Structural insights of stomatin from native human platelets by cryo-EM coupled with “Build and Retrieve” (BaR) data processing. Free

Background: Stomatin is an integral membrane protein known for cholesterol binding, membrane stiffening, regulating ion channel activity, and interacting with the cholesterol-rich lipid raft membrane domains. It primarily resides on α-granules as a key lipid raft component in resting platelets and translocates to the platelet surface following platelet activation. However, the structure of the full-length, membrane-associated platelet stomatin complex is unknown, and the mechanism by which stomatin helps organize and function in platelet α-granules remains unclear. Here, we determined the structure of stomatin from native platelet membranes using cryo-EM and Build and Retrieve (BaR) data processing to provide new insights into the complex's architecture. Further, our platform of advanced data processing allows solving high-resolution structures from crude preparations via in silico purification from large heterogeneous datasets.

Methods: Whole blood from a healthy donor was collected, and the platelets were isolated via gel filtration in HEPES-Tyrode's buffer. Gel-filtered platelets were activated with 10nM thrombin. Membrane proteins were solubilized with 1% DDM and assembled into lipidic nanodiscs. Following size exclusion chromatography, the mixture of proteins was directly applied to cryo-EM grids. Data were collected on a Titan Krios G3i cryogenic transmission electron microscope equipped with a BioQuantum K3 camera. All data processing was done in cryoSPARC using the BaR protocol. The near-atomic-resolution cryo-EM maps were used for protein identification via the DeepTracer server. The final protein models were built by Coot and refined by PHENIX.

Results: Using cryo-EM and the BaR method, we solved the first full-length platelet-derived stomatin structure from thrombin-activated human platelet membranes at 2.9 Å resolution. The structure reveals a cage-like oligomer with a wider shoulder bottom (147.5 Å) and a narrower top cap (83.2 Å). The cap folds inward to form a narrow β-barrel hydrophobic pore (23.5 Å). Since the protein is extracted directly from its native source, some unassigned density near the pore suggests a potential cofactor that may contribute to the selectivity for small molecules. The complex is formed by 16 31-kDa monomers via conserved salt bridges, notably between E59 and R70, R205 and E262, K220 and E227, K235 and E243. Further, our structure shows stomatin anchors to the membrane via a unique mechanism involving H1 and H2 helices at the N-terminus. The proline between the two helices, P47, defines the angle at which the two helices form a hairpin element to interact with the membrane. Three cysteines, C30, C53, and C87, serve as conserved palmitoylation sites that further support membrane attachment. This mechanism allows stomatin to anchor to a curvature-resistant membrane, supporting the existence of preassembled α-granular lipid rafts on human platelets.

Conclusions:We used cryo-EM with an iterative bottom-up data processing method, BaR, to uncover novel structures of proteins at near-atomic resolution directly from human platelet membranes. The native-state structure of platelet-derived stomatin provides unprecedented insights into its functions. We are now positioned to determine stomatin's roles in platelet and hemostasis. More broadly, our approach further opens new avenues for an in-depth systems biology approach with structural biology that will uncover how the native environment regulates platelet function at the molecular level.