Human FEZ1 Protein Forms a Disulfide Bond Mediated Dimer: Implications for Cargo Transport

Abstract

The human proteins FEZ1 (fasciculation and elongation protein zeta 1) and FEZ2 are orthologs of the protein UNC-76 from C. elegans, involved in the growth and fasciculation of the worms axon. Pull down assays showed that the protein FEZ1 interacts with other proteins (e.g., the protein SCOCO, short coiled-coil protein), mitochondria, and vesicles. These components may therefore represent cargoes to be transported along the microtubule, and the transport may be mediated through FEZ1 reported binding to kinesins (KIF3A). We previously showed that FEZ1 dimerizes in its N-terminal region and interacts with other proteins, including the candidate cargoe proteins, through its C-terminus. Here, we studied the fragment FEZ1(92-194) as well as full-length 6xHis-FEZ1 (1-392) in vitro and endogenous FEZ1 isolated from HEK 293 cells and were able to demonstrate the formation of an intermolecular disulfide bond through FEZ1 Cys-133, which appears to be essential for dimerization. This disulfide bond may be important for the FEZ1 role as a dimeric and bivalent transport adaptor molecule, since it establishes a strong link between the monomers, which could be a prerequisite for the simultaneous binding of two cargoes.

Crystal Structure of the Human Short Coiled Coil Protein and Insights into SCOC-FEZ1 Complex Formation

“…SEC-MALLS measurements yielded a molecular weight of 120.2±4 kDa for the SCOC-FEZ1cc complex and showed that the complex is homogeneous (Figure 5B, C). FEZ1 is a dimer in solution [3,5,32]. Assuming that both proteins are dimers and that they interact with a 1:1 stoichiometry, there would be six copies of each protein in the SCOC-FEZ1 complex.…”

Structural Analysis of Intermolecular Interactions in the Kinesin Adaptor Complex Fasciculation and Elongation Protein Zeta 1/ Short Coiled-Coil Protein (FEZ1/SCOCO)

“…Previous work from our group demonstrated that FEZ1 has regions with characteristics of natively unfolded protein [17], and forms disulfide-bridge stabilized homo-dimers in solution [18]. The data suggest that FEZ1 dimerizes through its N-terminal region but exposes its C-terminal regions, which contain the coiled-coil interaction modules.…”

“…The cloning of FEZ1 (92-194) used in NMR studies is described in Alborghetti et al (2010) [18]. pET-FEZ1 (92-194) plasmid was transformed into BL21(DE3) Escherichia coli which weregrown at 37°C in M9 minimal medium supplemented with 30 µg/mL kanamycin, 4 g/L [13C]glucose and 1 g/L [15N]ammonium chloride.…”

Implementação de uma abordagem híbrida utilizando modelagem comparativa e ab initio para predição de estruturas tridimensionais de proteínas contendo múltiplos domínios com conectores flexíveis

“…The cloning of FEZ1 (92-194) used in NMR studies is described in Alborghetti et al (2010) [18]. pET-FEZ1 (92-194) plasmid was transformed into BL21(DE3) Escherichia coli which weregrown at 37°C in M9 minimal medium supplemented with 30 µg/mL kanamycin, 4 g/L [13C]glucose and 1 g/L [15N]ammonium chloride.…”

“…pET-FEZ1 (92-194) plasmid was transformed into BL21(DE3) Escherichia coli which weregrown at 37°C in M9 minimal medium supplemented with 30 µg/mL kanamycin, 4 g/L [13C]glucose and 1 g/L [15N]ammonium chloride. Expression was induced at an OD 600 of 0.8-1.0 by the addition of 0.2 mM IPTG for 24 h. The cells were harvested by centrifugation at 5000 g for 10 min and the purification methodology was previously described in Alborghetti et al (2010) [18]. Fractions containing pure 6xHis-FEZ1 (92-194) were pooled and concentrated to 0.3 mM in buffer containing 20 mM phosphate, 50mM NaCl, pH 6.2.…”