Hotspots in an Obligate Homodimeric Anticancer Target. Structural and Functional Effects of Interfacial Mutations in Human Thymidylate Synthase

Abstract

Human thymidylate synthase (hTS), a target for antiproliferative drugs, is an obligate homodimer. Single-point mutations to alanine at the monomer–monomer interface may enable the identification of specific residues that delineate sites for drugs aimed at perturbing the protein–protein interactions critical for activity. We computationally identified putative hotspot residues at the interface and designed mutants to perturb the intersubunit interaction. Dimer dissociation constants measured by a FRET-based assay range from 60 nM for wild-type hTS up to about 1 mM for single-point mutants and agree with computational predictions of the effects of these mutations. Mutations that are remote from the active site retain full or partial activity, although the substrate KM values were generally higher and the dimer was less stable. The lower dimer stability of the mutants can facilitate access to the dimer interface by small molecules and thereby aid the design of inhibitors that bind at the dimer interface.

Deoxyribonucleotide metabolism, mutagenesis and cancer

“…As an example, Cannaza et al 136 have used the crystal structure of human thymidylate synthase to design peptides that inhibit the essential dimerization of the protein, and the same laboratory is working on non-peptide analogues that bind to the same target but that resist degradation and can be taken up into tumour cells 137 . A related approach 138 involves the identification of amino acid residues that are essential to dimerization, leading to the design of dimerization inhibitors that target these sites. These studies suggest that the future of cancer chemotherapy involving enzymes of deoxyribonucleotide metabolism may focus less on metabolic complexities and more on protein structure and enzyme function.…”

Prediction of quaternary structure by analysis of hot spot residues in protein‐protein interfaces: the case of anthranilate phosphoribosyltransferases

“…The importance of protein oligomerization can be studied by comparing the catalytic properties of the monomeric and oligomeric enzyme forms. An approach to examine the role of quaternary structures is to artificially create the monomers by identifying putative hot spot residues in the oligomerization interface and harnessing these hot spot residues to design mutants with perturbed intersubunit interactions . In the present study, we followed a complementary approach by identifying hot spot residues in the dimer interface of known AnPRT variants by means of a comprehensive sequence comparison.…”

“…Indeed, an increase in oligomerization state is one of the stabilization strategies observed in proteins from thermophilic organisms . Aberrant interactions in the assembly of the homomer complex can result in disease and homomeric complexes are regarded as potential drug targets in pharmacology …”

“…interactions in the assembly of the homomer complex can result in disease 8 and homomeric complexes are regarded as potential drug targets in pharmacology. 9,10 The specific characteristics of an oligomeric protein interface depend on the composition and the duration of the interaction. However, some general features of interfaces have been worked out:…”

Noncovalent Intermediate of Thymidylate Synthase: Fact or Fiction?

“…4 Consequently, alternative strategies to improve selectivity and find new classes of TSase inhibitors are pursued. 4–6 In this respect mechanistic studies of TSase-catalyzed reaction can provide invaluable insights.…”