[SCAN] Integrative structural biology of full-length, flexible and oligomeric multidomain proteins involved in pathogenicity.

Title: Integrative structural biology of full-length, flexible and oligomeric multidomain proteins involved in pathogenicity.

Speaker: Tiago Gomes

Abstract:The transforming growth factor β pathway (TGFβ) plays decisive roles in embryo development, tissue homeostasis, and immune responses. When unregulated, it can promote tumor invasion and fibrosis. Recently, it has also become apparent the involvement of TGFβ in enforcing coronaviruses pathogenicity, namely SARS-CoV-2. The TGFβ pathway is hyperactivated, in a SARS-CoV-2 infection, leading to tissue fibrosis and immune deregulation, with a poor patient prognosis.

Smad (mothers against decapentaplegic) proteins are TGFβ principal effectors. Until now, no structural information regarding full-length Smad proteins has been put forward. Using an integrative structural biology approach unifying nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS) and mass spectrometry, we have characterized the structures of full-length Smad2 and Smad4 in solution.

We show that Smads inter-domain linker behaves as an intrinsically disordered protein (IDP) tethering the MH1 (DNA binding) and MH2 (protein binding) globular domains within flexible multidomain Smad2 and Smad4. In solution, Smads display different oligomeric states. Smad4 is monomeric and not predominantly in an auto-inhibited conformation as previously postulated. Smad2 is an oligomeric protein in a monomer-dimer-trimer equilibrium, shaped by phosphorylation and its MH1 domain.

Smad proteins interact with many human and pathogen targets. The Nucleocapsid (N) protein from coronaviruses is postulated to interact with Smads, driving aberrant TGFβ signaling. We have shown that the N protein is also a flexible and oligomeric protein that undergoes liquid-liquid phase separation (LLPS) phenomena, though to be crucial for pathogenicity.

By providing insights into Smads and N protein full-length structures we have established a methodology to tackle the structure of large and flexible multidomain proteins. This will be extremely valuable in subsequent studies targeting the SARS-CoV-2 interactome, and other multidomain  proteins.