PCV - Programme interdiciplinaire en physique et chimie du vivant

Determinants of specificity and affinity in partner recognition by intrinsically disordered proteins – AFF-IDP

Submission summary

Recent studies have shown that as much as one third of eukaryotic proteins contain long intrinsically disordered regions, with 12% of them being fully disordered. Intrinsically disordered proteins (IDPs) are functional proteins that fulfill essential biological functions while lacking highly populated constant secondary and tertiary structure under physiological conditions. Most IDPs are involved in functions that imply multiple partner interactions, such as molecular recognition, molecular assembly (and amyloidogenesis), cell cycle regulation, signal transduction and transcription. As such, IDPs are implicated in the development of several pathological conditions, including cancer and cardiovascular diseases, and have been shown to be promising new targets for rational drug design. A distinguishing feature of IDPs is their ability to bind multiple partners while maintaining specificity. Although their promiscuity is thought to be encoded by the high plasticity conferred by the lack of a rigid scaffold, no systematic study has been undertaken so far to address the molecular features that encode the selectivity of IDPs and their binding efficiency. The understanding of the mechanisms by which regions of disorder mediate recognition of multiple partners is therefore relevant to human health and will contribute to extend and improve our present comprehension of the molecular basis of protein-protein interactions and protein networks, an ambitious goal of current efforts in systems biology. The primary goal of this project is to identify the molecular determinants of specificity and affinity of partner recognition by IDPs. To reach this goal, we will combine in vitro, in silico and in vivo approaches. We will use an in vitro evolution approach to generate amino acid diversity within an intrinsically disordered protein domain, and we will assess how these amino acid substitutions affect its partner recognition profile and protein function in a relevant biological system. The model system used in this study will be the intrinsically disordered C-terminal domain of the MeV nucleoprotein (NTAIL), a well characterized disordered domain that was shown by Partner 1 to undergo a-helical induced folding upon interaction with the MeV phosphoprotein X domain (XD), and to display both partner multiplicity and selectivity. A library of NTAIL mutants will be generated by using random mutagenesis. Generation of this sequence diversity will be coupled to an efficient screening method based on reconstitution of the green fluorescent protein (GFP), thus allowing the easy identification of clones with an altered interaction pattern. The NTAIL coding regions brought by these clones will be sequenced and a subset of these mutated NTAIL proteins will be purified and characterized in terms of their (in)ability to interact with the "natural partner" (i.e. MeV XD) and three model proteins that will serve as the "non-natural" partners: the Sendai virus X domain (SeV XD), the intrinsically disordered N-terminal domain of MeV P (MeV PNT), and the regulatory domain of IRF3 (IRF3-RD). The rationale for the choice of these latter proteins as "non-natural" partners, rather than of fully irrelevant proteins, resides in the fact that MeV PNT and IRF3-RD are true potential partners in infected cells and that the functional impact of their interaction with NTAIL can be assessed in a cellular context. As for SeV XD, its high structural similarity with MeV XD affords a way of investigating the role of specific protein-protein contacts within a similar structural scaffold, with the functional impact of its interaction with NTAIL being also accessible to investigation in a cellular context. The interaction between NTAIL variants and the partner proteins will be investigated using a plethora of spectroscopic approaches that Partner 1 has already proven to be well-suited to document the MeV NTAIL-XD interaction, as well as surface plasmon resonance and isothermal titration calorimetry. The complexes between NTAIL variants and their structured partners (i.e. MeV XD, SeV XD and IRF3-RD) will be studied by using both direct structural (X-ray crystallography and NMR) and in silico (molecular dynamics simulation) approaches. The functional impact of NTAIL substitutions will also be assessed in vivo in the context of MeV infection. A library of N variants will be generated by incorporating the NTAIL mutated gene fragments in the N gene. The constructs will be used to transfect infected cells where the expression of endogenous N has been silenced. The impact of the NTAIL mutations on the rates of transcription and replication, and on the production of functional nucleocapsid template will be assessed. To reach this goal, three teams are combining their complementary skills and expertise in a highly multidisciplinary collaborative project, gathering biochemistry, molecular and cellular biology, structural biology and physics.

Project coordination

Sonia LONGHI (Université)

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

INSERM DR RHONE-ALPES AUVERGNE

Help of the ANR 490,000 euros
Beginning and duration of the scientific project: - 48 Months

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