Poised for Allostery: Specific Interactions Underlie Precise Positioning of the C-Terminal Tail in Phosphofructokinase-1

Abstract Disordered terminal segments often regulate enzymes, but how such flexible elements achieve the spatial precision required for allostery remains poorly understood. Using human phosphofructokinase-1 as a model system, we combine enhanced-sampling molecular dynamics simulations, conservation-guided spatial analysis, prior allosteric network analysis, and coarse-grained modeling to show that allosteric positioning of the C-terminal tail, defined by residue-level contacts with the catalytic and regulatory domains, is maintained by a few key hydrogen bonding and hydrophobic interactions. This positioning enables the conserved tail residues L766 and E767 to dynamically couple the tail to the active site through a conserved surface region and to bridge the catalytic and regulatory domains in the T-state, experimentally associated with ATP inhibition. In contrast, coarse-grained simulations lacking explicit directional representation of hydrogen-bonding interactions fail to maintain stable tail docking, whereas including selected OLIVES-derived native hydrogen-bond terms improves the stability of the T-state-like docking pose. These findings support sequence-specific surface docking of a disordered enzyme tail as a physicochemical mechanism for allosteric regulation.