Elucidating the Structural Dynamics and Native Modifications of α2-Macroglobulin from Native Platelet Samples By Cryo-EM Coupled with "Build and Retrieve" (BaR) Data Processing

Background:

α2-macroglobulin (α2M) is a broad-spectrum protease inhibitor that regulates both pro- and anti-coagulant processes. Additionally, it captures and neutralizes cytokines that influent hemostasis. These multifaceted roles have made it challenging to truly evaluate the contribution of α2M on hemostasis and thrombosis. The presence of α2M in platelets was first observed in 1976 and confirmed with proteomic tools in 2021. Despite the numerous studies on the α2M mechanism of action, its role in platelets is still unclear. Further, the native membrane environment provides essential regulatory cues that impact the protein structure and mechanism of action. Here, we have determined the structure of α2M from native platelet membranes using single-particle cryogenic electron microscopy (cryo-EM) coupled with Build and Retrieve (BaR) data processing. Our recent breakthrough in data processing makes it possible to obtain high-resolution protein structures from crude preparations in their native environments by performing in silico purification, image sorting, and model building from large heterogeneous datasets. This approach provides new insights on the function of α2M.

Methods:

Whole blood was collected, and the platelet membrane proteins were solubilized by Styrene maleic acid lipid particles (SMALPs) 200 to isolate proteins from their endogenous source. Following size exclusion chromatography, the mixture of proteins was directly applied on cryo-EM grids. Data were collected on 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. The final protein models were built by Coot and refined by PHENIX.

Results:

We have used cryo-EM followed by the BaR method to solve the first full-length platelet-derive protease inhibitor α2-macroglobulin (α2M)₄ structure directly from resting human platelet membranes. These structures are in two distinct functional states, terms native I and II, at 2.9Å and 3.6Å, respectively. Our results agree with previous studies that concentrated fractions obtained from solubilized human platelet membranes contain platelet-derived tetrameric α2M. We were able to assign all eight endogenous N-linked glycan sites in these two structures that were built based on the atomic-resolution cryo-EM maps. This revealed how macro- and micro-heterogeneity of glycosylation naturally regulates (α2M)₄ dynamics and function on resting human platelets. The highly reactive thioester bond formed by the side chains of C972 and Q975 was deeply buried in a hydrophobic pocket created by the thioester motif domain (TED) and the receptor binding domain (RBD) to protect it from premature hydrolysis. Finally, we also observed the highly dynamic of the Native II state, which led to three distinct conformations in its natural environment.

Conclusions:

We demonstrate the power of using cryo-EM with the BaR data processing to uncover novel structures directly from natural sources. This iterative bottom-up methodology has allowed us to solve the structures of several key platelet membrane proteins at a near-atomic resolution from samples extracted straight from human platelet membranes. Here, the structures of platelet-derived α2M in its native states provide unprecedented insights into its functions on platelets 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, including lipid and metal binding, post-translational modifications, and co-factor associations regulate platelet function at the molecular level.