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

<div><p>Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in <i>C. elegans</i>), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.</p> </div

Purification of the FEZ1-SCOCO complex and GST-SCOCO and SAXS experimental

<p>data A) SDS-PAGE 10% of the 6His-FEZ1 (1-392) and GST-SCOCO (2-82) protein complex. The complex was analyzed by SAXS at 1.10 mg/mL in PBS buffer solution. The complex poly-dispersity was 28,0% according to DLS assay. B) SAXS (Small Angle X-ray Scattering) experiments of 6His-FEZ1 (1-392) interacting with GST-SCOCO (2-82). C) SDS-PAGE 10% of the GST-SCOCO (2-82) protein. D) The protein was analyzed by SAXS at 0.84 mg/mL in PBS buffer solution. The figure shows the experimental intensity points (empty symbols) and the theoretical fit (continuous line) obtained with the GNOM program package for both samples. Insets in both panels (B) and (C) show the linear behavior of the data in the Guinier region. R_g and D_max values obtained from the GNOM fitting for GST-SCOCO and FEZ1-SCOCO samples were (28.7 ±1, approx. 95 Ǻ) and (107±1, approx. 340 Ǻ), respectively. SAXS data of FEZ1 protein were previously published [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076602#B17" target="_blank">17</a>].</p

Interaction between FEZ1 and SCOCO.

<p>A) Purified recombinant proteins FEZ1 and SCOCO were incubated, chemically cross-linked, digested with trypsin, and analyzed by MS. MS/MS spectra were manually validated for b and y ion series of the α (peptide of FEZ1) and β (peptide of SCOCO) chains. B) General scheme of FEZ1 and SCOCO proteins cross-linked. Coiled-coils: box, alpha-helix prediction: gray. Amino acids 261-279 in FEZ1 correspond to the mininal interaction region of UNC-69/SCOCO in UNC-76/FEZ1.C) Best conformation based on both cross-link distance and energy value of the in silico modeled complex. FEZ1 is colored in green, and SCOCO is depicted in deep blue. The peptides identified in the MS analysis are shown in orange and the lysine residue in red. DSS is represented in yellow.</p

FEZ1 homodimerization involves few amino acids residues.

<p>A) Amino acid sequence of human FEZ1 protein (92-194). The numbers indicate the position of each amino acid residue within the full-length sequence. The first three residues, unnumbered, are generated by the recombinant protein’s cleavage with TEV protease. B) 15N-HSQC FEZ1 (92-194) Nuclear Magnetic Resonance (NMR) spectra. HSQC shows chemical shifts in reduced monomeric protein (black) and non-reduced dimeric protein (red). The spectrum was obtained in spectrometer 600 MHz. For the series of experiments, isotope 15N was introduced in minimal medium for growth of bacteria and induction of protein expression. C) steady-state heteronuclear NOE experiments with dimers and monomers. Heteronuclear NOEs intensities of the monomer were subtracted from those of the dimer, resulting in the differential pattern of relaxation corresponding to amino acids probably present in the region of homodimerization.</p

General scheme of UNC-76/FEZ1 (<i>C. elegans / H. sapiens</i>) and UNC-69/SCOCO (<i>C. elegans / H. sapiens</i>) proteins.

<p>Mutations observed in <i>C. elegans</i>, and the corresponding region in the human protein, are indicated by asterisks. Regions of interaction with other proteins are indicated. Coiled-coils: box, alpha-helix prediction: gray.</p

FEZ1 is an anti-parallel dimer.

<p>15N-edited NOESY-HSQC of dimers and monomers of FEZ1 (92-194) shows contacts between side chains of amino acids that occur in the homodimer, preferably in an anti-parallel topology. These contacts occur between residues 123 and 143, 127 and 139, 130 and 137, and are indicated in the sequence shown at the bottom of the figure.</p