Fig. 2.
The Ig1–Ig2 interface in Dlar and mouse LAR. (a) Stereo view of the interface between Ig domains 1 and 2 in Dlar(Ig1-2). Residues at the interface between the two domains are shown as ball-and-sticks and colored gold (Ig1) or brown (Ig2). This view is in the same orientation as the right view in Fig. 1c. Transparent gray spheres and dashed lines denote residues involved in van der Waals contacts and potential hydrogen bonds and salt bridges, respectively. (b) Stereo view of the interface between Ig domains 1 and 2 in LAR(Ig1-2). Residues at the interface between the two domains are shown as ball-and-sticks and colored cyan (Ig1) or blue (Ig2). This view is in the same orientation as the right view in Fig. 1d. (c) Alignment of amino acid sequences of Ig domains 1–2 of Dlar and mouse type IIa RPTPs. Strictly conserved residues are shaded in black and similar residues are colored gray. The numbering refers to Dlar. Cysteine residues involved in disulfide bridges are numbered in green below the sequences. Gold and brown triangles indicate the residues involved in interactions between Ig domains 1 and 2 of Dlar that are unique to Dlar. Cyan and blue triangles indicate the residues involved in interactions between Ig domains 1 and 2 of mouse LAR that are unique to LAR. Red stars indicate the positions of residues involved in interactions between Ig1 and Ig2 of both Dlar and mouse LAR. (d) Analysis of interference-free SAXS curve for LAR(Ig1-2). The experimental scattering profile (black) for LAR(Ig1-2) and the theoretical scattering (red line, χ2=1.2) calculated from the LAR(Ig1-2) crystal structure are shown on the left panel. The right panel shows the Guinier plots with linear fit (red line). (e) Crystal structure of LAR(Ig1-2) used to calculate the theoretical scattering profile in which the red regions indicate disordered residues at the N- and C-termini that have been added in extended form and optimized by BILBOMD 41. These residues are GPGSSRG at the N-terminus and VRRVAPRFS at the C-terminus. SAXS data were collected at the ALS beamline 12.3.1 LBNL Berkeley, California 42. Tunable wavelength λ 1.0–1.5 Å and the sample-to-detector distances were set to 1.5 m resulting in scattering vectors, q, ranging from 0.01 Å−1 to 0.32 Å−1. The scattering vector is defined as q = 4π sinθ/λ, where 2θ is the scattering angle. All experiments were performed at 20 °C and data was processed as described 42. Data acquired at short and long time exposure (0.5 and 5 s) were merged for calculations using the entire scattering profile. The experimental SAXS data for different protein concentrations were investigated for aggregation using Guinier plots 43. The radius of gyration RG is 20.5 ± 0.1 Å and was derived by the Guinier approximation I(q) = I(0) exp(−q2RG 2/3) with the limits qRG < 1.6. The theoretical SAXS profile and the corresponding fit to the experimental data were calculated using the program FoXS 44. (f) Fusion proteins of hGH with wild-type Dlar(Ig1-2) (Dlar(Ig1-2), WT) and a mutant form of Dlar(Ig1-2) with cysteine residues at positions 52 and 219 (Dlar(Ig1-2), Cys)) were analyzed by SDS-PAGE (9% gel) under reducing and non-reducing conditions followed by immunoblotting against hGH. (g) Schematic drawing of the Drosophila larval central nervous system, illustrating the positions of the two brain hemispheres (BH) and the ventral nerve cord (VNC). (h) Confocal micrographs of the VNC region shown in (g) (dotted box). Fc fusion proteins of histidine-tagged wild-type and mutant Dlar(Ig1-2) were expressed in HEK293 cells and purified from conditioned media by cobalt-affinity chromatography. The brain/VNC complex was dissected from second instar larvae in phosphate-buffered saline (PBS) and incubated with the indicated fusion proteins for 30 minutes. After fixation for 30 minutes in PBS/formaldehyde, the larval VNC was washed with PBS with 0.05% (v/v) Triton X-100 and incubated with protein G-Alexa Fluor 488. Both wild-type Dlar(Ig1-2) (left panel) and its cysteine mutant (middle panel) showed a staining pattern consistent with ventral nerve cord staining that was not observed in mock (cobalt-affinity chromatography eluate of conditioned media from untransfected HEK293 cells, right panel).