Within a crowded cellular milieu, inherent transient and dynamic functional ensembles are hallmarks of enzymes involved in the key metabolic pathways that sustain life. Interactions between macromolecules and their cellular environments sometimes facilitate sampling of “hidden” structural states along enzymatic mechanisms. These states are not revealed or populated within the time-resolution of traditional in-vitro experiments. Our recent glycolytic enzyme simulations within specially constructed human cell cytoplasm models are now addressing a key challenge in enzyme structural studies - capturing the atomic details at the exact moment when enzymes are undergoing transformations into functional and biologically conformers, often occurring rapidly at the micro-second time-scales. We resolved two types of functional structures of glycolytic enzymes that were occurring in a human cytoplasm model simulation due to protein-protein driven cellular interactions. Three enzymes sequential along the glycolysis pathway – glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase (PGK), and phosphoglycerate mutase – were seen coming together to form transient multi-enzyme complexes with lifetimes on the order of about 1 to 5 microseconds. These complexes were characteristic of metabolons, multi-enzyme complexes that form along metabolic pathways to facilitate both compartmentalization of the pathways in cells and substrate channeling between enzymes. While metabolons were seen to have evolved from the weak, but attractive, “sticking” interactions between neighboring proteins in the cell, repulsive cellular crowding interactions were likely behind large-scale hinge-bending motions up to 28 degrees seen occurring in PGK across our cytoplasm simulation trajectory. These motions were causing the enzyme to shift from the more energetically favorable and enzymatically inactive conformation to the active state. Simulations uncovering hidden structural states in cell can be a powerful tool for complementing experimental structural biology characterizations of enzymes.

Cell cytoplasm models reveal “hidden” states in glycolytic enzymes

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