Dynamic allostery in substrate binding by human thymidylate synthase

Abstract

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.

Making sense of chaos: uncovering the mechanisms of conformational entropy

“…A beautiful example of how entropy influences positive cooperativity is in the dimeric enzyme human thymidylate synthase (hTS) 19,32 . Careful calorimetric measurements at multiple protein concentrations demonstrated positive cooperativity between sites, with the second binding event requiring ~1.5kcal/mol less energy than the first.…”

“…However, this population change is insufficient to explain the large swing in entropy. The loss of slow dynamics and dramatic change in side chain dynamics can be reconciled through population shuffling, where the fast dynamics undergo reorganization due to the influence of slower movements 19,33 . Remarkably, the entropy-driven positive cooperativity is highly specific to hTS's substrate dUMP.…”

“…Additionally, some side chains exhibit increased disorder even when the overall trend is towards unfavorable conformational entropy (and vice versa). Furthermore, different ligands bound to the same receptor can have drastically different impacts on conformational entropy 10,19,20 . While NMR order parameters provide the extent of the disorder, many equivalent side chain conformational ensembles can produce a given order parameter value, as seen in the packing void example (Figure 1D,E) 17 .…”

Allostery Frustrates the Experimentalist

“…Indeed, allostery is sensitive to small changes in structure. [48][49][50][51] Therefore, the smaller the perturbation, the more likely is the experimental result and its interpretation correct. However, even conservative point mutations may change the structure and the allosteric network.…”

Mixed, nonclassical behavior in a classic allosteric protein

“…However, the switching dynamics between these end states has not been directly observed in detail, and hence questions about ligand-independent switching, the concertedness and synchrony of switching, extent of spontaneous switching, and the role of effectors have gone unanswered in these systems. In addition, some allosteric proteins appear to access three or more conformations ( 10 – 14 ), utilize only dynamics (entropy) and not conformational change to drive allostery ( 13 , 15 – 17 ), or reveal the same ligand can act as an agonist in some contexts and as antagonists in others ( 18 ), raising the question of whether the simple models are adequate. Many of these finer details were revealed by NMR and its unique ability to provide site-specific structural and dynamic information ( 19 ).…”