Cryogenic electron microscopy (cryo-EM) resolves the structures of proteins and their complexes under near-native conditions that are often inaccessible to other structural techniques. Cryo-EM has recently contributed significant advances in basic knowledge to the field of coagulation. New paradigms regarding individual domain role in protein-protein interactions have emerged, casting the main reactions of the coagulation cascade in a much-needed structural context. This review focuses on recent advances in the study of coagulation factors using cryo-EM.

The conversion of the inactive zymogen prothrombin to the active protease thrombin in the common pathway of the coagulation cascade is the molecular event responsible for the pathophysiology of hemostasis and thrombosis. The conversion entails 2 proteolytic cleavages at R320 and R271 by the prothrombinase complex composed of the enzyme factor Xa (fXa), the cofactor fVa, Ca2+, and phospholipids. A recent cryogenic electron microscopy (cryo-EM) structure revealed how cleavage at R320 generates the active intermediate meizothrombin in the first step of the activation pathway. Here we present the 3.8 Å resolution cryo-EM structure of a truncated form of meizothrombin (mzTΔF1) bound to fVa and fXa that reveals how the second cleavage at R271 generates thrombin. The cleavage is brokered by molecular contacts that involve mostly the protease domains of mzTΔF1 and fXa and largely validate the results from biochemical studies. The switch in cleavage site from R320 to R271 involves a significant reorientation rather than conformational transitions of the protease domain of mzTΔF1 that moves the guanidinium group of R271 more than 20 Å into the primary specificity pocket of fXa. The findings complete the cryo-EM structural analysis of prothrombin activation along the meizothrombin pathway and advance our molecular understanding of a reaction critical to the pathophysiology of blood coagulation.

Coagulation factor (F)VIII is the inactive precursor of FVIIIa, an essential component of the intrinsic tenase complex. Despite numerous structures of recombinant FVIII in solution, the structural basis of the procofactor-membrane interface remains poorly defined. Traditional liposome platforms require special grids and often suffer from heterogeneous particle distribution and overcrowding, precluding high-resolution single-particle analysis, while membrane mimetics-nanodiscs-are too planar, posing significant biophysical challenges.

Estrogen-related receptors (ERRs) are master regulators of mitochondrial metabolism and exercise-responsive transcription, yet only a limited number of synthetic agonists with suitable potency and drug-like properties have been reported. SLU-PP-332 is a well-established exercise mimetic and widely used chemical probe for ERR activation; however, the structural features governing its potency, efficacy, selectivity, and drug-like properties have not been systematically elucidated. Here, we report the first comprehensive structure-activity relationship (SAR) analysis of the SLU-PP-332 scaffold, integrating chemical synthesis, cell-based functional assays, downstream gene-expression profiling, and computational modeling. Through iterative modification of core pharmacophoric elements, we identify key structural determinants that control ERRα and ERRγ agonism, transcriptional efficacy, ligand efficiency, and physicochemical properties. While SLU-PP-332 remains a strong benchmark for ERR activation, several analogues achieve comparable or context-dependent transcriptional responses while exhibiting improved ligand efficiency, solubility, or metabolic stability. Computational docking and molecular dynamics simulations reveal how subtle structural modifications influence ERR engagement and signaling outcomes. Together, this work defines design principles for tuning ERR agonism and provides a foundational SAR roadmap for the rational development of next-generation ERR agonists and exercise-mimetic therapeutics.

Coagulation factor Va (FVa) is the cofactor component of the prothrombinase complex required for rapid generation of thrombin from prothrombin in the penultimate step of the coagulation cascade. In addition, FVa is a target for proteolytic inactivation by activated protein C (APC). Like other protein-protein interactions in the coagulation cascade, the FVa-APC interaction has long posed a challenge to structural biology and its molecular underpinnings remain unknown. A recent cryogenic electron microscopy (cryo-EM) structure of FVa has revealed the arrangement of its A1-A2-A3-C1-C2 domains and the environment of the sites of APC cleavage at R306 and R506. Here, we report the cryo-EM structure of the FVa-APC complex at 3.15 Å resolution in which the protease domain of APC engages R506 in the A2 domain of FVa through electrostatic interactions between positively charged residues in the 30-loop and 70-loop of APC and an electronegative surface of FVa. The auxiliary γ-carboxyglutamic acid and epidermal growth factor domains of APC are highly dynamic and point to solvent, without making contacts with FVa. Binding of APC displaces a large portion of the A2 domain of FVa and projects the 654VKCIPDDDEDSYEIFEP670 segment as a “latch,” or exosite ligand, over the 70-loop of the enzyme. The latch induces a large conformational change of the autolysis loop of APC, which in turn promotes docking of R506 into the primary specificity pocket. The cryo-EM structure of the FVa-APC complex validates the bulk of existing biochemical data and offers molecular context for a key regulatory interaction of the coagulation cascade.