Dynamic allostery in substrate binding by human thymidylate synthase
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 s...
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2022-10-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/79915 |
| _version_ | 1811196101254971392 |
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| author | Jeffrey P Bonin Paul J Sapienza Andrew L Lee |
| author_facet | Jeffrey P Bonin Paul J Sapienza Andrew L Lee |
| author_sort | Jeffrey P Bonin |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T00:53:07Z |
| format | Article |
| id | doaj.art-f5ce217a3fdc496e9cb1e1f35094fea8 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T00:53:07Z |
| publishDate | 2022-10-01 |
| publisher | eLife Sciences Publications Ltd |
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| spelling | doaj.art-f5ce217a3fdc496e9cb1e1f35094fea82022-12-22T03:54:41ZengeLife Sciences Publications LtdeLife2050-084X2022-10-011110.7554/eLife.79915Dynamic allostery in substrate binding by human thymidylate synthaseJeffrey P Bonin0https://orcid.org/0000-0002-2138-4440Paul J Sapienza1https://orcid.org/0000-0001-5279-3653Andrew L Lee2https://orcid.org/0000-0003-2783-1907Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina, Chapel Hill, United StatesDivision of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, United StatesDepartment of Biochemistry and Biophysics, School of Medicine, University of North Carolina, Chapel Hill, United States; Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, United StatesHuman 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.https://elifesciences.org/articles/79915allosteryprotein dynamicsNMRconformational entropycooperativitythymidylate synthase |
| spellingShingle | Jeffrey P Bonin Paul J Sapienza Andrew L Lee Dynamic allostery in substrate binding by human thymidylate synthase eLife allostery protein dynamics NMR conformational entropy cooperativity thymidylate synthase |
| title | Dynamic allostery in substrate binding by human thymidylate synthase |
| title_full | Dynamic allostery in substrate binding by human thymidylate synthase |
| title_fullStr | Dynamic allostery in substrate binding by human thymidylate synthase |
| title_full_unstemmed | Dynamic allostery in substrate binding by human thymidylate synthase |
| title_short | Dynamic allostery in substrate binding by human thymidylate synthase |
| title_sort | dynamic allostery in substrate binding by human thymidylate synthase |
| topic | allostery protein dynamics NMR conformational entropy cooperativity thymidylate synthase |
| url | https://elifesciences.org/articles/79915 |
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