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Model of the Interaction between the NF-#B Inhibitory protein p100 and the E3 ubiquitin ligase #-TrCP based on NMR and Docking Experiments Maxime Melikian, Baptiste Eluard, Gildas Bertho, Veronique Baud, and Nathalie Evrard-Todeschi J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.5b00409 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on December 29, 2016

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Journal of Chemical Information and Modeling

Model of the Interaction between the NF-κB Inhibitory protein p100 and the E3 ubiquitin ligase β-TrCP based on NMR and Docking Experiments Maxime Melikian1,a, Baptiste Eluard2,a, Gildas Bertho1;Véronique Baud2* and Nathalie EvrardTodeschi1* 1

Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601-CNRS, 45 rue des Saint-Pères, 75006 Paris, Université Paris Descartes, Sorbonne Paris Cité,France 2 NF-kB, Differentiation and Cancer, Université Paris Descartes, Sorbonne Paris, Cité, 4 Avenue de l'Observatoire, 75006, Paris. a

M.M. and B.E. contributed equally to this work

* To whom correspondence should be addressed: - Nathalie Evrard, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601-CNRS, 45 rue des Saint-Pères, 75006 Paris, France Email: [email protected] - Véronique Baud,2NF-kB, Differentiation and Cancer, Université Paris Descartes, Sorbonne Paris, Cité, 4 Avenue de l'Observatoire, 75006, Paris Email: [email protected]

Abstract NF-κB is a major transcription factor whose activation is triggered through two main activation pathways: the canonical pathwayinvolving disruption of IκB-α/NF-κB complexes and the alternative pathway whoseactivationrelies on the inducible proteolysis of theinhibitory protein p100. One centralstep controlling p100 processing consists in the interaction of the E3 ubiquitin ligase β-TrCP with p100, thereby leading to its ubiquitinylation and subsequent either complete degradation or partial proteolysis by the proteasome. However, the interaction mechanismbetween p100 and β-TrCP is still poorly defined. In this work, a diphosphorylated 21-mer p100 peptide model containing the phosphodegron motif was used to characterize the interaction withβ-TrCPby NMR. In parallel, docking simulations were performed in order to obtain a model of the 21P-p100/β-TrCP complex. STD experiments were performed in order to highlight the residues of p100 involved in the interaction with the β-TrCPprotein. These results highlighted the importance of pSer865 and pSer869 residues in the interaction with β-TrCP and particularly the Tyr867 that fits inside the hydrophobe β-TrCP cavity with theArg474 guanidinium group. Four otherarginines, Arg285, Arg410, Arg431, and Arg521 were found essential in the stabilization of p100 on the βTrCPsurface. Importantly, the requirement for these five arginine residues of β-TrCPfor the interaction with p100 was further confirmedin vivo, thereby validating the docking model through a biological approach.

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Introduction NF-κB is a major transcription factor that plays a crucial role in the control of the inflammatory response1 as well as other stressful situations2. NF-κB is also involved in both the innate immune system3, building the first line of defense against pathogens, and in the acquired one, through its anti-apoptotic properties4. Chronic inflammatory diseases are also characterized by abnormally high constitutive activation of NF-κB, so that NF-κB became one of the favorite targets for the development of a new generation of anti-inflammatory molecules. Activation of NF-κB is also involved in many cancers, such as solid tumors (e.g. breast cancer, ovarian, colon, pancreas), as well as in B and T2cell lymphomas5, by promoting tumor cell survival thus reducing the efficiency of conventional anticancer therapies6. Hence, targeting NF-κB activation pathways would help to develop new anticancer drugs and/or new treatments potentiating the sensitivity of tumors to radiotherapy and chemotherapy. Proteasome inhibition has already been shown to block the chemotherapy-induced activation of NF-κBin vitro, and has been correlated with enhanced chemosensitivity and increased apoptosis in xenografted tumor cells in mice7. Similarly, inhibition of NF-κB activation increases radiation induced apoptosis and enhances the radiosensitivity of cancer cells, such as colorectal cancer cells both in vitro and in vivo8. The mammalian NF-κB transcription factor family is composed of five members: RelA (p65), RelB, cRel, NF-κB1 (p105/p50) and NF-κB2 (p100/p52)9. These proteins form homodimeric and heterodimeric complexes that, under non-stimulated conditions, are sequestered in the cytoplasm through interactions with the inhibitory proteins of the IκB family. The IκB protein family includes, at least, p100, p105, IκBα, IκBβ, IκBγ, IκBε. Following stimulation with a broad range of stimuli such as cytokines, virus, genotoxic agents and radiations, the inhibitory IκBα are phosphorylated by the IκB kinase complexe (IKK) at specific serine residues (Figure 1), leading to their interaction with and next ubiquitination by the SCF-βTrCP, and finally degradation by the proteasome pathway. NF-κB dimers are subsequently released and free to translocate into the nucleus to activate the transcription of their target genes10. Since IκB-βTrCP protein-protein interaction plays an essential role in controlling NF-κB activation, targeting IκB/βTrCP proteinprotein interaction has arisen as a promising new approach to inhibit NF-κB activity limiting unspecific events responsible for the high toxicity of proteasome inhibitors. It is well known that IκBα, Vpu, and β catenin share a common phosphorylated motif, DpSGXXpS called the phosphodegron motif, that is sufficient for interaction with β-TrCP11 (Table 1). Previous results on mapping the binding epitope of these βTrCP ligands have been published12–14. NMR studies using a transfer NOE technique such as STD NMR experiments have been established as an important technique to identify binding activities and characterize binding epitope of ligands. Here, we have investigated NMR and Molecular docking studies of p100 with βTrCP using a 21 mer p100 peptide containing the phosphodegron motif (here after referenced as 21P-p100). These results, compared to those obtained with IκBα allowed us to predict ligand-binding modes and consider βTrCP protein structural variations in ligand binding.

Results NMR assignment of 21P-p100 peptide containing the phosphodegron motif 2 ACS Paragon Plus Environment

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From the 2D experiments HH-TOCSY and HH NOESY acquired, it was possible to assign all the proton resonances from the 21P-p100 peptide (Table 2).The chemical shift data of the free 21P-p100 peptide is shown in Table S1 of the supplementary data, and the temperature change allowed us to obtain information about intramolecular hydrogen bonded or solvent shielded. Amide proton resonances are located in a small area in the aromatic region of the spectrum between 9.13 and 8.27ppm. The assignment strategy consisted in starting from specific resonances from unique residues in the sequence. The unique tyrosine residue in the sequence is also the unique aromatic residue. It has two isolated resonances respectively at 3.00 and 3.12 ppm which can be unambiguously identified as HB2 and HB3 protons. This residue is neighbor to a unique glycine residue and to an alanine which both have specific resonances. Another starting point consisted in identifying the isolated HB2 and HB3 spins of the Asp864 at 2.65 and 2.78 ppm. By following the HN-HN correlations in the NOESY spectrum from the triplet initially identified Ala866, Tyr867, Gly868, it was possible to determine the sequential assignment of the peptide. The complete side-chain assignment was determined by the correlations in the TOCSY spectrum. All assignments are reported in Table 2. It is interesting to note that the two amide protons from the phosphoserine residues have the most deshielded proton chemical shift respectively at 9.13 and 9.07 ppm separated from the rest of amide resonance by a significant gap of 0.2 ppm. Among the 21 amide proton resonances in the peptide, the 19 amide protons from the non-phosphoserine residues cluster in a small area of 0.56 ppm wide. Therefore, this region is very crowded and a significant overlap is observed. This particular property of amide chemical shift from phosphoserine residues was also observed previously in other peptides derived from β-TrCP substrates13,15–17which also contain a recognition motif including two phosphoserines (Table 1). Epitope mapping of 21P-p100 in interaction with β-TrCP From the NMR assignment, the main 21P-p100 residues involved in β-TrCP binding can be identified. To investigate the interaction of the 21P-p100peptide with β-TrCP, we performed STD18–22 and TRNOE NMR experiments23,24. In the STD experiment, the subtraction of saturated resonances of the protein spectrum from a reference spectrum allows us to observe the enhancements of the resonance of protons in close contact with the protein. We can thus define the amino acid of the peptide in interaction with the protein. In order to confirm the STD results, we made the same experiment without any β-TrCP protein. So no ligand signals are observed because saturation transfer does not occur without the protein. The Figure S1 shows clearly that STD signal is only observed for the sample containing the β-TrCP protein. The amide protons spectral region is well resolved and can be used to classify the amino acid residues relevant for interaction with the protein. By overlapping the spectrum obtained by STD with the 1D reference spectrum and rescaling intensities, a STD ratio can be determined for each residue. By definition, STD ratio corresponds to the following formula: STD ratio = (Ionresonance - Ioff resonance) / (Ion resonance). Since the difference can be directly obtained on the spectrum without splitting, an alternative approach consists in comparing STD spectrum with 1D spectrum. From the observable data: ISTD = Ion resonance - Ioffresonance ; STD ratio = (ISTD) / (I1D) x SNR (SNR: signal to noise ratio). Then, a normalization of 100% is applied to the residue experiencing the highest enhancement or amplification. The corresponding STD ratio values for each residue are shown in Figure.2. The pSer865 HN proton, the largest signal of the 21P-p100 peptide was selected and set to 100%. All other signals were normalized to this signal and we can thus evaluate the STD effect. Among the amino acids experiencing the highest STD ratio (above 70%), four of them -pSer865, 3 ACS Paragon Plus Environment


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Tyr867, Gly868 and pSer869- are included inside the 864DpSAYGpS869 consensus recognition βTrCP motif. The closest proximity of this group to the β-TrCP surface might be an indication that this part of the molecule is buried deeper into the β-TrCP ensemble forming hydrogen bond interactions or involved charged group. The lowest intensities (0.5Å. PyMOL (http://www.pymol.org/) was used for the analysis and presentation of the results of structure determination. Docking Docking simulations were carried out using the Surflex-Dock program35. As previously described12, we used a L-0.3-1 protomol (L=ligand; proto-thresh=0.3 and proto-bloat=1) for docking studies of 21P-p100. Surflex-Dock scores36were expressed in -log(Kd) units to represent binding affinities. During the docking procedure, the β-TrCP protein frame was fixed, whereas the 21P-p100 peptide was allowed to move. The results were analyzed according to the energy terms, intramolecular and intermolecular interactions The STD-NMR data allow us to define the different atoms in interaction with the protein and then the buried surface. We can observe a lot of hydrogen bonds and hydrophobic contacts. Building of mutants The five mutants were built with the “build mutants” protocol which mutates R285, R410, R431, R474 and R521 to Glutamic acid (E) and optimizes the conformation of both the mutated residues and any neighboring residues. The model with the best score was selected for the docking simulation. All calculations were performed in Discovery Studio. Flexible ligand-rigid protein docking was performed using CDOCKER 37. Using a binding site sphere we can specify 10 ACS Paragon Plus Environment

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the ligand placement in the active site. The pose showing the lower energy was retained for the molecular dynamic simulation. Molecular Dynamics The molecular dynamic (MD) simulation was performed with the NAMD 2.9 program implemented in DS 4.1 (Biovia) 38 and the system was modeled with CHARMM36 forcefield39. The crystal structure (PDB code 1P22) was placed in the center of an orthorhombic box solvated by a solution of 0.15M of NaCl in water followed by minimization, and the protonation state of ionizable residues was set to a neutral pH (pH=7)40. Periodic boundary conditions were used and the simulation was performed in NPT ensemble. The solvation was simulated by added 11455 water molecules, 36 sodium ions and 30 chloride ions. Then a minimization was perform using Adopted Basis Newton-Raphson algorithm until a minimization RMS gradient of 1.0 kCal/(mol*Å2) or a minimization maximum step of 500 cycles. A 20ns MD simulation was performed for data acquisition using NAMD with NPT (Constant-temperature, constant-pressure ensemble) which classically mimics experimental conditions. The temperature (300K) and the pressure were controlled using Langevin dynamic and Piston.200 conformations were obtained and analyzed. The root mean square deviation (RMSD)of the backbone was calculated during the simulation using the first frame as a reference. Residues involved in ligand-protein interaction were displayed using the LigPlot program41. Acknowledgements This work was supported by grants from Agence Nationale pour la Recherche (ANR OncoKappaB) and postdoctoral funding from ANR (to MM). We thank Antonio Cuadrado (Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM, Madrid, Spain) for wild-type β-TrCP and R285E, R410E, R431E, R474E, R521E point mutants. We thank Carol Burgess, the Biovia support for her help in the molecular modeling. Supporting Information Supporting Information Available: Table S1. 1H NMR chemical Shifts of the free peptide 21P-p100 in ppm from TSP-d4 and amide signal Shift temperature coefficients (∆(δ NH)/∆T) in ppb K-1 Table S2. Noesy peaklist used to calculate distance restraints for structure calculation Table S3. Comparison of amide chemical shifts of β-TrCP partners during interaction Figure S1. Superposition of the region containing resonances of the amide protons of the 21Pp100.A) 1D 1H STD-NMR of 21P-p100 alone, B) 1D 1H STD-NMR of 21P-p100/β-TrCP, C) 1D 1 H of 21P-p100. Figure S2A. Superposition of the low energy structure of the 21P-p100 corresponding to the two clusters. Figure S2B. . Structure of the complex of β -TrCP with the 21P-p100. The residues with a higher RMSF are represented in Ball and stick. Figure S3. Amide chemical shifts comparison of different β-TrCP partners during interaction around the conserved D-pS-(AG)-long-X-pS motif This material is available free of charge on the ACS Publications website



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FIGURE LEGENDS FIGURE1.Canonical and non-canonical NF-κB pathways.In the classical or canonical pathway, activation of IKK complex leads to the phosphorylation by IKKβ of two specific serines near the N terminus of IκB-α, which targets IκBα for ubiquitination (generally by a complex called βTrCP) and degradation by the 26S proteasome. In the non-canonical (or alternative) pathway, the p100-RelB complex is activated by phosphorylation of the C-terminal region of p100, which leads to ubiquitination followed by degradation of the p100 IκB-like C-terminal sequences to generate p52-RelB. In either pathway, the unmasked NF-κB complex can then enter the nucleus to activate target gene expression. FIGURE 2. STD intensity ratio plot measured on 21P-p100 peptide in presence of β-TrCP. The integral value of the largest signal of the 21P-p100 peptide, pSer865 HN proton, was set to 100%. The degree of saturation for the individual protons normalized to that of the pSer865 can be used to compare the STD effect. FIGURE 3.Structures of the 21P-p100 peptide bound to β-TrCPand analysis of the docking interactions. (A)Of 50 TRNOE-derived structures, the five lowest-energy conformations are shown with a superimpositionfrom residues864 to 869.(B) The seven lowest-score configurations obtained by docking are representative of the bound structures of p100. The superimposition was made on the DpSAYGpS motif.(C) Best p100 docked structure (cyan) superimposed on the TRNOE bound structure (red) on the DpSAYGpS motif. FIGURE 4.(A)Root mean square deviation (RMSD)of backbone atoms. (B)Root mean square fluctuation (RMSF) of the 20 ns molecular dynamic simulation for the 21P-p100/β-TrCP complex. (C) Potential energy values of 20 ns molecular dynamic (MD) simulation. FIGURE 5.(A) IKKα potentiates β-TrCP-p100 interaction. HEK293 cells were transfected with either wild type Flag-β-TrCP and p100, or wild type Flag-β-TrCP and p100 along with HAIKKα. Whole cell extracts were subjected to immunoprecipitation (IP) with M2 (FLAG) antibody, and analyzed for associated p100 by immunoblot (IB).(B)In vivo validation that R285, R410, R431, R474 and R521 within β-TrCP are required for interaction with p100. HEK293 cells were transfected with equivalent amounts of the indicated expression plasmids and whole cell extracts were subjected to immunoprecipitation (IP) with M2 (FLAG) antibody, and analyzed by immunoblot (IB) for associated p100. FIGURE 6.. (A) Docking result: Top view β-TrCP residues that participate in hydrophilic intermolecular interactions including Tyr271, Arg285, Ser325, Leu351, Lys365, Asn394, Arg410, Arg431, Ser448, Arg474, Tyr488 and Arg521 (green). H-bonds are displayed with dashed black lines. (B) Molecular dynamic result: 2D diagram plot underline the atom and residues which are crucial in stabilizing the complex. The different contacts, hydrogen bonds, electrostatic and hydrophobic interaction are represented by dotted lines. FIGURE 7.Results of docking studies starting with the NMR bound structure of IκB-α. (A) Close-upview of the interface between the β-TrCP and IκB-α. The IκB-α peptide is represented in pink. (B)Portion of the seven β-propeller structure of the β-TrCP protein corresponding to the 15 ACS Paragon Plus Environment


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active site is shown with the IκB-α peptide. The different contacts, hydrogen bonds, electrostatic and hydrophobic interaction are represented by dotted lines.


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Figure 1.



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Figure 2.

*: Not determined due to signal overlapping


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Figure 3 A.

pSer869

pSer865 Tyr867

B.

Gln870 Asp864 Ala866 pSer869

pSer865

Gly868 Tyr867

C.

pSer865 pSer869

Tyr867



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Figure 4 A

B

C


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Figure 5



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Figure 6 A.

B.


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Figure 7 A

B.



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Table 1: Comparison between target recognition sequences of the different β-TrCP partners. Highlighted serine residues in DSXXXS conserved motif require phosphorylation prior recognition by β-TrCP. Name 21P-p100 24P-IκB-α 22P-Vpu 11P-β -Catenin

Sequence number 858-878 21-44 41-62 30-40

Sequence

STEVKEDSAYGSQSVEQEAEK KKERLLDDRHDSGLDSMKDEEYEQ LIDRLIERAEDSGNESEGEISA YLDSGIHSGAT

Table 2.Proton chemical shift of 21P-p100 peptide in presence of β-TrCP. Residue 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878

Ser Thr Glu Val Lys Glu Asp Sep Ala Tyr Gly Sep Gln Ser Val Glu Gln Glu Ala Glu Lys

δHN

δHα

δHβ

8.56 8.49 8.51 8.46 8.65 8.81 8.65 9.13 8.53 8.27 8.33 9.07 8.74 8.34 8.36 8.63 8.59 8.65 8.52 8.53 8.42

4.54 4.42 4.33 4.10 4.40 4.32 4,60 4.44 4.32 4.61 4.02 4.47 4.45 4.45 4.17 4.27 4.32 4.23 4.34 4.24 4.27

3.89 4.33 1.94 2.04 1.70 2.08 2.65 4.06 1.33 3.00

3.94

4.13 2.24 3.91 2.12 2.04 2.14 1.99 1.45 2.04 1.92

4.10 2.02 3.98

δHγ

2.06 1.81 1.93 2.78

1.24 2.23 0.96 1.45 2.30

δHδ

δHε

1.72

3.04

6.88

7.17

2.31

3.12

3.98 2.43

2.00 2.03 2.05

0.97 2.31 2.42 2.32

1.83

2.33 1.55


1.46

1.73

7.94

6.91

7.71

7.01

3.03

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Non-canonical NF-κB pathways and Side view of 21P-p100 in interaction with the β-TrCP protein.

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