TY  - JOUR
A1  - Kuekenshoener, Tim
A1  - Wohlwend, Daniel
A1  - Niemoeller, Christoph
A1  - Dondapati, Padmarupa
A1  - Speck, Janina
A1  - Adeniran, Adebola V.
A1  - Nieth, Anita
A1  - Gerhardt, Stefan
A1  - Einsle, Oliver
A1  - Mueller, Kristian M.
A1  - Arndt, Katja Maren
T1  - Improving coiled coil stability while maintaining specificity by a bacterial hitchhiker selection system
JF  - Journal of structural biology
N2  - 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 alpha-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 (2.1 angstrom) 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. (C) 2014 Elsevier Inc. All rights reserved.
KW  - Basic helix-loop-helix leucine zipper
KW  - Coiled coils
KW  - Microphthalmia associated transcription factor
KW  - Selection and design
KW  - Twin arginine translocation pathway
Y1  - 2014
U6  - https://doi.org/10.1016/j.jsb.2014.03.002
SN  - 1047-8477
SN  - 1095-8657
VL  - 186
IS  - 3
SP  - 335
EP  - 348
PB  - Elsevier
CY  - San Diego
ER  -