N-terminal horseshoe conformation of DCC is functionally required for axon guidance and might be shared by other neural receptors

Abstract

Deleted in colorectal cancer (DCC) is a receptor for the axon guidance cues netrin-1 and draxin. The interactions between these guidance cues and DCC play a key role in the development of the nervous system. In the present study, we reveal the crystal structure of the N-terminal four Ig-like domains of DCC. The molecule folds into a horseshoe-like configuration. We demonstrate that this horseshoe conformation of DCC is required for guidance-cue-mediated axonal attraction. Structure-based mutations that disrupt the DCC horseshoe indeed impair its function. A comparison of the DCC horseshoe with previously described horseshoe structures has revealed striking conserved structural features and important sequence signatures. Using these signatures, a genome-wide search allows us to predict the N-terminal horseshoe arrangement in a number of other cell surface receptors, nearly all of which function in the nervous system. The N-terminal horseshoe appears to be evolutionally selected as a platform for neural receptors.

Fig. 1.

Structure of DCC horseshoe. (A) Ribbon drawing of the crystal structure of the DCC N-terminal four Ig-like domains. The molecule folds into a horseshoe configuration with a six-residue linker between domains D2 and D3. Also shown is how the conserved Asn329 and Gln361 of D4 form hydrogen bonds to the main-chain of D1 to create a specific D1/D4 interface, which defines the unique shape of the horseshoe. (B) The D2–D3 junction. At the C-terminus of D2 (in red) the last residue of D2, Leu193, participates in a pair of hydrogen bonds to Phe114 and Met115. At the N-terminus of D3 (in cyan) the first residue Arg200 is involved in a main-chain hydrogen bond with Tyr228. This clearly defines a six-residue linker (in green) from Ser194 to His199. (C) The D1–D2 junction. There is no linker present here. The last D1 residue (Ala99; in green) is still located in a part of the β sheet. The first D2 residue (Gly100; in red) is also an integrated part of D2 as it engages in a complicated hydrogen bond network. (D) The D3–D4 junction. There is no linker between these two domains either. The last D3 residue (Leu290; in cyan) is involved in a β sheet hydrogen bond network, whereas the first D4 residue (Val291; in orange) forms two main-chain hydrogen bonds with Lys319, which is on the BC loop next to the cis-Pro320. This kind of junction is commonly seen in many IgSF structures with two abutting Ig-like domains (Wang and Springer, 1998).

Fig. 2.

A comparative sequence alignment of peptide sequences containing D3–D4 transition (upper panel) and the D2–D3 transition (middle panel) shows the existence of a linker in D2–D3. The structure-based alignment was made by Dali pairwise comparison. Secondary structural elements are marked in accordance with the structure of DCC. A notable feature is the β turns between the F and G strands of the preceding domain and those between the A and B strands of the following domain. At the AB turn in particular there is a conserved Gly (shaded in orange), whereas N-terminal to the FG turn is a conserved Asn, which forms a complicated hydrogen bond network, exemplified by Asn178 shown in Fig. 1C. The end of the preceding domain can be defined by the alternate hydrophobic (shaded in gray) and hydrophilic residues at the C-terminus of the G strand. All of these make domain size at this area much less variable and the domain boundary easy to identify. In the D3–D4 transition, the interval between the conserved Cys (shaded in yellow) on the F strand of D3 and the Cys (shaded in yellow) on the B strand of D4 is 41–43 residues. By contrast, in the D2–D3 transition, this interval is 48–53 residues, which is indicative of a linker. Included at the very bottom of the alignment is the transition between D1 and D2 of UNC5 (bottom panel), which suggests a bend-over of D1/D2 with a five-residue linker.

Fig. 3.

The D1/D4 interface. (A) Ribbon drawing of a local area of the D1/D4 interface of DCC. A conserved Asn329 forms two hydrogen bonds with the amide group of Phe359 in D4 (in green) and the amide group of Ser88 in D1 (in orange), respectively, to bridge the two domains together. Another conserved residue of D4 (Gln361) contributes two hydrogen bonds to the main-chain of Ile90 in D1. These specific hydrophilic interactions bring the end of the G strand of the D1 dock into a precise area of D4, allowing for hydrophobic contacts between Met327 and Phe359 of D4 to Ile90 and Ile89 of D1, respectively. This creates a defined D1/D4 interface, conserved among DCC, Dscam, and axonin, and possibly in many other neural receptors predicted to have N-terminal horseshoe arrangements. (B) Schematic of D1/D4 interfaces for DCC (left-hand panel), Dscam (middle panel), and axonin (right-hand panel) with known structures. Residues in rectangles are conserved. Asn/Asp and Gln, the key residues that contribute specific hydrogen bonds, are in a bold red font. The specific hydrogen bonds Asn/Asp and Gln mediated are drawn. The contacting hydrophobic residues are shaded with the same colors. In DCC they are the Met327-Ile90 pair (shaded in purple) and the Phe359-Ile89 pair (shaded in brown). (C) Structure-based sequence alignment of the D1/D4 interface of DCC, Dscam, axonin, neurofascin and hemolin, made by Dali pairwise comparison. The secondary structural elements are marked in accordance with the structure of DCC. The key residues Asn/Asp and Gln are in a bold red font. The conserved Cys, Tyr and Trp are shaded in yellow, light green and blue, respectively. The contacting hydrophobic pairs are shaded with the same colors, purple and brown, respectively.

Fig. 4.

Measurement of axon guidance. From the center of the neuronal cell body to the center of the coated bead, a line was drawn as axis x. Axis y is perpendicular to x. Therefore x and y marked four quadrants: I, II, III and IV. Only the axons ending (no matter where the initiation point is) at the same side as the bead were measured (e.g. in the example diagram, quadrants I and II, but not III and IV). Then a line was drawn from the initiating point of the axon to the bead (line a). A line was drawn from the end point of the axon to the bead (line b). The angle (α) between a and b was measured. A value of α≤5 was taken as ‘towards the bead’ (A, not B, in the example diagram).

Fig. 5.

DCC mutants had reduced axon guidance induced by netrin. (A) Examples of axons growing and the netrin-coated beads (white circle). Scale bar: 20 µm. (B) Compared with WT, the numbers of axons growing towards netrin-coated beads in N329P, Q361S and Δlinker2-3 DCC mutants were significantly decreased. In addition, the length of axons was reduced in neurons microinjected with mutant DCC. Beads coated with PBS or BSA did not attract axons. Data are means±s.e.m. (n = 6). **P<0.01. (C) In COS-7 cells, netrin-binding assays showed binding ability of netrin to non-transfected cells, vector only, WT, N329P, Q361S and Δlinker2-3 DCC mutants at the cell surface. Scale bar: 20 µm.

Fig. 6.

DCC mutants had reduced axon guidance induced by draxin. (A) Compared with WT, the numbers of axons growing toward draxin-coated beads in N329P, Q361S and Δlinker2-3 DCC mutants were significantly decreased. In addition, the length of axons was reduced in neurons microinjected with mutant DCC. Data are means±s.e.m. (n = 6). **P<0.01. (B) In COS-7 cells, draxin-binding assays showed binding ability of draxin to vector only, WT, N329P, Q361S and Δlinker2-3 DCC mutants at the cell surface. The control medium was the culture medium without draxin treatment. Scale bar: 20 µm.