Human FEZ1 has characteristics of a natively unfolded protein and dimerizes in solution.
The fasciculation and elongation protein Zeta 1 (FEZ1) is the mammalian orthologue of the Caenorhabditis elegans protein UNC-76, which is necessary for axon growth. Human FEZ1 interacts with Protein Kinase C (PKC) and several regulatory proteins involved in functions ranging from microtubule associated transport to transcriptional regulation. Theoretical prediction, circular dichroism, fluorescence spectroscopy, and limited proteolysis of recombinant FEZ1 suggest that it contains disordered regions, especially in its N-terminal region, and that it may belong to the group of natively unfolded proteins. Small angle X-ray scattering experiments indicated a mainly disordered conformation, proved that FEZ1 is a dimer of elongated shape and provided overall dimensional parameters for the protein. In vitro pull down experiments confirmed these results and demonstrated that dimerization involves the N-terminus. Ab-initio 3D low resolution models of the full-length conformation of the dimeric constructs 6xHis-FEZ1(1-392) and 6xHis-FEZ1(1-227) were obtained. Furthermore, we performed in vitro phosphorylation assays of FEZ1 with PKC. The phosphorylation occurred mainly in its C-terminal region, and does not cause any significant conformational changes, but nonetheless inhibited its interaction with the FEZ1 interacting domain of the protein CLASP2 in vitro. The C terminus of FEZ1 has been reported to bind to several interacting proteins. This suggests that FEZ1 binding and transport function of interacting proteins may be subject to regulation by phosphorylation.
Lanza
Daniel C F
DC
Laboratório Nacional de Luz Síncrotron, Campinas, SP, Brasil.
Silva
Julio C
JC
Assmann
Eliana M
EM
Quaresma
Alexandre J C
AJ
Bressan
Gustavo C
GC
Torriani
Iris L
IL
Kobarg
Jörg
J
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D017510:Protein Folding; D011493:Protein Kinase C / Q000378:metabolism; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
D048868:Adaptor Proteins, Signal Transducing; C497851:CLASP2 protein, human; C528597:FEZ1 protein, human; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C073885:protein kinase C (19-31); D011493:Protein Kinase C