The effects of different polymer nanodiscs on extracting the full-length complexes of platelet receptor GPVI

Secondary Supervisor(s): Dr Hoor Ayub

University of Registration: University of Birmingham

BBSRC Research Themes: Understanding the Rules of Life (Structural Biology)

Project Outline

Glycoprotein VI (GPVI) is the major signalling receptor for collagen on platelets. GPVI is also activated by additional ligands including fibrin, fibronectin, histones and snake venom toxins. Ligand binding to the extracellular domains of GPVI results in platelet activation via an intracellular Syk dependent phosphorylation cascade. GPVI associates with the FcRγ-chain in the membrane, and this association is critical for signal activation. GPVI is a promising anti-thrombotic target due having major roles in thrombosis but limited roles in haemostasis. Despite its importance in platelet activation and thrombosis, we have only a rudimentary understanding for the mechanisms of how the receptor complex is activated. This is largely due to structural studies being limited to the isolated extracellular domains which provides little context for the structure of full-length GPVI in the membrane and how intracellular signalling is induced. All we know is that clustering plays a role in GPVI activation.

In attempts to improve our mechanistic understanding for GPVI activation in the membrane, we have used styrene maleic acid (SMA) polymer to extract the full-length GPVI:FcRγ complex from the cell membrane into nanoscale membrane discs (‘SMALPs’) and are using cryo-electron microscopy (EM) to study the full-length structure (Wang et al, 10.1016/j.abb.2024.109944). Furthermore, we have developed GPVI specific multivalent nanobodies, that potently activate GPVI (Martin et al, 10.1016/j.jtha.2023.09.026) and aim to use these ligands to extract the active receptor complex and study structural changes that occur during activation. Extractions have been performed using SMA200, and it is unclear if this polymer is successfully extracting the larger GPVI complexes as no clear increases in molecular weight have been visualised upon addition of the multivalent nanobody. To successfully extract the activated GPVI clusters we must test alternative ‘SMALP’ discs of different sizes.

Objectives

In this project, we will test different polymers of varying compositions and sizes in their ability to extract the full-length GPVI:FcRγ complex in resting and activated states for use in downstream biochemical and structural studies. The overall aim is to determine the structural events that occur upon GPVI activation. This will provide insight into the mechanisms for how this receptor complex induces downstream signalling and platelet activation.

Methods and experimental approach

This work will be a collaboration between the Universities of Birmingham and Coventry. Birmingham will be the primary host-institute, with membrane solubilisations being performed at Coventry. GPVI and FcR-γ will be transfected into mammalian cells and membranes will be isolated. Membranes will be solubilised with different SMA-like polymers with different styrene:maleic acid ratios, and diisobutylene-maleic acids (DIBMA). The solubilised complex will be purified by affinity chromatography and the complex size monitored by size exclusion chromatography, native page electrophoresis and analytical ultra centrifugation. To ensure the receptor complex remains active in the solubilised particles, binding assays to natural ligands including collagen will be performed. Purified complexes will be visualised using negative stain EM and, if particle quality is good enough, taken forward to cryo-EM for structural determination.

In addition to transfected cells, the membranes of washed platelets, in the presence and absence of nanobody agonists, will be solubilised using different SMA-like co-polymers. The relative ratio of GPVI:FcR-γ and nanobody will be determined by mass spectrometry. Furthermore, the presence of downstream signalling proteins that associate with GPVI, including Src and Syk kinases, will also be measured which will reveal which polymer is best for extracting the large GPVI signalosome and provide structural insight for how these larger complexes assemble.