Peptide-based NTA(Ni)-nanodiscs for studying membrane enhanced FGFR1 kinase activities
Juanjuan Liu,
Lei Zhu,
Xueli Zhang,
Bo Wu,
Ping Zhu,
Hongxin Zhao,
Junfeng Wang
Affiliations
Juanjuan Liu
High Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, China
Lei Zhu
High Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, China
Xueli Zhang
National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
Bo Wu
High Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, China
Ping Zhu
National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
Hongxin Zhao
High Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, China
Junfeng Wang
High Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, China
Tyrosine autophosphorylation plays a crucial regulatory role in the kinase activities of fibroblast growth factor receptors (FGFRs), and in the recruitment and activation of downstream intracellular signaling pathways. Biophysical and biochemical investigations of FGFR kinase domains in membrane environments offer key insights into phosphorylation mechanisms. Hence, we constructed nickel chelating nanodiscs based on a 22-residue peptide. The spontaneous anchoring of N-terminal His6-tagged FGFR1c kinase domain (FGFR1K) onto peptide nanodiscs grants FGFR1K orientations occurring on native plasma membranes. Following membrane incorporation, the autophosphorylation of FGFR1K, as exemplified by Y653 and Y654 in the A-loop and the total tyrosine phosphorylation, increase significantly. This in vitro reconstitution system may be applicable to studies of other membrane associated phenomena.
