Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent

Abstract E. coli purine nucleoside phosphorylase is a homohexamer, which structure, in the apo form, can be described as a trimer of dimers. Earlier studies suggested that ligand binding and kinetic properties are well described by two binding constants and two sets of kinetic constants. However, mo...

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Autores principales:	Marta Narczyk, Łukasz Mioduszewski, Aleksandra Oksiejuk, Maria Winiewska-Szajewska, Beata Wielgus-Kutrowska, Adrian Gojdź, Joanna Cieśla, Agnieszka Bzowska
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Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/496f274ca99b4a1f857d7a1fc80b317d
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spelling	oai:doaj.org-article:496f274ca99b4a1f857d7a1fc80b317d2021-12-02T15:49:42ZSingle tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent10.1038/s41598-021-90472-42045-2322https://doaj.org/article/496f274ca99b4a1f857d7a1fc80b317d2021-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-90472-4https://doaj.org/toc/2045-2322Abstract E. coli purine nucleoside phosphorylase is a homohexamer, which structure, in the apo form, can be described as a trimer of dimers. Earlier studies suggested that ligand binding and kinetic properties are well described by two binding constants and two sets of kinetic constants. However, most of the crystal structures of this enzyme complexes with ligands do not hold the three-fold symmetry, but only two-fold symmetry, as one of the three dimers is different (both active sites in the open conformation) from the other two (one active site in the open and one in the closed conformation). Our recent detailed studies conducted over broad ligand concentration range suggest that protein–ligand complex formation in solution actually deviates from the two-binding-site model. To reveal the details of interactions present in the hexameric molecule we have engineered a single tryptophan Y160W mutant, responding with substantial intrinsic fluorescence change upon ligand binding. By observing various physical properties of the protein and its various complexes with substrate and substrate analogues we have shown that indeed three-binding-site model is necessary to properly describe binding of ligands by both the wild type enzyme and the Y160W mutant. Thus we have pointed out that a symmetrical dimer with both active sites in the open conformation is not forced to adopt this conformation by interactions in the crystal, but most probably the dimers forming the hexamer in solution are not equivalent as well. This, in turn, implies that an allosteric cooperation occurs not only within a dimer, but also among all three dimers forming a hexameric molecule.Marta NarczykŁukasz MioduszewskiAleksandra OksiejukMaria Winiewska-SzajewskaBeata Wielgus-KutrowskaAdrian GojdźJoanna CieślaAgnieszka BzowskaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-16 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Marta Narczyk Łukasz Mioduszewski Aleksandra Oksiejuk Maria Winiewska-Szajewska Beata Wielgus-Kutrowska Adrian Gojdź Joanna Cieśla Agnieszka Bzowska Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent
description	Abstract E. coli purine nucleoside phosphorylase is a homohexamer, which structure, in the apo form, can be described as a trimer of dimers. Earlier studies suggested that ligand binding and kinetic properties are well described by two binding constants and two sets of kinetic constants. However, most of the crystal structures of this enzyme complexes with ligands do not hold the three-fold symmetry, but only two-fold symmetry, as one of the three dimers is different (both active sites in the open conformation) from the other two (one active site in the open and one in the closed conformation). Our recent detailed studies conducted over broad ligand concentration range suggest that protein–ligand complex formation in solution actually deviates from the two-binding-site model. To reveal the details of interactions present in the hexameric molecule we have engineered a single tryptophan Y160W mutant, responding with substantial intrinsic fluorescence change upon ligand binding. By observing various physical properties of the protein and its various complexes with substrate and substrate analogues we have shown that indeed three-binding-site model is necessary to properly describe binding of ligands by both the wild type enzyme and the Y160W mutant. Thus we have pointed out that a symmetrical dimer with both active sites in the open conformation is not forced to adopt this conformation by interactions in the crystal, but most probably the dimers forming the hexamer in solution are not equivalent as well. This, in turn, implies that an allosteric cooperation occurs not only within a dimer, but also among all three dimers forming a hexameric molecule.
format	article
author	Marta Narczyk Łukasz Mioduszewski Aleksandra Oksiejuk Maria Winiewska-Szajewska Beata Wielgus-Kutrowska Adrian Gojdź Joanna Cieśla Agnieszka Bzowska
author_facet	Marta Narczyk Łukasz Mioduszewski Aleksandra Oksiejuk Maria Winiewska-Szajewska Beata Wielgus-Kutrowska Adrian Gojdź Joanna Cieśla Agnieszka Bzowska
author_sort	Marta Narczyk
title	Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent
title_short	Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent
title_full	Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent
title_fullStr	Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent
title_full_unstemmed	Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent
title_sort	single tryptophan y160w mutant of homooligomeric e. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/496f274ca99b4a1f857d7a1fc80b317d
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Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent

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