www.sciencemag.org/cgi/content/full/science.1255149/DC1

Supplementary Materials for

Structures of netrin-1 bound to two receptors provide insight into its axon guidance mechanism

Kai Xu, Zhuhao Wu, Nicolas Renier, Alexander Antipenko, Dorothea Tzvetkova-Robev, Yan Xu, Maria Minchenko, Vincenzo Nardi-Dei, Kanagalaghatta R. Rajashankar, Juha

Himanen, Marc Tessier-Lavigne,* Dimitar B. Nikolov*

*Corresponding author. E-mail: nikolovd@mskcc.org (D.B.N.); marctl@mail.rockefeller.edu (M.T.-L.)

Published 29 May 2014 on Science Express DOI: 10.1126/science.1255149

This PDF file includes:

Materials and Methods Figs. S1 to S9 Table S1 References

Submitted Manuscript: Confidential 18 May 2014

Supplementary Materials: www.sciencemag.org

Materials and Methods Figures S1-S9

Tables S1 References (31-35)

Materials and Methods:

Mouse lines

The genotyping of Netrin-1-/- (2), Dcc-/- (21) and Neo1-/- (31) mutant lines, as well as mutant

embryos, were previously described. Double mutant embryos were generated from intercrosses of

double-heterozygous parents, and E0.5 designates the noon of the plug date.

Dcc-/-, Neo1-/- and Dcc-/-;Neo1-/- mutants are in the same C57Bl6 genetic background. Netrin-1-/-

mutants are from a CD1 background. To check whether the genetic background could explain the

differences seen in Dcc-/- and Netrin-1-/- mutants embryos, we back-crossed Dcc-/- mice on a CD1

background, and didn’t see any effect on the phenotypes described. Similarly, Netrin-1-/-

phenotypes were not affected when back-crossed with C57Bl6. However we found that

commissural thickness does change with the progression of embryonic development. Therefore we

have only taken size-matched littermates from the intercross of compound heterozygous animals

for comparison. The genetic backgrounds for the ones shown and quantified are C57Bl6/CD1 for

figure 1A, and C57Bl6 for figure 1B and 1D.

Histology and Immunohistochemistry

Mouse embryos were harvested at E11, fixed in PBS/4% PFA overnight at 4°C, cryopreserved in

PBS/10% sucrose for 2 days at 4°C. 20µm frozen sections were cut on a cryostat. The following

primary antibodies were used: goat anti-human Robo3 (1:400, R&Dsystems), goat anti-mouse/rat

TAG1 (1:1000, R&Dsystems), 4D7 mouse IgM monoclonal anti-TAG1 (1:50, DSHB), rabbit anti-

NF-M (1:1000, Covance), mouse or rabbit anti-neuronal ßIII-tubulin (1:1000, Covance), goat anti-

mouse DCC (1:400, R&D), goat anti-mouse neogenin (1:400, R&Dsystems). The following

secondary antibodies were used: donkey anti-goat, anti-mouse, anti-rabbit coupled to Alexa 488,

568 or 647 (1:1000, Invitrogen). Sections were examined with a fluorescent microscope (Eclipse

90i, Nikon) coupled to a Nikon QiMc camera or a confocal microscope (Sp8, Leica).

Quantification of the commissural bundle to the spinal cord size ratios where done for each

embryo on 5 evenly spaced transverse spinal cord sections taken at brachial levels. Ratios where

normalized to the WT control values. To minimalize the variations due to the embryonic stages

and growth speeds across different litters and genetic backgrounds, only littermate embryos were

compared. One-way ANOVA analysis with Bonferroni post-test was performed to compare the

means across genotypes. Robo3-positive axons deviated to the motor column were counted from

the same set of sections, and total numbers for each embryo were presented.

Dorsal Spinal Cord explant cultures

Spinal cords were dissected from E11.5 embryos as previously described (12) in ice-cold L-15

media (Gibco), and dorsal part of the spinal cord were cut into 160 to 180 µm segments (DSC

explants) from a open-book preparation. DSC explants were embedded in rat-tail collagen (BD

Bioscience) and cultured in DMEM/F12 (Gibco) supplemented with 5% FBS, 1% D-glucose, L-

glutamine and penicillin/streptomycin (Invitrogen), in a 5% CO2, 95% humidity incubator at 37°C

for 24 hours. Recombinant Netrin-1 proteins (R&D) were added at the indicated concentrations to

the culture media. Explants were visualized by immunostaining with mouse anti-neuronal ßIII-

tubulin antibody and signal intensities were quantified using ImageJ software (Rasband WS,

ImageJ, U.S. National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/, 1997–2009).

Signals from the axonal area were normalized with signals from the center cell mass region for

each explant, and compiled data is expressed as mean and SEM. Statistical significance was

calculated using a two-way ANOVA with Bonferroni post-test. The cultures were repeated 3 times

and the results were blindly evaluated.

Expression and purification of Netrin-1, DCC, and Neogenin

Chicken Netrin-1 (NP_990750.1), containing the VI (LN) and V (LE1, LE2, LE3) laminin-like

regions (residues 26-458) was cloned in both the pMA152a baculovirus vector and the

pCDNA3.1+ mammalian expression vector (Invitrogen). pMA152a is based on the pAcGP67B

vector (BD Biosciences) but has an incorporated, removable Fc-tag (human). Constructs of mouse

Neogenin (NP_032710.2), containing the FN4-5 domains (residues 765-980), and constructs of

mouse DCC (NP_031857.2), containing FN domains 1-5, 4, 4-5, 4-6, 5 and 5-6 (residues 410-941,

721-839, 721-941, 721-1035, 839-941 and 839-1035, respectively) were cloned in the pMA152a

vector. Secreted recombinant proteins were produced by baculovirus-infected Hi5 insect cell and

stably transfected HEK293 cell following the protocols provided by the companies (BD

Biosciences and Invitrogen). Hi5 insect cell expressed Netrin-1 protein was used for unbound-

netrin crystallization, while HEK293 expressed Netrin-1 protein and Fn domains 4-5 of both

Neogenin and DCC were used for crystallization of the netrin/receptors complexes. To facilitate

crystallization of the complexes, the shorter isoforms of neogenin (Isoform-3, lacking residues

863-878, Uniport identifier: P97798-3) and DCC (Isoform-C, lacking residues 819-838, Uniport

identifier: P70211-2), containing shorter linker region between FN4 and FN5, were chosen. A

predicted N-link glycosylation site (N924) on neogenin FN5 domain was mutated to glutamine for

the same purpose. Purification of all recombinant proteins was facilitated by the removable (by

thrombin protease) C-terminal Fc tag. Protein-A affinity chromatography and SD-200 size-

exclusion chromatography (GE Biosciences) were used to obtain the final purified recombinant

proteins.

Binding assays for recombinant netrin and DCC/neogenin constructs

The binding affinity of the Netrin-1 protein (LN-LE3) to different domains of DCC, as well as to

the FN 4-5 regions of both the longer and shorter isoforms of neogenin and DCC, were measured

by bio-layer interferometry on a BLItz instrument (ForteBio). To evaluate the role of the netrin-

bound Ca++, 10 mM EDTA was added in one of the measurements. ProteinA biosensors were used

to immobilize the Fc tagged proteins (Neogenin and DCC). Binding affinities (Kd) were calculated

from a non-linear fit of the data using the BLItz software.

Crystallization and data collection:

All purified proteins for crystallization were kept in 20mM Hepes buffer saline with pH 7.2 and

0.5M NaCl and concentrated to around 10mg/ml. netrin/receptors complexes were obtained by

mixing Netrin-1 with receptors in 1:1.5 molar ratio and purified by size-exclusion chromatography.

Netrin-1 (LN-LE1-3) migrates as a monomer, the Netrin-1/neogenin complex migrates as a 2:2

heterotetramer, while the Netrin-1/DCC complex migrates as a complex larger than 1:1, but

somewhat smaller than the Netrin-1/neogenin complex. Initial crystal screening was performed by

both manual hanging drop vapor diffusion and robot (TTP LabTech’s Mosquito) sitting-drop vapor

diffusion methods. The crystallization conditions were 0.1M Na-Cacodylate pH 6.5, 0.5M KCl and

1 M Na-Acetate (unbound netrin); 0.1M Hepes pH 7.0, 12% PEG3350 (netrin/neogenin complex);

and 0.05 M Ammonium sulfate, 0.05 M BIS-TRIS pH 6.5, 30% v/v Pentaerythritol ethoxylate

(15/4 EO/OH) (netrin/DCC complex). Crystals were optimized by Additive Screen and Detergent

Screen (Hampton Research). Crystals were cryo-protected by adding 25% glycerol. Crystal

diffraction data were collected at NSLS beamline X9B of the Brookhaven National Lab and APS

beamline ID-24 of the Argonne National Laboratory.

Structure determination and refinement:

Data images were processed using the program HKL2000 (32). All structures were determined by

Molecular Replacement with the program Phaser (33). The laminin a5 chain N-terminal fragment

structure (PDB-ID 2Y38) was used as the search template for the unbound Netrin-1 structure

determination. The Netrin-1/neogenin and Netrin-1/DCC complex structures were determined

using the structure of unbound Netrin-1 and the solution structures of the individual FN domain of

the receptors (PDB-ID 1X5I, 1X5J, 2EDB and 2EDD) as search models. The models were built

and refined iteratively using Coot (34) and PHENIX Refine (35). A summary of data collection

and refinement statistics is presented in Supplementary Table S1.

Illustrations

All molecular representations were produced with PyMOL (Delano Scientific LLC). Figures were

prepared using Adobe Illustrator and Adobe Photoshop.

Supplementary Figures：

Fig. S1.

(A) Transverse sections of brachial spinal cord of E11 wild type, or Tag-1-/- litermate mouse

embryos stained for TAG-1 with the 4D7 mouse monoclonal IgM antibody and the goat

polyclonal antibody. (B) Transverse sections of brachial spinal cord of E11 wild type, Dcc-/- or

Netrin-1-/- mouse embryos stained for TAG-1 with the monoclonal and polyclonal antibodies.

The monoclonal antibody gives a weaker labeling than the polyclonal antibody (arrows),

however it shows more residual commissural axons in Netrin-1-/- than in Dcc-/- embryos.

Fig. S2.

A) Immunostating for Dcc and Neogenin in E11 spinal cord cross-sections in wild type and Dcc

mutants, or wild type and Neo1 mutants respectively. Dcc and Neogenin are detected in

commissural axons. B) Cross sections of E11 wild-type, Neo1-/-, Dcc-/-, Dcc-/-; Neo1-/- and

Netrin-1-/- littermate mouse embryos at the level of brachial spinal ganglia, stained for Robo3,

showing details of the motor column. Both Netrin-1-/- and Dcc-/-; Neo1-/- double mutants have

numerous axons in the motor column. C) Schematics showing the path taken by commissural

axons across genotypes. C : Commissural neurons, lf : lateral funiculus, fp : floor plate, mn :

motor neurons. D) Number of Robo3+ axons found in motor column of E11 Dcc-/-, Neo1-/- and

Dcc-/-; Neo1-/- embryos. The quantification shows the total number of axons counted on both

sides on 5 sections cut at the level of the brachial spinal cord.

Scale bars are 200µm (A) and 100µm (B).

cNet1: 1 MPRRGAEGPLALLLAAAWLAQPLRGGYPGLNMFAVQTAQPDPC----YDEHGLPR---RC hNet1: 1 M-MRAVWEALAALAAVACLVGAVRGG-PGLSMFAGQAAQPDPC----SDENGHPR---RC hNet3: 1 MPGW----PWGLLLTAGTL-------FAALS--PGPPAPADPC----HDEGGAPR---GC hNet4: 1 MGS----CARLLLLWGCTVVAAGLSGVAGVS---------SRC----------EK---AC hNet5: 1 MPV-----TFALLLLLG-------------------QATADPC----YDPQGRPQ---FC hNetG1: 1 MYLSRFLSIHALWVTVSSVMQP----------YPLVWGHYDLCKTQIYTEEGKVWDYMAC hNetG2: 1 M-----LHLLALF------LHC----------LPLASGDYDICKSWVTTDEGPTWEFYAC mLam5: 1 MAKRG--GQLCAGSAPGALGPRSPAPRPLLLLLAGLALVGEA-RTPGGDGFSLHPPYFN- S0 S0’ S1 S1 S2 cNet1: 54 IPDFVNSAFGKEVKVSS---TCGKPPSR-YCVV------TEKGEE--QVRSCHLCNASDP hNet1: 52 IPDFVNAAFGKDVRVSS---TCGRPPAR-YCVV------SERGEE--RLRSCHLCNASDP hNet3: 41 VPGLVNAALGREVLASS---TCGRPATR-------------------------ACDASDP hNet4: 35 NPRMGNLALGRKLWADT---TCGQNATELYCFY------SENTDLTCRQPKCDKCNAAYP hNet5: 30 LPPVTQLA-----AVAA---SCPQA-----CAL------SP--GN--HLGARETCNGS-- hNetG1: 51 QPESTDMTKYLKVKLDPPDITCGDPPET-FCAM------GNP--YMCN----NECDASTP hNetG2: 40 QPKVMRLKDYVKVKVEPSGITCGDPPER-FCSH------ENP--YLCS----NECDASNP mLam5: 57 LAEGARITASATCGEEAPTRSVSRPTEDLYCKLVGGPVAGGDPNQTIQGQYCDICTAANS S3 S4 cNet1: 102 KRAHPPSFLTDLNNPHNLTCWQSDSYVQYP--HNVTLTLSLGKKFEVT-YVSLQFC-SPR hNet1: 99 KKAHPPAFLTDLNNPHNLTCWQSENYLQFP--HNVTLTLSLGKKFEVT-YVSLQFC-SPR hNet3: 73 RRAHSPALLTSPGGTASPLCWRSESLPRAP--LNVTLTVPLGKAFELV-FVSLRFC-SAP hNet4: 86 HLAHLPSAMADSSFRFPRTWWQSAEDVH-----REKIQLDLEAEFYFT-HLIVMFK-SPR hNet5: 65 ------------------------------------LTLALGGPFLLT-SVSLRFC-TPG hNetG1: 98 ELAHPPELMFDFEGRHPSTFWQSATWKEYPKPLQVNITLSWSKTIELTDNIVITFE-SGR hNetG2: 87 DLAHPPRLMFDKEEEGLATYWQSITWSRYPSPLEANITLSWNKTVELTDDVVMTFE-YGR mLam5: 117 NKAHPVSNAIDGTER----WWQSPPLSRGLEYNEVNVTLDLGQVFHVA-YVLIKFANSPR S5 S6 H1 S7 cNet1: 158 PESMAIYKSMDYGKTWVPFQFYST---QCRKMYNK-PSRAAITKQNEQ-EAICTDSHTD- hNet1: 156 PESMAIYKSMDYGRTWVPFQFYST---QCRKMYNR-PHRAPITKQNEQ-EAVCTDSHTD- hNet3: 129 PASVALLKSQDHGRSWAPLGFFSS---HCDLDYGRLPAPANGPAGPGP-EALCFPAPLA- hNet4: 139 PAAMVLDRSQDFGKTWKPYKYFAT---NCSATFG------LEDDVVKK-GAICTSKYSS- hNet5: 87 PPALILSAAWASGGPW-------------RLLWHRPAWPGAL------------------ hNetG1: 157 PDQMILEKSLDYGRTWQPYQYYAT---DCLDAFHMDPKSVKDLSQHTVLEIICTEEYST- hNetG2: 146 PTVMVLEKSLDNGRTWQPYQFYAE---DCMEAFGMSARRARDMSSSSAHRVLCTEEYSRW mLam5: 172 PDLWVLERSTDFGHTYQPWQFFASSKRDCLERFG----PRTLERITQDDDVICTTEYS-R

S8 H2 H2 H3 S9 cNet1: 203 VRPLSGGLIAFSTLD-----GRPTAHD-------FDNSPVLQDWVTATDIKVTFSRLHTF hNet1: 210 MRPLSGGLIAFSTLD-----GRPSAHD-------FDNSPVLQDWVTATDIRVAFSRLHTF hNet3: 184 -QPDGSGLLAFSMQD-----SSPPGLD-------LDSSPVLQDWVTATDVRVVLTRPSTA hNet4: 188 PFPCTGGEVIFKAL------SPPYDTE-------NPYSAKVQEQLKITNLRVQLLKRQSC hNet5: 116 --------------------GGPERVT-------FHSTPGPKATVAASHLRVEF------ hNetG1: 213 GYTTNSKIIHFEIKDRFAFFAGPRLRNMASLYGQLDTTKKLRDFFTVTDLRIRLLRP-AV hNetG2: 203 AGSKKEKHVRFEVRDRFAIFAGPDLRNMDNLYTRLESAKGLKEFFTLTDLRMRLLRP-AL mLam5: 227 IVPLENGEIVVSLVN-----GRPGALN-------FSYSPLLRDFTKATNIRLRFLRTNTL H4 S10 S11 S12 cNet1: 259 GDE---NEDDSELARDSYFYAVSDLQVGGRCKCNGHASRCVRDRDDNLV----------- hNet1: 258 GDE---NEDDSELARDSYFYAVSDLQVGGRCKCNGHAARCVRDRDDSLV----------- hNet3: 231 GDP---RDMEAVVP---YSYAATDLQVGGRCKCNGHASRCLLDTQGHLI----------- hNet4: 235 PCQ---RNDLNEEPQHFTHYAIYDFIVKGSCFCNGHADQCIPVHGFRPVKAPGTFHMVHG hNet5: 143 GGQ---AGLAAAGLR-------------GRCQCHGHAARCA-ARARPPR----------- hNetG1: 272 GEI---FVDELHLAR--YFYAISDIKVRGRCKCNLHATVCVYDNSKL------------- hNetG2: 262 GGT---YVQRENLYK--YFYAISNIEVIGRCKCNLHANLCSMREGSL------------- mLam5: 275 LGHLMGKALRDPTVTRRYYYSIKDISIGGRCVCHGHADVC--DAKDPL----DPFRL--- S12 S13 S14 S15 cNet1: 306 -CDCKHNTAGPECDRCKPFHYDRPWQRAT---ARE--ANECVA-------CNCNLHARRC hNet1: 304 -CDCRHNTAGPECDRCKPFHYDRPWQRAT---ARE--ANECVA-------CNCNLHARRC hNet3: 274 -CDCRHGTEGPDCGRCKPFYCDRPWQRAT---ARE--SHACLA-------CSCNGHARRC hNet4: 292 KCMCKHNTAGSHCQHCAPLYNDRPWEAAD---GKTGAPNECRT-------CKCNGHADTC hNet5: 175 -CHCRHHTTGPGCESCRPSHRDWPWRPAT---PRH--PHPCLP-------CSCNQHARRC hNetG1: 314 TCECEHNTTGPDCGKCKKNYQGRPWSPGSYLPIPKGTANTCIPSISSIGNCECFGHSNRC hNetG2: 304 QCECEHNTTGPDCGKCKKNFRTRSWRAGSYLPLPHGSPNAC-ATAGSFGNCECYGHSNRC mLam5: 326 QCACQHNTCGGSCDRCCPGFNQQPWKPAT-----TDSANECQS-------CNCHGHAYDC S15 H5 S16 S17 S18 S19 S20 cNet1: 353 RFNMELYKLSGRKS-------GGVCLNCRHNTAGRHCHYCKEGFYRDLSKPISHRKACKE hNet1: 350 RFNMELYKLSGRKS-------GGVCLNCRHNTAGRHCHYCKEGYYRDMGKPITHRKACKA hNet3: 321 RFNMELYRLSGRRS-------GGVCLNCRHNTAGRHCHYCREGFYRDPGRALSDRRACRA hNet4: 342 HFDVNVWEASGNRS-------GGVCDDCQHNTEGQYCQRCKPGFYRDLRRPFSAPDACKP hNet5: 223 RFNSELFRLSGGRS-------GGVCERCRHHTAGRHCHYCQPGFWRDPSQPIFSRRACRA hNetG1: 374 SY-IDLLNTV-------------ICVSCKHNTRGQHCELCRLGYFRNASAQLDDENVCIE hNetG2: 363 SY-IDFLNVV-------------TCVSCKHNTRGQHCQHCRLGYYRNGSAELDDENVCIE mLam5: 374 YYDPEVDRRNASQNQDNVYQGGGVCLDCQHHTTGINCERCLPGFFRAPDQPLDSPHVCRP

S21 S22 S23 S23 cNet1: 406 CDCHPVGAAGQT------CNQTTGQCPCKDGVTGITCNRCAKG-Y-------------QQ hNet1: 404 CDCHPVGAAGKT------CNQTTGQCPCKDGVTGITCNRCAKG-Y-------------QQ hNet3: 374 CDCHPVGAAGKT------CNQTTGQCPCKDGVTGLTCNRCAPG-F-------------QQ hNet4: 395 CSCHPVGSAVLPANSVTFCDPSNGDCPCKPGVAGRRCDRCMVG-YWGFGDYGCRPCDCAG hNet5: 275 CQCHPIGATGGT------CNQTSGQCTCKLGVTGLTCNRCGPG-Y-------------QQ hNetG1: 420 CYCNPLGSIHDRCNGSGFCE-------CKTGTTGPKCDECLPGNSW---HYGCQPNVCDN hNetG2: 409 CNCNQIGSVHDRCNETGFCE-------CREGAAGPKCDDCLPTHYW---RQGCYPNVCDD mLam5: 434 CDCESDFTDGT-------CEDLTGRCYCRPNFTGELCAACAEG-YTDF------------ S24 cNet1: 446 SRSPIAP-CIKIPAA----PPPTAASSTEEPAD-------CDSY--------CKASKGKL hNet1: 444 SRSPIAP-CIKIPVA----PPTTAASSVEEPED-------CDSY--------CKASKGKL hNet3: 414 SRSPVAP-CVKTPIP----GPTEDSSPVQ-PQD-------CDSH--------CKPARGSY hNet4: 454 SCDPITGDCISSHTDIDWYHEVPDFRPVHNKSEPAWEWEDAQGFSALLHSGKCECKEQTL hNet5: 315 SRSPRMP-CQRIPEA----TTTLATTPGAYSSDP-----QCQNY--------CNMSDTRV hNetG1: 470 ------------------------------------ELLHCQNGGTCHNNVRCL------ hNetG2: 459 ------------------------------------DQLLCQNGGTCLQNQRCA------ mLam5: 474 ------PHCYPLPS----FPHNDTREQVL----PAGQIVNCDCNAAGTQGNACR-KDPRL cNet1: 486 KINMKKYCKKDYAVQIHI--LKAEKNADWWKFTVNIISVYKQGSNRLRRGDQ-TLWVHA- hNet1: 484 KINMKKYCKKDYAVQIHI--LKADKAGDWWKFTVNIISVYKQGTSRIRRGDQ-SLWIRS- hNet3: 453 RISLKKFCKKDYAVQVAV-GARGEARGAWTRFPVAVLAVFRSGEERARRGSS-ALWVPA- hNet4: 514 G-NAKAFCGMKYSYVLKIKILSAHDKGTHVEVNVKIKKVLKSTKLKIFRGKR-TLYPESW hNet5: 357 HMSLRRYCQQDHVLRAQV-LASEAAGPAWQRLAVRVLAVYKQRAQPVRRGDQ-DAWVPR- hNetG1: 488 -------CPAAYT----------------------------------------------- hNetG2: 477 -------CPRGYT----------------------------------------------- mLam5: 519 G---RCVCKPNF------------------------------------RGAHCELCAPGF cNet1: 542 KDIACK-CPKVKPMKKYLLLGSTEDSPDQS------GIIADKSSLVIQWRDTWARRLRKF hNet1: 540 RDIACK-CPKIKPLKKYLLLGNAEDSPDQS------GIVADKSSLVIQWRDTWARRLRKF hNet3: 510 GDAACG-CPRLLPGRRYLLLGGGPGAAAGGAGGRGPGLIAARGSLVLPWRDAWTRRLRRL hNet4: 572 TDRGCT-CPILNPGLEYLVAGHEDI--------RTGKLIVNMKSFVQHWKPSLGRKVMDI hNet5: 414 ADLTCG-CLRLQPGTDYLLLGSAVGDPDPTR------LILDRHGLALPWRPRWARPLKRL hNetG1: 494 -GILCE-KLRCE---E---AGSCGSDSGQGAPPHGSPALLLLTTLL-------------- hNetG2: 483 -GVRCE-QPRCDPADD---DGGLDCDRAPGAAPR-PATLLGCLLLL-------------- mLam5: 540 HGPSCHPCQCSSPG-----VANSLCDPESGQCMCRTGFEGDRCDHCAL—GYFHFPLCQL Fig. S3. Sequence alignment of various netrins and mouse Laminin-a5. Secondary structure elements are shown as arrows (strands) or blocks (helices) and colored blue for LN, green for LE1, pink for LE2, and red for LE3 to match the colors in Fig. 3C. Residues that are part of protein-protein interfaces are highlighted as follows: red, netrin-LN/neogenin-FN4; yellow, netrin-LE3/neogenin-FN5; cyan, netrin/netrin. Conserved cysteines are boxed.

A B C D E m-Neo-s: 765 DETRVPEVPSSLHVRPLVTSIVVSWTPPENQNIVVRGYAIGYGIGSPHAQTIKVDYKQRY m-DCC-s: 721 DESQVPDQPSSLHVRPQTNCIIMSWTPPLNPNIVVRGYIIGYGVGSPYAETVRVDSKQRY m-Neo-l: 745 DETRVPEVPSSLHVRPLVTSIVVSWTPPENQNIVVRGYAIGYGIGSPHAQTIKVDYKQRY m-DCC-l: 721 DESQVPDQPSSLHVRPQTNCIIMSWTPPLNPNIVVRGYIIGYGVGSPYAETVRVDSKQRY E F G G’ m-Neo-s: 825 YTIENLDPSSHYVITLKAFNNVGEGIPLYESAVTRPHT-------------------VPD m-DCC-s: 781 YSIERLESSSHYVISLKAFNNAGEGVPLYESATTRSITD--------------------V m-Neo-l: 805 YTIENLDPSSHYVITLKAFNNVGEGIPLYESAVTRPHTDT--SEVDLF-VINAPYTPVPD m-DCC-l: 781 YSIERLESSSHYVISLKAFNNAGEGVPLYESATTRSITDPTDP-VDYYPLLDDFPTSGPD A B C D m-Neo-s: 866 -PTPMMPPVGVQASILSHDTIRITWADNSLPKHQKITDSRYYTVRWKTNIPANTKYKNAN m-DCC-s: 821 -STPMLPPVGVQAVALTHEAVRVSWADNSVPKNQKTSDVRLYTVRWRTSFSASAKYKSED m-Neo-l: 862 -PTPMMPPVGVQASILSHDTIRITWADNSLPKHQKITDSRYYTVRWKTNIPANTKYKNAN m-DCC-l: 840 VSTPMLPPVGVQAVALTHEAVRVSWADNSVPKNQKTSDVRLYTVRWRTSFSASAKYKSED E F G G’ m-Neo-s: 925 ATTLSYLVTGLKPNTLYEFSVMVTKGRRSSTWSMTAHGAT m-DCC-s: 879 TTSLSYTATGLKPNTMYEFSVMVTKNRRSSTWSMTAHATTYEA m-Neo-l: 921 ATTLSYLVTGLKPNTLYEFSVMVTKGRRSSTWSMTAHGAT m-DCC-l: 900 TTSLSYTATGLKPNTMYEFSVMVTKNRRSSTWSMTAHATTYEA

Fig. S4. Sequence alignment of the FN4 and FN5 domains of mouse neogenin and DCC (both the short and long isoforms). Secondary structure elements (strands) are colored red for the FN4 domain and green for the FN5 domain. Netrin-binding residues are highlighted in magenta if part of the netrin-LN/neogenin-FN4 interface and in yellow if part of the netrin-LE3/neogenin-FN5 interface. The human and zebrafish neogein and DCC isoforms are highly homologous to their human counterparts (>80% sequence identity) and have identical lengths of the FN4-FN5 linkers.

Fig. S5. Superimposition of the structures netrin-1 (marine), netrin-G1 (grey), 3ZYJ, (RMSD is

1.4 Å between 207 corresponding C-alpha atoms), netrin-G2 (orange), 3TBD, (RMSD is 1.3 Å

between 183 corresponding C-alpha atoms), Laminin-alpha (magenta), 2Y38, (RMSD is 1.4 Å

between 266 corresponding C-alpha atoms), Laminin-beta (green), 4AQS, (RMSD is 1.6 Å

between 283 corresponding C-alpha atoms), Laminin-gamma (yellow), 4AQT, (RMSD is 0.8 Å

between 301 corresponding C-alpha atoms).

Fig. S6. Representative electron density maps for the reported refined structures. The 2Fo-Fc

maps were contoured at 1.5 sigma. A) Unbound Netrin-1. B) The Netrin-1/neogenin complex. C)

The Netrin-1/DCC complex, D) The ordered, extended linker region between neogenin domains

FN4 and FN5 in the Netrin-1/neogenin complex.

Fig. S7.

Folding topology diagrams for netrin (top) and neogenin (bottom). The netrin secondary

structure elements are colored according to domains (as in Fig. 3C): LN, blue; LE1, green, LE2,

pink, LE3, red. The boundaries of the secondary structure elements are indicated. The N- and C-

termini are shown as yellow boxes.

Fig. S8.

Schematic representation of all residues and contacts at the different protein-protein interfaces in

the Netrin-1/DCC and Netrin-1/neogenin complexes. Residues with hydrophobic side chains are

represented as grey ovals, with positively charged side chains – blue, negatively charged – red,

aromatic – magenta, polar – green, glycines and prolines – orange, cysteines – yellow.

Fig. S9.

The continuous netrin/DCC assembly as observed in the netrin/DCC crystals. Netrin is in blue,

DCC-FN4 is in red and DCC-FN5 is in green.

Table S1. Data-collection and structure-refinement statistics. Netrin Netrin/Neogenin Netrin/DCC Wavelength (Å) 0.9792 0.9792 0.9792 Resolution range (Å) 50 - 2.8 (2.91 - 2.8) 50 - 3.2 (3.31 - 3.2) 50 - 2.9 (2.95 - 2.9) Space group P 32 2 1 P 21 P 21 21 21 Unit cell 62.627 62.627

269.788 90 90 120 79.07 130.211 126.037

90 99.98 90 49.478 72.939 285.159

90 90 90 Total reflections 92454 154909 93328 Unique reflections 17681 40738 22820 Multiplicity 5.2 (5.1) 3.8 (3.5) 4.1 (3.1) Completeness (%) 99.13 (97.36) 97.24 (92.32) 95.87 (84.00) Mean I/sigma(I) 14.2 (3.2) 4.90 (1.1) 10.50 (1.4) Wilson B-factor 64.94 60.77 70.14 R-merge 0.095 (0.734) 0.194 (0.524) 0.123 (0.675) R-work 0.1923 (0.3428) 0.1963 (0.3177) 0.2235 (0.3567) R-free 0.2571 (0.3907) 0.2392 (0.3865) 0.2876 (0.3853) Number of atoms 3386 9827 4869 macromolecules 3255 9741 4729 ligands 46 86 68 water 85 0 72 Protein residues 419 1243 604 RMS(bonds) 0.009 0.009 0.009 RMS(angles) 1.40 1.31 1.40 Ramachandran favored (%)

96 96 96

Ramachandran outliers (%)

0 0 0

Clashscore 8.57 8.77 12.82 Average B-factor 75.60 26.00 42.10 macromolecules 75.60 25.90 42.10 ligands 112.40 36.40 51.70 solvent 56.00 31.10

Statistics for the highest-resolution shell are shown in parentheses.

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