Structural and dynamics studies of a truncated variant of CI repressor from bacteriophage TP901-1

Kim Krighaar Rasmussen; Kristian Erik Høpfner Frandsen; Elisabetta Boeri Erba; Margit Pedersen; Anders Kokkenborg Varming; Karin Hammer; Mogens Kilstrup; Peter Waaben Thulstrup; Martin Blackledge; Malene Ringkjøbing Jensen; Leila Lo Leggio

doi:10.1038/srep29574

Structural and dynamics studies of a truncated variant of CI repressor from bacteriophage TP901-1

Kim Krighaar Rasmussen, Kristian Erik Høpfner Frandsen, Elisabetta Boeri Erba, Margit Pedersen, Anders Kokkenborg Varming, Karin Hammer, Mogens Kilstrup, Peter Waaben Thulstrup, Martin Blackledge, Malene Ringkjøbing Jensen, Leila Lo Leggio

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Abstract

The CI repressor from the temperate bacteriophage TP901-1 consists of two folded domains, an N-terminal helix-turn-helix DNA-binding domain (NTD) and a C-terminal oligomerization domain (CTD), which we here suggest to be further divided into CTD₁ and CTD₂. Full-length CI is a hexameric protein, whereas a truncated version, CIΔ58, forms dimers. We identify the dimerization region of CIΔ58 as CTD 1 and determine its secondary structure to be helical both within the context of CIΔ58 and in isolation. To our knowledge this is the first time that a helical dimerization domain has been found in a phage repressor. We also precisely determine the length of the flexible linker connecting the NTD to the CTD. Using electrophoretic mobility shift assays and native mass spectrometry, we show that CIΔ58 interacts with the O L operator site as one dimer bound to both half-sites, and with much higher affinity than the isolated NTD domain thus demonstrating cooperativity between the two DNA binding domains. Finally, using small angle X-ray scattering data and state-of-the-art ensemble selection techniques, we delineate the conformational space sampled by CIΔ58 in solution, and we discuss the possible role that the dynamics play in CI-repressor function.

Originalsprog	Engelsk
Artikelnummer	29574
Tidsskrift	Scientific Reports
Vol/bind	6
Antal sider	12
ISSN	2045-2322
DOI	https://doi.org/10.1038/srep29574
Status	Udgivet - 12 jul. 2016

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10.1038/srep29574Licens: CC BY

Rasmussen_2016_Structural_and_dynamicsForlagets udgivne version, 1,68 MBLicens: CC BY

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title = "Structural and dynamics studies of a truncated variant of CI repressor from bacteriophage TP901-1",

abstract = "The CI repressor from the temperate bacteriophage TP901-1 consists of two folded domains, an N-terminal helix-turn-helix DNA-binding domain (NTD) and a C-terminal oligomerization domain (CTD), which we here suggest to be further divided into CTD1 and CTD2. Full-length CI is a hexameric protein, whereas a truncated version, CIΔ58, forms dimers. We identify the dimerization region of CIΔ58 as CTD 1 and determine its secondary structure to be helical both within the context of CIΔ58 and in isolation. To our knowledge this is the first time that a helical dimerization domain has been found in a phage repressor. We also precisely determine the length of the flexible linker connecting the NTD to the CTD. Using electrophoretic mobility shift assays and native mass spectrometry, we show that CIΔ58 interacts with the O L operator site as one dimer bound to both half-sites, and with much higher affinity than the isolated NTD domain thus demonstrating cooperativity between the two DNA binding domains. Finally, using small angle X-ray scattering data and state-of-the-art ensemble selection techniques, we delineate the conformational space sampled by CIΔ58 in solution, and we discuss the possible role that the dynamics play in CI-repressor function.",

author = "Rasmussen, {Kim Krighaar} and Frandsen, {Kristian Erik H{\o}pfner} and {Boeri Erba}, Elisabetta and Margit Pedersen and Varming, {Anders Kokkenborg} and Karin Hammer and Mogens Kilstrup and Thulstrup, {Peter Waaben} and Martin Blackledge and Jensen, {Malene Ringkj{\o}bing} and {Lo Leggio}, Leila",

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T1 - Structural and dynamics studies of a truncated variant of CI repressor from bacteriophage TP901-1

AU - Rasmussen, Kim Krighaar

AU - Frandsen, Kristian Erik Høpfner

AU - Boeri Erba, Elisabetta

AU - Pedersen, Margit

AU - Varming, Anders Kokkenborg

AU - Hammer, Karin

AU - Kilstrup, Mogens

AU - Thulstrup, Peter Waaben

AU - Blackledge, Martin

AU - Jensen, Malene Ringkjøbing

AU - Lo Leggio, Leila

PY - 2016/7/12

Y1 - 2016/7/12

N2 - The CI repressor from the temperate bacteriophage TP901-1 consists of two folded domains, an N-terminal helix-turn-helix DNA-binding domain (NTD) and a C-terminal oligomerization domain (CTD), which we here suggest to be further divided into CTD1 and CTD2. Full-length CI is a hexameric protein, whereas a truncated version, CIΔ58, forms dimers. We identify the dimerization region of CIΔ58 as CTD 1 and determine its secondary structure to be helical both within the context of CIΔ58 and in isolation. To our knowledge this is the first time that a helical dimerization domain has been found in a phage repressor. We also precisely determine the length of the flexible linker connecting the NTD to the CTD. Using electrophoretic mobility shift assays and native mass spectrometry, we show that CIΔ58 interacts with the O L operator site as one dimer bound to both half-sites, and with much higher affinity than the isolated NTD domain thus demonstrating cooperativity between the two DNA binding domains. Finally, using small angle X-ray scattering data and state-of-the-art ensemble selection techniques, we delineate the conformational space sampled by CIΔ58 in solution, and we discuss the possible role that the dynamics play in CI-repressor function.

AB - The CI repressor from the temperate bacteriophage TP901-1 consists of two folded domains, an N-terminal helix-turn-helix DNA-binding domain (NTD) and a C-terminal oligomerization domain (CTD), which we here suggest to be further divided into CTD1 and CTD2. Full-length CI is a hexameric protein, whereas a truncated version, CIΔ58, forms dimers. We identify the dimerization region of CIΔ58 as CTD 1 and determine its secondary structure to be helical both within the context of CIΔ58 and in isolation. To our knowledge this is the first time that a helical dimerization domain has been found in a phage repressor. We also precisely determine the length of the flexible linker connecting the NTD to the CTD. Using electrophoretic mobility shift assays and native mass spectrometry, we show that CIΔ58 interacts with the O L operator site as one dimer bound to both half-sites, and with much higher affinity than the isolated NTD domain thus demonstrating cooperativity between the two DNA binding domains. Finally, using small angle X-ray scattering data and state-of-the-art ensemble selection techniques, we delineate the conformational space sampled by CIΔ58 in solution, and we discuss the possible role that the dynamics play in CI-repressor function.

U2 - 10.1038/srep29574

DO - 10.1038/srep29574

M3 - Journal article

C2 - 27403839

SN - 2045-2322

VL - 6

JO - Scientific Reports

JF - Scientific Reports

M1 - 29574

ER -

Structural and dynamics studies of a truncated variant of CI repressor from bacteriophage TP901-1

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