Latent TGF-Β Structure and Activation

ARTICLE doi:10.1038/nature10152

Latent TGF-b structure and activation

Minlong Shi1, Jianghai Zhu1, Rui Wang1,XingChen1, Lizhi Mi1, Thomas Walz2 & Timothy A. Springer1

Transforming growth factor (TGF)-b is stored in the extracellular matrix as a latent complex with its prodomain. Activation of TGF-b1 requires the binding of av integrin to an RGD sequence in the prodomain and exertion of force on this domain, which is held in the extracellular matrix by latent TGF-b binding proteins. Crystals of dimeric porcine proTGF-b1 reveal a ring-shaped complex, a novel fold for the prodomain, and show how the prodomain shields the growth factor from recognition by receptors and alters its conformation. Complex formation between avb6 integrin and the prodomain is insufficient for TGF-b1 release. Force-dependent activation requires unfastening of a ‘straitjacket’ that encircles each growth-factor monomer at a position that can be locked by a disulphide bond. Sequences of all 33 TGF-b family members indicate a similar prodomain fold. The structure provides insights into the regulation of a family of growth and differentiation factors of fundamental importance in morphogenesis and homeostasis.

The TGF-b family is key to specifying the body plan during metazoan Crystal structure development1,2. Members of this family, including nodal, activins, The structure of pro-TGF-b1 at 3.05 A˚ (Fig. 1a–c and Supplementary inhibins, bone morphogenetic proteins (BMPs) and growth differ- Table 1) was solved using multi- and single-wavelength anomalous entiation factors (GDFs), specify the anterior/posterior and dorsal/ diffraction. Electron density maps (Supplementary Fig. 1) were ventral axes, endoderm, mesoderm and ectoderm, left–right asym- improved by multi-crystal, multi-domain averaging over four mono- metry and details of individual organs. TGF-b1, TGF-b2 and TGF-b3 mers per asymmetric unit. In a ring-like shape, two prodomain arm are important in development, wound healing, immune responses domains connect at the elbows to crossed ‘forearms’ formed by the and tumour-cell growth and inhibition1,3. two growth-factor monomers and by prodomain ‘straitjacket’ ele- Although TGF-b synthesis and expression of its receptors are wide- ments that surround each growth-factor monomer (Fig. 1a–c and spread, activation is localized to sites where TGF-b is released from 4a). The centre of the ring contains solvent. The arms come together latency. TGF-b family members are synthesized with large amino- at the neck, where they are disulphide-linked in a bowtie, and RGD terminal prodomains, which are required for the proper folding and motifs locate to each shoulder (Fig. 1a). On the opposite side of the dimerization of the carboxy-terminal growth-factor domain4. Despite ring where the straitjacketed forearms cross, LTBP would be linked to intracellular cleavage by furin, after secretion, noncovalent asso- straitjacket residue Cys 4, which is mutated to serine in the crystal- ciation persists between the dimeric growth-factor domain and pro- lization construct (Fig. 1a–c). domain of TGF-b, and of an increasingly recognized number of other The arm domain, residues 46–242, has a novel fold12 with unusual family members. The prodomain is sufficient to confer latency on features. Its two anti-parallel, four-stranded b-sheets bear extensive some family members and it also targets many members for storage hydrophobic faces but these overlap only partially in the hydrophobic in the extracellular matrix, in complex with latent TGF binding core (Fig. 1a, e). The hydrophobic faces are extended by long meanders proteins (LTBPs) or fibrillins5,6. between the two sheets and burial by the a2, a3 and a4 helices. The prodomains of TGF-b1 and TGF-b3 contain an RGD motif b-strands b8andb9 extend on the two-fold pseudo-symmetry axis to that is recognized by av integrins. Mice with the integrin-binding link the two arm domains in a bowtie at the neck (Fig. 1a). The bow is RGD motif mutated to RGE recapitulate all major phenotypes of tied with reciprocal inter-prodomain disulphide bonds, Cys 194– TGF-b1-null mice, including multi-organ inflammation and defects Cys 196 and Cys 196–Cys 194, and by hydrophobic residues (Fig. 1a, e). in vasculogenesis, thus demonstrating the essential role of integrins in Arg 215 of the RGD motif locates to a disordered loop (residues 7 TGF-b activation . Among av integrins, the phenotypes of integrin 209–215) following the bowtie b9 strand. Partially ordered Gly 216 b6-null and integrin b8-null mice demonstrate the particular import- and Asp 217 of the RGD motif (Fig. 1a, d) begin the long, 12-residue ance of the avb6 and avb8 integrins for activation of TGF-b1 and TGF- meander across the hydrophobic face of the neck-proximal b-sheet b3 in vivo8,9. that connects to b10 in the forearm-proximal b-sheet. Integrin binding alone is not sufficient for TGF-b activation. The straitjacket, residues 1–45, is formed by the a1helixandthe Activation by avb6 integrin requires incorporation of TGF-b1intothe latency lasso (Fig. 1a–c and 2a). The latency lasso, an extended loop that extracellular matrix, by association with LTBP, and association of the b6 connects the a1anda2 helices, has little contact with the remainder of cytoplasmic domain with the actin cytoskeleton5,10,11. Furthermore, con- the prodomain while encircling the tip of each TGF-b monomer tractile force is necessary for TGF-b activation by myofibroblasts8.Thus, (Fig. 1a, b, f). Six proline residues and three aliphatic residues make tensile force exerted by integrins across the LTBP–prodomain–TGF-b hydrophobic contacts with an extensive array of growth-factor aro- complex is hypothesized to change the conformation of the prodomain matic and aliphatic residues, and help these to stabilize the conforma- and to free TGF-b forreceptorbinding5,8. Here, we describe the struc- tion of the latency lasso (Fig. 1f). ture of latent TGF-b, mechanisms for latency and integrin-dependent A highly hydrophobic face of the amphipathic a1 helix, bearing activation, and broad implications for the regulation of bioactivity in the isoleucine and leucine residues, interacts with Trp 279 and Trp 281 TGF-b family. and with aliphatic side chains on one growth-factor monomer (Fig. 1f,

1Immune Disease Institute, Children’s Hospital Boston and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA. 2Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.

a d e αVβ6 αVβ6 G216 L218 Bowtie β9 D217 L131 β9 8 I221 D197 β RGD R189 β8 Arm β2 β7 domain H193 E140 β4 β4 β4 β7 β5 β5 β5 α4 D217 β3 β10 β6 α3 α4 β1 β3 β6 β2 α2 TGF-β monomers Y52 β10 β1 α4 α2 Fingers α3 α5 Furin Y52 Furin Latency V48 α3 lasso R238 Latency Y74 lasso Y75 α5 Fastener Strait- α3 jacket Fastener α1

N C4S N f P45 α2 90º LTBP linkage Latency C4S lasso Y52 V347 b V338

E348 α1 W279 P34 L28 W281 α1 P33 L25 α5 L30

α1

C4S c g h SS SS α β α β RGD RGD Y74 V 6 V 6 Y307 SS Y299 SS Arm Arm S351 RGD RGD Y75 Arm Arm Free TGF-β1 K27 Unfastened straitjacket 1 27 TGF-β1 α W279 Cytoskeletal force Straitjacket 76 I17 R238 S S S S I24 S S S S LTBP LTBP E348

Figure 1 | Architecture of proTGF-b1. Arm, straitjacket and TGF-b1 yellow. c, Schematic of the structure and activation mechanism. SS, disulphide monomer segments are coloured differently. a, b, Overall structure. Spheres bonds. d, Hydrophobic residues near Asp 217 of the RGD motif. e,Arm mark the last residue visible in density in the prodomain and the first residue of domain. Side chains for the hydrophobic core are shown in gold (also marked the growth factor. Disordered segments are dashed. Red arrows show the in Fig. 2 and Supplementary Fig. 2), conserved a2-helix residues that interact directions of forces during activation by integrins. Key side chains are shown in with the growth factor are in pink, fastener residues are in silver and bowtie stick representation, including Asp of the RGD motif in cyan and CED residues are in light green for aliphatics and yellow for Cys. f–h, Straitjacket and mutations in white. Disulphide bonds and the Cys 4 mutation to Ser are in fastener details.

344 | NATURE | VOL 474 | 16 JUNE 2011 ©2011 Macmillan Publishers Limited. All rights reserved ARTICLE RESEARCH g). The tryptophan residues are further covered by lasso residues and Tyr 75 also secure the straitjacket. Notably, Lys 27, Tyr 74 and Leu 30, Pro 33 and Pro 34 (Fig. 1f). Notably, the a1 helix is buried Tyr 75 are invariant among TGF-b1, TGF-b2 and TGF-b3 (Sup- deeply in an interface between the two growth-factor monomers plementary Fig. 2). Fastening is reinforced by backbone hydrogen (Fig. 1a, b, g). The interface on the second monomer includes three bonds between arm b1-strand residues 77 and 78 and growth-factor tyrosine residues (Fig. 1g). b-fingers, which join the prodomain and the growth factor in a super- Together with the straitjacket, the arm domain completes the en- b-sheet (Fig. 1a). circlement of each growth-factor monomer. The a2 helix buries The TGF-b dimer forms the forearms, although TGF-b monomers Val 338 and Val 347 of the TGF-b finger (Fig. 1f). The a5 helix pro- have also been described as hand-like13–15 (Fig. 3). Each monomer has jects from the base of the arm domain (Fig. 1a and e) and in it, no hydrophobic core, aside from the cystine knot motif in which one prodomain residue Arg 238 forms a salt bridge to Glu 348 in TGF- disulphide passes between two polypeptide segments bridged by two b1 (Fig. 1f, h). These two residues are invariant in proTGF-b1, other disulphides. proTGF-b2 and proTGF-b3 (Supplementary Fig. 2). Prodomain-bound TGF-b1 differs markedly from previous TGF-b The straitjacket is fastened to arm residues 74–76 (Fig. 1a, h). A structures in both the orientation between monomers and the posi- backbone hydrogen bond between the nitrogen of Ala 76 and the tion of elements within monomers (Fig. 3 and Supplementary Fig. 3). oxygen of Lys 27 caps the C-terminal end of the a1 helix (Fig. 1h). The Ca root-mean-squared deviations over all 112 residues are 7 A˚ , Moreover, the carbonyl oxygen of Ala 76 forms a hydrogen bond to and 2 A˚ over the most similar 85 residues. The largest differences are Arg 238 in the a5 helix (Fig. 1h). Lys 27 is a key fastener residue. Its imposed by the prodomain a1 helix. It occupies a similar position to side chain forms a p-cation bond to the side chain of Tyr 74, a hydro- the growth-factor a3 helix in mature TGF-b1 (Supplementary Fig. 3). gen bond to the backbone of Tyr 74, and hydrogen bonds to the Intercalation of the prodomain a1 helix between the growth-factor backbone and sidechain of Ser 351 (Fig. 1h). Van der Waals contacts monomers reduces the total area buried between monomers from between the bulky side chains of the fastener residues Lys 27, Tyr 74 850 A˚ 2 to 335 A˚ 2. The large conformational changes in TGF-b1 are

a α α ..1 . Latency lasso . 2 TGF-β1 ----LSTCKTIDMELVKRKRIEA I RGQI LSKLRLASPPSQGDV------PPGPLPEAV 48 Myost ( 1 3 ) GL CNACTWRQNT KSSR I E A IKIQI LSKLRLETAPNI SKDVI RQLL-PKAPPLREL 67 Inh-α ------CQGLELARELVLAKVRALFLDAL---GPPAVTREGGD----PG------V 37 BMP4 (19)QGHAGGRRSGQ-SHELLRDFEATLLQMFGLRRRPQ------PSKSAVIPDYM 64 Lefty ------LTGEQLLGSLLRQLQLKEVPTLDRADMEELVIPTHVRAQYV 41 . α2 . . Fastener β.1 . TGF-β1 LALYNSTRDRVAGESVEPEPEPEADYYAKEVTRVLMV- - - ESGNQI YDKFKGT- - - - - P 99 Myost I DQYDVQRDD- SSDGSL ED- - - - - DDYHAT TET I I TMPT ESDF LMQVD------G 110 Inh-α RRLPRRHALGGFTHRGSEP------EEEEDVSQAILFPATDASCEDKSAARGLAQEAEE 90 BMP4 RDLYRLQSGEEEEEQI HSTGLEYPERPASRANTVRSFHHEEHLEN IPGTSE------N 116 Lefty AL L QRSHGDRSRGKRFSQS------FREVAGRFLALE------A 73 β β β . 2 ..α3 . 3 . 4 TGF-β1 HSLYMLFNTSELREAVPEPVLLSRAELRLLR------LKLKVEQHVELYQK 144 Myost KPKCCF FKF SSK I QYNK----VVKAQLWIYL------RPVETPTTVFVQILRL 153 Inh-α GLFRYMFRPSQHTRSRQ----VTSAQLWFHT------GLDRQGTAASNSSEPLLGLLAL 139 BMP4 SAFRFLFNLSSIPENEV----ISSAELRLFR------EQVDQGPDWERGF - HR I N I YEV 164 Lefty S THL L V FGMEQRL PPNSE---LVQAVLRLFQEPVPKAALHRHGRLSPRSARARVTVEWL 129 β β α β ...5 6 4 ..7 TGF-β1 YSND------SWRYLSNRLLA---PSDSPEWLSFDVTGVVRQWLTRRE- - A I EGFRL 190 Myost I KPMKDG------TRYTGIRSLKLDMNPGT GI WQS I DVKTVL QNWLKQPE- - SNL G I E I 204 Inh-α SPG------GPVAVPMSLG----HAPPHWAVL HLAT SAL SL LT HPV- - L VL L L RC 182 BMP4 MKPPAEVVPGHL I TRLLDTRLVH- - - - HNVTRWETFDVSPAVLRWTREKQ- - PNYGL A I 217 Lefty RVRDDGS----NRTSLIDSRLVS----VHESGWKAFDVTEAVNFWQQL SRPRQPL L L QV 180 β β β β α 7 8 . 9 . . .10 5. TGF-β1 SAHCSCDSKDNT L HVE I NGFNSGRRGDLAT IHGMNRPF L L LMATPL ERAQHL HSSRHRR 249 Myost KAL DENGHDL AVT F PG------PGEDGLNPFLEVKVTD------TPKRSRR 243 Inh-α PLCTCSA------RPEATPFLVAHTRTRPP---SGGERARR 214 BMP4 EVTHLHQTRTHQGQHVRI S- - - RSLPQGSGNWAQLRPLLVTFGHDGRG---HALTRRRR 270 Lefty SVQREHL GPL ASGAHKL V-----RFASQGAPAGLGEPQLELHTLDLGDYGAQGD----- 229

b GDF9 BMP3 GDF10 TGF-β1 TGF-β2 BMP15 TGF-β3 GDF11 Myostatin BMP8A,B Inhibin-βA BMP6 Inhibin-βB BMP7 Inhibin-βC BMP5 Inhibin-βE BMP4 Lefty1 BMP2 GDF15 Lefty2 Nodal Anti-Müllerian GDF2 hormone BMP10 α GDF7 GDF1 Inhibin- GDF6 GDF5 GDF3 Figure 2 | The TGF-b family. a, Sequence alignment of five representative mark decadal residues. Vertical dashed lines mark cleavage sites. prodomains. Orange circles mark the core hydrophobic residues shown in b, Phylogenetic tree of the TGF-b family, based on the alignment in Fig. 1e. Inh-a, inhibin-a; Myost, myostatin. Black dots over TGF-b1 sequence Supplementary Fig. 2.

a b Prodomain Mature TGF-β monomer monomer

Latent TGF-β monomer β3 Latency c d β10 lasso β1 R type I TGF-β α3 α5

α1 R type II e f g 123 2,000 Mock αVβ6

αV 1,500

β6 1,000 LAP dimer 500 Luciferase activity 0

TGF-β1 WT WT Mock Y74AY74TY74FY74KY75AY75SY75FY75TY75CL28CMock Figure 3 | Shielding from receptor binding. ProTGF-b1 and TGF-b1in dimer complex with its receptors (R type I and II) (ref. 15) were superimposed on the K27C/Y75C K27C/Y75C TGF-b dimers. For clarity, only one monomer of each is shown. The receptors are shown as transparent molecular surfaces. Elements of the prodomain that hi Anti-LAP Biotin labelled clash with the receptors are labelled.

Y75C Y75C K27C Mock WT K27C/ L28C Mock Y75C L28C K27C/ Y75C WT ProTGF-β driven by an intimate interaction between the growth-factor and LAP prodomain dimers, which buries a total area of 2,440 A˚ 2. LAP

Figure 4 | ProTGF-b1 complexes with LTBP and avb6 integrin, and Implications for biosynthesis activation of TGF-b.a–d, Representative negative-stain electron microscopy Folding and secretion of active TGF-b1 and activin A requires the co- class averages of proTGF-b (a), the complex of proTGF-b with a fragment of expression of their prodomains4, whereas the TGF-b1 prodomain can LTBP1 containing TGF-binding domain 3–EGF–EGF–TGF-binding domain 4 be biosynthesized in the absence of the growth-factor domain16. These (b) and complexes of proTGF-b1 with avb6 integrin, prepared with an excess of proTGF-b1(c)oranexcessofavb6 integrin (d). Scale bars, 100 A˚ . e, Non- findings suggest that the C-terminal growth-factor domain folds either reducing SDS–PAGE of the complex peak from S200 gel filtration used for concomitantly with, or subsequently to, the N-terminal prodomain. electron microscopy in d (lane 1), avb6 integrin (lane 2) and proTGF-b (lane 3). The kinetics of biosynthesis of TGF-b1, activin and anti-Mu¨llerian LAP, latency-associated protein (prodomain). f, Activation of TGF-b1. 293T hormone are slow and for anti-Mu¨llerian hormone, folding of the cells stably transfected with avb6 integrin or a mock control were additionally growth-factor domain is rate-limiting17. Regions of the prodomain that transfected with the indicated wild-type (WT) or mutant proTGF-b1 may be particularly important in templating the folding of the growth- constructs, or with empty vector (mock), and co-cultured with TGF-b indicator factor domain include the b1 strand that forms a supersheet with the cells5. g, Material made by the indicated mutants in 293T cells was heated at TGF-b fingers and the a1anda2 helices, which pack against extensive 80 uC and assayed with indicator cells. Error bars in f and g show the s.e.m. of hydrophobic interfaces on opposite sides of the growth-factor fingers 3–9 samples from 1–3 representative experiments. h, i, Western blots of b a proTGF- 1 secreted by the indicated transfectants, using an antibody to the (Fig. 1a, f, g). Residues Ile 17, Ile 24, Leu 25 and Leu 28 in the 1-helix prodomain (h) or streptavidin to detect biotinylated cysteines (i). interface, and Leu 30 in the lasso interface (Fig. 1f, g), have been specifically identified as important for TGF-b1 association18. The embrace of the fingers of each growth-factor monomer may complement the This assignment indicates a swap in the growth-factor monomer correct formation of the cystine knot and inter-monomer disulphide that each prodomain monomer embraces, with the intimate interac- bonds in TGF-b. The structure of proTGF-b1 makes these disulphides tions described above occurring between prodomains and growth- accessible to disulphide isomerases during biosynthesis (Fig. 1b). factor domains that are present on different polypeptide chains in A definitive assignment of which growth-factor and prodomain the precursor in the endoplasmic reticulum (for example, the gold monomers derive from the same polypeptide chain is not possible and green domains in Fig. 1). Thus, the surface area buried between because of intracellular cleavage by furin and lack of density for resi- the growth-factor domains and prodomains of different precursor 2 dues 243–249. However, cleavage is incomplete and the small amount monomers (900 A˚ ) is substantially larger than that within the same 2 of uncleaved proTGF-b that is present in protein preparations co- precursor monomer (370 A˚ ) and adds substantially to the inter- 2 crystallizes with cleaved proTGF-b (Supplementary Fig. 1d), indi- monomer interfaces of the growth factor (330 A˚ ) and prodomain 2 cating that there is no major conformational change after cleavage. (600 A˚ ). Swapping may be important in regulating the proportion of A long prodomain–growth-factor connection through the centre of growth-factor heterodimers, which have unique functions in settings the ring, spanning ,50 A˚ , would require substantial conformational such as dorsoventral patterning19. change and would limit access to furin. Therefore, we have assigned the shorter ,30 A˚ connection, between the C terminus of the prodomain Complex with LTBP and activation and the N terminus of the growth factor, located on the same side of the In the large latent complex, a single LTBP molecule is disulphide- ring (for example, the magenta and gold spheres in Fig. 1a, b). bonded to two proTGF-b monomers. We confirmed this unusual 1:2

346 | NATURE | VOL 474 | 16 JUNE 2011 ©2011 Macmillan Publishers Limited. All rights reserved ARTICLE RESEARCH stoichiometry by multi-angle light-scattering mass measurements of be resisted by the b1 strand. The b1 and b10 strands are each parallel the complex with an LTBP fragment. The LTBP-crosslinking Cys 4 to applied force and adjacent in a b-sheet (Fig. 1) and are thus in the residues in each monomer (serines in our construct) are 40 A˚ apart most force-resistant structural geometry known, the hydrogen-bond (Fig. 1b). Their linkage to LTBP will reinforce the straitjacket by clamp23. By contrast, the topologies and geometries of the a-helices fastening together the forearms (Fig. 1c). The large 40 A˚ separation and the long latency lasso of the straitjacket are ill-suited to resist may represent a mechanism for preventing disulphide linkage force. Force on Cys 4 will apply leverage to the C-terminal end of between the two Cys 4 residues. In contrast, the two cognate cysteines the a1 helix and weaken interactions with fastener residues. After in TGF-binding domain 3 of LTBP become linked to one another in the unfastening, the long latency lasso, which has no stabilizing hydro- absence of complex formation. These cysteines are surface-exposed gen-bond interactions, will be easily elongated and straightened by the and surrounded by acidic residues that interact with proTGF-b (refs applied tensile force. Thus, freed by opening of its straitjacket, TGF-b 20, 21). In agreement with this, the straitjacket a1-helices bear basic will be released from the prodomain and activated for receptor bind- residues (Fig. 1b). Moreover, between the two prodomain a1-helices, a ing (Fig. 1c). concave growth-factor surface that bears numerous hydrophobic resi- The prodomain not only holds TGF-b in a markedly different dues, including proline and disulphide-linked cysteine, is available for conformation from when it is free or bound to receptors, it also blocks interaction with LTBP (Fig. 1b). receptor access completely. TGF-b family members are recognized by Negative-stain electron microscopy class averages of proTGF-b are two type I receptors and two type II receptors that surround the in excellent agreement with our crystal structure (Fig. 4a and Sup- growth-factor dimer (Supplementary Fig. 3e)15. Binding of the type plementary Fig. 4). A complex with an LTBP fragment that contains II receptor to the finger-tips of the growth factor is blocked by the TGF-b-binding domains 3 and 4 and two intervening EGF domains latency lasso, and binding of the type I receptor to the body of the shows no major conformational change in the proTGF-b moiety growth-factor domain is blocked by the prodomain a1 helix, a5 helix, (Fig. 4b). An additional density corresponding to the LTBP fragment the fastener and the ends of b-strands 1, 3 and 10 (Fig. 3). Although is present on the periphery of the ring, as expected from our crystal straitjacket removal might be sufficient to allow binding of type II structure, and causes the ring to lie at an angle to the substrate in some receptors, type I receptor interactions overlap with so many interac- class averages (Fig. 4b, middle panel and Supplementary Fig. 4c). tions between TGF-b and the arm domain (Fig. 3) that complete The RGD motifs in the shoulders of proTGF-b are highly accessible release from the prodomain would be required for receptor binding. for integrin binding (Fig. 1a). In contrast to many RGD motifs that are The structure thus shows that integrins could not expose TGF-b present in extended loops, the position of Asp 217 is stabilized by sufficiently for receptor activation if it remained bound to the prodo- burial of Leu 218 and by a 218–131 backbone hydrogen bond main, and that other explanations should be sought for the greater (Fig. 1d). Exposed hydrophobic side chains that are nearby on the activity of integrin-activated TGF-b on neighbouring cells than on body of the arm domain (Fig. 1d) may increase affinity for integrins. distant cells10. The integrin avb6 ectodomain, with a C-terminal clasp, formed To test the importance of unfastening in TGF-b activation, key complexes with proTGF-b1inCa21 and Mg21 that could be isolated residues were mutated. Non-conservative substitutions of the fastener by gel filtration, demonstrating unusually tight binding for an integrin residues Tyr 74 and Tyr 75 resulted in spontaneous, non-integrin- (Fig. 4c–e and Supplementary Fig. 4a). Different input ratios of dependent TGF-b activation (Fig. 4f). Among the different amino proTGF-b1 and avb6 integrin yielded 1:1 (Fig. 4c) and 1:2 (Fig. 4d, acids to which Tyr 74 and Tyr 75 were mutated, only phenylalanine e) complexes. Binding to ligand stabilized extension of the integrin was not activating. As a control, mutation of nearby Leu 28, in a 22 legs and the open conformation of the avb6 headpiece (Fig. 4c, d and hydrophobic interface with TGF-b, was not activating (Fig. 4f). Supplementary Fig. 4d, e). Integrins were bound to proTGF-b at the These results are consistent with the importance to fastening of interface between a large density, corresponding to the av b-propeller p-bonding and of van der Waals interactions of the aromatic tyrosine domain, and a small density, corresponding to the b6 bI domain, with side chains. their legs extending away from proTGF-b. The spacing between the In the fastener, the Ca carbons of Lys 27 and Tyr 75 are only 4.1 A˚ binding sites on the ring seen by electron microscopy (40–50 A˚ ) was apart, permissive for disulphide bond formation in a K27C/Y75C appropriate for that between the two RGDs in the crystal structure mutant, as confirmed by free-cysteine labelling of the Y75C mutant (45 A˚ ). No major conformational change in proTGF-b1 was apparent, but not the K27C/Y75C mutant (Fig. 4h, i). The K27C mutation even with two integrins bound (Fig. 4d), and SDS–polyacrylamide gel greatly reduced expression (Fig. 4h). Similarly, a K27A mutation electrophoresis (SDS–PAGE) confirmed the presence of TGF-b1in greatly reduces expression, and also releases free TGF-b1 (ref. 18). the complex (Fig. 4e). These biochemical and structural studies dem- The Y75C mutant was constitutively active (Fig. 4f). The K27C/Y75C onstrate that integrin binding to proTGF-b1 is not sufficient for double mutation rescued expression compared to K27C, prevented release of TGF-b1, consistent with previous cell-biological assays5,8,10. the spontaneous release of TGF-b that was seen with Y75C and, com- The requirements of (1) attachment of proTGF-b through LTBP to pared to wild type, made proTGF-b completely resistant to integrin- the extracellular matrix; (2) integrin attachment to the cytoskeleton avb6-dependent activation (Fig. 4f, h). Denaturants such as heat can and (3) cellular contraction indicate that the generation of tensile unfold proteins by pathways distinct from applied force23. Heat released force across proTGF-b1 is required for activation of TGF-b5,8,10,11. comparable amounts of active TGF-b from wild type and the K27C/ The structure enables the overall mechanism of TGF-b1 activation Y75C mutant (Fig. 4g). Thus, a disulphide bond can fasten the strait- by applied force to be readily predicted (Fig. 1c). Tensile force applied jacket permanently and prevent integrin-dependent activation. These to the RGD motifs in the shoulders by av integrins attached to the results support the hypothesis that tensile force applied to the prodo- actin cytoskeleton will be resisted at the opposite end of the ring, main by integrins can release TGF-b, and emphasise the importance of where the Cys 4 residues in the straitjacket are disulphide-linked to straitjacket unfastening in integrin-dependent activation. LTBP, which is tightly associated with the extracellular matrix. Pulling force will be applied in the directions shown by red arrows in Fig. 1a. Mutations in disease The direction of the pulling force and fold topology strongly influ- Camurati–Engelmann disease (CED), which is characterized by ence the unfolding pathway and resistance to force23. b-Sheet proteins thickening of the shafts of the long bones with pain in muscle and are the most force-resistant and thus the arm domain will be the most bone, is caused by mutations in the prodomain of TGF-b1 that force-resistant portion of the prodomain. Pulling against the RGD increase its release18,24. Among CED mutations, Y52H disrupts an motif will be transmitted through the long meander to the b10 strand. a2-helix residue that cradles the TGF-b fingers (Fig. 1a, f). The Force transmitted from the Cys 4 residues through the straitjacket will charge-reversal E140K and H193D mutations disrupt a pH-regulated

16 JUNE 2011 | VOL 474 | NATURE | 347 ©2011 Macmillan Publishers Limited. All rights reserved RESEARCH ARTICLE salt bridge between Glu 140 and His 193 in the dimerization interface the second site is in the disordered loop bearing the arginine of RGD of the prodomain (Fig. 1a). ‘Hotspot’ residue Arg 189 is substantially in TGF-b1 (Fig. 2a). Loss of the central b10 strand between the two buried: it forms a cation-p bond with Tyr 142 and salt bridges across cleavage sites results in loss of binding of the BMP4 prodomain to its the dimer interface with bowtie residue Asp 197 (Fig. 1a). Moreover, growth factor27. CED mutations in Cys 194 and Cys 196 demonstrate the importance The prodomain of Nodal, which binds to Cripto, targets Nodal for of the bowtie disulphide bonds. cleavage by proteases secreted by neighbouring cells29. Anti-Mu¨llerian hormone is secreted largely uncleaved and association with the pro- Implications for the large TGF-b family domain greatly potentiates its activity in vivo30. Lefty protein, which is The TGF-b family consists of 33 members (Fig. 2b)2. Although involved in establishing bilateral asymmetry, is not cleaved between growth-factor domains are highly conserved, prodomains vary in the arm and growth-factor domains, and is cleaved instead between length from 169 to 433 residues, and are variously described as unre- the a2 helix and the fastener31 (Fig. 2). Notably, release of the strait- lated in sequence or low in homology. However, alignment shows that jacket should be sufficient to enable access of type II receptors to all prodomains have a similar fold (Fig. 2a and Supplementary Fig. 2). growth-factor domains. Deeply buried hydrophobic residues in core secondary-structure elements of the arm domain, that is, the a2 helix and b-strands 1–3, 6, 7 Concluding perspective and 10, are conserved in all members (gold side chains in Fig. 1e and We have described the structure of latent TGF-b1 and a force-dependent orange circles in Fig. 2 and Supplementary Fig. 2). mechanism for its activation by av integrins. It is notable that so many Most family members also contain clear sequence signatures for the members of the TGF-b family associate with fibrillins or with LTBPs, amphipathic C-terminal portion of the a1 helix that inserts intimately which co-assemble in the elastic fibres of connective tissues6.Forces between the two growth-factor monomers (Fig. 2 and Supplementary acting on elastic fibres would extend fibrillins and LTBPs, and we Fig. 2). A similar insertion in inhibin-a and inhibin-bA has been speculate that this could weaken their association with TGF-b family demonstrated by mapping disruptive mutations to the equivalents members, enabling release and activation. It is thus possible that force- b 25 of Ile 24 and Leu 28 in TGF- (Fig. 1f, g) . Many family members dependent regulation of TGF-b family activation could extend beyond also contain proline-rich latency lasso loops with lengths that are integrin-dependent mechanisms and could be important in a wide compatible with encirclement of the growth-factor b-finger (Fig. 2 variety of contexts, including regulation of bone and tissue growth. and Supplementary Fig. 2). Thus, a prodomain structure similar to Although prodomains in the TGF-b family are diverse, their sequences that of proTGF-b, including a portion of the straitjacket, is widespread are highly conserved between species2. Further studies are required to in the TGF-b family. However, the low sequence identity and many address the diversity of mechanisms by which prodomains regulate insertions and deletions indicate substantial specializations. latency and activation in the TGF-b family. Differences in prodomain dimerization among family members are indicated by variations in cysteine positions. The bowtie (b-strands 8 and 9) and its disulphides are specializations. Inhibin-a and -b sub- METHODS SUMMARY units have cysteines in similar positions, whereas other family mem- Porcine proTGF-b1 with an N-terminal tag, C4S and N147Q mutations was bers either have cysteine residues in the b7 strand or lack cysteines expressed in CHO-Lec 3.2.8.1 cells. The protein was purified, treated with 3C protease to remove tags and then crystallized. Diffraction-quality crystals were altogether in this region (Fig. 2 and Supplementary Fig. 2). obtained in 6.5–7.5% (w/v) polyethylene glycol 3500, 17–18% isopropanol, 4–5% The interface between the two arm domains in the b4 and b5 glycerol and 0.1 M sodium citrate, pH 5.6. Structures were solved by Se multi- strands is modest in size and lacks hydrophobic and conserved resi- wavelength anomalous dispersion and Hg single-wavelength anomalous disper- dues. GDF1 and GDF15 specifically lack the b4 and b5 strands, which sion. Maps were improved by multi-crystal averaging. The structure was built are adjacent in sequence and structure, on the edge of a b-sheet manually and refined to Rwork and Rfree factors of 27.4% and 31.1%, respectively. (Fig. 1a and Supplementary Fig. 2). Therefore, arm-domain dimeriza- The avb6 ectodomain was expressed using C-terminal a-helical coiled-coils and tion seems to be variable or absent in some family members. tags, purified and subjected to negative-stain electron microscopy. TGF-b assays The close relatives of TGF-b, myostatin and GDF11, which are also used 293T cells stably transfected with avb6 integrin and transiently transfected latent, show conservation of the fastener residues Lys 27 and Tyr 75 with mutant human proTGF-b1, then co-cultured with indicator cells expressing (Fig. 2a and Supplementary Fig. 2). Myostatin regulates muscle mass a luciferase gene under the control of a TGF-b1-inducible promoter. and is stored in the extracellular matrix, bound to LTBP3. Release of Full Methods and any associated references are available in the online version of myostatin and GDF11 from latency requires cleavage of the prodo- the paper at www.nature.com/nature. main between Arg 75 and Asp 76 by BMP1/tolloid metalloproteinases (reviewed in ref. 26). This cleavage is between the a2 helix and the Received 20 December 2010; accepted 27 April 2011. fastener (Fig. 2a). Thus at least two different methods of unfastening 1. Wu, M. Y. & Hill, C. S. TGF-b superfamily signaling in embryonic development and the straitjacket, force and proteolysis, can release family members homeostasis. Dev. Cell 16, 329–343 (2009). from latency. 2. Derynck, R. & Miyazono, K. in The TGF-b Family (eds Derynck, R. & Miyazono, K.) Ch. An increasingly large number of TGF-b family members are recog- 2, 29–43 (Cold Spring Harbor Laboratory Press, 2008). nized to remain associated with their prodomains after secretion, 3. Blobe, G. C., Schiemann, W. P. & Lodish, H. F. Role of transforming growth factor b in human disease. N. Engl. J. Med. 342, 1350–1358 (2000). including BMP4, BMP7, BMP10, GDF2, GDF5 and GDF8 (ref. 27). 4. Gray, A. M. & Mason, A. J. Requirement for activin A and transforming growth Furthermore, many of these prodomains bind with high affinity to factor-b 1 pro-regions in homodimer assembly. Science 247, 1328–1330 (1990). fibrillin-1 and fibrillin-2. Targeting by the prodomain to the extra- 5. Annes, J. P., Chen, Y., Munger, J. S. & Rifkin, D. B. Integrin aVb6-mediated activation of latent TGF-b requires the latent TGF-b binding protein-1. J. Cell Biol. 165, cellular matrix may be of wide importance in regulating bioactivity in 723–734 (2004). 6 the TGF-b family . Moreover, binding to LTBPs or fibrillins seems to 6. Ramirez, F. & Sakai, L. Y. Biogenesis and function of fibrillin assemblies. Cell Tissue strengthen the prodomain–growth-factor complex6. Thus, although Res. 339, 71–82 (2010). only a limited number of TGF-b family members are latent as 7. Yang, Z. et al. Absence of integrin-mediated TGFb1 activation in vivo recapitulates the phenotype of TGFb1-null mice. J. Cell Biol. 176, 787–793 (2007). prodomain–growth-factor complexes, the concept of latency may 8. Wipff, P. J. & Hinz, B. Integrins and the activation of latent transforming growth extend to other members when their physiologically relevant com- factor b1 — an intimate relationship. Eur. J. Cell Biol. 87, 601–615 (2008). plexes with LTBPs and fibrillins are considered. 9. Aluwihare, P. et al. Mice that lack activity of aVb6-andaVb8-integrins reproduce the The signalling range of BMP4 in vivo is increased by extracellular abnormalities of Tgfb1-andTgfb3-null mice. J. Cell Sci. 122, 227–232 (2009). 10. Munger, J. S. et al. The integrin aVb6 binds and activates latent TGF b1: A cleavage of the prodomain by furin-like proteases at a second site mechanism for regulating pulmonary inflammation and fibrosis. Cell 96, upstream of the prodomain–growth-factor cleavage site28. Notably, 319–328 (1999).

11. Yoshinaga, K. et al. Perturbation of transforming growth factor (TGF)-b1 25. Walton, K. L. et al. A common biosynthetic pathway governs the dimerization and association with latent TGF-b binding protein yields inflammation and tumors. secretion of inhibin and related transforming growth factor b (TGFb)ligands.J. Biol. Proc. Natl Acad. Sci. USA 105, 18758–18763 (2008). Chem. 284, 9311–9320 (2009). 12. Holm, L., Kaariainen, S., Rosenstrom, P. & Schenkel, A. Searching protein structure 26. Anderson, S. B., Goldberg, A. L. & Whitman, M. Identification of a novel pool of databases with DaliLite v.3. Bioinformatics 24, 2780–2781 (2008). extracellular pro-myostatin in skeletal muscle. J. Biol. Chem. 283, 7027–7035 13. Daopin, S., Piez, K. A., Ogawa, Y. & Davies, D. R. Crystal structure of transforming (2008). growth factor-b2: an unusual fold for the superfamily. Science 257, 369–373 27. Sengle, G. et al. Targeting of bone morphogenetic protein growth factor complexes (1992). to fibrillin. J. Biol. Chem. 283, 13874–13888 (2008). 14. Schlunegger, M. P. & Grutter, M. G. An unusual feature revealed by the crystal 28. Cui, Y. et al. The activity and signaling range of mature BMP-4 is regulated by structure at 2.2 A˚ resolution of human transforming growth factor-b2. Nature 358, sequential cleavage at two sites within the prodomain of the precursor. Genes Dev. 430–434 (1992). 15, 2797–2802 (2001). 15. Radaev, S. et al. Ternary complex of transforming growth factor-b1 reveals isoform- 29. Blanchet, M. H. et al. Cripto recruits Furin and PACE4 and controls Nodal trafficking specific ligand recognition and receptor recruitment in the superfamily. J. Biol. during proteolytic maturation. EMBO J. 27, 2580–2591 (2008). Chem. 285, 14806–14814 (2010). 30. Wilson, C. A. et al. Mu¨llerian inhibiting substance requires its N-terminal domain for maintenance of biological activity, a novel finding within the transforming growth 16. Gentry, L. E. & Nash, B. W. The pro domain of pre-pro-transforming growth factor factor-b superfamily. Mol. Endocrinol. 7, 247–257 (1993). b1 when independently expressed is a functional binding protein for the mature 31. Ulloa, L. et al. Lefty proteins exhibit unique processing and activate the MAPK growth factor. Biochemistry 29, 6851–6857 (1990). pathway. J. Biol. Chem. 276, 21387–21396 (2001). 17. Belville, C. et al. Mutations of the anti-Mu¨llerian hormone gene in patients with persistent Mu¨ llerian duct syndrome: biosynthesis, secretion, and processing of Supplementary Information is linked to the online version of the paper at the abnormal proteins and analysis using a three-dimensional model. Mol. www.nature.com/nature. Endocrinol. 18, 708–721 (2004). Acknowledgements We thank P. Sun for porcine TGF-b1 cDNA, K. Koli for human 18. Walton, K. L. et al. Two distinct regions of latency-associated peptide coordinate TGF-b1 and LTBP1 cDNAs, D. Rifkin for human LTBP1 cDNA and transformed mink stability of the latent transforming growth factor-b1 complex. J. Biol. Chem. 285, lung epithelial cells, and the staff of the Advanced Photon Source General Medical 17029–17037 (2010). Sciences and National Cancer Institute (APS GM/CA-CAT) beamline 23-ID. 19. Little, S. C. & Mullins, M. C. Bone morphogenetic protein heterodimers assemble heteromeric type I receptor complexes to pattern the dorsoventral axis. Nature Cell Author Contributions M.S. cloned, expressed and purified proTGF-b1, crystallized the Biol. 11, 637–643 (2009). protein, collected and processed X-ray data, refined and analysed the structure, 20. Lack, J. et al. Solution structure of the third TB domain from LTBP1 provides insight designed and performed biochemical assays and wrote the paper. J.Z. collected and into assembly of the large latent complex that sequesters latent TGF-b. J. Mol. Biol. processed X-ray data, refined and analysed the structure and performed electron 334, 281–291 (2003). microscopy studies. R.W. designed and performed TGF-b1 assays. X.C. performed 21. Chen, Y. et al. Amino acid requirements for formation of the TGF-b-latent TGF-b electron microscopy studies. L.M. processed X-ray data. T.W. provided electron binding protein complexes. J. Mol. Biol. 345, 175–186 (2005). microscopy supervision. T.A.S. designed and supervised the project, refined and 22. Luo, B.-H., Carman, C. V. & Springer, T. A. Structural basis of integrin regulation and analysed the structure and wrote the paper. signaling. Annu. Rev. Immunol. 25, 619–647 (2007). Author Information X-ray structures have been deposited in the Protein Data Bank 23. Forman, J. R. & Clarke, J. Mechanical unfolding of proteins: insights into biology, under the accession number 3RJR. Reprints and permissions information is available structure and folding. Curr. Opin. Struct. Biol. 17, 58–66 (2007). at www.nature.com/reprints. The authors declare no competing financial interests. 24. Janssens, K. et al. Camurati-Engelmann disease: review of the clinical, radiological, Readers are welcome to comment on the online version of this article at and molecular data of 24 families and implications for diagnosis and treatment. www.nature.com/nature. Correspondence and requests for materials should be J. Med. Genet. 43, 1–11 (2005). addressed to T.A.S. ([email protected]).

METHODS CCP4, and NCS matrices for each domain between molecules and crystals were ProTGF-b1. The porcine proTGF-b1 construct with the rat serum albumin computed by LSQKAB in CCP4. Rigid-body refinements were carried out by leader sequence (MKWVTFLLLLFISGSAFS), followed by eight histidine resi- PHENIX for each lattice, on the basis of the averaged maps. The new models for dues, a streptavidin-binding peptide (TTGWRGGHVVELAGELEQLRARLEHH each lattice were then used to calculate a set of new NCS matrices for the next PQGQREP)32 and a HRV-3C protease site (LEVLFQGP) was amplified from cycle of DMMULTI. These steps were cycled twice. 43 pcDNA-GS-TGF-b1 (ref. 33). Porcine proTGF-b1 with the C4S mutation was Model building in COOT was based on multi-crystal, multi-domain averaged amplified from the latter construct. The C4S mutation increases proTGF-b1 electron density maps and 2Fo 2 Fc maps. NCS restraints and translation- expression33 and avoids inappropriate disulphide bond formation34. No crystals libration-screw (TLS) groups were used in refinement with PHENIX. The were obtained with this construct. One or two N-linked sites were deleted in the sequence-to-structure register was confirmed using Se anomalous maps. The N147Q and N107Q/N147Q constructs, which were expressed similarly to wild multi-crystal, multi-domain averaging was repeated using the refined structure type35. The best crystals were obtained with N147Q; the N107Q/N147Q mutant at an Rfree of about 33% and no major differences were found. Two residues from yielded needles that could not be optimized. CHO-Lec 3.2.8.1 cells were trans- the 3C protease site remain at the N terminus after cleavage. The structures include fected by electroporation and cultured with 10 mgml21 puromycin. Clones were residues 0–62, 70–208, 216–241, 250–361 and one N-acetylglucosamine (NAG) screened for expression using a sandwich enzyme-linked immunosorbent assay residue (chain A); residues 1–62, 70–208, 216–242, 250–299, 310-361 and two (ELISA) with a capture antibody to prodomain-1 (R&D Systems) and a biotiny- NAG residues (chain B); residues 1–62, 68–208, 216–241, 250–361 and three NAG lated detection antibody to the His tag (Qiagen). The clone with highest expres- residues (chain C) and residues 0–62, 69–208, 216–242, 250–361 and two NAG sion of proTGF-b1(,2mgl21) was expanded and cultured in roller bottles with residues (chain D). Validation and Ramachandran statistics used 44 J/J medium and 5% fetal bovine serum (FBS). Supernatants were collected every MOLPROBITY . All structure figures were generated using Pymol (DeLano 5 d, clarified by centrifugation, concentrated tenfold with tangential flow filtra- Scientific). tion (Vivaflow 200, Sartorius Stedim), diluted fivefold with 10 mM Tris-HCl, In the asymmetric unit of the crystal, a second pro-TGF dimer extends each 0.14 M NaCl (TBS, pH 8.0), then concentrated fivefold. Material was adjusted two-stranded b-ribbon to form a four-stranded, inter-dimer super-b-sheet in to 0.2 M NaCl and purified using Ni-NTA agarose (Qiagen) (25 ml per 5 l of which Leu 203 forms a hydrophobic lattice contact. In its absence, Leu 203 may culture supernatant), then washed with three column volumes of 0.6 M NaCl, mediate hydrophobic interactions within the bowtie. 0.01 M Tris (pH 8.0) and eluted with 0.25 M imidazole in TBS. Material was Mutagenesis. Wild-type human proTGF-b1 was inserted into the pEF1-puro adjusted to pH 7.4, applied to Strep-tactin agarose (IBA) (3 ml per 5 l of culture plasmid. Site-directed mutagenesis was performed using QuikChange supernatant) and washed with TBS (pH 7.4). Then 4 ml of recombinant His- (Stratagene). All mutations were confirmed by DNA sequencing. tagged HRV-3C protease (Novagen, 100 U mg21, 1 mg ml21), diluted 20-fold Free cysteine labelling and prodomain detection. HEK-293T cells were trans- in TBS (pH 7.4) with 10% glycerol, was applied to the column, which was held fected using Polyfect reagent (Qiagen) according to the manufacturer’s instruc- at 4 uC for 16 h. The flow-through with two column volumes of TBS (pH 7.4), tions, using 2 mg of proTGF-b1 cDNA per 6-cm dish of cells at 70–80% containing untagged proTGF-b1, was concentrated to 1 ml and applied to confluency. The cells were then cultured in FreeStyle serum-free medium MonoQ and Superdex S200 columns connected in series and equilibrated with (Invitrogen) for 3 d. Supernatant was reacted with 450 mM biotin-BMCC TBS (pH 7.5). Purified proTGF-b1 was concentrated to about 15 mg ml21 in (1-biotinamido-4-(49-(maleimidoethylcyclohexane)-carboxamido)butane) (Pierce) 10 mM Tris (pH 7.5), 75 mM NaCl for crystal screening in 96-well Greiner micro- for 60 min at 22u C, followed by the addition of 40 mM N-ethylmaleimide. plates (100 nl hanging-drop vapour diffusion format) using a mosquito crystal- ProTGF-b1 was immunoprecipitated with 1.5 mg anti-human-prodomain-1 lization robot (Molecular Dimensions) at 20 uC. Hits were optimized in 24-well (LAP-1) antibody (R&D Systems) and Protein A Sepharose beads (GE plates using hanging-drop vapour diffusion. However, better-diffracting crystals Healthcare) at 4 uC for 2 h, then subjected to reducing SDS 10% PAGE. After could only be obtained from sitting drops containing equal volumes of 12–15 ml transfer to polyvinylidene difluoride membranes (Millipore), biotin was detected protein and well solution under the optimized conditions of 6.5–7.5% PEG 3500, using streptavidin-horseradish peroxidase with the ECL-plus western blotting kit 17–18% isopropanol and 0.1 M sodium citrate (pH 5.6), with the addition of (GE Healthcare). Total proTGF-b1 was similarly detected on a separate blot using 4–5% glycerol to slow crystal growth and improve crystal size and shape. biotinylated human prodomain-1 (LAP-1) antibody. Maximum single-crystal dimensions reached 450 mm 3 150 mm 3 40 mm. TGF-b1 activation assay. Transformed mink lung epithelial cells (TMLCs) stably Before cooling the crystals to 100 K in liquid nitrogen, three rounds of increases transfected with a luciferase construct under plasminogen activator inhibitor in PEG 3350 concentration (12 h for each increase of 8% per cycle) were carried promoter 1 (ref. 5) were provided by D. Rifkin (New York University). HEK- 36 out in the mother liquor . The final PEG 3350 concentration of about 31% was 293T cell transfectants stably expressing av and b6 were selected with puromycin sufficient for cryoprotection. Crystals are summarized in Supplementary Table 1. and G418. Clones expressing high levels of integrin avb6 were selected by immu- There are two complexes per asymmetric unit, with a Matthews coefficient of nofluorescent flow cytometry using an anti-b6 antibody. Cells stably transfected 2.9 A˚ 3 Da21, giving a solvent content of 57.8%. with empty vector were used as a control. These cells were subsequently transi- To prepare Se-Met proTGF-b1, cells were washed with PBS (pH 7.4), supple- ently transfected with human wild-type or mutant proTGF-b1, using lipofecta- mented with 1% FBS, then incubated for 8 h with methionine-free a-MEM (SAFC mine with 0.4 mg plasmid DNA per well in a 48-well plate. After 16–24 h, each well Biosciences) supplemented with 50 mg l21 L-Se-Met (Sigma) and 10% dialyzed was used to seed 3 wells of a 96-well plate with about 15,000 cells, which were co- FBS. After replacement with the same medium, cells were cultured for 4 d. Se-Met cultured with 15,000 TMLCs in 100 ml DMEM with 0.1% BSA for 16–24 h. TGF- proTGF-b1, at a yield of 1.5 mg l21, was purified and crystallized identically to b1-induced luciferase activity in cell lysates was measured using the luciferase native proTGF-b1. Furthermore, a heavy-atom derivative was obtained by soak- assay system (Promega). To assess heat-releasable TGF-b1, cells were transfected ing crystals in mother liquor containing 0.4 mM HgBr2 for 4 h. as above except that polyfect was used in 6-well plates. After 2 d, cells were Structure determination and refinement. Native Se multiple-wavelength anom- collected and heated in 150 ml of DMEM with 0.1% BSA at 80 uC for 10 min. alous dispersion (MAD) and single-wavelength anomalous dispersion (SAD) Hg TGF-b1 activity in 50 ml aliquots was measured using the luciferase assay with derivative data were collected at 100 K at beamline 23-ID, then processed using TMLCs. HKL2000 (ref. 37) and XDS38. Statistics are in Supplementary Table 1. Initial Negative-stain electron microscopy. A large latent-complex fragment was iso- experimental phases were determined independently using Se-MAD and Hg- lated from supernatants of 293T cells transiently co-transfected with native SAD, with 19 out of 24 Se sites and 14 Hg sites in the asymmetric unit located human proTGF-b and a human LTBP fragment containing the same N- using PHENIX39. Electron density maps from Se-Met phasing, calculated after terminal tag as was used above on proTGF-b. The LTBP fragment contained fourfold non-crystallographic symmetry (NCS) averaging, clearly defined the the TB3 and TB4 domains and two intervening EGF-like domains (residues orientation of each monomer. The mature TGF-b1 homodimer was easily docked Thr 1333–Asn 1578, immature numbering). Multi-angle light scattering gave 40 into the map using model 1KLC with MOLREP in CCP4 (ref. 41). The prodo- an Mr of 119,400, compared to a calculated Mr of 120,400 for a 2:1 proTGF- main was built into the map manually. A crude model of proTGF-b1 was b:LTBP fragment (2 3 46,400 for proTGF-b, including 2 3 7,500 for three obtained after rigid-body refinement by PHENIX, with both domains as one rigid high-mannose N-linked sites, plus 27,600 for the LTBP fragment). The avb6 body. The same model was docked into Hg-SAD density for the two homodimers, ectodomain was expressed using C-terminal a-helical coiled-coils and tags; puri- using MOLREP. fied, then subjected to negative-stain electron microscopy as previously To improve the phases and extend them to higher resolution, multi-crystal described45. Purified proTGF-b1 or proTGF-b1 in complex with an LTBP frag- averaging (two crystals in total: Se-MAD and Hg derivative), multi-domain aver- ment (20 mg), proTGF-b1 (30 mg) in molar excess over clasped avb6 (20 mg), or aging (with separate masks for each prodomain and TGF-b monomer) and clasped avb6 (60 mg) in excess over proTGF-b (10 mg) were subjected to Superdex solvent flattening and histogram matching were performed using DMMULTI42 S200 chromatography in TBS (pH 7.5) with 1 mM Ca21 and 1 mM Mg21. Peak from the CCP4 suite. The mask for each domain was calculated by NCSMASK in fractions corresponding to the purified proteins or complexes were subjected to

©2011 Macmillan Publishers Limited. All rights reserved ARTICLE RESEARCH negative-stain electron microscopy. Particle selection, alignment, classification 38. Kabsch, W. in International Tables for Crystallography, Vol. F: Crystallography of and averaging were conducted as previously described46. Biological Macromolecules (eds Rossmann, M. G. & Arnold, E. V.) Ch. 25.2.9 XDS, 730–734 (Springer, 2001). 32. Keefe, A. D., Wilson, D. S., Seelig, B. & Szostak, J. W. One-step purification of 39. Adams, P. D. et al. PHENIX: building new software for automated crystallographic recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, structure determination. Acta Crystallogr. D 58, 1948–1954 (2002). the SBP-Tag. Protein Expr. Purif. 23, 440–446 (2001). 40. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D 33. Zou, Z. & Sun, P. D. Overexpression of human transforming growth factor-b1using 66, 22–25 (2010). a recombinant CHO cell expression system. Protein Expr. Purif. 37, 265–272 41. Bailey, S. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D (2004). 50, 760–763 (1994). 34. Gentry, L. E. et al. Type 1 transforming growth factor beta: amplified expression 42. Cowtan, K. Recent developments in classical density modification. Acta and secretion of mature and precursor polypeptides in Chinese hamster ovary Crystallogr. D 66, 470–478 (2010). cells. Mol. Cell. Biol. 7, 3418–3427 (1987). 43. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta 35. Brunner, A. M. et al. Site-directed mutagenesis of glycosylation sites in the Crystallogr. D 60, 2126–2132 (2004). transforming growth factor-beta 1 (TGF beta 1) and TGF beta 2 (414) precursors 44. Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and of cysteine residues within mature TGF beta 1: effects on secretion and and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007). bioactivity. Mol. Endocrinol. 6, 1691–1700 (1992). 45. Takagi, J., Petre, B. M., Walz, T. & Springer, T. A. Global conformational 36. Heras, B. & Martin, J. L. Post-crystallization treatments for improving diffraction rearrangements in integrin extracellular domains in outside-in and inside-out quality of protein crystals. Acta Crystallogr. D 61, 1173–1180 (2005). signaling. Cell 110, 599–611 (2002). 37. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in 46. Chen, X. et al. Requirement of open headpiece conformation for activation of oscillation mode. Methods Enzymol. 276, 307–326 (1997). leukocyte integrin aXb2. Proc. Natl Acad. Sci. USA 107, 14727–14732 (2010).

A B

TGF β1B TGF β1A

LAP1A LAP1B

LAP1 C LAP1D

TGF β1 TGF β1D C

C D 1 M 2

Tyr75 49.5kDa

Lys27 36.7kDa Tyr74 Ser26

Gln23 12.8kDa

Figure S1. Multi-crystal averaged experimental electron density and crystal protein. A: Multi-crystal average density at 1 σ for the two SLC homodimers in the asymmetric unit. B: Anomalous Fourier map calculated from the Se-Met data, contoured at 4.2 σ. Electron density is shown as black mesh. Met sidechains are shown as white sticks, with Se(S) atoms as gold spheres. C: Multi-crystal average electron density at 1 σ around the clasp region of chain D. D: SDS-PAGE of the SLC preparation used for crystallization (lane 1) and a washed crystal (lane 2).

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α1 TGF1po ------LSTCKTIDM••ELVKRKRIEAIRGQILSKLR•LASPP 34 TGF1hu ------LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPP 34 TGF2 ------SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPP 35 TGF3 ------SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPP 37 myosta ------NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLETAP 47 GDF11 ------AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEAP 69 Inh_bA ------SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHILNMLHLKKRP 52 Inh_bB ------SPTPPPTPAAPPPPPPPGSPGGSQDTCTSCGGFRRPEELGRVDGDFLEAVKRHILSRLQMRGRP 64 Inh_bC ------TPRAGGQCPACGG--PTLELESQRELLLDLAKRSILDKLHLTQRP 43 Inh_bE ------QGTGSVCPSCGG--SKLAPQAERALVLELAKQQILDGLHLTSRP 42 Inh_a ------CQGLELARELVLAKVRALFLDAL---GPP 26 BMP3 ------ERPKPPFPELRKAVPGDRTAGGGP 24 GDF10 ------SHRAPAWSALPAAADGLQGDRDLQRHP 27 BMP15 ------MEHRAQMAEGGQSSIALLAEAPTLPLIEEL-LEESP 35 GDF9 ------SQASGGEAQIAASAELESGAMPWSLLQHIDERDRAGLLPALFKVLSV 47 GDF1 ------PVPPGPAAALLQALGLRDEP 20 GDF3 ------QEYVFLQFLGLDKAP 15 BMP10 ------SPIMNLEQSPLEEDMSLFGDVFSEQDGVDFNTLLQSMKDEFLKTLNLSDIP 51 GDF2 ------KPLQSWGRGSAGGNAHSPLGVPGGGLPEHTFNLKMFLENVKVDFLRSLNLSGVP 54 Nodal ------TVATALLRTRGQPSSP 16 BMP2 ------LVPELGRRKFAAASSGRPSSQPSDEVLSEFELRLLSMFGLKQRP 44 BMP4 ------GASHASLIPETGKKKVAEIQGHAGGRRSGQ-SHELLRDFEATLLQMFGLRRRP 52 BMP5 ------DNHVHSSFIYRRLRNHERREIQREILSILGLPHRP 35 BMP7 ------DFSLDNEVHSSFIHRRLRSQERREMQREILSILGLPHRP 39 BMP6 ------CCGPPPLRPPLPAAAAAAAGGQLLGDGGSPGRTEQPPPSPQSSSGFLYRRLKTQEKREMQKEILSVLGLPHRP 73 BMP8 ------GGGPGLRPPPGCPQRRLGARERRDVQREILAVLGLPGRP 39 GDF5 ------APDLGQRPQGTRPGLAKAEAKERPPLARNVFRPGGHSYGGGATNANARAKGGTGQTGGLTQPKKDEPKKLPPRPGGPEPKPGHPPQTRQATARTVTPKGQLPGGKAPPKAGSVPSSFLLKKAREPGPP 128 GDF6 ------FQQASISSSSSSAELGSTKGMRSRKEGKMQRAPRDSDAGREGQEPQPRPQDEPRAQQP 58 GDF7 ------RDGLEAAAVLRAAGAGPVRSPGGGGGGGGGGRTLAQAAGAAAVP 44 GDF15 ------LSLAEAS 7 LEFTY1 ------LTGEQLLGSLLRQLQLKEVP 20 LEFTY2 ------LTEEQLLGSLLRQLQLSEVP 20 AMH LLGTEALRAEEPAVGTSGLIFREDLDWPPGIPQEPLCLVALGGDSNGSSSPLRVVGALSAYEQAFLGAVQRARWGPRDLATFGVCNTGDRQAALPSLRRLGAWLRDPGGQRLVVLHLEEVTWEPTPSLRFQEPP 134 NRTN ------IWMCREGLLLSHRLGPALVPLHRLPRTLDARIARLAQYRALLQGA 45 ARTN ------SLGSAPRSPAPREGPPPVLASPAGHLPGGRTARWCSGRARRPPP 44 PSPN ------WGPDARGVP 9 GDNF ------FPLPAGKRPPEAPAEDRSLGRRRAP 25

α2 β1 TGF1po SQGDV------P•P------GPLPEAVLA•LYNSTRDRVA•GESVEPE------PE••PEADYYAKEVTRVLMV---ESGN•Q 90 TGF1hu SQGEV------PP------GPLPEAVLALYNSTRDRVAGESAEPE------PEPEADYYAKEVTRVLMV---ETHNE 90 TGF2 EDY------PE------PEEVPPEVISIYNSTRDLLQEKASRRA------AACERERSDEEYYAKEVYKIDMPPFFPSENA 98 TGF3 E------PT------VMTHVPYQVLALYNSTRELLEEMHGERE------EGCTQENTESEYYAKEIHKFDMIQGLAEHNE 99 myosta NISKDVIRQLL------PK------APPLRELIDQYDVQRDD-SSDGSLED------DDYHATTETIITMPTESDFLM 106 GDF11 NISREVVKQLL------PK------APPLQQILDLHDFQGDALQPEDFLEE------DEYHATTETVISMAQETDPAV 129 Inh_bA DVTQPV------PK------AALLNAIRKLHVGKVGENGYVEI------EDDIGRRAEMNELMEQTSEIITFAESG---- 110 Inh_bB NITHAV------PK------AAMVTALRKLHAGKVREDGRVEI------PHLDGHASPGADGQERVSEIISFAETDGLA- 125 Inh_bC TLNRPV------SR------AALRTALQHLHGVPQGALLEDNR------EQECEIISFAETGLST- 90 Inh_bE RITHPP------PQ------AALTRALRRLQPGSVAPGN------GEEVISFATVTDSTS 84 Inh_a AVTREGGD------PG------VRRLPRRHALGGFTHRGSEP------EEEEDVSQAILFPATDASCE 76 BMP3 DSELQ------PQ------DKVSEHMLRLYDRYSTVQAARTPGSL------EGGSQPWRPRLLREGNTVRSFRAAAAETL 86 GDF10 GDAAATLG------PS------AQDMVAVHMHRLYEKYSRQGARPGG------GNTVRSFRARLEVV- 76 BMP15 GEQPRK------PR------LLGHSLRYMLELYRRSADSHGHPRENRT------IGATMVRLVKPLTSVAR 88 GDF9 GRGGSPRLQ------PD------SRALHYMKKLYKTYATKEGIPKSNRS------HLYNTVRLFTPCTRHKQ 101 GDF1 QGA------PR------LRPVPPVMWRLFRRRDPQETRSGSRRT------SPGVTLQPCHVEELGVAGNIVRHIPDRGAPTR 84 GDF3 S------PQ------KFQPVPYILKKIFQDREAAATTGVSRDL------CYVKELGVRGNVLRFLPDQGFFLY 70 BMP10 TQDSAKVDP------PE------YMLELYNKFATDRTS------MPSANIIRSFKNEDLFSQ 95 GDF2 SQDKTRVEP------PQ------YMIDLYNRYTSDKSTT------PASNIVRSFSMEDAISI 98 Nodal S------PL------AYMLSLYR------DPLPRADIIRSLQAEDVAVD 47 BMP2 T------PS------RDAVVPPYMLDLYRRHSGQPGSPAPDHR------LERAASRANTVRSFHHEESLEE 97 BMP4 Q------PS------KSAVIPDYMRDLYRLQSGEEEEEQIHST------GLEYPERPASRANTVRSFHHEEHLEN 109 BMP5 R------PF---SPGKQASSAPLFMLDLYNAMTN---EENPEESEYSVRASLAEETRGARKGYPASPNGYPRRIQLSRTTPLTTQSPPLASLHDTNFLNDADMVMSFVNLVERDK 138 BMP7 R------PH---LQGKH-NSAPMFMLDLYNAMAV---EEGGGPGGQGFSYPYKAVFS------TQGPPLASLQDSHFLTDADMVMSFVNLVEHDK 115 BMP6 RPLHGLQQPQPPALRQQEEQQQQQQLPRGEPPPGRL-KSAPLFMLDLYNALSADNDEDGASEGERQQSWPHEAASSSQRRQPPPGAAHPLNRKSLLAPGSGSGGASPLTSAQDSAFLNDADMVMSFVNLVEYDK 206 BMP8 R------PRAPPAASRLPASAPLFMLDLYHAMAGDDDEDGAPAE------QRLGRADLVMSFVNMVERDR 97 GDF5 REPKEPFRPPPIT------PH------EYMLSLYRTLSDADRKGGNSSV------KLEAGLANTITSFIDKGQDDR 186 GDF6 RAQEPPGRGPRVV------PH------EYMLSIYRTYSIAEKLGINASF------FQSSKSANTITSFVDRGLDDL 116 GDF7 AAAVPRARAARRAAGSGFRNGSVV--PH------HFMMSLYRSLAGRAPAGAAAVS------ASGHGRADTITGFTDQATQDE 113 GDF15 RASFPG------PS------ELHSEDSRFRELRKRYEDLLTRLRANQS------WEDSNTDLVPAPAVRI 59 LEFTY1 TLDRADMEELVI------PT------HVRAQYVALLQRSHGDRSRGKRFSQS------FREVAGRFLALE---- 72 LEFTY2 VLDRADMEKLVI------PA------HVRAQYVVLLRRSHGDRSRGKRFSQS------FREVAGRFLASE---- 72 AMH ------PG------GAGPPELALLVLYPGPGPEVTVTRAGLP--GAQSLCPSRD------TRYLVLAVDRPAGAWRGSGLALTLQPRGEDSR 206 NRTN ------PD------AMELRELTPWAGRPPGP------64 ARTN ------QP------SRPAPPPPA------55 PSPN ------VA------DGEFSSEQVAKAGGTWLGTHR------32 GDNF ------FALSS----DSNMPEDYPDQFDDVMDFIQATIKRLKRSPD------61 α3β2 β3 β4 β5 β6 TGF1po IYDKFKGT------P•HSLYMLFNTS•ELREAVPEP--V•LLSRAELRLL•R------LKLKVEQHV•ELYQKYSND------S•WRYLSNRLLA----•PSDSPEWLSF•DV------171 TGF1hu IYDKFKQS------THSIYMFFNTSELREAVPEP--VLLSRAELRLLR------LKLKVEQHVELYQKYSNN------SWRYLSNRLLA----PSDSPEWLSFDV------171 TGF2 IPPTFYR------PYFRIVRFDVSAMEKNASN-----LVKAEFRVFR------LQNPKARVPEQRIELYQILKSKDLT------SPTQRYIDSKVVK----TRAEGEWLSFDV------184 TGF3 LAVCPKG------ITSKVFRFNVSSVEKNRTN-----LFRAEFRVLR------VPNPSSKRNEQRIELFQILRPDEHI------AKQRYIGGKNLP----TRGTAEWLSFDV------184 myosta QVD------GKPKCCFFKFSSKIQYNK------VVKAQLWIYL------RPVETPTTVFVQILRLIKPMKDG------TRYTGIRSLKLD-MNPGTGIWQSIDV------185 GDF11 QTD------GSPLCCHFHFSPKVMFTK------VLKAQLWVYL------RPVPRPATVYLQILRLKPLTGEGTA------GGGGGGRRHIRIRSLKIE-LHSRSGHWQSIDF------216 Inh_bA ------TARKTLHFEISKEGSDLSV-----VERAEVWLFL------KVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVD-----ARKSTWHVFPV------203 Inh_bB ------SSRVRLYFFISNEGNQNLF-----VVQASLWLYL------KLLPYVLEKGSRRKVRVKVYFQEQGH------GDRWNMVEKRVD-----LKRSGWHTFPL------203 Inh_bC ------INQTRLDFHFSSDRTAGDRE----VQQASLMFFV------QLPSNTTWTLKVRVLVLGPHN------TNLTLATQYLLE-----VDASGWHQLPL------164 Inh_bE ------AYSSLLTFHLSTPRSHH------LYHARLWLHV------LPTLPGTLCLRIFRWGPRRRRQ------GSRTLLAEHHI------TNLGWHTLTL------154 Inh_a DKSAARGLAQEAE-EGLFRYMFRPSQHTRSRQ------VTSAQLWFHT------GLDRQGTAASNSSEPLLGLLALSPG------GPVAVPMSLG-----HAPPHWAVLHL------163 BMP3 ------ERKGLYIFNLTSLTKSEN------ILSATLYFCIGELGNISLSCPVSGGCSHHAQRKHIQIDLSAWTLK------FSRNQSQLLGHLSVDMAKSHRDIMSWLSKDI------180 GDF10 ------DQKAVYFFNLTSMQDSEM------ILTATFHFYS-EPPRWPRALEVLCKPRAKNASGRPLPLGPPTRQHLLFR------SLSQNTATQGLLRGAMAL--APPPRGLWQAKDI------173 BMP15 PHRGTWHIQILGFPLRPNRGLYQLVRATV------VYRHHLQLTR------FNLSCHVEPWVQKNPTN------HFPSSEGDSSKPSLMSNAWKEMDI------168 GDF9 APGDQVTGI-----LPSVELLFNLDRITTVEH------LLKSVLLYNI------NNSVSFSSAVKCVCNLMIKEPKSSSR------TLGRAPYSFTFNSQFEF-----GKKHKWIQIDV------192 GDF1 ASEPASAAGH----CPEWTVVFDLSAVEPAER------PSRARLELRFAAA------AAAAPEGGWELSVAQAGQGA 145 GDF3 PKKISQASS-----CLQKLLYFNLSAIKEREQ------LTLAQLGLDL------GPNSYYNLGPELELALFLVQEPHVWGQ------TTPKPGKMFVLRSVPWPQ-----GAVHFNLLDV------162 BMP10 PVSFNG------LRKYPLLFNVS-IPHHEE------VIMAELRLYT------LVQRDRMIYDGVDRKITIFEVLESKGDN------EGERNMLVLVSGEIYG-----TNSEWETFDV------182 GDF2 TATEDFP------FQKHILLFNIS-IPRHEQ------ITRAELRLYV------SCQNHVDPSHDLKGSVVIYDVLDGTDAW------DSATETKTFLVSQDIQD------EGWETLEV------185 Nodal ------GQNWTFAFDFSFLSQQED------LAWAELRLQL------SSPVDLPTEGSLAIEIFHQPKPDTEQ------ASDSCLERFQMDLFTVTLSQVTFSLGSMVLEV------133 BMP2 LPETSG------KTTRRFFFNLSSIPTEEF------ITSAELQVFR------EQMQDALGNNSSFHHRINIYEIIKPATAN------SKFPVTRLLDTRLVN-----QNASRWESFDV------186 BMP4 IPGTSE------NSAFRFLFNLSSIPENEV------ISSAELRLFR------EQVDQGPDWERGF-HRINIYEVMKPPAEV------VPGHLITRLLDTRLVH-----HNVTRWETFDV------198 BMP5 DFSHQR------RHYKEFRFDLTQIPHGEA------VTAAEFRIYK------DRSNNRFENETIKISIYQIIKEYTNR------DADLFLLDTRKAQ-----ALDVGWLVFDI------222 BMP7 EFFHPR------YHHREFRFDLSKIPEGEA------VTAAEFRIYK------DYIRERFDNETFRISVYQVLQEHLGR------ESDLFLLDSRTLW-----ASEEGWLVFDI------199 BMP6 EFSPRQ------RHHKEFKFNLSQIPEGEV------VTAAEFRIYK------DCVMGSFKNQTFLISIYQVLQEHQHR------DSDLFLLDTRVVW-----ASEEGWLEFDI------290 BMP8 ALGHQE------PHWKEFRFDLTQIPAGEA------VTAAEFRIYK------VPSIHLLNRTLHVSMFQVVQEQSNR------ESDLFFLDLQTLR-----AGDEGWLVLDV------180 GDF5 GPV------VRKQRYVFDISALEKDG------LLGAELRILRKKPSDTAK-PAAPGGGRAAQLKLSSCPSGR------QPASLLDVRSVPG----LDGSGWEVFDI------269 GDF6 SHTP------LRRQKYLFDVSMLSDKEE------LVGAELRLFR------QAPSAPWGPPAGPLHVQLFPCLSP------LLLDARTLDPQ--GAPPAGWEVFDV------195 GDF7 SAA------ETGQSFLFDVSSLNDADE------VVGAELRVLRRGSPE----SGPGSWTSPPLLLLSTCPGAA------RAPRLLYSRAAEP----LVGQRWEAFDV------194 GDF15 LT------PEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLS------PTASRSWDVTRP------111 LEFTY1 ------ASTHLLVFGMEQRLPPNSE-----LVQAVLRLFQEPVPKAALHRHGRLSPRSARARVTVEWLRVRDDGS------NRTSLIDSRLVS-----VHESGWKAFDV------159 LEFTY2 ------ASTHLLVFGMEQRLPPNSE-----LVQAVLRLFQEPVPKAALHRHGRLSPRSAQARVTVEWLRVRDDGS------NRTSLIDSRLVS-----VHESGWKAFDV------159 AMH LST------ARLQALLFGDDHRCFTR------MTPALLLLPR------SEPAPLPAHGQLDTVPFPPPR------PSAELEESPPS------ADPFLETL------276 NRTN ------64 ARTN ------55 PSPN ------32 GDNF ------61 Figure S2 (first page)

2 | WWW.NATURE.COM/NATURE SUPPLEMENTARY INFORMATION RESEARCH

α4 β7 β8 β9 β10 α5

TGF1po ------TGVVRQWL•TRRE--AIEGFR•LSAHCSCDSKD------N TLHVEING•FN------SGRRGDLA•TIHGMNRPFL•LLMATPLERA•QH------241 TGF1hu ------TGVVRQWLSRGG--EIEGFRLSAHCSCDSRD------NTLQVDINGFT------TGRRGDLATIHGMNRPFLLLMATPLERAQH------241 TGF2 ------TDAVHEWLHHKD--RNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQ------275 TGF3 ------TDTVREWLLRRE--SNLGLEISIHCPCHTFQP-NGDILENIHEVMEIKFKGVDNED---DHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNP------271 myosta ------KTVLQNWLKQPE--SNLGIEIKALDENGHDLAVTFPG------PGEDGLNPFLEVKVTD------236 GDF11 ------KQVLHSWFRQPQ--SNWGIEINAFDPSGTDLAVTSLG------PGAEGLHPFMELRVLE------267 Inh_bA ------SSSIQRLLDQGK--SSLDVRIACEQCQESGASLVLLGKKKKKEE------EGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSED------282 Inh_bB ------TEAIQALFERGE--RRLNLDVQCDSCQELAVVPVFV------DPGEESHRPFVVVQARLGD------256 Inh_bC ------GPEAQAACSQGH--LTLELVLEGQVAQSSVIL------GGAAHRPFVAARVRVG------210 Inh_bE ------PSSGLRGEKS-GVLKLQLDCRPLEGNSTV------TGQPRRLLDTAGHQQPFLELKIRANEP------209 Inh_a ------ATSALSLLTHPV--LVLLLRCPLCTCSA------RPEATPFLVAHTRTRPP------206 BMP3 ------TQLLRKAKENEE--FLIGFNITSK------GRQLPKRRLPFPEPYILVYANDAAISEPESVVSSLQGHRNFPTGTVPK 250 GDF10 ------SPIVKAARRDGE--LLLSAQLDSE------ERDPGVPRPSPYAPYILVYANDLAISEPNSVAVTLQRYDPFPAG---- 239 BMP15 ------TQLVQQRFWNNKGHRILRLRFMCQQQKD------SGGLELWHGTSSLDIAFLLLYFNDTHKSIRKAKFLPRGMEEFMER----- 241 GDF9 ------TSLLQPLVASNK--RSIHMSINFTCMKDQLEHP------SAQNGLFNMTLVSPSLILYLNDTSAQAYHSWYSLHYKRRPSQGPDQER 271 GDF1 GADPGPVLLRQLVPALGPPVRAELLGAAWARNASWPRSLRLALALR------PRAPAACARLAEASLLLVTLDPRLC------216 GDF3 ------AKDWNDNPR--KNFGLFLEILVKEDRDSGVNFQPEDT------CARLRCSLHASLLVVTLNPD------217 BMP10 ------TDAIRRWQKSGS--STHQLEVHIESKHDEAEDAS------SGRLEIDTSAQNKHNPLLIVFSDD------238 GDF2 ------SSAVKRWVRSDSTKSKNKLEVTVE------SHRKGCDTLDISVPPGSRNLPFFVVFSNDH------239 Nodal ------TRPLSKWLKR-----PGALEKQMS------RVAGECWPRPPTPPATNVLLMLYSNL------178 BMP2 ------TPAVMRWTAQGH--ANHGFVVEVAHLEEKQGVSK------RHVRISRSLHQDEHSWSQIRPLLVTFGHDGKG------250 BMP4 ------SPAVLRWTREKQ--PNYGLAIEVTHLHQTRTHQG------QHVRISRSLPQGSGNWAQLRPLLVTFGHDGRG------262 BMP5 ------TVTSNHWVINPQ--NNLGLQLCAETGDGRSINVKSAGL------VGRQGPQSKQPFMVAFFKASEVLLRSVR------286 BMP7 ------TATSNHWVVNPR--HNLGLQLSVETLDGQSINPKLAGL------IGRHGPQNKQPFMVAFFKATEVHFRSIR------263 BMP6 ------TATSNLWVVTPQ--HNMGLQLSVVTRDGVHVHPRAAGL------VGRDGPYDKQPFMVAFFKVSEVHVRTTR------354 BMP8 ------TAASDCWLLKRH--KDLGLRLYVETEDGHSVDPGLAGL------LGQRAPRSQQPFVVTFFRASPSPIRTPR------244 GDF5 ------WKLFRNFKNS------AQLCLELEAWERGRAV------DLRGLGF---DRAARQVHEKALFLVFGRT------321 GDF6 ------WQGLRHQPWK------QLCLELRAAWGELDAGEAEARARGPQQPPPP------DLRSLGF---GRRVRPPQERALLVVFTRS------262 GDF7 ------ADAMRRHRREPR--PPRAFCLLLRAVAGPVPSPL------ALRRLGFGWPGGGGSAAEERAVLVVSSRT------255 GDF15 ------LRRQLSLARPQAPALHLRLSPPPSQSDQLL------AESSSARPQLELHLRP------157 LEFTY1 ------TEAVNFWQQLSRPRQPLLLQVSVQREHLGPLASGAHKLV------RFASQGAPAGLGEPQLELHTLDLGDYGAQGD------229 LEFTY2 ------TEAVNFWQQLSRPRQPLLLQVSVQREHLGPLASGAHKLV------RFASQGAPAGLGEPQLELHTLDLRDYGAQGD------229 AMH ------TRLVRALRVPPARASAPRLALDPDALAGFPQGLVNLSDPAALERLLDG------EEPLLLLLRPTAATTGDPAPLHDPTSAPWATALARRV 361 NRTN ------64 ARTN ------55 PSPN ------32 GDNF ------61

α1 * β1 TGF1po ------LHSSRHRR------ALDTNYCFSS•• TEKNCCVR 267 TGF1hu ------LQSSRHRR------ALDTNYCFSSTEKNCCVR 267 TGF2 ------QTNRRKKR------ALDAAYCFRNVQDNCCLR 301 TGF3 ------GQGGQRKKR------ALDTNYCFRNLEENCCVR 298 myosta ------TPKRSRR------DFGLDCDEHSTESRCCRY 261 GDF11 ------NTKRSRR------NLGLDCDEHSSESRCCRY 292 Inh_bA ------HPHRRRRR------GLECDGKVNICCKK 304 Inh_bB ------SRHRIRKR------GLECDGRTNLCCRQ 278 Inh_bC ------GKHQIHRR------GIDCQGGSRMCCRQ 232 Inh_bE ------GAGRARRR------TPTCEPATPLCCRR 231 Inh_a ------SGGERARR------STPLMSWPWSPSALRLLQRPPEEPAAHANCHRV 247 BMP3 WDSHIRAAL--SIERRKKRSTGVLLPLQNNELPGAE------YQYKKDEVWEERKPYKTLQA-QAPEKSKNKK------KQRKGPHRKSQTLQFDEQTLKKARRKQWIEPRNCARR 351 GDF10 -DPEPRAAPNNSADPRVRRAAQATGPLQDNELPGLDERPPRAHAQHFHKHQLWPS--PFRALKP-RPGRKDRRKK------GQEV-FMAASQVLDFDEKTMQKARRKQWDEPRVCSRR 346 BMP15 ------ESLLRRTR------QADGISAEVTA------SSSKHSGPENNQCSLH 276 GDF9 SLSAYPVGEEAAEDGR------SSHHRHRR------GQETVSSELKKPLGPASFNLSEYFRQFLLPQNECELH 332 GDF1 ------HPLARPRRD------AEPVLGGGPGGACRAR 241 GDF3 ------QCHPSRKRRA------AIPVPKLSCKNLCHRH 243 BMP10 ------QSSDKERKEELNEMISHEQLPELDNLGLDSFSSGPGEEALLQMRSNIIYDS--TARIRRNAKGNYCKRT 305 GDF2 ------SSGTKETRLELREMISHEQESVLKKLSKDGSTEAGESSHEEDTDGHVAAGSTLARRKRSAGAGSHCQKT 308 Nodal ------SQEQRQLGGSTLLWEAESSWRAQEGQLSWEWGKR------HRRHHLPDRSQLCRKV 228 BMP2 ------HPL--HKREKR------QAKHKQRKRLKSSCKRH 276 BMP4 ------HALTRRRRAKR------SPKHHSQRARKKNKNCRRH 292 BMP5 ------AANKRKNQNRNK------SSSHQDSSRMSS-VGDYNTSEQKQACKKH 326 BMP7 ------STGSKQRSQNRSK------TPKNQEALRMAN-VAENSSSDQRQACKKH 304 BMP6 ------SASSRRRQQSRNR------STQSQDVARVSS-ASDYNSSELKTACRKH 395 BMP8 ------AVRPLRRRQP-KK------SNELPQANRLPGIFDDVRGSHGRQVCRRH 285 GDF5 ------KKRDLFFNEIKAR------SGQDDKTVYEYLFSQRRKRRAPLATR------QGKR------PSKNLKARCSRK 376 GDF6 ------QRKNLFAEMREQ---LGSAEAAGPGAGAEGSWPPPSGAPDARPWLP-SPGRRRRRTAFASR------HGKRHGKK------SRLRCSKK 335 GDF7 ------QRKESLFREIRAQARALGAALASEPL------PDPGTGTASPRAV-IGGRRRRRTALAGTRTAQGSGGGAGRGHGRR------GRSRCSRK 333 GDF15 ------QAARGRRRARAR------NGDHCPLGPGRCCRLH 185 LEFTY1 ------CDPEAPMTEGTRCCRQ 245 LEFTY2 ------CDPEAPMTEGTRCCRQ 245 AMH AAELQAAAAELRSLPGLPPATAPLLARLLALCPGGPGGLGDPLRALLLLKALQGLRVEWRGRDP-RGPGRAQRSA------GATAADGPCALR 447 NRTN ------RRRAGPRRRRAR------ARLGARPCGLR 87 ARTN ------PPSALPRGGRAARAGG------PGSRARAAGARGCRLR 87 PSPN ------PLARLRRALSGPCQLW 48 GDNF ------KQMAVLPRRERNRQAAAANPENSRG------KGRRGQRGKNRGCVLT 102 β2 α2 β3 β4 β5 β6 β7 β8 . * * * . . * * TGF1po QL•YIDFRKDLGW--•KWIHEPKGYH••ANFCLGPCPYIWSLD-----T----QYSK•VLALYNQH--N-P---•GASAAPC--CVP•QALEPLPIVYYVG-----RKPKVEQ•LSNMIVRSCKCS------361 TGF1hu QLYIDFRKDLGW--KWIHEPKGYHANFCLGPCPYIWSLD-----T----QYSKVLALYNQH--N-P---GASAAPC--CVPQALEPLPIVYYVG-----RKPKVEQLSNMIVRSCKCS------361 TGF2 PLYIDFKRDLGW--KWIHEPKGYNANFCAGACPYLWSSD-----T----QHSRVLSLYNTI--N-P---EASASPC--CVSQDLEPLTILYYIG-----KTPKIEQLSNMIVKSCKCS------395 TGF3 PLYIDFRQDLGW--KWVHEPKGYYANFCSGPCPYLRSAD-----T----THSTVLGLYNTL--N-P---EASASPC--CVPQDLEPLTILYYVG-----RTPKVEQLSNMVVKSCKCS------392 myosta PLTVDFE-AFGW--DWIIAPKRYKANYCSGECEFVFLQK--YPHT------HLVHQA--N-P---RGSAGPC--CTPTKMSPINMLYFNGK----EQIIYGKIPAMVVDRCGCS------352 GDF11 PLTVDFE-AFGW--DWIIAPKRYKANYCSGQCEYMFMQK--YPHT------HLVQQA--N-P---RGSAGPC--CTPTKMSPINMLYFNDK----QQIIYGKIPGMVVDRCGCS------383 Inh_bA QFFVSFK-DIGW-NDWIIAPSGYHANYCEGECPSHIAGTSGSSLS----FHSTVINHYRMRGHS-P---FANLKSC--CVPTKLRPMSMLYYDDG----QNIIKKDIQNMIVEECGCS------406 Inh_bB QFFIDFR-LIGW-NDWIIAPTGYYGNYCEGSCPAYLAGVPGSASS----FHTAVVNQYRMRGLN-P----GTVNSC--CIPTKLSTMSMLYFDDE----YNIVKRDVPNMIVEECGCA------379 Inh_bC EFFVDFR-EIGW-HDWIIQPEGYAMNFCIGQCPLHIAGMPGIAAS----FHTAVLNLLKANTAA-G---TTGGGSC--CVPTARRPLSLLYYDRD----SNIVKTDIPDMVVEACGCS------334 Inh_bE DHYVDFQ-ELGW-RDWILQPEGYQLNYCSGQCPPHLAGSPGIAAS----FHSAVFSLLKAN--N-P---WPASTSC--CVPTARRPLSLLYLDHN----GNVVKTDVPDMVVEACGCS------331 Inh_a ALNISFQ-ELGW-ERWIVYPPSFIFHYCHGGCGLHIPPN--LSLP----VPGAPPTPAQPY--S-L---LPGAQPCCAALPGTMRPLHVRTTSDGG---YSFKYETVPNLLTQHCACI------348 BMP3 YLKVDFA-DIGW-SEWIISPKSFDAYYCSGACQFPMPKS--LKPS----NHATIQSIVRAV--GVV---PGIPEPC--CVPEKMSSLSILFFDEN----KNVVLKVYPNMTVESCACR------450 GDF10 YLKVDFA-DIGW-NEWIISPKSFDAYYCAGACEFPMPKI--VRPS----NHATIQSIVRAV--GII---PGIPEPC--CVPDKMNSLGVLFLDEN----RNVVLKVYPNMSVDTCACR------445 BMP15 PFQISFR-QLGW-DHWIIAPPFYTPNYCKGTCLRVLRDG--LNSP----NHAIIQNLINQL--V-D---QSVPRPS--CVPYKYVPISVLMIEAN----GSILYKEYEGMIAESCTCR------374 GDF9 DFRLSFS-QLKW-DNWIVAPHRYNPRYCKGDCPRAVGHR--YGSP----VHTMVQNIIYEK--L-D---SSVPRPS--CVPAKYSPLSVLTIEPD----GSIAYKEYEDMIATKCTCR------430 GDF1 RLYVSFR-EVGW-HRWVIAPRGFLANYCQGQCALPVALS--GSGGPPALNHAVLRALMHAA--A-P---GAADLPC--CVPARLSPISVLFFDNS----DNVVLRQYEDMVVDECGCR------343 GDF3 QLFINFR-DLGW-HKWIIAPKGFMANYCHGECPFSLTIS--LNSS----NYAFMQALMHAV--D-P----EIPQAV--CIPTKLSPISMLYQDNN----DNVILRHYEDMVVDECGCG------340 BMP10 PLYIDFK-EIGW-DSWIIAPPGYEAYECRGVCNYPLAEH--LTPT----KHAIIQALVHLK--N-S---QKASKAC--CVPTKLEPISILYLDKG----VVTYKFKYEGMAVSECGCR------403 GDF2 SLRVNFE-DIGW-DSWIIAPKEYEAYECKGGCFFPLADD--VTPT----KHAIVQTLVHLK--F-P---TKVGKAC--CVPTKLSPISVLYKDDM---GVPTLKYHYEGMSVAECGCR------407 Nodal KFQVDFN-LIGW-GSWIIYPKQYNAYRCEGECPNPVGEE--FHPT----NHAYIQSLLKRY--Q-P---HRVPSTC--CAPVKTKPLSMLYVDN-----GRVLLDHHKDMIVEECGCL------325 BMP2 PLYVDFS-DVGW-NDWIVAPPGYHAFYCHGECPFPLADH--LNST----NHAIVQTLVNSV----N---SKIPKAC--CVPTELSAISMLYLDE----NEKVVLKNYQDMVVEGCGCR------373 BMP4 SLYVDFS-DVGW-NDWIVAPPGYQAFYCHGDCPFPLADH--LNST----NHAIVQTLVNSV----N---SSIPKAC--CVPTELSAISMLYLDE----YDKVVLKNYQEMVVEGCGCR------389 BMP5 ELYVSFR-DLGW-QDWIIAPEGYAAFYCDGECSFPLNAH--MNAT----NHAIVQTLVHLM--F-P---DHVPKPC--CAPTKLNAISVLYFDD----SSNVILKKYRNMVVRSCGCH------424 BMP7 ELYVSFR-DLGW-QDWIIAPEGYAAYYCEGECAFPLNSY--MNAT----NHAIVQTLVHFI--N-P---ETVPKPC--CAPTQLNAISVLYFDD----SSNVILKKYRNMVVRACGCH------402 BMP6 ELYVSFQ-DLGW-QDWIIAPKGYAANYCDGECSFPLNAH--MNAT----NHAIVQTLVHLM--N-P---EYVPKPC--CAPTKLNAISVLYFDD----NSNVILKKYRNMVVRACGCH------493 BMP8 ELYVSFQ-DLGW-LDWVIAPQGYSAYYCEGECSFPLDSC--MNAT----NHAILQSLVHLM--K-P---NAVPKAC--CAPTKLSATSVLYYDS----SNNVILRKHRNMVVKACGCH------383 GDF5 ALHVNFK-DMGW-DDWIIAPLEYEAFHCEGLCEFPLRSH--LEPT----NHAVIQTLMNSM--D-P---ESTPPTC--CVPTRLSPISILFIDS----ANNVVYKQYEDMVVESCGCR------474 GDF6 PLHVNFK-ELGW-DDWIIAPLEYEAYHCEGVCDFPLRSH--LEPT----NHAIIQTLMNSM--D-P---GSTPPSC--CVPTKLTPISILYIDA----GNNVVYKQYEDMVVESCGCR------433 GDF7 PLHVDFK-ELGW-DDWIIAPLDYEAYHCEGLCDFPLRSH--LEPT----NHAIIQTLLNSM--A-P---DAAPASC--CVPARLSPISILYIDA----ANNVVYKQYEDMVVEACGCR------431 GDF15 TVRASLE-DLGW-ADWVLSPREVQVTMCIGACPSQFRAA-----N----MHAQIKTSLHRL--K-P---DTVPAPC--CVPASYNPMVLIQKTD-----TGVSLQTYDDLLAKDCHCI------279 LEFTY1 EMYIDLQ-GMKWAENWVLEPPGFLAYECVGTCRQPPEAL------AFKWP---FLGPRQC---IASETDSLPMIVSIKEG-GRTRPQVVSLPNMRVQKCSCASDGALVPRRLQP 345 LEFTY2 EMYIDLQ-GMKWAKNWVLEPPGFLAYECVGTCQQPPEAL------AFNWP---FLGPRQC---IASETASLPMIVSIKEG-GRTRPQVVSLPNMRVQKCSCASDGALVPRRLQP 345 AMH ELSVDLR-A----ERSVLIPETYQANNCQGVCGWPQSDR-----NPRYGNHVVLLLKMQVR--G-A---ALARPPC--CVPTAY-AGKLLISLSE----ERISAHHVPNMVATECGCR------542 NRTN ELEVRVS-ELGL---GYASDETVLFRYCAGACEAAARVY------DLGLRRLRQRRRLRRE---RVRAQPC--CRPTAYEDEVSFLDA------HSRYHTVHELSARECACV------178 ARTN SQLVPVR-ALGL---GHRSDELVRFRFCSGSCRRARSPH------DLSLASLLGAGALRPPPGSRPVSQPC--CRPTRY-EAVSFMDV------NSTWRTVDRLSATACGCLG------181 PSPN SLTLSVA-ELGL---GYASEEKVIFRYCAGSCPRGARTQ------HGLALARLQGQ----G---RAHGGPC--CRPTRY-TDVAFLDD------RHRWQRLPQLSAAACGCGG------135 GDNF AIHLNVT-DLGL---GYETKEELIFRYCSGSCDAAETTY------DKILKNLSRNRRLV-S---DKVGQAC--CRPIAFDDDLSFLDD------NLVYHILRKHSAKRCGCI------192 Figure S2. Alignment of all 37 human TGF-β superfamily members and porcine TGF-β1. BMP8A and B are almost identical so only BMP 8A is shown. Sequence alignment with the MAFFT version 6 server (http://mafft.cbrc.jp/alignment /server/) used the E-INS-i option. Minor manual adjustments were made to the alignment in the β1 and β2 strands. Gap lengths were shortened when this broke alignment of residues in only a small number of sequences. Any gaps in secondary structure were moved to neighboring loops. The four GDNF-like factors lacked arm domain sequence signatures, and were manually aligned with the straightjacket region. Dots mark decadal residues in TGF-β1. Gold circles mark hydrophobic core residues of the arm domain. The coloring scheme for sequence conservation is modified from Clustal.

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A B Fingers a3 a1 a3 a3

a3 a3

D proa1

a3 proa1

proa2 prob1

Mature Prodomain TGF-b

Latent b3b10 b1 TGF-b R type I

a1 R type II

Latency lasso Figure S3. Conformational changes relative to mature TGF-β1. A and B. Superposition of TGF-β1 monomers from proTGF-β (chain C, gold and chain D, cyan) and from a receptor complex (magenta) (3KFD). C-E. Superposition of TGF-β dimers. In C and D, TGF-β 1 from a receptor complex (C) is shown in the same orientation as TGF-β1 from proTGF-β (D), with vertical displacement on the page. Dashed black lines show the change in position of representative residues in the α3-helix and long β-finger. Interacting prodomain secondary structure elements are shown in silver in D. E. Similar to main text Fig. 3, except the dimeric rather than monomeric unit is shown. Receptors are shown as transparent surfaces.

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A B proTGF-b, 2862 particles

proTGF-b

proTGF-b + aVb6 (1:1)

C proTGF-b complex with LTBP fragment, 3652 particles

proTGF-b + aVb6 (1:2)

aVb6

D Excess proTGF-b + aVb6 (1:1), 2710 particles

Figure S4. Gel filtration profiles and complete class averages for EM experiments. A. S200 gel filtration with an excess of aVb6 (cyan trace) or excess of proTGF-b (magenta trace). The two runs were done 10 months apart, on different columns of same nominal size (1 x 30 cm). B-E. The complete sets of EM class averages. Scale bars are 100 Å - see figure 4 part 2 on next page.

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E proTGF-b and excess aVb6 (1:2), 2069 particles

Figure S4 part 2

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GDF9 BMP3 GDF10 TGF-b1 TGF-b2 TGF-b3 BMP15 GDF11 myostatin (GDF8)

BMP8A,B Inhibin-bA BMP6 Inhibin-bB BMP7 Inhibin-bC BMP5 Inhibin-bE

Lefty1 BMP4 Lefty2 BMP2

GDF15 Nodal GDF2 Anti- BMP10 Mullerian hormone GDF7 GDF6 GDNF GDF5 NRTN ARTN PSPN GDF3 GDF1 Inhibin-a

Figure S5. TGF-b Superfamily tree. The TGF-β superfamily consists of 33 true family members that signal through type I and II receptors, and four ligands related to glial-cell derived neurotrophic factor (GDNF) that signal in the nervous system through the tyrosine kinase RET and co-receptors specific for each ligand (GDNF, NRTN, PSPN, and ARTN).

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Table S1: Diffraction dataa and refinement statistics for SLC1 structure.

Data sets Native Se MAD Hg SAD Peak Inflection Remote Peak Space group P21 P21 P21 Unit Cell a (Å) 54.7 54.2 50.9 b (Å) 127.1 128.3 128.0 c (Å) 138.2 138.6 138.7 β (°) 96.7 96.7 95.5 Wavelength (Å) 0.97931 0.97941 0.97959 0.95667 1.0070 Resolution (Å) 50 – 3.05 50 – 3.8 50 – 3.8 50 – 3.8 50 – 3.5 Completeness (%) 97.9 (96.5) 96.0 (78.2) 92.5 (57.9) 93.1 (59.3) 99.8 (100.0) I/σ(I) 16.4 (1.8) 10.9 (2.3) 14.2 (2.3) 15.6 (2.0) 7.0 (2.0) b Rmerge (%) 4.7 (75.2) 7.9 (35.5) 6.2 (30.2) 6.5 (35.3) 9.3 (58.6) Redundancy 5.6 (4.2) 3.8 (3.3) 3.7 (2.8) 4.0 (2.8) 5.3 (5.3) Solvent content 57.8 57.8 55.2 Number of reflections 199,523/ 67,691/ 63,150/ 70,060/ 117,999/ Total/Unique 21,876 18,018 17,260 17,501 17,253 c d Rwork /Rfree (%) 27.4/31.1 RMS Bond (Å) 0.002 Angle (°) 0.488 Ramanchandran Favored/Outliere (%) 89.78/0.45 PDB code 3RJR

aNumbers in parentheses correspond to the outermost resolution shell. b Rmerge=ΣhΣi|Ii(h)−|/ΣhΣiIi(h), where Ii(h) and are the ith and mean measurement of the intensity of reflection h. c Rwork=Σh||Fobs(h)|−|Fcalc(h)||/Σh|Fobs(h)|, where Fobs(h) and Fcalc(h) are the observed and calculated structure factors, respectively. No I/σ cutoff was applied. d Rfree is the R value obtained for a test set of reflections consisting of a randomly selected ~3% subset of the data set excluded from refinement. eResidues in favored and outlier regions of the Ramachandran plot as reported by MOLPROBITY.

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