLL</td><td></td><td>399</td></tr><tr><td>mouse</td><td>RQTAAKAVQHFYSVKLQGKVMHKLFLEMLEAKV</td><td>433</td><td></td></tr><tr><td>human</td><td>RQTAAKAVQHFYSVKLQGKVMHKLFLEMLEAKV</td><td>433</td><td></td></tr><tr><td>goat</td><td>RQTAAKAVQHFYSVKLQGKVMHKLFLEMLEAKV</td><td>454</td><td></td></tr><tr><td>whale</td><td>RQTAAKAVQHFYSIKLQGKVMHKLFLEMLEAKV</td><td>454</td><td></td></tr><tr><td>chicken</td><td>RQTAAKAVQHFYSIKLQGKVMHKLFLEMLEAKV</td><td>454</td><td></td></tr><tr><td>fish</td><td>RQTATKAVQHFYSIKVQGKVMHKLFLEMLEAKV</td><td>433</td><td></td></tr></tbody></table>
### 3.5.3. Disorder prediction

<figure><img src="image_27.png" /></figure>

### 3.5.4. Coding DNA sequences, produced protein constructs

#### Esrrb-h_aal-102_C12A-C72A-C91A

**Synthesized sequence:**

Ccgcggtgagaacctgtacttccaggcatgagcagcgaagatcgtcacctgggtagcagcgcggcagctttattaaaaccgagccgagcag
cccgagcagcggtattgatgcgctgagccaccatagcccgagcggtagcagcgatgcgagcggtggcttcggtattgcgctgagcacccatgcg
aacggtctggatagcccgccgatgtttgcgggtgcgggcctgggtggcaaccggcgcgctaaaagctacgaagactgcaccagcggcatcatgg
aggatagcgcgattaaggcggaatatatgctgaacgcgattccgaaacgtctgtaaaagctt

**Translates into:**

AGENLYFQGMSSEDRHLGSSAGSFIKTEPSSPSSGIDALSHHSPSGSSDASGGFGIALSTHANGLDSPPMFAGAGLGGNPARKSYEDCTSGIME
DSAIKAEYMLNAIPKRL*

**Expressed peptide:**

GST-His-Tev-Esrrb(aa1-102)_C12A-C72A-C91A

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMSAIIRYIADKHNMLGGCPKERAEISM
LEGAVLDIRYGVSRAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQ
IDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGSTSGGHHHHHSAGENLYFQGMSSEDRHLGSSAGSFIKTEPSSPSSGIDALSHHSPSGSS
DASGGFGIALSTHANGLDSPPMFAGAGLGGNPARKSYEDCTSGIMEDSAIKAEYMLNAIPKRL

**After Tev-cleavage (leaving 1 Gly in N-ter):**

GMSSEDRHLGSSAGSFIKTEPSSPSSGIDALSHHSPSGSSDASGGFGIALSTHANGLDSPPMFAGAGLGGNPARKSYEDCTSGIMEDSAIKAEY
MLNAIPKRL

G MSSEDRHLGS SAGSFIKTEP SSPSSGIDAL SHHSPSGSSD ASGGFGIALS THANGLDSP
70 80 90 100
MFAGAGLGGN PARKSYEDCT SGIMEDSAIK AEYMLNAIPK RL
## Esrrb-h aa1-102_C12A-C91A

### Coding sequence (mutated from Esrrb-h aa-102_C12A-C72A-C91A):

Ccgcggtgagaacctgtacttccagggcatgagcagcgaagatcgtcacctgggtagcagcgcgggcagctttattaaaaccgagccgagcagcccgagcagcggtattgatgcgctgagccaccatagcccgagcggtagcagcgatgcgagcggtggcttcggtattgcgctgagcacccatgcgaacggtctggatagcccgcgatgtttgcvgggtgcgggcctgggtggcaacccgtccgtaaaaagctacgaagactgcaccagcggcatcatgaggatagcgcgattaaggcggaatatatgctgaacgcgattccgaaacgtctgtaaaagctt

### Translates into:

AGENLYFQGMSSERHLGSSAGSFIKTEPSSPSSGIDALSHHSPSGSSDASGGFGIALSTHANGLDSPPMFAGAGLGGNPCRKSYEDCTSGIME
DSAIKAEYMLNAIPKRL*

### After Tev-cleavage (leaving 1 Gly in N-ter):

GMSSERHLGSSAGSFIKTEPSSPSSGIDALSHHSPSGSSDASGGFGIALSTHANGLDSPPMFAGAGLGGNPCRKSYEDCTSGIMEDSAIKAEY
MLNAIPKRL

10 20 30 40 50 60
G MSSEDRHLGS SAGSFIKTEP SSPSSGIDAL SHHSPSGSSD ASGGFGIALS THANGLDSP
70 80 90 100
MFAGAGLGGN PCRKSYEDCT SGIMEDSAIK AEYMLNAIPK RL

## Esrrb-h aa1-102_C12A-C72A-C91A_AviTag-His6

### Synthesized sequence:

Ccgcggtgagaacctgtacttccagggcatgagcagcgaagatcgtcacctgggtagcagcgcgggcagctttattaaaaccgagccgagcagcccgagcagcggtattgatgcgctgagccaccatagcccgagcggtagcagcgatgcgagcggtggcttcggtattgcgctgagcacccatgcgaacggtctggatagcccgcgatgtttgcvgggtgcgggcctgggtggcaacccggcgcgtaaaaagctacgaagactgcaccagcggcatcatgaggatagcgcgattaaggcggaatatatgctgaacgcgattccgaaacgtctgtaaaagctt

### Translates into:

MSSEDRHLGSSAGSFIKTEPSSPSSGIDALSHHSPSGSSDASGGFGIALSTHANGLDSPPMFAGAGLGGNPARKSYEDCTSGIMEDSAIKAEYM
LNAIPKRLGLNDIFEAQKIEWHEGAGLE

### Expressed peptide:

MSSEDRHLGSSAGSFIKTEPSSPSSGIDALSHHSPSGSSDASGGFGIALSTHANGLDSPPMFAGAGLGGNPARKSYEDCTSGIMEDSAIKAEYM
LNAIPKRLGLNDIFEAQKIEWHEGAGLEHHHHHH*

### Expressed peptide:

MSSEDRHLGSSAGSFIKTEPSSPSSGIDALSHHSPSGSSDASGGFGIALSTHANGLDSPPMFAGAGLGGNPARKSYEDCTSGIMEDSAIKAEYM
LNAIPKRLGLNDIFEAQKIEWHEGAGLEHHHHHH

10 20 30 40 50 60
MSSEDRHLGS SAGSFIKTEP SSPSSGIDAL SHHSPSGSSD ASGGFGIALS THANGLDSP
70 80 90 100
MFAGAGLGGN PARKSYEDCT SGIMEDSAIK AEYMLNAIPK RL GLNDIFEAQKIEWHEGAGLEHHHHHH
## Esrrb-h aa1-102_C12A-C72A_AviTag-His6

### Coding sequence (mutated from Esrrb-h aa-102_C12A-C72A-C91A_AviTag-His6):

Catatgagcagcgaagaccgtcacctgggtagcagcgcgggtagctttattaagaccgaaccgagcagcccgagcagcggcattgatgcgctga gccatcatagcccgagcggtagcagcgatgcgagcggtggtcttcggtattgcgctgagcacccatgcgaacggtctggatagcccgccgatgtt tgCGGGTGCGGGCCTGGGTGGAACCCGGCGCGTAAGAGCTACGAGGACTGCACCAGCGGCATCATGGAGGATAGCGCGATTAAGTGCGAATAT atgctgaACCGGATTCGAAACGCTGGGCCTGAACGACATTTTGAAGCGCAGAAGATTGAGTGGCATGAGGGTGCGGGCCTCGAG

### Translates into:

MSSEDRHLGSSAGSFIKTEPSSPSSGIDALSHHSPSGSSDASGGFGIALSTHANGLDSPPMFAGAGLGGNPARKSYEDCTSGIMEDSAIKCEYM LNAIPKRLGLNDIFEAQKIEWHEGAGLE

### Expressed peptide:

MSSEDRHLGSSAGSFIKTEPSAPSSGIDALSHHSPSGSSDASGGFGIALSTHANGLDSPPMFAGAGLGGNPARKSYEDCTSGIMEDSAIKCEYM LNAIPKRLGLNDIFEAQKIEWHEGAGLEHHHHHH

10 20 30 40 50 60
MSSEDRHLGS SAGSFIKTEP SSPSSGIDAL SHHSPSGSSD ASGGFGIALS THANGLDSP
70 80 90 100
MFAGAGLGGN PARKSYEDCT SGIMEDSAIK CEYMLNAIPK RL GLNDIFEAQKIEWHEGAGLEHHHHHH

## Esrrb-h aa1-102_C12A-C72A-C91A-S22A_AviTag-His6

### Coding sequence (mutated from Esrrb-h aa-102_C12A-C72A-C91A_AviTag-His6):

Catatgagcagcgaagaccgtcacctgggtagcagcgcgggtagctttattaagaccgaaccgagcagcgcggcattgatgcgctga gccatcatagcccgagcggtagcagcgatgcgagcggtggtcttcggtattgcgctgagcacccatgcgaacggtctggatagcccgccgatgtt tgCGGGTGCGGGCCTGGGTGGAACCCGGCGCGTAAGAGCTACGAGGACTGCACCAGCGGCATCATGGAGGATAGCGCGATTAAGCGGAATAT atgctgaACCGGATTCGAAACGCTGGGCCTGAACGACATTTTGAAGCGCAGAAGATTGAGTGGCATGAGGGTGCGGGCCTCGAG

### Translates into:

MSSEDRHLGSSAGSFIKTEPSAPSSGIDALSHHSPSGSSDASGGFGIALSTHANGLDSPPMFAGAGLGGNPARKSYEDCTSGIMEDSAIKCEYM LNAIPKRLGLNDIFEAQKIEWHEGAGLE

### Expressed peptide:

MSSEDRHLGSSAGSFIKTEPSAPSSGIDALSHHSPSGSSDASGGFGIALSTHANGLDSPPMFAGAGLGGNPARKSYEDCTSGIMEDSAIKCEYM LNAIPKRLGLNDIFEAQKIEWHEGAGLEHHHHHH*

10 20 30 40 50 60
MSSEDRHLGS SAGSFIKTEP SAPSSGIDAL SHHSPSGSSD ASGGFGIALS THANGLDSP
70 80 90 100
MFAGAGLGGN PARKSYEDCT SGIMEDSAIK AEYMLNAIPK RL GLNDIFEAQKIEWHEGAGLEHHHHHH

## References

[1] W. Lee, M. Tonelli, J. L. Markley, *Bioinformatics*, 2015, **31**, 1325-1327.

[2] F.-X. Theillet, C. Smet-Nocca, S. Liokatis, R. Thongwichian, J. Kosten, M.-K. Yoon, R. W. Kriwacki, I. Landrieu, G. Lippens, *P. Selenko, J. Biomol. NMR*, 2012, **54**, 217-236.

[3] F.-X. Theillet, H. M. Rose, S. Liokatis, A. Binolfi, R. Thongwichian, M. Stuiver, P. Selenko, *Nat. Protoc.*, 2013, **8**, 1416-1432.

[4] A. Mylona, F.-X. Theillet, C. Foster, T. M. Cheng, F. Miralles, P. A. Bates, P. Selenko, R. Treisman, *Science*, 2016, **354**, 233-237.

[5] M. Julien, C. Bouguechtouli, A. Alik, R. Ghouil, S. Zinn-Justin, F.-X. Theillet, in *Intrinsically Disordered Proteins: Methods and Protocols* (B. B. Kragelund, K. Skriver, eds.), Springer US, New York, NY, 2020, 793-817.

[6] J.-P. Lambert, M. Tucholska, T. Pawson, A.-C. Gingras, *J. Proteomics*, 2014, **100**, 55-59.

[7] P. Poullet, S. Carpentier, E. Barillot, *Proteomics*, 2007, **7**, 2553-2556.
[8] M. The, M. J. MacCoss, W. S. Noble, L. Käll, *J. Am. Soc. Mass Spectrom*, 2016, **27**, 1719-1727.

[9] B. Valot, O. Langella, E. Nano, M. Zivy, *Proteomics*, 2011, **11**, 3572-3577.

[10] Y. Perez-Riverol, J. Bai, C. Bandla, D. García-Seisdedos, S. Hewapathirana, S. Kamatchinathan, D. J. Kundu, A. Prakash, A. Frericks-Zipper, M. Eisenacher, M. Walzer,

S. Wang, A. Brazma, J. A. Vizcaíno, *Nucleic Acids Res.*, 2022, **50**, D543-D552.

[11] C. Smet-Nocca, H. Launay, J.-M. Wieruszeski, G. Lippens, I. Landrieu, *J. Biomol. NMR*, 2013, **55**, 323-337.

[12] K. Tamiola, F. A. A. Mulder, *Biochem. Soc. Trans.*, 2012, **40**, 1014-1020.

[13] J. T. Nielsen, F. A. A. Mulder, *J. Biomol. NMR*, 2018, **70**, 141-165.

[14] C. Camilloni, A. De Simone, W. F. Vranken, M. Vendruscolo, *Biochemistry*, 2012, **51**, 2224-2231.