14-3-3 proteins are a group of seven dimeric adapter proteins that exert
their biological function by interacting with hundreds of phosphorylated
proteins, thus influencing their sub-cellular localization, activity or
stability in the cell. Due to this remarkable interaction network, 14-3-3
proteins have been associated with several pathologies and the protein-protein
interactions established with a number of partners are now considered promising
drug targets. The activity of 14-3-3 proteins is often isoform specific and to
our knowledge only one out of seven isoforms, 14-3-3$\zeta$, has been assigned.
Despite the availability of the crystal structures of all seven isoforms of
14-3-3, the additional NMR assignments of 14-3-3 proteins are important for
both biological mechanism studies and chemical biology approaches. Herein, we
present a robust backbone assignment of 14-3-3$\sigma$, which will allow
advances in the discovery of potential therapeutic compounds. This assignment
is now being applied to the discovery of both inhibitors and stabilizers of
14-3-3 protein-protein interactions

Boll, Emmanuelle

Cantrelle, François-Xavier

Hanoulle, Xavier

Landrieu, Isabelle

Merzougui, Hamida

Neves, João

English

arXiv

International audience14-3-3 proteins are a group of seven dimeric adapter proteins that exert their biological function by interacting with hundreds of phosphorylated proteins, thus influencing their sub-cellular localization, activity or stability in the cell. Due to this remarkable interaction network, 14-3-3 proteins have been associated with several pathologies and the protein-protein interactions established with a number of partners are now considered promising drug targets. The activity of 14-3-3 proteins is often isoform specific and to our knowledge only one out of seven isoforms, 14-3-3ζ, has been assigned. Despite the availability of the crystal structures of all seven isoforms of 14-3-3, the additional NMR assignments of 14-3-3 proteins are important for both biological mechanism studies and chemical biology approaches. Herein, we present a robust backbone assignment of 14-3-3σ, which will allow advances in the discovery of potential therapeutic compounds. This assignment is now being applied to the discovery of both inhibitors and stabilizers of 14-3-3 protein-protein interactions

Neves, João,

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Backbone chemical shift assignments of human 14-3-3σ
João Neves, Isabelle Landrieu, Hamida Merzougui, Emmanuelle Boll, Xavier
Hanoulle, François-Xavier Cantrelle
To cite this version:
João Neves, Isabelle Landrieu, Hamida Merzougui, Emmanuelle Boll, Xavier Hanoulle, et al.. Back-
bone chemical shift assignments of human 14-3-3σ. Biomolecular NMR Assignments, Springer, 2019,
13 (1), pp.103-107. ￿10.1007/s12104-018-9860-1￿. ￿hal-02107858￿
1 
Backbone chemical shift assignments of human 14-3-3σ 
 
João Filipe Neves
1
, Isabelle Landrieu
1
*, Hamida Merzougui
1
, Emmanuelle Boll
1
, Xavier 
Hanoulle
1
, François-Xavier Cantrelle
1
 
 
 
1
UMR 8576 CNRS-Lille University, 59000 Lille, France 
 
ORCID ID for authors: 
JFN: 0000-0003-0582-1193 
IL: 0000-0002-4883-2637 
XH: 0000-0002-3755-2680 
 
*Corresponding author: Isabelle Landrieu, isabelle.landrieu@univ-lille1.fr 
+33 (0)3 62 53 17 02 
 
Abstract 
14-3-3 proteins are a group of seven dimeric adapter proteins that exert their biological 
function by interacting with hundreds of phosphorylated proteins, thus influencing their sub-
cellular localization, activity or stability in the cell. Due to this remarkable interaction 
network, 14-3-3 proteins have been associated with several pathologies and the protein-
protein interactions established with a number of partners are now considered promising drug 
targets. The activity of 14-3-3 proteins is often isoform specific and to our knowledge only 
one out of seven isoforms, 14-3-3ζ, has been assigned. Despite the availability of the crystal 
structures of all seven isoforms of 14-3-3, the additional NMR assignments of 14-3-3 proteins 
are important for both biological mechanism studies and chemical biology approaches. 
Herein, we present a robust backbone assignment of 14-3-3σ, which will allow advances in 
the discovery of potential therapeutic compounds. This assignment is now being applied to 
the discovery of both inhibitors and stabilizers of 14-3-3 protein-protein interactions.  
 
Keywords 
Protein-Protein Interactions, 14-3-3 proteins, Drug discovery, NMR resonance assignments 
 
 
 
 
2 
Biological context 
The 14-3-3 family includes seven different isoforms (β, γ, ε, η, σ, τ and ζ) expressed in all 
eukaryotic organisms. 14-3-3 proteins are adapter proteins, expressed in an ubiquitous 
manner in all tissues and cell compartments. Despite the considerably high structural 
homology between different isoforms, 14-3-3 activity is often isoform specific (Fu et al. 
2000). To our knowledge, 14-3-3ζ is the only isoform of 14-3-3 whose assignment is reported 
(Killoran et al. 2015). 14-3-3 proteins exert their biological functions through the modulation 
of the activity of hundreds of phosphorylated proteins. This remarkable interactome makes 
14-3-3 proteins influent actors in many cellular events and, by consequence, in several 
pathologies like cancer, Alzheimer’s disease, Parkinson’s disease, among others (Kaplan et 
al. 2017) . With the rise of the interest on the modulation of Protein-Protein Interactions 
(PPIs) in modern drug discovery, 14-3-3 proteins are being seen as promising targets for 
several pathologies. Especially over the last 10 years, there has been a remarkable effort 
towards the discovery of compounds capable of inhibiting or stabilizing 14-3-3 PPIs (Stevers 
et al. 2018). In particular, we have been interested in modulating the Tau/14-3-3 interaction, 
which is thought to have detrimental effects in neuronal cells in Alzheimer’s disease (Joo et 
al. 2015). Peptidomimetic compounds have been designed and shown to inhibit this PPI 
(Milroy et al. 2015; Andrei et al. 2018). Assignment of 14-3-3σ spectra will further help the 
development of these promising compounds. 
In physiological conditions, 14-3-3 proteins exist as functional dimers, both as hetero and 
homodimers (Fu et al. 2000). They are acidic proteins and each monomer has a molecular 
weight (MW) of around 30 kDa, being composed by nine α-helices, commonly named αA-I, 
disposed in an antiparallel fashion. At the C-terminus of the last helix of 14-3-3 proteins (αI), 
there is a flexible and highly acidic tail, which is the less conserved region of 14-3-3 (Obsil 
and Obsilova 2011).  
Here we report the backbone assignments (
13
Cα, 
13
Cβ, 
13
CO, 
15
N and 
1
H
N
) of 14-3-3σ (a 
truncated form comprising residues 1-231 out of 248). Due to the large number of partners of 
14-3-3σ, the assignment of this protein opens new possibilities for the study of hundreds of 
PPIs. Our assignment is currently being applied to drug screening and to the study of the 
effect of small molecules on the modulation of 14-3-3 PPIs. In the future, it may have a role 
on the development of new 14-3-3 PPI modulators that can be employed as tool compounds 
and hopefully, be the basis for the development of new therapeutic molecules.  
  
 
3 
Sample preparation 
The plasmids used for protein expression were kindly provided by Dr. Christian Ottmann 
from the Eindhoven University of Technology, The Netherlands. The cDNA coding for 
human 14-3-3σ (residues 1-248) or 14-3-3σΔC17 (residues 1-231) was cloned into the 
pProExHtb vector between the BamHI and NotI sites. A Tobacco Etch Virus (TEV) cleavage 
site followed by a linker (GAMGS) are present between the regions coding for the N-terminal 
His6-tag and for the protein, yielding a recombinant expression plasmid with the following 
configuration: His6 tag-TEV cleavage site-Linker-14-3-3σ/14-3-3σΔC17. Therefore, 14-3-3σ 
and 14-3-3σΔC17 were both expressed as N-terminally His6-tagged proteins. 
15
N
13
C
2
H labeled 14-3-3σ or 14-3-3σΔC17 were expressed in E. coli BL21 (DE3) cells. A 20 
ml pre-culture in Luria–Bertani (LB) medium containing 100 mg/L ampicillin was grown 
overnight at 310 K and was used to inoculate 1 L of deuterated M9 minimal medium 
supplemented with 2 g/L 
13
C6
2
H7 D-Glucose, 1 g/L 
15
N Ammonium Chloride, 0.4 g/L Isogro 
15
N
13
C
2
H  Powder – Growth Medium (Sigma Aldrich) and 100 mg/L ampicillin. The culture 
was grown at 310 K to an OD600 of 0.9 and induced with 0.5 mM Isopropyl-β-D-
Thiogalactopyranoside (IPTG). Incubation was continued for 15h at 301 K with vigorous 
shaking. Cells were harvested by centrifugation, resuspended in a buffer containing 50 mM 
Tris-HCl, 500 mM NaCl, 5% (v/v) glycerol, 25 mM imidazole, 2 mM β-Mercaptoethanol 
(BME), pH 8.0 and lysed with a homogenizer. The cellular debris were then eliminated by 
centrifugation and the supernatant was loaded into a Ni-NTA column (GE Healthcare). 
Elution of proteins was performed with a buffer containing 50 mM Tris-HCl, 500 mM NaCl, 
5% (v/v) glycerol, 250 mM imidazole, 2 mM BME, pH 8.0. The N-terminal His6-tag was 
then cleaved by the TEV protease for 2 hours at 293 K, followed by 12 hours at 277 K, while 
being dialyzed against 100 mM sodium phosphate, pH 6.8 and 50 mM NaCl (NMR buffer). 
The molar ratio between 14-3-3σ or 14-3-3σΔC17 and TEV protease was 50:1. The protein 
without His6-tag was collected in the flow-through of a Ni-NTA column, using NMR buffer 
for the elution, while the uncleaved fraction and the His6-tagged peptide were retained on the 
column. The protein was then concentrated to 2 mM, aliquoted, flash frozen and stored at 193 
K. Typical yields were in the range of 45-70 mg of protein per liter of culture. 
NMR spectroscopy 
All NMR experiments were recorded using a 900 MHz Bruker Avance spectrometer, 
equipped with a cryoprobe. Samples, in a buffer containing 100 mM sodium phosphate, pH 
6.8, 50 mM NaCl, 1mM DTT (Dithiothreitol), EDTA-free Protease Inhibitor Cocktail 
4 
(Roche, Switzerland) and 10% (v/v) D2O, were transferred to Shigemi tubes and experiments 
were acquired at 305 K. The concentration of 
15
N
13
C
2
H labeled protein for assignment 
experiments ranged from 0.8 mM to 1.0 mM. Backbone assignments were obtained from 
TROSY-HNCACB, TROSY-HN(CO)CACB, BEST-TROSY-HNCO and 
1
H-
15
N-NOESY-
HMQC spectra on 
15
N
13
C
2
H  labeled 14-3-3σ or 14-3-3σΔC17 samples. The mixing time of 
the 
1
H-
15
N-NOESY-HMQC spectrum was 120 ms. Non-Uniform Sampling was employed 
for the acquisition of all 3D spectra. The reference for the 
1
H chemical shift was relative to 
Trimethyl silyl propionate. The 
15
N and 
13
C chemical shifts were referenced indirectly. All 
spectra were collected and processed with Topspin 3.5 (Bruker Biospin) and analyzed with 
Sparky 3.12 (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San 
Francisco). Automatic assignments were performed with MARS version 1.2 (Jung and 
Zweckstetter 2004) and with PINE-NMR server (Bahrami et al. 2009).  
Assignments and data deposition 
For sequence numbering purposes, the residues of the linker left after TEV cleavage 
(GAMGS) were not considered. Accordingly, residue 1 corresponds to the first Methionine 
residue of 14-3-3σ. Similarly to what has been reported for the assignment of 14-3-3ζ 
(Killoran et al. 2015), we also observed the presence of highly intense peaks in the 
1
H-
15
N 
TROSY-HSQC spectrum of the full-length 14-3-3σ (residues 1-248). Because these intense 
signals were overlapping with signals corresponding to the ordered core-region, we decided 
to use a construct of 14-3-3σ without the C-terminal flexible region (14-3-3σΔC17, 
containing residues 1-231) for the backbone assignment. The elimination of the resonances 
corresponding to this flexible region indeed allowed to detect more resonances on the 
1
H-
15
N 
TROSY-HSQC spectrum and additionally improved the overall quality of the 3D spectra. 
Removal of the disordered terminal tail did not affect the structure of the protein in solution 
as resonances in the 2D spectra of the protein with or without the tail superimposed. The final 
construct, after cleavage of the N-terminus by the TEV protease, yielded a 14-3-3σΔC17 
protein with a MW of 26.51 KDa. Given its association as a dimer and therefore the 
formation of a biomolecule with a MW higher than 50 KDa, it was necessary to produce a 
deuterated protein, to use a high-field spectrometer and to extend acquisition times. A good 
coverage of the protein assignment was reached although it should be noted that not all the 
amide peaks of the protein were detected on the 
1
H-
15
N TROSY-HSQC (shown in figure 1 a) 
and in some cases, backbone signals in the 
13
C dimension were of low intensity. The 
5 
incomplete 
2
H/
1
H exchange may have been a factor that prevented the detection of some 
resonances, in particular those corresponding to residues located at the dimer interface. 
Overall, we were able to obtain a robust assignment of 14-3-3σΔC17 comprising 177 out of 
226 
1
H
N
 and 
15
N resonances (78%), 200 out of 231 
13
Cα resonances (87%), 187 out of 219 
13
Cβ resonances (85%) and 176 out of 231 
13
CO resonances (77%). The assignment covers 
homogeneously the protein’s sequence (figure 1 b). The assigned chemical shifts were 
deposited into the Biological Magnetic Resonance Database (http://www.bmrb.wisc.edu/) 
under the BMRB accession number 27563. 
Secondary structure 
The obtained chemical shift assignments were further used for the analysis of the secondary 
structure of 14-3-3σ by two different methods. On one hand, the ensemble of backbone 
chemical shifts (
13
Cα, 
13
Cβ, 
13
CO, 
15
N and 
1
H
N
) was submitted to CSI 3.0 sever (Hafsa et al. 
2015) in order to obtain the Chemical Shift Indexes predictive of the secondary structure. On 
the other hand, the 
13
Cα and 
13
Cβ chemical shift values were used to calculate the Secondary 
Structure Propensity (SSP) scores (Marsh et al. 2006). Prediction by SSP scores allowed 
comparison with the secondary structure analysis of the previously assigned 14-3-3 isoform 
(Killoran et al. 2015). 
The results of the predictions made by both softwares are summarized in figure 2. Indeed, the 
results obtained from both methods are coherent and well correlated with what is reported in 
the crystal structure of 14-3-3σ (PDB ID: 1YZ5). The SSP scores of 14-3-3σ presented here 
are almost identical to the ones of 14-3-3ζ showing that the secondary structures of both 
isoforms in solution are very similar. The only remarkable difference is the length of helix 
αD, which is shorter in 14-3-3σ at its N-terminus due to the replacement of Q79 of 14-3-3ζ by 
a proline residue.  
  
6 
Acknowledgments 
We thank MSc. Eline Sijbesma and Dr. Christian Ottmann from the Eindhoven University of 
Technology for kindly providing us the plasmids of both 14-3-3σ and 14-3-3σΔC17. We also 
thank Dr. Elian Dupré from Lille University for his help with the automatic assignments 
software. 
The research is supported by funding from the European Union through the TASPPI project 
(H2020-MSCA-ITN-2015, grant number 675179) and by the LabEx (Laboratory of 
Excellence) DISTALZ (ANR, ANR-11-LABX- 009). The NMR facilities were funded by the 
Nord Region Council, CNRS, Institut Pasteur de Lille, the European Community (ERDF), the 
French Ministry of Research and the University of Lille and by the CTRL CPER cofunded by 
the European Union with the European Regional Development Fund (ERDF), by the Hauts 
de France Regional Council (contract n°17003781), Métropole Européenne de Lille (contract 
n°2016_ESR_05), and French State (contract n°2017-R3-CTRL-Phase 1). We acknowledge 
support for the NMR facilities from TGE RMN THC (CNRS, FR-3050) and FRABio (Univ. 
Lille, CNRS, FR-3688).  
Conflict of interest 
The authors declare that they have no conflict of interest. 
 
  
7 
References 
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Peptides Operating via Two Different Binding Modes. ACS Chem Neurosci. doi: 
10.1021/acschemneuro.8b00118 
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10.1371/journal.pcbi.1000307 
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Annual review of pharmacology and toxicology 40:617–647 
Hafsa NE, Arndt D, Wishart DS (2015) CSI 3.0: a web server for identifying secondary and super-
secondary structure in proteins using NMR chemical shifts. Nucleic Acids Res 43:W370–
W377. doi: 10.1093/nar/gkv494 
Joo Y, Schumacher B, Landrieu I, et al (2015) Involvement of 14-3-3 in tubulin instability and 
impaired axon development is mediated by Tau. The FASEB Journal 29:4133–4144. doi: 
10.1096/fj.14-265009 
Jung Y-S, Zweckstetter M (2004) Mars - robust automatic backbone assignment of proteins. J Biomol 
NMR 30:11–23. doi: 10.1023/B:JNMR.0000042954.99056.ad 
Kaplan A, Ottmann C, Fournier AE (2017) 14-3-3 adaptor protein-protein interactions as therapeutic 
targets for CNS diseases. Pharmacological Research 125:114–121. doi: 
10.1016/j.phrs.2017.09.007 
Killoran RC, Fan J, Yang D, et al (2015) Structural Analysis of the 14-3-3ζ/Chibby Interaction 
Involved in Wnt/β-Catenin Signaling. PLOS ONE 10:e0123934. doi: 
10.1371/journal.pone.0123934 
Marsh JA, Singh VK, Jia Z, Forman-Kay JD (2006) Sensitivity of secondary structure propensities to 
sequence differences between α- and γ-synuclein: Implications for fibrillation. Protein Sci 
15:2795–2804. doi: 10.1110/ps.062465306 
Milroy L-G, Bartel M, Henen MA, et al (2015) Stabilizer-Guided Inhibition of Protein-Protein 
Interactions. Angewandte Chemie International Edition 54:15720–15724. doi: 
10.1002/anie.201507976 
Obsil T, Obsilova V (2011) Structural basis of 14-3-3 protein functions. Seminars in Cell & 
Developmental Biology 22:663–672. doi: 10.1016/j.semcdb.2011.09.001 
Stevers LM, Sijbesma E, Botta M, et al (2018) Modulators of 14-3-3 Protein–Protein Interactions. J 
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Zhang H, Neal S, Wishart DS (2003) RefDB: A database of uniformly referenced protein chemical 
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8 
 
Fig. 1 Assignment of 14-3-3σ is robust and covers homogeneously the protein's surface. a) 
Annotated 
1
H-
15
N TROSY-HSQC spectrum of 
15
N
13
C
2
H labeled 14-3-3σΔC17 0.8 mM 
acquired at 305 K in a buffer containing 100 mM sodium phosphate, pH 6.8, 50 mM NaCl, 
1mM DTT, EDTA-free Protease Inhibitor Cocktail (Roche, Switzerland) and 10% (v/v) D2O. 
The spectrum was acquired with 3426 complex points in the 
1
H dimension and 256 complex 
points in the 
15
N dimension with 64 scans per increment. The spectral window was between 
4.72 ppm and 14.64 ppm for the 
1
H dimension and between 80.2 ppm and 140.1 ppm for the 
15
N dimension. The selected window contains all the assigned backbone resonances. b) 
Cartoon representation of the crystal structure of 14-3-3σ (PDB ID: 1YZ5, gray cartoon) with 
the assigned residues colored in red. The structure is shown both as a dimer and as a 
monomer of 14-3-3σ.  
9 
 
 
Fig. 2 Secondary Structure analysis of 14-3-3σ by calculation of the SSP scores and by the 
CSI 3.0 server reveals that 14-3-3σ is composed by nine α-helices, as expected from the 
crystal structure. a) SSP scores calculation for 14-3-3σ. The y axis of the plot represents the 
score (~ 1 for α-helix, ~ 0 for random coil, ~ -1 for β-strand) for each residue of 14-3-3σ 
(represented on the x axis). Yellow bars correspond to non-assigned residues or to residues 
preceding prolines, which were not considered for the SSP score calculation. Random coil 
chemical shift values used for the calculation of SSP scores were taken from RefDB database 
(Zhang et al. 2003). The plot is divided in 3 lines. b) Output of the CSI 3.0 server in a residue 
specific-way (blue cartoon stands for α-helix, red arrow stands for β-strand and black line 
stands for random coil). The plot is divided in 3 lines. The identification of the helices of 14-
3-3σ (αA-αI) is shown above the output of the CSI 3.0 server. 


Backbone chemical shift assignments of human 14-3-3σ

Backbone chemical shift assignments of human 14-3-3$\sigma$

Backbone chemical shift assignments of human 14-3-3 $\sigma$

Abstract

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Archive Ouverte en Sciences de l'Information et de la Communication

Backbone chemical shift assignments of human 14-3-3σ\sigmaσ

Abstract

Similar works

Full text

Available Versions

Archive Ouverte en Sciences de l'Information et de la Communication

Backbone chemical shift assignments of human 14-3-3 $\sigma$