title: Structural and calorimetric studies demonstrate that the hepatocyte nuclear factor 1β (HNF1β) transcription factor is imported into the nucleus via a monopartite NLS sequence.
abstract: The transcription factor hepatocyte nuclear factor 1β (HNF1β) is ubiquitously overexpressed in ovarian clear cell carcinoma (CCC) and is a potential therapeutic target. To explore potential approaches that block HNF1β transcription we have identified and characterised extensively the nuclear localisation signal (NLS) for HNF1β and its interactions with the nuclear protein import receptor, Importin-α. Pull-down assays demonstrated that the DNA binding domain of HNF1β interacted with a spectrum of Importin-α isoforms and deletion constructs tagged with eGFP confirmed that the HNF1β (229)KKMRRNR(235) sequence was essential for nuclear localisation. We further characterised the interaction between the NLS and Importin-α using complementary biophysical techniques and have determined the 2.4Å resolution crystal structure of the HNF1β NLS peptide bound to Importin-α. The functional, biochemical, and structural characterisation of the nuclear localisation signal present on HNF1β and its interaction with the nuclear import protein Importin-α provide the basis for the development of compounds targeting transcription factor HNF1β via its nuclear import pathway.
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK; Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.
authors.forename: Mareike M
authors.initials: MM
authors.lastname: Wiedmann
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Aibara
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: David R
authors.initials: DR
authors.lastname: Spring
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK. Electronic address: ms@mrc-lmb.cam.ac.uk.
authors.forename: Murray
authors.initials: M
authors.lastname: Stewart
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.
authors.forename: James D
authors.initials: JD
authors.lastname: Brenton
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D001665:Binding Sites; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D018360:Crystallography, X-Ray; D057809:HEK293 Cells; D051539:Hepatocyte Nuclear Factor 1-beta / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D019913:Nuclear Localization Signals; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D020539:Sequence Analysis, Protein; D029867:Xenopus Proteins / Q000737:chemistry; D014982:Xenopus laevis; D028901:alpha Karyopherins / Q000737:chemistry
chemical_list: C496495:HNF1B protein, human; D019913:Nuclear Localization Signals; D029867:Xenopus Proteins; D028901:alpha Karyopherins; D051539:Hepatocyte Nuclear Factor 1-beta
keywords: Hepatocyte nuclear factor-1β (HNF1β); Importin-α; Isothermal titration calorimetry; Nuclear import pathway; Nuclear localisation signal sequence (NLS); Site-directed mutagenesis; X-ray crystallography