TY - JOUR TI - Dynamic allostery in substrate binding by human thymidylate synthase AU - Bonin, Jeffrey P AU - Sapienza, Paul J AU - Lee, Andrew L A2 - Andreotti, Amy H A2 - Dötsch, Volker A2 - Venditti, Vincenzo VL - 11 PY - 2022 DA - 2022/10/06 SP - e79915 C1 - eLife 2022;11:e79915 DO - 10.7554/eLife.79915 UR - https://doi.org/10.7554/eLife.79915 AB - Human thymidylate synthase (hTS) is essential for DNA replication and therefore a therapeutic target for cancer. Effective targeting requires knowledge of the mechanism(s) of regulation of this 72 kDa homodimeric enzyme. Here, we investigate the mechanism of binding cooperativity of the nucleotide substrate. We have employed exquisitely sensitive methyl-based CPMG and CEST NMR experiments enabling us to identify residues undergoing bifurcated linear 3-state exchange, including concerted switching between active and inactive conformations in the apo enzyme. The inactive state is populated to only ~1.3%, indicating that conformational selection contributes negligibly to the cooperativity. Instead, methyl rotation axis order parameters, determined by 2H transverse relaxation rates, suggest that rigidification of the enzyme upon substrate binding is responsible for the entropically-driven cooperativity. Lack of the rigidification in product binding and substrate binding to an N-terminally truncated enzyme, both non-cooperative, support this idea. In addition, the lack of this rigidification in the N-terminal truncation indicates that interactions between the flexible N-terminus and the rest of the protein, which are perturbed by substrate binding, play a significant role in the cooperativity—a novel mechanism of dynamic allostery. Together, these findings yield a rare depth of insight into the substrate binding cooperativity of an essential enzyme. KW - allostery KW - protein dynamics KW - NMR KW - conformational entropy KW - cooperativity KW - thymidylate synthase JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -