Robo1 Forms a Compact Dimer-of-Dimers Assembly

Abstract

Roundabout (Robo) receptors provide an essential repulsive cue in neuronal development following Slit ligand binding. This important signaling pathway can also be hijacked in numerous cancers, making Slit-Robo an attractive therapeutic target. However, little is known about how Slit binding mediates Robo activation. Here we present the crystal structure of Robo1 Ig1-4 and Robo1 Ig5, together with a negative stain electron microscopy reconstruction of the Robo1 ectodomain. These results show how the Robo1 ectodomain is arranged as compact dimers, mainly mediated by the central Ig domains, which can further interact in a "back-to-back" fashion to generate a tetrameric assembly. We also observed no change in Robo1 oligomerization upon interaction with the dimeric Slit2-N ligand using fluorescent imaging. Taken together with previous studies we propose that Slit2-N binding results in a conformational change of Robo1 to trigger cell signaling.

Keywords: X-ray crystallography; electron microscopy; receptor; robo; slit.

Graphical abstract

Figure 1

Structure of Human Robo1 Ig1-4 and Ig5 (A) The domain composition of Robo1 and Slit2. The Robo1 Ig and Fn domains are colored green and blue, respectively. The Slit2 LRR, EGF, Lamin, and CTCK domains are colored orange, yellow, red, and brown, respectively. The cleave site of Slit2 is indicated by a dashed line. (B) Robo1 Ig1-4 domains adopt an extended structure. The N-glycosylation at N160 is shown in stick representation. (C) The major crystallographic contacts are mediated by Ig1, Ig3, and Ig4. Interface 1 is symmetric and mediated by Ig4 (blue); interface two is mediated by Ig2-3 (yellow) and Ig4 (orange); and interface three is mediated by Ig3 (red) and Ig4 (salmon); interface four is mediated by Ig1 (light and dark green) and overlaps the Slit2 D2 binding site, illustrated as a ribbon representation (N- and C-terminal caps colored in magenta and cyan, respectively, and LRR colored in orange). (D) The Robo1 Ig5 domain structure showing a canonical I-set fold. (E) A superposition of Robo1 Ig domains with Ig1, Ig2, Ig3, Ig4, and Ig5 colored in red, gray, cyan, blue and green, respectively. One potential conformation of K137 and R136 (disordered) is shown in stick representation to highlight the Robo1 Ig1 heparin binding region (βE-βF loop).

Figure 2

EM Reconstruction of the Robo1 ECD (A) A representative negative stain EM micrograph is shown. Scale bar, 50 nm. (B) Comparison of 2D class averages after alignment and classification with the back projections of the final refined volume. (C) Gold-standard Fourier shell correlation (FSC) curves indicating a resolution of 16 Å according to FSC = 0.143 criterion. (D) 3D reconstruction and domain assignment of Robo1 ECDs. Three orientations are shown to illustrate the “dimer-of-dimers” configuration. The monomers (A, B, C, and D) form inactive dimers (A/B and C/D), which further assemble in a “back-to-back” fashion as a tetramer. (E) Schematic overview of the major Robo1 back-to-back dimer conformation in similar orientations to (D). For illustration purposes the membrane insertion region was included, one dimer was paled, and only one dimer shown in the final orientation. The Ig and Fn domains are labeled and colored green and blue or red and yellow, respectively, for each dimer.

Figure 3

Robo1 Remains Oligomeric on the Cell Surface upon Addition of Slit2-N (A) Schematic overview of the in situ fluorescence-based PLA assay used to detect Robo1 oligomerization on the cell surface. (B) An interaction between Robo1-EGFP and -mCherry tagged proteins on the cell surface of HEK293T cells was detected by PLA. (C) This interaction is maintained in the presence of Slit2-N (0.1 μM). Each green dot represents the detection of a Robo1-EGFP and Robo1-mCherry interaction complex, and nuclei were counterstained with DAPI (blue). Single optical slices were acquired with a laser scanning confocal microscope. Robo1-mCherry, PLA interaction, and DAPI are colored red, green and blue, respectively. Scale bars, 10 μm.

Figure 4

Putative Arrangements of Robo1 on the Cell Surface (A) Compact inactive Robo1 dimers on the cell surface can (B) undergo a conformational change but remain intact upon stimulation with Slit2-N.