TY - JOUR

T1 - Flexible linker modulates the binding affinity of the TP901-1 CI phage repressor to DNA

AU - Varming, Anders Kokkenborg

AU - Rasmussen, Kim Krighaar

AU - Zong, Zhiyou

AU - Thulstrup, Peter Waaben

AU - Kilstrup, Mogens

AU - Lo Leggio, Leila

N1 - Funding Information: We thank Alina Vitaliyivna Kulakova for help with analyzing the SAXS data and Yusuf Theibich for help with running the IEF electrophoresis. We also thank Karin Hammer for helpful discussions throughout the TP901‐1 switch project. AKV was funded by a PhD scholarship from the Lundbeck Foundation (grant R249‐2017‐977). ZZ was funded by a grant from Novo Nordisk Foundation (NNF17OC0027698) to LLL. Additional funding for materials was provided by a grant from the Danish Council for Independent Research (4002‐00107) to LLL). Molecular dynamics simulations were performed at the Danish National Supercomputer for Life Sciences Computerome 2.0, installed at the National Life Science Center at Technical University of Denmark. We acknowledge the European Synchrotron Radiation Facility for provision of beam time on BM29, and we would like to thank the beamline staff for assistance with collecting SAXS data. Travel to synchrotrons was supported by the DANSCATT program, funded by the Danish Ministry for Higher Education and Science and the European Community's Seventh Framework Programme (FP7/2007–2013) under BioStruct‐X (grant agreement no. 283570). Publisher Copyright: © 2021 Federation of European Biochemical Societies

PY - 2022

Y1 - 2022

N2 - Temperate bacteriophages can switch between two life cycles following infection of a host bacterium: the lytic or lysogenic life cycle. The choice between these is controlled by a bistable genetic switch. We investigated the genetic switch of the lactococcal temperate bacteriophage, TP901-1, which is controlled by two regulatory proteins, the Clear 1 (CI) repressor and modulator of repression (MOR) antirepressor. CI consists of a DNA-binding N-terminal domain and a C-terminal domain responsible for oligomerization, connected by a flexible interdomain linker. Full-length CI is hexameric, whereas the truncated version CI with 58 C-terminal residues truncated (CIΔ58), missing the second C-terminal subdomain, is dimeric, but binds with the same affinity as full-length CI to the OL operator site, responsible for lytic genes transcription repression. Three variants of CIΔ58 with shorter, longer, and PP substituted linkers were produced and confirmed by circular dichroism spectroscopy and nanodifferential scanning fluorimetry to be well folded. With small-angle X-ray scattering, we delineated the conformational space sampled by the variants and wild-type in solution and found that shortening and lengthening the linker decrease and increase this, respectively, as also substantiated by molecular dynamics and as intended. Isoelectric focusing electrophoresis confirmed that all variants are able to bind to the MOR antirepressor. However, using electrophoretic mobility shift assays, we showed that shortening and lengthening the linker lead to a 94 and 17 times decrease in affinity to OL, respectively. Thus, an appropriate linker length appears to be crucial for appropriate DNA-binding and subsequent TP901-1 genetic switch function.

AB - Temperate bacteriophages can switch between two life cycles following infection of a host bacterium: the lytic or lysogenic life cycle. The choice between these is controlled by a bistable genetic switch. We investigated the genetic switch of the lactococcal temperate bacteriophage, TP901-1, which is controlled by two regulatory proteins, the Clear 1 (CI) repressor and modulator of repression (MOR) antirepressor. CI consists of a DNA-binding N-terminal domain and a C-terminal domain responsible for oligomerization, connected by a flexible interdomain linker. Full-length CI is hexameric, whereas the truncated version CI with 58 C-terminal residues truncated (CIΔ58), missing the second C-terminal subdomain, is dimeric, but binds with the same affinity as full-length CI to the OL operator site, responsible for lytic genes transcription repression. Three variants of CIΔ58 with shorter, longer, and PP substituted linkers were produced and confirmed by circular dichroism spectroscopy and nanodifferential scanning fluorimetry to be well folded. With small-angle X-ray scattering, we delineated the conformational space sampled by the variants and wild-type in solution and found that shortening and lengthening the linker decrease and increase this, respectively, as also substantiated by molecular dynamics and as intended. Isoelectric focusing electrophoresis confirmed that all variants are able to bind to the MOR antirepressor. However, using electrophoretic mobility shift assays, we showed that shortening and lengthening the linker lead to a 94 and 17 times decrease in affinity to OL, respectively. Thus, an appropriate linker length appears to be crucial for appropriate DNA-binding and subsequent TP901-1 genetic switch function.

KW - bacteriophage

KW - flexible linker

KW - genetic switch

KW - lysogeny

KW - repressor

U2 - 10.1111/febs.16238

DO - 10.1111/febs.16238

M3 - Journal article

C2 - 34665941

AN - SCOPUS:85118330867

VL - 289

SP - 1135

EP - 1148

JO - F E B S Journal

JF - F E B S Journal

SN - 1742-464X

IS - 4

ER -