Disordered N-Terminal Tail "Wags the Dog" in Human Thymidylate Synthase.

The utility of intrinsically disordered regions (IDRs) in protein function has become an area of broad interest in recent years given their known roles in signaling, biomolecular condensates, and protein activity regulation. The central challenge, however, is that disorder is difficult to characterize, and thus, there is a need to develop approaches to monitor how IDRs influence proteins. Here, we employ a strategy to restrict the freedom of the intrinsically disordered N-terminal tail (ID-tail) of human thymidylate synthase (hTS), which, together with tail truncation, enables testing aspects of how the ID-tail modulates enzyme function. The highly flexible hTS tail is known to impact allosteric substrate cooperativity and conformational switching dynamics, although how the tail brings about these effects has not been determined. We attempted to restrict native tail dynamics by chemically linking the dimer tails and tested linked forms with activity, binding, and nuclear magnetic resonance heteronuclear single quantum coherence (NMR HSQC) spectra. Truncating four N-terminal residues eliminated cooperativity in dUMP binding to apo-hTS, but because a similar result was obtained from the linked full-length protein, complete tail freedom appeared to be important for intersubunit communication. NMR analysis was aided by referencing the inactive M190K mutant, which roughly defined regions of active-inactive conformational switching that were disrupted by tail linkage. Collectively, these results point toward a role of the ID-tail to influence the ordered core of the protein. Moreover, while the natural ID-tail is extremely flexible, it likely possesses specific physiochemical movement dynamics and positioning attributes that facilitate its impact on allosteric cooperativity and conformational dynamics.