Cargando…

High-pressure protein crystal structure analysis of Escherichia coli dihydrofolate reductase complexed with folate and NADP(+)

A high-pressure crystallographic study was conducted on Escherichia coli dihydrofolate reductase (ecDHFR) complexed with folate and NADP(+) in crystal forms containing both the open and closed conformations of the M20 loop under high-pressure conditions of up to 800 MPa. At pressures between 270 and...

Descripción completa

Detalles Bibliográficos
Autores principales: Nagae, Takayuki, Yamada, Hiroyuki, Watanabe, Nobuhisa
Formato: Online Artículo Texto
Lenguaje:English
Publicado: International Union of Crystallography 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130465/
https://www.ncbi.nlm.nih.gov/pubmed/30198899
http://dx.doi.org/10.1107/S2059798318009397
_version_ 1783353937172103168
author Nagae, Takayuki
Yamada, Hiroyuki
Watanabe, Nobuhisa
author_facet Nagae, Takayuki
Yamada, Hiroyuki
Watanabe, Nobuhisa
author_sort Nagae, Takayuki
collection PubMed
description A high-pressure crystallographic study was conducted on Escherichia coli dihydrofolate reductase (ecDHFR) complexed with folate and NADP(+) in crystal forms containing both the open and closed conformations of the M20 loop under high-pressure conditions of up to 800 MPa. At pressures between 270 and 500 MPa the crystal form containing the open conformation exhibited a phase transition from P2(1) to C2. Several structural changes in ecDHFR were observed at high pressure that were also accompanied by structural changes in the NADP(+) cofactor and the hydration structure. In the crystal form with the closed conformation the M20 loop moved as the pressure changed, with accompanying conformational changes around the active site, including NADP(+) and folate. These movements were consistent with the suggested hypothesis that movement of the M20 loop was necessary for ecDHFR to catalyze the reaction. In the crystal form with the open conformation the nicotinamide ring of the NADP(+) cofactor undergoes a large flip as an intermediate step in the reaction, despite being in a crystalline state. Furthermore, observation of the water molecules between Arg57 and folate elucidated an early step in the substrate-binding pathway. These results demonstrate the possibility of using high-pressure protein crystallography as a method to capture high-energy substates or transient structures related to the protein reaction cycle.
format Online
Article
Text
id pubmed-6130465
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher International Union of Crystallography
record_format MEDLINE/PubMed
spelling pubmed-61304652018-09-17 High-pressure protein crystal structure analysis of Escherichia coli dihydrofolate reductase complexed with folate and NADP(+) Nagae, Takayuki Yamada, Hiroyuki Watanabe, Nobuhisa Acta Crystallogr D Struct Biol Research Papers A high-pressure crystallographic study was conducted on Escherichia coli dihydrofolate reductase (ecDHFR) complexed with folate and NADP(+) in crystal forms containing both the open and closed conformations of the M20 loop under high-pressure conditions of up to 800 MPa. At pressures between 270 and 500 MPa the crystal form containing the open conformation exhibited a phase transition from P2(1) to C2. Several structural changes in ecDHFR were observed at high pressure that were also accompanied by structural changes in the NADP(+) cofactor and the hydration structure. In the crystal form with the closed conformation the M20 loop moved as the pressure changed, with accompanying conformational changes around the active site, including NADP(+) and folate. These movements were consistent with the suggested hypothesis that movement of the M20 loop was necessary for ecDHFR to catalyze the reaction. In the crystal form with the open conformation the nicotinamide ring of the NADP(+) cofactor undergoes a large flip as an intermediate step in the reaction, despite being in a crystalline state. Furthermore, observation of the water molecules between Arg57 and folate elucidated an early step in the substrate-binding pathway. These results demonstrate the possibility of using high-pressure protein crystallography as a method to capture high-energy substates or transient structures related to the protein reaction cycle. International Union of Crystallography 2018-09-03 /pmc/articles/PMC6130465/ /pubmed/30198899 http://dx.doi.org/10.1107/S2059798318009397 Text en © Nagae et al. 2018 http://creativecommons.org/licenses/by/2.0/uk/ This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.http://creativecommons.org/licenses/by/2.0/uk/
spellingShingle Research Papers
Nagae, Takayuki
Yamada, Hiroyuki
Watanabe, Nobuhisa
High-pressure protein crystal structure analysis of Escherichia coli dihydrofolate reductase complexed with folate and NADP(+)
title High-pressure protein crystal structure analysis of Escherichia coli dihydrofolate reductase complexed with folate and NADP(+)
title_full High-pressure protein crystal structure analysis of Escherichia coli dihydrofolate reductase complexed with folate and NADP(+)
title_fullStr High-pressure protein crystal structure analysis of Escherichia coli dihydrofolate reductase complexed with folate and NADP(+)
title_full_unstemmed High-pressure protein crystal structure analysis of Escherichia coli dihydrofolate reductase complexed with folate and NADP(+)
title_short High-pressure protein crystal structure analysis of Escherichia coli dihydrofolate reductase complexed with folate and NADP(+)
title_sort high-pressure protein crystal structure analysis of escherichia coli dihydrofolate reductase complexed with folate and nadp(+)
topic Research Papers
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130465/
https://www.ncbi.nlm.nih.gov/pubmed/30198899
http://dx.doi.org/10.1107/S2059798318009397
work_keys_str_mv AT nagaetakayuki highpressureproteincrystalstructureanalysisofescherichiacolidihydrofolatereductasecomplexedwithfolateandnadp
AT yamadahiroyuki highpressureproteincrystalstructureanalysisofescherichiacolidihydrofolatereductasecomplexedwithfolateandnadp
AT watanabenobuhisa highpressureproteincrystalstructureanalysisofescherichiacolidihydrofolatereductasecomplexedwithfolateandnadp