Blanc SVSE 8 - Blanc - SVSE 8 - Biochimie, biologie moléculaire et structurale

Structural and functional investigations of the intrinsically disordered C-terminal domain of ErbB2 – ERBB2CTER

Structural and functional investigation of the C-terminal intrinsically disordered cytosolic fragment of ErbB2

Most previous studies of ErbB2 focused on the extracellular and trans-membrane domains of the protein and yielded an efficient anti-ErbB2 therapy against beast cancer. However, only one-third of ErbB2-positive tumors respond to trastuzumab treatment and those that do acquire resistance within three years after the initiation of treatment. The implication of the C-terminal cytosolic tail of ErbB2 (CtErbB2) in signaling pathways was established but is still poorly understood.

Gain structural and functional insights on the C-terminal cytosolic tail of ErbB2 (CtErbB2), by combining structural and cellular biology tools.

One of the challenge of this project will be to characterize in detail the structural features of this large intrinsically disordered domain (275 amino acids) free and bound to some of its identified cellular partners. We will more specifically analyze how the prolines , which are overrepresented in the sequence, and the phophorylation of the tyrosines, which is directly linked to downstream signaling processes, impact the structure, the dynamics and the function of the domain. <br />Taking into account our final objectives to analyze a supramolecular complex associated to the C-terminal domain of ErbB2, the present project contains five tasks. Task1 and Task2 will focus on the study of ErbB2 C-terminal derivative fragments in complex with various ErbB2 adaptors/kinase domains, in order to identify specific structuration of phosphorylated tyrosines-containing fragments upon the binding of different partners and investigate the importance of proline residues in adaptor/kinase domains recognition and in the flexibility of the unstructured domain. Task3 and Task4 will aim at the analysis of the influence of the binding of the adaptor/kinase domains at a longer range along the entire intrinsically disordered CtErbB2 by NMR and will address the question of the cooperativity and avidity of the adaptors interactions. Finally, Task5 will be an in vivo study using mutants of CtErbB2 and will be conducted in parallel to Tasks 1-4. The impact of the mutations on adaptor protein docking, intracellular signaling pathways and cellular functions (proliferation, survival and migration) will be determinant to propose an integrated structural model of ErbB2 C-terminal/adaptor complex.

The efficiency of our multidisciplinary approach is provided by the three teams including a group of structural biologists well equipped with very high field NMR spectrometers and expert in the analysis of protein dynamics and of large complexes involving intrinsically disordered proteins by NMR, a group of cell biologists, specialists of ErbB2 adaptors and a team of biophysicists well established in developing thermodynamics and structural studies of macromolecular complexes and who were previously involved in structural analysis of CtErbB2 fragments.
Classical NMR, ITC and SPR experiments will be used for tasks 1-4. The analysis of long, intrinsically disordered CtErbB2 fragments in tasks will necessitate very high field spectrometers and NMR experiments dedicated to disordered proteins. The structures ensemble of the various fragments produced will be inferred from (i) the analysis of the chemical shifts, (ii) relaxation data (iii) RDCs (Residual Dipolar Couplings) measured using orienting media such as PEG-hexanol or stretched polyacrylamide gels, (iii) PRE (Paramagnetic Relaxation Enhancement) experiments. These techniques are now well described in the literature. For PRE experiments, cysteine residues will be introduced at different places in the long fragments through point mutations by directed mutagenesis. In task 5, point mutations or small deletions of the Pro-rich regions surrounding specific Tyr residues will be performed by directed mutagenesis. Second, we will construct cellular models. Then, we will perform biological assays to measure the effect of the mutations on cell signalling, cell proliferation and cell migration.

Main results at december 15th 2014
• Production of the entire C-terminal domain for NMR studies (15N, 13C labeled)
• Assignment of most of the resonances of the backbone (and Cb) of CtErbB2
• On-going characterization of the structural features of the state ensemble of CtErbB2, from NMR relaxation experiments, RDCs and PRE. Although largely disordered, the chemical shifts analysis indicates that the domain is not fully random coil.
• First phosphorylation tests with ErbB2 kinase (small scale, mass-spectrometry analysis)
• Finalisation of the interaction study of a peptide fragment of CtErbB2 (1146-RPQPPSPRE) with the SH3 domain of Fyn (see publication)

1- Complete the structural and dynamic analysis of CtErbB2 using NMR (RDCs, PREs, relaxation) and SAXS
2- Mutagenesis of proline residues
3- Finalize the interaction study between FynSH3 and CtErbB2
4- Phosphorylation of CtErbB2 - Use of specific cells.
5- Pulldown experiments, first with the non-phosphorylated CtErbB2 to identify partners that do not interact with phosphorylated tyrosines, then with phosphorylated tyrosine(s). The histag construct will be used.
6- Set-up of in-cellulo experiments on cell lines that don't express ErbB2 (T47D) and that overexpress ErbB2 (SkBr3): Cter deletion will be a first step. Then, depending on the structural data, mutants will be designed and tested in order to analyze their impact on the signaling pathways.
7- Analysis of the interactions between CtErbB2 and Grb2(SH2SH3 or SH3SH3SH2), MEMO and Shc. This will necessitate the control of the tyrosine phosphorylations.
8- The results of these interactions studies will drive further mutants analysis in vivo.

Bornet O, Nouailler M, Feracci M, Sebban-Kreuzer C, Byrne D, Halimi H, Morelli X, Badache A, GuerlesquinF. Identification of a Src kinase SH3 binding site in the C-terminal domain of the human ErbB2 receptor tyrosine kinase. (2014) FEBS Lett. 588(12):2031-6.

Overexpression of ErbB2 results in constitutive ligand-independent activation of tyrosine kinase signaling. Experimental studies have revealed that ErbB2 shows many features of a therapeutic target: ErbB2 is able to confer many characteristics of a cancerous cell, including uncontrolled proliferation, resistance to apoptosis and increased motility. Most of the previous studies focused on the extracellular and trans-membrane domains of the protein and yielded in particular to an efficient anti-ErbB2 therapy against beast cancer. However, only one-third of ErbB2-positive tumors respond to trastuzumab treatment and those that do acquire resistance within three years after the initiation of treatment. Our purpose is here to gain structural and functional insights on the C-terminal cytosolic tail of ErbB2 (CtErbB2), whose implication in signaling pathways is established but still poorly understood. One of the challenge of this project will be to characterize in detail the structural features of this large intrinsically disordered domain (275 amino acids) free and bound to some of its identified cellular partners. We will more specifically analyze how the prolines, which are overrepresented in the sequence, impact the structure, the dynamics and the function of the domain. The use of NMR, SAXS and modeling approaches will be essential to carry out this project and our extensive background in ErbB2 signaling pathway, reinforced by cellular analyses including cell proliferation and cell migration, will allow integrating the structural data in a cellular context.
The efficiency of this multidisciplinary approach is provided by the three teams including a group of structural biologists well equipped with very high field NMR spectrometers and expert in the analysis of protein dynamics and of large complexes involving intrinsically disordered proteins by NMR, a group of cell biologists, specialists of ErbB2 adaptors and a team of biophysicists well established in developing thermodynamics and structural studies of macromolecular complexes and who were previously involved in structural analysis of CtErbB2 fragments.
Taking into account our final objectives to analyze a supramolecular complex associated to the C-terminal domain of ErbB2, the present project contains five tasks. Task1 and Task2 will focus on the study of ErbB2 C-terminal derivative fragments in complex with various ErbB2 adaptors/kinase domains, in order to identify specific structuration of phosphorylated tyrosines-containing fragments upon the binding of different partners and investigate the importance of proline residues in adaptor/kinase domains recognition and in the flexibility of the unstructured domain. Task3 and Task4 will aim at the analysis of the influence of the binding of the adaptor/kinase domains at a longer range along the entire intrinsically disordered CtErbB2 by NMR and will address the question of the cooperativity and avidity of the adaptors interactions. Finally, Task5 will be an in vivo study using mutants of CtErbB2 and will be conducted in parallel to Tasks 1-4. The impact of the mutations on adaptor protein docking, intracellular signaling pathways and cellular functions (proliferation, survival and migration) will be determinant to propose an integrated structural model of ErbB2 C-terminal/adaptor complex.

Project coordination

Carine VAN HEIJENOORT (Institut de Chimie des Substances Naturelles)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRS DR12 -LISM Centre National de la Recherche Scientifique Délégation Provence et Corse _ Laboratoire d'Ingenierie des Systèmes Macromoléculaires
INSERM-CRCM Centre de Recherche en Cancérologie de Marseille
CNRS-ICSN Institut de Chimie des Substances Naturelles

Help of the ANR 319,561 euros
Beginning and duration of the scientific project: December 2013 - 42 Months

Useful links

Explorez notre base de projets financés

 

 

ANR makes available its datasets on funded projects, click here to find more.

Sign up for the latest news:
Subscribe to our newsletter