Crystal structures of the Ig1–Ig2 domains of Dlar and mouse LAR. (a) Schematic representation of type IIa RPTPs. Ig: immunoglobulin-like domains, FNIII: fibronectin type III domains, TM: transmembrane region, PTP: protein tyrosine phosphatase domains. The Ig domains that were crystallized are shaded. (b) Identification of a heparin-binding region within the Ig domains of Dlar. Fragments of Dlar were fused to human growth hormone (hGH) and expressed transiently in HEK293 cells. Conditioned media were incubated with heparin-sepharose. Bound fusion proteins were visualized by immunoblotting against hGH. (c) Ribbon diagram of Dlar(Ig1-2). The letters N and C indicate the N- and C-termini, respectively. Each β-strand is labeled. Disulfide bonds are shown as orange ball-and-stick models. The first and second Ig domains are colored gold and brown, respectively. (d) Ribbon diagram of mouse LAR(Ig1-2). The first and second Ig domains are colored cyan and blue, respectively. Two bound sulfate ions are shown in ball-and-stick representation. Oxygen and sulfur atoms are colored red and orange, respectively. Structural images were prepared with PYMOL (www.pymol.org).

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 R_G is 20.5 ± 0.1 Å and was derived by the Guinier approximation I(q) = I(0) exp(−q²R_G ²/3) with the limits qR_G < 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).

Structural comparisons of the antiparallel arrangement of Ig domains 1 and 2 of Dlar with other horseshoe-like structures. (a) Comparison of the Dlar(Ig1-2) structure (left, colored gold and brown) with three structures of tandem Ig repeats that adopt a horseshoe-like conformation. The distinct conformations of horseshoe-like arrangements of these tandem β-sandwiches repeats arise from interactions between residues found in distinct strands, which are listed below each structure. In each of the compared structures, the N-terminal and C-terminal Ig repeats are colored pink and magenta, respectively. Disulfide bonds are shown as orange ball-and-stick models. The letters N and C indicate the N- and C-termini, respectively. These views of Dlar(Ig1-2), p58(Ig1-2), CNTN4(Ig2-3) and Dscam(Ig5-6) were obtained by superimposing their N-terminal Ig repeats in an effort to highlight the distinct positions of the C-terminal Ig repeats that result from changes in the contacting faces in each of the horseshoe-like structures. (b) Topology diagrams for I set Ig domains and FNIII domains highlighting the similarities between the two folds ⁴⁵. (c) Ribbon diagram depicting the horseshoe-like arrangement observed for the tandem FNIII repeats of Drosophila iHog. The first and second FNIII repeat of iHog are colored pink and magenta, respectively. This view was obtained by superimposing the 1st FNIII iHog and the 1st Ig repeat of Dlar using secondary structure matching. (d) Overlay of the Ig1–Ig2 fragment of Dlar with the first (pink) and second (magenta) FNIII domains of Drosophila iHog. The HSPG-binding site for iHog and the presumed glycosaminoglycan-binding site for Dlar partially overlap and are shown in the boxed view.

(a) Electrostatic surface representation of Dlar(Ig1-2). This view is related to the left view on panel c by a counterclockwise rotation of 120° along a vertical axis. Regions with negative electrostatic potential are colored red and regions with positive electrostatic potential are colored blue (scale ± 5 e/kT). (b) Electrostatic surface representation of mouse LAR(Ig1-2). Electrostatic potentials were calculated with DELPHI ^{46, 47}. (c) Interactions between mutants of Dlar(Ig1-2) and heparin. Fragments of Dlar fused to hGH were expressed transiently in HEK293 cells. These fragments include wild-type Dlar(Ig1-2) (labeled Dlar(Ig1-2), WT), Dlar(Ig1-2) with cysteine residues at positions 52 and 219 (Dlar(Ig1-2), Cys), with asparagine residues at positions 52 and 218 to introduce N-linked carbohydrates (Dlar(Ig1-2), Asn), Dlar(Ig1) and Dlar(Ig2). The left panel shows wild-type Dlar(Ig1-2) along with its cysteine and asparagine mutants to illustrate the difference in size between the proteins upon introduction of consensus N-linked glycosylation sites in Dlar(Ig1-2). Samples were resolved by SDS-PAGE (9% gel) and fusion proteins were visualized by immunoblotting against hGH. The right panel shows the results of heparin affinity isolation assays. Conditioned media were incubated with heparin-sepharose in PBS with 1 % (v/v) Tween-20 for one hour at room temperature. Resins were washed in the same buffer and samples were resolved by SDS-PAGE (12% gel). Bound fusion proteins were visualized by immunoblotting against hGH.