ROLE OF A FLEXIBLE, ALLOSTERIC LOOP IN REGULATING PROTEIN ACTIVITY

Allostery is a key driver of protein function and behavior in biological systems. While structure-based classical models and protein dynamics have greatly advanced our understanding of allostery, our knowledge of the underlying mechanisms of allosteric communication remains limited. This is evidenced by the ongoing challenges in engineering allosteric proteins and therapeutics, underscoring the need for deeper insights into the mechanisms governing allostery. Here, we investigate the allosteric protein chorismate mutase (CM), a homodimeric enzyme critical for the biosynthesis of aromatic amino acids. Although CM is differentially regulated by tryptophan and tyrosine through a shared pocket located over 25 Å from the active site, both TrpCM and TyrCM yield nearly identical NMR spectra corresponding to a “tense” T-state. TrpCM shows minor excursions into an excited state that aligns well with a “relaxed” R-state; however, it remains unclear whether this T-to-R pre-equilibrium is essential for CM function. In this work, we demonstrate that the R-state population in TrpCM can be substantially reduced by modifying buffer conditions. Lowering the salt concentration decreases the R-state population by more than half without significantly impairing enzymatic activity. Furthermore, we present a constitutively active CM variant fixed in the T-state without access to the R-state or other alternative microstates, suggesting that a pre-existing switching to the R-state may not be strictly required for catalytic activity. Subsequently, we demonstrate that a mutation within a structurally invisible and highly flexible loop, loop 11-12, located far from the active site, drastically alters CM’s activity landscape. Through paramagnetic labeling of the loop, we show that loop 11-12 undergoes transient excursions toward the active site only in the presence of the activator Trp, which binds over 20 Å away. Furthermore, employing a novel NMR approach, we show that loop 11-12 modulates CM’s electrostatics, potentially influencing charge distribution to optimize enzymatic activity. Our findings support a sophisticated allosteric process in which a flexible, distal loop is functionally coupled to both the effector binding region and the active site. This mechanism provides new insights into the diverse ways proteins achieve allosteric regulation and may contribute to understanding flexible regions in other allosteric systems.