Hotspots in an obligate homodimeric anticancer target. Structural and functional effects of interfacial mutations in human thymidylate synthase.

Outi M H Salo-Ahen,Cecilia Pozzi,Robert M Stroud,Maria Paola Costi,Anna Tochowicz,Stefano Mangani,Stefania Ferrari,Daniela Cardinale,Rebecca C Wade,Hannu Myllykallio,Puneet Saxena,Glauco Ponterini,Yap Boum

doi:10.1021/acs.jmedchem.5b00137

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.

Highlights

Protein–protein interfaces are important but difficult targets for inhibitor design
R176 is directly involved in deoxyuridine 5′-monophosphate (dUMP) binding, it was not selected as a putative hotspot because neither FoldX nor Robetta identified it as a hotspot
We provide evidence that hotspot mutations affect the stability of the protein fold, the protein monomer–dimer equilibrium, the active–inactive conformational equilibrium of the dimer, and the binding of substrates in the active site to varying extents

Summary

Introduction

Protein–protein interfaces are important but difficult targets for inhibitor design. Mutagenesis provides a means to probe protein–protein interfaces to identify potential sites for interface-perturbing drugs. Obligate homodimeric enzymes in which, by definition, residues of both subunits contribute to and are essential for activity present a special class of protein–protein interfaces where disruption of the interface results in inhibition of the enzyme In such cases, the subunits of the dimer generally bind each other with high affinity. A single point mutation can influence the affinity for inhibitors designed to bind at the interface to destabilize the homodimer or to stabilize an inactive form of the enzyme. We address these issues by identifying and characterizing interfacial hotspots in the obligate homodimeric enzyme, human thymidylate synthase (hTS), with the goal of providing new sites for drug targeting of an essential function in the cell

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Conclusion