Fig. 2: NELL2 EGF2–3 bind Robo3 FN1 to mediate complex formation.
From: NELL2-Robo3 complex structure reveals mechanisms of receptor activation for axon guidance
a, b Domains mediating NELL2–Robo3 interactions were mapped using a COS-7-based AP-fusion protein binding assay. NELL2 EGF2 and EGF3 together are necessary and sufficient for binding to Robo3 (a). Robo3 FN1 is necessary and sufficient for NELL2 binding (b). c Size-exclusion chromatography of the hNELL2 EGF1–6 and hRobo3 FN1–3 complex. hNELL2 EGF1–6, hRobo3 FN1–3, and a molar 1:1 mixed complex samples were injected in a Superdex 200 Increase 10/300 column, and the elution profile was recorded by following absorbance at 280 nm with a pathlength of 0.2 cm. Dark blue: hNELL2 EGF1–6 + hRobo3 FN1–3 complex sample; Cyan: hRobo3 FN1–3; and Red: hNELL2 EGF1–6. AU: Absorbance units. SPR sensorgrams (d) and equilibrium response fitting to a Langmuir 1:1 binding model (e) for biotinylated hRobo3 FN1–3 as ligand on an NLC/neutravidin chip with hNELL2 EGF1–6 as the analyte (mobile phase) collected on a ProteOn XPR36. Legend on the right in (d) refers to concentration of the analyte injected on the SPR chip. Black curve in (e) is the Langmuir model fit to response values measured at equilibrium. ± refers to standard error of the fit. Scale bar, 100 μm, (a, b).
