Abstract
The Slit-Robo (sr) GTPase-activating protein (GAPs) are important components in the intracellular pathway mediating Slit-Robo signaling in axon guidance and cell migration. We report the first crystal structure of the srGAP1 SH3 domain at 1.8-A resolution. The unusual side chain conformation of the conserved Phe-13 in the P1 pocket renders the ligand binding pocket shallow and narrow, which contributes toward the low binding affinity. Moreover, the opposing electrostatic charge and the hydrophobic properties of the P3 specificity pocket are consistent with the observed binding characteristics of the srGAP1 SH3 domain to its ligand. Surface plasmon resonance experiments indicate that the srGAP1 SH3 domain interacts with its natural ligand inaCtoN orientation. The srGAP1 SH3 domain can bind to both the CC2 and CC3 motifs in vitro. The N-terminal two acidic residues in the CC3 motif recognition site are necessary for srGAP1 SH3 domain binding. A longer CC3 peptide (CC3-FL) binds with greater affinity than its shorter counterpart, suggesting that the residues surrounding the proline-rich core are important for protein-peptide interactions. Our study reveals previously unknown properties of the srGAP-Robo interaction. Our data provide a structural basis for the srGAP-Robo interaction, consistent with the role of the Robo intracellular domain in interacting with other downstream signaling molecules and mediating versatile and dynamic responses to axon guidance and cell migration cues.
Highlights
In the developing central nervous system, the axon must navigate through a complex terrain consisting of various cell types, distinct neuronal processes and many extracellular matrix molecules
Extracellular application of Slit to primary neurons increases the intracellular binding of Robo and srGAP1, and inhibits Cdc42 activity in a Robo- and srGAP-dependent manner, which emphasizes the importance of the cytoplasmic motif 3 (CC3) motifs in Robo1 and the SH3 domain of srGAPs in Slit-Robo signaling pathway [11]
We have investigated the recognition site in the intracellular domain of Robo recognized by the srGAP1 SH3 domain using BIAcore experiments
Summary
Protein Expression and Purification—The DNA fragments encoding the srGAP SH3 domains (srGAP1 SH3, srGAP2 SH3, and srGAP3 SH3) from Ala-9 to Val-60 were amplified by PCR using the full-length srGAP sequences (KIAA1304, KIAA0456, and KIAA0411) as templates. The recombinant srGAPs SH3 domains were stored in 10 mM Tris, pH 8.0, 100 mM NaCl and concentrated to 15 mg/ml. The best diffracting crystals of srGAP1 SH3 domain were obtained at 18 °C from a protein solution at 15 mg/ml in 10 mM Tris, pH 8.0, 100 mM NaCl, and a reservoir solution containing 1.5 M lithium sulfate, pH 7.5.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
