NESIAC · Nanobody-enabled Structural Investigation of a G Protein-Coupled Receptor-Arrestin Complex
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2015-07-01 → 2017-06-30
- EU contribution
- €160,800
- Participants
- 1
- Scheme
- MSCA-IF
Lines connect the coordinator with its partners.
Results in brief
Nanobody-enabled Structural Investigation of a G Protein-Coupled Receptor-Arrestin Complex
The Steyaert lab pioneered the use of nanobodies (Nbs) for chaperone-assisted X-ray crystallography, aiming for the highest hanging fruits of structural biology. The host lab has several patents pertaining to the use of Nbs as tools that stabilize protein conformations or protein complexes. In 2014 the Flemish government funded the lab to establish Nanobodies4Instruct, extending the cutting-edge technology and scientific expertise to Instruct members. In the host lab the Nb platform is routinely used for purification purposes, crystallization and structure determination of stabilized conformers or protein complexes with applications extending to drug discovery, as research tools, as well as for diagnosis and treatment of diseases. In July 2014 the research fellow, Dr Róise Mc Govern, joined the Steyaert lab. After initial training in the cell biology techniques and biophysical methods -central to the Nb platform and this project- the fellow quickly began working independently. Róise applied the Steyaert lab’s state of the art nanotechnology to several new high profile protein complexes. The goal of which was to identify nanobodies for the stabilisation and structural characterisation of chanllenging PPIs including: 1. β2AR•Arrestin-2 complex 2. Rho•Visual Arrestin 3. Arrestin-2•pERK2 & Arrestin-2•pERK2•V2R-pp 4. KLB•FGFR1•FGF19
Data: CORDIS, © European Union
Project objective
Although many thousands of transient protein-protein interactions (PPIs) are known, there is a disturbing paucity of high-resolution structures of the resulting complexes and the difficulties involved in experimentally determining these atomic structures remain essentially unaddressed.The Steyaert lab has shown that cross-linking transiently interacting proteins, followed by immunization of llama’s with this cross-linked antigen, causes the maturation of single domain antibodies called Nanobodies (Nbs). The Nbs bind composite conformational epitopes unique to the transient complex. Highly efficient selection methods can discriminate Nbs that exclusively bind the transient (non-cross-linked) complex from binders that bind to the dissociated monomers. Such Nbs will be instrumental to purify and solve the structures of PPIs that have been resistant to investigation by X-ray, NMR, SAXS or EM and for the functional analysis of these complexes within a living cell. This ground-breaking technology will be validated with well-chosen case studies covering key PPIs of the GPCR transmembrane signaling pathway including parts of the arrestin interactome. During this project the fellow will establish a unique research niche by systematically applying her arsenal of learnt techniques to develop the next generation antibody‐enabled methods for the structural investigation of the GPCR-arrestin targets. The ultimate goal of NESIAC is to determine the atomic structures of the most relevant transient associations of these signaling proteins. This will open up a new platform for realising the structural basis of the elusive GPCR regulation by arrestins.
Original text from CORDIS.
Participants
- VIB VZW · ZWIJNAARDE - GENTCoordinatorBelgium
Links
- View on CORDIS
- DOI: 10.3030/660753
- https://web.archive.org/web/20170515143705/http://steyaertlab.structuralbiology.be/Nanobodies
Data: CORDIS, © European Union