The binding of DCC-P3 motif and FAK-FAT domain mediates the initial step of netrin-1/DCC signaling for axon attraction

Abstract

Netrin-1 plays a key role in axon guidance through binding to its receptor, Deleted in Colorectal Cancer (DCC). The initial step of signaling inside the cell after netrin-1/DCC ligation is the binding of DCC cytoplasmic P3 motif to focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK). Here we report the crystal structure of P3/FAT complex. The helical P3 peptide interacts with a helix-swapped FAT dimer in a 2:2 ratio. Dimeric FAT binding is P3-specific and stabilized by a calcium ion. Biochemical studies showed that DCC-P3 motif and calcium ion could facilitate FAT dimerization in solution. Axon guidance assays confirm that the DCC/FAK complex is essential for netrin-1-induced chemoattraction. We propose that netrin-1/DCC engagement creates a small cluster of P3/FAT for FAK recruitment close to the cell membrane, which exerts a concerted effect with PIP2 for FAK signaling. We also compare P3/FAT binding with paxillin/FAT binding and discuss their distinct recognition specificity on a common FAT domain for axon attraction versus integrin signaling, respectively.

Fig. 1. Crystal structure of DCC-P3 motif in complex with FAK-FAT domain.

a Domain diagram of DCC and FAK, with the region present in the crystal structure in red. b Two views of the overall structure of the DCC-P3/FAK-FAT complex. For the helix-swapped FAT dimer, one FAT domain is colored in cyan and the other in green. The two P3 motifs are colored in orange. For one of the FAT domains, the secondary structure elements are marked. c A representative simulated-annealing composite omit 2F_o–F_c electron density map (contoured at 1.0σ level) for P3 motif. The Q¹⁴³⁶ residue that is important for the binding specificity is labeled

Fig. 2. Characteristic features of the P3-bound FAT dimer.

a Comparison of the FAT 4-helix bundle structures in monomer and dimer. Upper and lower panels show the 4-helix bundles in the 2:2 P3/FAT dimer and the FAT monomer (apo-form) (PDB code 1K40), respectively. Residues on the H1/H2 loop are shown with ball-and-stick models and labeled. The secondary structure elements of the FAT domains are labeled. The color coding for the 2:2 P3/FAT dimer is the same as in Fig. 1b. The apo-FAT monomer is colored in salmon. b Dimer interface in the P3/FAT complex. In the left panel, the secondary structure elements of the FAT domains are labeled. Right panel is a close-up view of the dimer interface. The hydrogen bonds and metal ion coordination bonds are indicated with black dashed lines. The calcium ion is shown in gray sphere. Side-chain or main-chain atoms of the residues involved in stabilizing the dimer are shown with ball-and-stick models. For clarity, residues from one P3/FAT protomer are labeled in black and the other in magenta. The color coding for the P3/FAT complex is the same as in Fig. 1b. c SEC-MALS assay showed that addition of the DCC-P3 peptide and Ca²⁺ to FAT results in FAT dimerization in solution. Upper panel: The elution profile of FAT alone. Middle panel: The elution profile of FAT in the presence of DCC-P3 peptide. Lower panel: The elution profile of FAT in the presence of DCC-P3 peptide and Ca²⁺

Fig. 3. Comparison of the P3/FAT complex with LD/FAT complexes.

The crystal structures of DCC-P3/FAK-FAT (a), paxillin LD2/FAK-FAT (PDB code 1OW8) (b), paxillin LD4/FAK-FAT (PDB code 1OW6) (c), and leupaxin LD1/Pyk2-FAT (PDB code 4XEF) (d) are shown in the same orientation. The hydrogen bonds are indicated with black dashed lines. Side-chain or main-chain atoms of the residues involved in binding are shown with ball-and-stick models as is the side chain of DCC-P3 S¹⁴²⁴ that is equivalent to the invariant aspartate residues in the LD motifs. At the H2/H3 interface of the leupaxin LD1/Pyk2-FAT complex, side chains of leupaxin LD1 D⁵ and Pyk2-FAT R⁹¹⁸ are also shown as ball-and-stick models. Residues of P3 and LD motifs are labeled in black. Concerning the FAT domains, residues involved in hydrogen-bond interactions are labeled in marine for the P3/FAT complex and magenta for LD/FAT complexes. For FAT, the H2–H3–H4 body is colored in cyan while the H1 is colored in green in the dimeric form and in cyan in the monomeric form. The DCC-P3 motif and the LD motifs are colored in orange and olive, respectively. e Structure-based sequence alignment of P3 motifs of rat DCC and its homologs (mouse Neogenin and Drosophila Frazzled) and LD motifs of human paxillin and human leupaxin. The hydrophobic residues at the ‘a’ or ‘d’ positions of heptad repeats are shaded in red. The residue Q¹⁴³⁶, important for P3 and FAT-binding specificity, is in yellow. The resolved regions of the LD peptides in the crystal structures of paxillin LD2/FAK-FAT (PDB code 1OW8), paxillin LD4/FAK-FAT (PDB code 1OW6), leupaxin LD1/Pyk2-FAT (PDB code 4XEF), and leupaxin LD4/Pyk2-FAT (PDB code 4XEV) are indicated with cyan arrows. The starting and ending residues of the peptides used for crystallization are indicated with magenta arrows. The sequence numbers of each motif and the secondary structure elements of DCC-P3 motif and leupaxin LD4 motif are marked

Fig. 4. Axon guidance assay verifying the importance of DCC-P3/FAK-FAT binding in netrin-1-induced axon attraction.

a Representative images of axon guidance assays for WT and mutant DCC affecting DCC-P3/FAK-FAT binding. Phosphate-buffer saline was used as a negative control. The position of the bead coated with netrin-1 is marked with a circle. The injected cells are indicated by DTR (red). Cell bodies are dyed with DAPI (blue). Growing axons are marked with anti-AnkG antibody (green) and indicated by white arrows. The scale bar denotes 10 μm. b Statistics data showing the percentage of axons attracted by the beads coated with netrin-1. DCC mutants affecting DCC-FAK binding abolish attraction. Data represent mean ± SE (n = 50 for each group). One-way ANOVA followed by a post hoc Scheffe´’s test were performed. **p < 0.01 compared with wild type

Fig. 5. A model for netrin-1/DCC engagement leading to intracellular FAK/Src signaling via DCC-P3 and FAK-FAT binding (proposed signaling sequence step-wise from left to right).

DCC is expressed on the growth cone of the axon. (Step 1) Netrin-1 binds to DCC to cause DCC clustering. (Step2) Two P3 motifs in the DCC cytoplasmic tails specifically ligate FAT domain of FAK into H1-swapped dimeric form, which is further stabilized by netrin-1 induced Ca²⁺ influx. The small cluster of netrin-1/DCC pairs on the cell surface puts P3-facilitated FAK dimers in proximity, resulting in a local accumulation of FAK dimers, promoting PIP2-induced FAK clustering. This eventually gives rise to FAK activation, a process initiated by PIP2 to release auto-inhibition of the FAK kinase domain by the FAK FERM domain