Cargando…

Evidence of Destabilization of the Human Thymidylate Synthase (hTS) Dimeric Structure Induced by the Interface Mutation Q62R

In human cells, thymidylate synthase (TS) provides the only source of 2′-deoxythymidyne-5′-monophosphate (dTMP), which is required for DNA biosynthesis. Because of its pivotal role, human TS (hTS) represents a validated target for anticancer chemotherapy. Nonetheless, the efficacy of drugs blocking...

Descripción completa

Detalles Bibliográficos
Autores principales: Pozzi, Cecilia, Lopresti, Ludovica, Santucci, Matteo, Costi, Maria Paola, Mangani, Stefano
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6523895/
https://www.ncbi.nlm.nih.gov/pubmed/30987202
http://dx.doi.org/10.3390/biom9040134
_version_ 1783419440477503488
author Pozzi, Cecilia
Lopresti, Ludovica
Santucci, Matteo
Costi, Maria Paola
Mangani, Stefano
author_facet Pozzi, Cecilia
Lopresti, Ludovica
Santucci, Matteo
Costi, Maria Paola
Mangani, Stefano
author_sort Pozzi, Cecilia
collection PubMed
description In human cells, thymidylate synthase (TS) provides the only source of 2′-deoxythymidyne-5′-monophosphate (dTMP), which is required for DNA biosynthesis. Because of its pivotal role, human TS (hTS) represents a validated target for anticancer chemotherapy. Nonetheless, the efficacy of drugs blocking the hTS active site has limitations due to the onset of resistance in cancer cells, requiring the identification of new strategies to effectively inhibit this enzyme. Human TS works as an obligate homodimer, making the inter-subunit interface an attractive targetable area. Here, we report the design and investigation of a new hTS variant, in which Gln62, located at the dimer interface, has been replaced by arginine in order to destabilize the enzyme quaternary assembly. The hTS Q62R variant has been characterized though kinetic assay, thermal denaturation analysis and X-ray crystallography. Our results provide evidence that hTS Q62R has a reduced melting temperature. The effective destabilization of the TS quaternary structure is also confirmed by structural analysis, showing that the introduced mutation induces a slight aperture of the hTS dimer. The generation of hTS variants having a more accessible interface area can facilitate the screening of interface-targeting molecules, providing key information for the rational design of innovative hTS interface inhibitors.
format Online
Article
Text
id pubmed-6523895
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-65238952019-06-03 Evidence of Destabilization of the Human Thymidylate Synthase (hTS) Dimeric Structure Induced by the Interface Mutation Q62R Pozzi, Cecilia Lopresti, Ludovica Santucci, Matteo Costi, Maria Paola Mangani, Stefano Biomolecules Article In human cells, thymidylate synthase (TS) provides the only source of 2′-deoxythymidyne-5′-monophosphate (dTMP), which is required for DNA biosynthesis. Because of its pivotal role, human TS (hTS) represents a validated target for anticancer chemotherapy. Nonetheless, the efficacy of drugs blocking the hTS active site has limitations due to the onset of resistance in cancer cells, requiring the identification of new strategies to effectively inhibit this enzyme. Human TS works as an obligate homodimer, making the inter-subunit interface an attractive targetable area. Here, we report the design and investigation of a new hTS variant, in which Gln62, located at the dimer interface, has been replaced by arginine in order to destabilize the enzyme quaternary assembly. The hTS Q62R variant has been characterized though kinetic assay, thermal denaturation analysis and X-ray crystallography. Our results provide evidence that hTS Q62R has a reduced melting temperature. The effective destabilization of the TS quaternary structure is also confirmed by structural analysis, showing that the introduced mutation induces a slight aperture of the hTS dimer. The generation of hTS variants having a more accessible interface area can facilitate the screening of interface-targeting molecules, providing key information for the rational design of innovative hTS interface inhibitors. MDPI 2019-04-03 /pmc/articles/PMC6523895/ /pubmed/30987202 http://dx.doi.org/10.3390/biom9040134 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Pozzi, Cecilia
Lopresti, Ludovica
Santucci, Matteo
Costi, Maria Paola
Mangani, Stefano
Evidence of Destabilization of the Human Thymidylate Synthase (hTS) Dimeric Structure Induced by the Interface Mutation Q62R
title Evidence of Destabilization of the Human Thymidylate Synthase (hTS) Dimeric Structure Induced by the Interface Mutation Q62R
title_full Evidence of Destabilization of the Human Thymidylate Synthase (hTS) Dimeric Structure Induced by the Interface Mutation Q62R
title_fullStr Evidence of Destabilization of the Human Thymidylate Synthase (hTS) Dimeric Structure Induced by the Interface Mutation Q62R
title_full_unstemmed Evidence of Destabilization of the Human Thymidylate Synthase (hTS) Dimeric Structure Induced by the Interface Mutation Q62R
title_short Evidence of Destabilization of the Human Thymidylate Synthase (hTS) Dimeric Structure Induced by the Interface Mutation Q62R
title_sort evidence of destabilization of the human thymidylate synthase (hts) dimeric structure induced by the interface mutation q62r
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6523895/
https://www.ncbi.nlm.nih.gov/pubmed/30987202
http://dx.doi.org/10.3390/biom9040134
work_keys_str_mv AT pozzicecilia evidenceofdestabilizationofthehumanthymidylatesynthasehtsdimericstructureinducedbytheinterfacemutationq62r
AT loprestiludovica evidenceofdestabilizationofthehumanthymidylatesynthasehtsdimericstructureinducedbytheinterfacemutationq62r
AT santuccimatteo evidenceofdestabilizationofthehumanthymidylatesynthasehtsdimericstructureinducedbytheinterfacemutationq62r
AT costimariapaola evidenceofdestabilizationofthehumanthymidylatesynthasehtsdimericstructureinducedbytheinterfacemutationq62r
AT manganistefano evidenceofdestabilizationofthehumanthymidylatesynthasehtsdimericstructureinducedbytheinterfacemutationq62r