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Nanobody-enabled Structural Investigation of a G Protein-Coupled Receptor-Arrestin Complex

Periodic Reporting for period 1 - NESIAC (Nanobody-enabled Structural Investigation of a G Protein-Coupled Receptor-Arrestin Complex)

Reporting period: 2015-07-01 to 2017-06-30

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
Using these research tools, a milestone was met for β2AR●Arrestin-2 complex under the supervision of Prof Steyaert. By using yeast display in combination with FACS, the research fellow identified Nbs that stabilize the β2AR●Arrestin-2 complex. The project was carried out in close collaboration with the 2012 Noble Laureate, Robert Lefkowitz. The Lefkowitz lab supported additional characterization of the complexes. As the β2AR●Arrestin-2 complex is prized as a high risk / high gain target in structural biology the project continues to develop.

Prof. Vsevolod Gurevich is a leading expert in GPCR signal transduction pathways. Róise worked closely with the Gurevich lab and initiated FACS screens, in combination with yeast display, to enrich Nbs that stabilize the Rho•Visual Arrestin complex.

Róise focused significant attention on the Arrestin-2•pERK2 & Arrestin-2•pERK2•V2R-pp complexes. To initiate this project the research fellow undertook a collaborative stay at the Gurevich lab at the Vanderbilt University School of Medicine, USA. Here, Róise learned the technique for producing and purifying Arrestin-2 and brought the knowledge and skill back to the Steyaert lab. The fellow produced and purified activated MAP/ERK kinases from E.coli using a bi-cistronic vector, which was obtained the Cob lab. Róise spent a significant time 1) characterizing the Arrestin-2•pERK2 & Arrestin-2•pERK2•V2R-pp complexes using an array of biophysical methods and 2) cross linking the complexes for immunization.

From the library created from immunization of Endocrine FGF●FGFR●Klotho co-receptor signal transduction unit, the research fellow identified a number of different Nb families that bind singular or multiple components of the signal transduction unit using eth Nb platform. These include Klotho binders, FGFR1c binders and complex binders. The project was carried out in collaboration with Prof. Schlessinger’s Lab, Yale University School of Medicine Conneticut. Further characterisation of the binders is expected to yield new high profile protein complexes.

Additional responsibilities undertaken by the fellow include maintaining lab standards, planning and writing research practicals for MSc students. Róise joined the Keystone Symposia on G Protein-Coupled Receptors: Structure, Signaling and Drug Discovery in 2016 and undertook several training courses provided by VIB. These include Women in Science, VIB Leadership Program and ePDB workshop. Through these projects Róise has diversified her skill-set, managing research projects and broadening her professional network.
Using these research tools, a milestone was met for β2AR●Arrestin-2 complex under the supervision of Prof Steyaert. By using yeast display in combination with FACS, the research fellow identified Nbs that stabilize the β2AR●Arrestin-2 complex. The project was carried out in close collaboration with the 2012 Noble Laureate, Robert Lefkowitz. The Lefkowitz lab supported additional characterization of the complexes. As the β2AR●Arrestin-2 complex is prized as a high risk / high gain target in structural biology the project continues to develop.

From the library created from immunization of Endocrine FGF●FGFR●Klotho co-receptor signal transduction unit, the research fellow identified a number of different Nb families that bind singular or multiple components of the signal transduction unit using eth Nb platform. These include Klotho binders, FGFR1c binders and complex binders. The project was carried out in collaboration with Prof. Schlessinger’s Lab, Yale University School of Medicine Conneticut. Further characterisation of the binders is expected to yield new high profile protein complexes.
Nb binding profiles by FACS