title: Improving coiled coil stability while maintaining specificity by a bacterial hitchhiker selection system.
abstract: The design and selection of peptides targeting cellular proteins is challenging and often yields candidates with undesired properties. Therefore we deployed a new selection system based on the twin-arginine translocase (TAT) pathway of Escherichia coli, named hitchhiker translocation (HiT) selection. A pool of α-helix encoding sequences was designed and selected for interference with the coiled coil domain (CC) of a melanoma-associated basic-helix-loop-helix-leucine-zipper (bHLHLZ) protein, the microphthalmia associated transcription factor (MITF). One predominant sequence (iM10) was enriched during selection and showed remarkable protease resistance, high solubility and thermal stability while maintaining its specificity. Furthermore, it exhibited nanomolar range affinity towards the target peptide. A mutation screen indicated that target-binding helices of increased homodimer stability and improved expression rates were preferred in the selection process. The crystal structure of the iM10/MITF-CC heterodimer (2.1Å) provided important structural insights and validated our design predictions. Importantly, iM10 did not only bind to the MITF coiled coil, but also to the markedly more stable HLHLZ domain of MITF. Characterizing the selected variants of the semi-rational library demonstrated the potential of the innovative bacterial selection approach.
authors.affiliation: Molecular Biotechnology, Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany; Institute for Biology III, University of Freiburg, Freiburg im Breisgau, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Kükenshöner
authors.affiliation: Institute for Biochemistry, University of Freiburg, Freiburg im Breisgau, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Wohlwend
authors.affiliation: Institute for Biology III, University of Freiburg, Freiburg im Breisgau, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Niemöller
authors.affiliation: Molecular Biotechnology, Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany; Institute for Biology III, University of Freiburg, Freiburg im Breisgau, Germany.
authors.forename: Padmarupa
authors.initials: P
authors.lastname: Dondapati
authors.affiliation: Molecular Biotechnology, Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany; Institute for Biology III, University of Freiburg, Freiburg im Breisgau, Germany.
authors.forename: Janina
authors.initials: J
authors.lastname: Speck
authors.affiliation: Molecular Biotechnology, Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany; Institute for Biology III, University of Freiburg, Freiburg im Breisgau, Germany.
authors.forename: Adebola V
authors.initials: AV
authors.lastname: Adeniran
authors.affiliation: Institute for Biology III, University of Freiburg, Freiburg im Breisgau, Germany; Centre for Biological Signalling Studies (BIOSS), University of Freiburg, Freiburg im Breisgau, Germany; Freiburg Institute for Advanced Studies, University of Freiburg, Freiburg im Breisgau, Germany.
authors.forename: Anita
authors.initials: A
authors.lastname: Nieth
authors.affiliation: Institute for Biochemistry, University of Freiburg, Freiburg im Breisgau, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Gerhardt
authors.affiliation: Centre for Biological Signalling Studies (BIOSS), University of Freiburg, Freiburg im Breisgau, Germany; Freiburg Institute for Advanced Studies, University of Freiburg, Freiburg im Breisgau, Germany; Institute for Biochemistry, University of Freiburg, Freiburg im Breisgau, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Einsle
authors.affiliation: Molecular Biotechnology, Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany; Institute for Biology III, University of Freiburg, Freiburg im Breisgau, Germany; Cellular and Molecular Biotechnology, Faculty of Technology, Bielefeld University, Germany.
authors.forename: Kristian M
authors.initials: KM
authors.lastname: Müller
authors.affiliation: Molecular Biotechnology, Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany; Institute for Biology III, University of Freiburg, Freiburg im Breisgau, Germany; Centre for Biological Signalling Studies (BIOSS), University of Freiburg, Freiburg im Breisgau, Germany; Freiburg Institute for Advanced Studies, University of Freiburg, Freiburg im Breisgau, Germany. Electronic address: katja.arndt@uni-potsdam.de.
authors.forename: Katja M
authors.initials: KM
authors.lastname: Arndt
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D018360:Crystallography, X-Ray; D019286:Endopeptidase K / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D016350:Leucine Zippers; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D051739:Microphthalmia-Associated Transcription Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility
chemical_list: D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D051739:Microphthalmia-Associated Transcription Factor; D019151:Peptide Library; D011994:Recombinant Proteins; C408585:twin-arginine translocase complex, E coli; D019286:Endopeptidase K
keywords: Basic helix-loop-helix leucine zipper; Coiled coils; Microphthalmia associated transcription factor; Selection and design; Twin arginine translocation pathway