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: [email protected] (D.B.N.); [email protected] (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
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Supplementary Materials for - Science · 2014. 5. 28. · Published 29 May 2014 on Science Express . DOI: 10.1126/science.1255149 . This includes: ... The genetic backgrounds for
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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*
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,
Statistics for the highest-resolution shell are shown in parentheses.
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