Intersite communication in dimeric enzymes highlighted by structural and thermodynamic analysis of didansyltyrosine binding to thymidylate synthases

• Published in: Bioorganic chemistry Vol. 151; p. 107663
• Main Authors: Venturelli, Alberto, Guaitoli, Giambattista, Vanossi, Davide, Saitta, Francesca, Fessas, Dimitrios, Vitiello, Simone, Malpezzi, Giulia, Aiello, Daniele, Ferrari, Stefania, Tondi, Donatella, Ponterini, Glauco, Paola Costi, Maria
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2024
• Subjects: Binding Sites; Dimeric protein; Dose-Response Relationship, Drug; Enzyme Inhibitors - chemical synthesis; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Escherichia coli - enzymology; Fluorometric titration; Isothermal titration calorimetry; Lacticaseibacillus casei - enzymology; Models, Molecular; Molecular Structure; Structure-Activity Relationship; Subunit anti-cooperativity; Thermodynamics; Thymidylate synthase; Thymidylate Synthase - antagonists & inhibitors; Thymidylate Synthase - chemistry; Thymidylate Synthase - metabolism; Tyrosine - chemistry; Tyrosine - metabolism; Dimeric protein; Fluorometric titration; Isothermal titration calorimetry; Thymidylate synthase; Subunit anti-cooperativity
• ISSN: 0045-2068; 1090-2120; 1090-2120
• DOI: 10.1016/j.bioorg.2024.107663

A dansyl derivative binding triggers negative cooperativity between the active sites of the hTS dimer and allows the characterization of different TS features. [Display omitted] •Intersite communication in dimeric enzymes represents a challenge of underestimated importance in enzyme inhibition strategy.•Fluorometric and calorimetric evidence consistently reveal different binding thermodynamics of a dansyl derivative to the active site of two monomers of hTS.•A ligand induced negative cooperativity between the communicating active sites, avenues a more efficient enzyme inhibition with a lower inhibitor dose. Intersite communication in dimeric enzymes, triggered by ligand binding, represents both a challenge and an opportunity in enzyme inhibition strategy. Though often understestimated, it can impact on the in vivo biological mechansim of an inhibitor and on its pharmacokinetics. Thymidylate synthase (TS) is a homodimeric enzyme present in almost all living organisms that plays a crucial role in DNA synthesis and cell replication. While its inhibition is a valid strategy in the therapy of several human cancers, designing specific inhibitors of bacterial TSs poses a challenge to the development of new anti-infective agents. N,O-didansyl-l-tyrosine (DDT) inhibits both Escherichia coli TS (EcTS) and Lactobacillus casei TS (LcTS). The available X-ray structure of the DDT:dUMP:EcTS ternary complex indicated an unexpected binding mode for DDT to EcTS, involving a rearrangement of the protein and addressing the matter of communication between the two active sites of an enzyme dimer. Combining molecular-level information on DDT binding to EcTS and LcTS extracted from structural and FRET-based fluorometric evidence with a thermodynamic characterization of these events obtained by fluorometric and calorimetric titrations, this study unveiled a negative cooperativity between the DDT bindings to the two monomers of each enzyme dimer. This result, complemented by the species-specific thermodynamic signatures of the binding events, implied that communication across the protein dimer was triggered by the first DDT binding. These findings could challenge the conventional understanding of TS inhibition and open the way for the development of novel TS inhibitors with a different mechanism of action and enhanced efficacy and specificity.