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Structural Biology and Evolution of the TGF-b Family

Andrew P. Hinck,1 Thomas D. Mueller,2 and Timothy A. Springer3,4

1Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260 2Department of Plant Physiology and Biophysics, Julius-von-Sachs Institute of the University Wuerzburg, D-97082 Wuerzburg, Germany 3Program in Cellular and Molecular Medicine and Division of Hematology, Department of Medicine, Boston Children’s Hospital, Boston, Massachusetts 02115 4DepartmentofBiologicalChemistryandPharmacology,HarvardMedicalSchool,Boston,Massachusetts02115 Correspondence: [email protected]

We review the evolution and structure of members of the transforming growth factor b (TGF-b) family, antagonistic or agonistic modulators, and receptors that regulate TGF-b signaling in extracellular environments. The growth factor (GF) domain common to all family members and many of their antagonists evolved from a common cystine knot growth factor (CKGF) domain. The CKGF superfamily comprises six distinct families in primitive metazo- ans, including the TGF-b and Dan families. Compared with Wnt/Frizzled and Notch/Delta families that also specify body axes, cell fate, tissues, and other families that contain CKGF domains that evolved in parallel, the TGF-b family was the most fruitful in evolution. Complexes between the prodomains and GFs of the TGF-b family suggest a new paradigm for regulating GF release by conversion from closed- to open-arm procomplex confor- mations. Ternary complexes of the final step in extracellular signaling show how TGF-b GF dimers bind type I and type II receptors on the cell surface, and enable understanding of much of the specificity and promiscuity in extracellular signaling. However, structures suggest that when GFs bind repulsive guidance molecule (RGM) family coreceptors, type I receptors do not bind until reaching an intracellular, membrane-enveloped compartment, blurring the line between extra- and intracellular signaling. Modulator protein structures show how structurally diverse antagonists including follistatins, noggin, and members of the chordin family bind GFs to regulate signaling; complexes with the Dan family remain elusive. Much work is needed to understand how these molecular components assemble to form signaling hubs in extracellular environments in vivo.

ulticellular organisms require an elaborate diverse processes including repair of damaged Msystem of intercellular communication to tissues and regulation of immune responses. coordinate cellular actions. In developing em- In mammals, the 33 genes of the transforming bryos, the ability of cells to sense and respond to growth factor b (TGF-b) family each encode a such communication is vital for the establish- polypeptide comprising a secretion signal pep- ment of the overall body plan and to pattern tide, a 250-residue prodomain, and a 110- tissues, whereas, in adults, it is required for residue growth factor (GF) domain. Family

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A.P. Hinck et al.

members include bone morphogenetic pro- TGF-b GFs and many of their inhibitors are teins (BMPs), growth and differentiation fac- constructed evolved in early metazoans before tors (GDFs), activins, and TGF-bs. We discuss the development of bilateral symmetry (see the emerging concept that the distinctive activ- section on a Brief History of TGF-b Family ities of TGF-b family GFs are determined not Evolution). GFs are biosynthesized as dimeric just by signaling type I and type II receptors, prodomain–GF complexes, and the prodomain which show varying degrees of promiscuity has an important role in storage and release for GFs, but also by multiple binding pro- (activation) of the GF (see section on Structures teins and enzymes. These modulators greatly and Functions of TGF-b Family Procomplexes). diversify signaling activity by adding another Type I and type II receptors and coreceptors layer of signaling that occurs in extracellular bind and discriminate between GFs and signal environments. They control not only whether into cells (see section on Specificity and Promis- GFs reach their receptors on cells, but also cuity in GF-Receptor Interactions). Finally, a whether additional components are present large number of inhibitors form complexes within GF-receptor complexes. Receptors in- with the GFs to antagonize or modulate signal- clude not only the classical type I and type II ing (see section on Regulation of TGF-b/BMP receptors, but also type III coreceptors and re- Signaling by Modulator Proteins—BMPs as pulsive guidance molecule (RGM) coreceptors. Signaling Hubs). Binding proteins comprise the cognate prodo- mains, antagonists such as noggin, follistatin, A BRIEF HISTORY OF TGF-b FAMILY chordin, and Dan proteins, anchoring mole- EVOLUTION cules such as latent TGF-b binding protein (LTBP), and activators such as integrins. En- Function, three-dimensional structure, and zymes include proprotein convertases (PCs) amino acid sequence are all related. Thus, trac- that cleave the prodomain from the GF, either ing the evolution of the TGF-b family through intracellularly during biosynthesis or extracellu- its sequence relationships is an excellent intro- larly, and tolloid and matrix metalloproteases. duction to function. The TGF-b family and its Thus, molecular recognition in the TGF-b fam- receptors and antagonists evolved in parallel ily is not singularly achieved by GF-receptor with the Wnt/Frizzled and Delta/Notch path- interactions, but by a network of interactions ways. Together, these extracellular ligands and with multiple partners. Box 1 summarizes the receptors create the networks that establish mul- proteins, interactions, relationships, and terms ticellular animal body plans. They specify ante- used in this review. roposterior, dorsoventral (bilateral), and left– The complex network of interactions de- right axes, details of individual organs, and reg- scribed here parallels that found through high- ulate development and homeostasis. throughput mapping of interactomes (Li et al. 2004; Rual et al. 2005; Guruharsha et al. 2011), The CKGF Domain and Its Families which has shown that regulation is maintained by a protein–protein interaction network in The cystine knot (CK) growth factor (CKGF) which some proteins serve as hubs and interact domain has a specific three-dimensional fold with a larger number of interaction partners and sequence. Other proteins contain CKs and to regulate activity (Jeong et al. 2000). These thus the CK alone does not define the CKGF findings shift our understanding of “linear” sig- domain; the definition of this domain also naling cascades toward complex signaling net- includes the topological relationship between works, which may better explain the functional b-strand order in b-sheets and amino acid se- diversity encoded by TGF-b family proteins in quence. The CKGF domain can thus be recog- multicellular animals. nized both by sequence and by structure (Roch Our review has four sections following the and Sherwood 2014), similarly to epidermal introduction. The protein domain from which growth factor (EGF) or immunoglobulin (Ig)

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Structural Biology and Evolution of the TGF-b Family

BOX 1. PRODOMAIN, GROWTH FACTOR (GF), AND RECEPTOR STRUCTURES AND EVOLUTIONARY TREES

Structures are described in sections on Structures and Functions of TGF-b Family Procomplexes, and Specificity and Promiscuity in GF-Receptor Interactions. For evolutionary trees, sequences were aligned using MAFFT version 7 and the E-INS-i strategy with BLOSUM30 matrices and gap penalties of 1.53 to 2.5 (Katoh and Standley 2013). Trees were calculated as implemented on the same MAFFT server with the NJ method using all gap-free sites, the JTT model, estimation of a, and 1000 bootstrap samples. In each tree, branch lengths are to the scale shown in the bars.

TGF-β1 homodimer GF homodimer GF monomer Prodomain 2 GF GF Finger, loop 3 Prodomain 1 Bowtie monomer 1 monomer 2 Heel, loop 2 Fingers Palm RGD RGD Heel Arm Arm Palm domain domain Wrist Knuckle Cysteine knot α Finger, loop 1 2 α2 Thumb Fastener GF1 GF2 RIIThumb RI Cystine knot α5 Strait- β β C6 jacket β4-β5 4- 5 C5 Latency Latency loop α loop C2 C4 lasso 1 α1 lasso C3 Association C1 region Finger 1 Cys Finger 3 linkage Finger 1 Finger 3 Finger 2 Finger 2 TGF-β1 monomer Prodomain 1 Bowtie ThickVeins_Dm AMHRII TGF-β receptors ALK3 ALK6 RGD Wishful Thinking_Dm BMPRII Type II Type I ALK2 Arm ALK1 domain Punt_Dm Sax_Dm Furin ActRIIB ActRII 0.2 ALK5 cleavage ALK4 α2 Fastener TβRII ALK7 GF1 Baboon_Dm BMP antagonists α5 Nbl1/Dan Strait- Cerberus Noggin jacket Gremlin Latency α1 PRDC 0.2 lasso Coco Sclerostin Association USAG1 region Cys linkage Screw_Dm β GBB_Dm TGF- prodomains Inhα TGF-β GFs BMP8 Maverick_Dm BMP6 TGFβ1 α Myostatin Myoglianin_Dm Dawdle_Dm BMP7 Inh LEFTY2 BMP5 TGFβ2 GDF11 LEFTY1 AMH GDF15 Maverick_Dm β InhβE TGF 3 BMP2 TGFβ3 β GDF1 InhβB TGF 2 BMP4 LEFTY2 InhβC TGFβ1 DPP_Dm β LEFTY1 Nodal Inh A AMH GDF10 BMP9 Activin_Dm GDF15 BMP10 BMP10 BMP3 GDF6 BMP9 BMP5BMP6 Myoglianin_Dm GDF7 Nodal BMP15 BMP7 0.2 0.2 GDF1 GDF5 GDF3 DPP_Dm BMP8 GDF11 GDF10 GDF3 BMP clade BMP4 GBB_Dm Myostatin β BMP2 GDF7 Activin_Dme Inh A BMP3 m Dawdle_Dm GDF9 GDF6 SCW_D BMP15 GDF5 β InhβB Activin clade Inh E GDF9 InhβC

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A.P. Hinck et al.

domains. And just as the latter families have factors (VEGFs), and (6) the nerve growth fac- diverse functions, many proteins containing tors (NGFs) (Roch and Sherwood 2014). CKGF CKGF domains lack growth factor activity. domains are also found in noggin, in the CCN Most TGF-b family members act as differenti- family, and as carboxy-terminal CK domains. ation factors rather than growth factors and some such as inhibins and Leftys antagonize From Prebilaterians to the Emergence activities of other members. However, just as of TGF-b Itself in Deuterostomes TGF-b is eponymous for the family as a whole, its signaling moiety is also eponymous for what Of these six families, the first five are represent- we term the GF domain. All family members ed in prebilaterian phyla. Moreover, sequences contain a GF domain that can form a GF dimer; characteristic of specific Dan family members thus, we term this the GF domain whether or represented in Cnidaria, Porifera, Ctenophora, not it has signaling activity. and Placazoa are detectable in humans. In other The CKGF domain contains two long b-rib- words, statistically significant relationships be- bons (b-sheets with two antiparallel b-strands) tween sequences can be used to infer orthology and a CK (GF monomer, Box 1). Two closely so that prebilaterian Dan family members can spaced pairs of cysteines in adjacent, parallel be linked to specific proteins or subfamilies of b-strands disulfide-link to form a ring com- related proteins in humans, including gremlin, posed of two peptide backbone segments and sclerostin, and uterine sensitization-associated two disulfides. Another disulfide passes through gene 1 (USAG1) (Roch and Sherwood 2014). the ring, linking two additional polypeptide seg- TGF-b family members and their type I and ments (cystine knot, Box 1). Thus, the knot ties type II receptors are also present in nonbilater- together four polypeptide segments that are ians, but orthology with particular family distal in sequence, and forms a highly stable members in chordates is unclear. In contrast, CK core from which three long loops emanate Smad orthologues corresponding to Smad 1 (GF monomer, Box 1). or 5, Smad 2 or 3, the co-Smad Smad4, and The CKGF domain superfamily is present in inhibitory Smads (I-Smads) can be identified the earliest metazoans. Like the Wnt/Frizzled in nonbilaterians (Herpin et al. 2004; Humi- and Delta/Notch families, the CKGF family is niecki et al. 2009; Pang et al. 2011). not found in protists, and first appears in prim- The next step in evolution is marked by the itive metazoans that lack a bilateral axis. Repre- definition of a new body axis, the bilateral axis, sentatives are present in four distinct prebilater- which creates dorsoventral asymmetry and also ian phyla, that is, Porifera (sponges), Cnidaria enables development of left–right asymmetry. (coral and hydra), Ctenophora (comb jellies), Bilaterian phyla are grouped by nucleotide and and Placozoa (Trichoplax) (Roch and Sherwood protein sequence into three branches that ex- 2014). The CKGF superfamily is composed tend previous embryological classification of six groups (families) with the CKGF domain (Blair and Hedges 2005; Dunn et al. 2008; Hej- as the primary structural feature, that is, with nol et al. 2009). The protostome branch is now or without a prodomain and without any other subdivided into the Ecdysozoa (e.g., Caenorhab- folded domain (Adamska et al. 2007; Roch and ditis elegans and Drosophila melanogaster) and Sherwood 2014). These are (1) the TGF-b fam- Lophotrochozoa (e.g., molluscs and annelids) ily, (2) the Dan family of BMP antagonists, (3) branches. Deuterostomes represent a later the glycoprotein hormone family (GPH), for evolving group of phyla. Molecular phylogeny example, the pituitary hormones follicle-stim- confirms deuterostomes as a branch with three ulating hormone, luteinizing hormone, and phyla, Echinodermata (e.g., sea urchins), Chor- thyroid-stimulating hormone, (4) bursicon data (vertebrates, urochordates, and cephalo- hormones, which are limited to invertebrates, chordates), and Hemichordata (acorn worms). (5) the platelet-derived growth factor (PDGF) Key innovations in the evolution of deutero- family, including vascular endothelial growth stomes from lower bilaterians include the emer-

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Structural Biology and Evolution of the TGF-b Family

gence of pharyngeal gill slits and an inversion in TGF-b emerged in a context in which the key the dorsoventral axis of the body plan (Holland tasks of bilaterian axis and organ specification et al. 2015; Lowe et al. 2015). Notably, the BMP by complex signaling networks were well estab- and chordin gradients that establish dorsoven- lished. Within this context, TGF-b evolved to tral polarity also invert in deuterostomes (Lowe take advantage of a new regulatory mechanism, et al. 2015). in which integrins activate signaling. Such a Despite the presence of multiple TGF-b new mechanism may have been required be- family members, TGF-b family receptors, and cause the regulatory hubs involving BMPantag- Smads, TGF-b is clearly absent from early bilat- onists were already well established in the spec- erians. TGF-b itself first appears in the genome- ification of body axes and organs, and their use basedevolutionary recordwiththeemergenceof by a radical new CKGF member might have deuterostomes. Lowerdeuterostomes,including thrown a wrench into this machinery. sea urchins, tunicates, and hemichordates, have only a single TGF-b, and in all cases its prodo- Family Trees main has an RGD motif (Robertson and Rifkin 2013). Mammals have three TGF-bs. TGF-b1 The family trees in Box 1 show the diversifi- and -b3 contain RGD motifs and bind and are cation of family members in humans and activated by integrins. TGF-b2 diverged from D. melanogaster for TGF-b family GFs and pro- TGF-b3 after the divergence of TGF-b1. Thus, domains (separately), BMP antagonists, and the lackof the Arg of the RGD motif in TGF-b2is TGF-b family type I and type II receptors. These an acquired, not ancestral, characteristic. Fur- trees were constructed using rigorous methods thermore, the acidic Asp residue retained in appropriate for defining phylogenetic relation- TGF-b2 is the only key residue required for in- ships, as implemented in MAFFT (Katoh and tegrin binding, and thus it is possible that TGF- Standley 2013). Scale bars show the extent of b2 is activated by a yet unidentified integrin. sequence divergence, which for any two mem- These evolutionary events shed important bers is given by adding the lengths of the branch- light on the TGF-b family and answer one of es that separate them. However, the trees show the commonly asked questions in the field, ontogenetic relationships, not phylogenetic re- “Why are there so many antagonists of BMPs lationships. Thus, the trees in Box 1 show se- and activins but not TGF-bs?” Currently se- quence divergence as aconsequence of function- quenced nonbilaterian metazoans have as al and structural diversification, rather than as a many or more members of the CKGF Dan consequence of separation of animal species in family than of the CKGF TGF-b family itself. evolutionary time. Furthermore, the shapes of Therefore, in prebilaterians, BMP antagonist the treesin Box 1 provide insights into how func- and TGF-b family members coevolved in the tion and structure have driven diversification of presence of one another (Roch and Sherwood the subfamilies. Separate trees for the TGF-b 2014), and in the presence of other develop- family prodomains and GFs emphasize that the mental pathways such as Wnt (Adamska et al. prodomains are more divergent; when the scale 2007). These signaling families matured and bars in Box 1 are taken into account, the prodo- diversified much further in bilaterian inverte- mains are about fourfold more divergent than brates, to the point where members in Droso- the GFs. Thus, the prodomains have been under phila can be grouped with specific TGF-b sub- greater functional or structural pressure for di- families in humans (TGF-b GFs, Box 1). In vergencethantheGFs.Also,thesepressuresmust contrast, TGF-b itself evolved much later, long differ, because branch lengths and clustering after the diversification of bilaterian phyla, within subfamilies differ; however, because of at the emergence of deuterostomes. Integrins, highersequenceidentityfor theGFs,subfamilies like the CKGF superfamily, are present in both can be defined with more statistical significance. prebilaterian and early bilaterian metazoans, in Clustering using neighbor joining with which they are already well diversified. Thus, bootstrap calculations (here) or Bayesian anal-

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A.P. Hinck et al.

ysis (Pang et al. 2011) support significant rela- families coupling to R-Smad1, 5, 8 and the tionships among TGF-b family GF domains ALK4/5/7 subfamily coupling to R-Smad2 (color-coded in Box 1). A BMP clade includes and 3. Type II receptors also show three groups. six subfamilies. Human BMP-5, 6, 7, and 8 and BMPRII and AMHRII relate to Wishful think- Drosophila Glass bottom boat (Gbb) and Screw ing, and ActRII and ActRIIB ally with Punt. In cluster in one group that is significantly related contrast, among all type I and type II receptors, to the cluster with human BMP-2 and 4 and only TbRII lacks a Drosophila relative, consistent Drosophila Decapentaplegic. Four other clusters with the recent deuterostome origin of TGF-b. contain BMP-9 and 10; GDF-5, 6, and 7; GDF-9 Moreover, TbRII has no close human relatives. and BMP-15; and GDF-1 and 3. Nodal falls in The Dan family of antagonists against BMPs the same BMP clade. An activin clade includes and Wnts has seven members in humans and three subgroups: Drosophila activin and human shares some similarity with noggin in its CKGF inhibin b-subunits, which dimerize to form domain (BMP antagonists, Box 1). Although activins, GDF-11, myostatin (GDF-8) and pos- present in many prebilaterians, the Dan family sibly Drosophila Myoglianin, and BMP-3 and was lost in insects. GDF-10. Further family members in the left side of the GF family tree in Box 1 are difficult Did the Prodomain Contribute to the to place, but lie closer to the activin than BMP Evolutionary Success of the TGF-b Family? clade. All have unique features. TGF-bs and anti-Mu¨llerian hormone (AMH) each have Although the origins of the prodomain of TGF- unique type II receptors. AMH and GDF-15 b remain unclear, it may have been an impor- have atypical prodomains that are the longest tant contributor to the success of the TGF-b and shortest in the family, with 434 and 170 family branch of the CKGF superfamily. In evo- residues, respectively. Lefty 1 and 2 are cleaved lutionary terms, success may be defined not by furin after the a2-helix in the prodomain, only as lack of extinction, but also as diversifi- rather than between the prodomain and GF do- cation and radiation into a protein family with a main (Fig. 1), and are thus predicted to have large number of members in higher organisms. their type II receptor binding sites permanently By the criterion of number of members alone, blocked (Shi et al. 2011; Mi et al. 2015). Inhibin- the TGF-b family has been more successful a is inhibitory; when it heterodimerizes with than any of its kindred hormone or GF families inhibin b-subunits it forms inhibin. in the CKGF superfamily (Roch and Sherwood Type Iandtype II receptors for TGF-bfamily 2014). The TGF-b family also radiated more GFs have structurally homologous ligand-bind- than other extracellular protein families of par- ing ectodomains and homologous dual specific- allel importance in developmental specification. itykinasecytoplasmicdomains,andthusmaybe With 33 gene family members in mammals, compared in the same tree (TGF-b receptors, the TGF-b family is more numerous than the Box 1). The longer branches of type II receptors 19 Wnts, which have 10 Frizzled receptors, or show that they are more diverse than the type I the total of five Deltas and Jaggeds, which have receptors; the receptor trees reflect primarily the four Notch receptors. cytoplasmic domain because of its much greater The prodomain, including its arm domain number of aligned positions. The human type I and straitjacket appendages (TGF-b1 homo- receptors show three clades, each with a Droso- dimer, Box 1) is clearly defined in the sequences phila relative. Activin receptor-like kinase (ALK) of prebilaterian TGF-b family members 1 and 2 are closely related to Saxophone (Sax), (Adamska et al. 2007; Roch and Sherwood ALK3 and 6 are related to Thickveins, and ALK4, 2014). The CKGF domains of the TGF-b family 5 and 7 are relatives to Baboon. The ALK1/2, dimerize in a specific manner and associate with ALK3/6, and ALK4–7 subfamilies couple to two prodomain monomers; the prodomain two distinct groups of receptor-regulated Smads arm domain and straitjacket extensions togeth- (R-Smads), with the ALK1/2 and ALK3/6 sub- er wrap around the GF and block receptor and

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Structural Biology and Evolution of the TGF-b Family

Prodomain Dpp_Dr ED I SQRF I AA I APVAAH I PLASASGSGSGRSGSRSVGASTSTALAKAFNPFSEPASFSDSDKSHRSKTNKKPSKSDANRQFNEVHKPRTDQLENS 95 GDF-5 ------APDLGQRPQGTRPGLAKAEAKERPPLARNVFRPGGHSYGGGATNANARAKGGTGQ 55 AMH ------LLGTEALRAEEPAVGTSGLIFREDLDWPPGIPQEPLCLVALGGDSNGSSSPLRVVGALSAY 61 Association α1 Latency lasso α2

TGF-β1 ------LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEV------PP------GPLPEAVLA 50 TGF-β2 ------SLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDY------PE------PEEVPPEVIS 50 TGF-β3 ------SLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPE------PT------VMTHVPYQVLA 51 myosta ------NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLETAPN ISKDVIRQLL------PK------APPLRELID 69 GDF-11 - - - - AEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVCVWRQHSRELRLES I KSQ I LSKLRLKEAPN ISREVVKQLL------PK------APPLQQILD 91 Inh-βA ------SPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHILNMLHLKKRPDVTQPV------PK------AALLNAIRK 69 Inh-βB ------SPTPPPTPAAPPPPPPPGSPGGSQDTCTSCGGFRRPEELGRVDGDFLEAVKRHILSRLQMRGRPN ITHAV------PK------AAMVTALRK 81 Inh-βC ------TPRAGGQCPACGG--PTLELESQRELLLDLAKRSILDKLHLTQRPTLNRPV------SR------AALRTALQH 60 Inh-βE ------QGTGSVCPSCGG--SKLAPQAERALVLELAKQQILDGLHLTSRPRITHPP------PQ------AALTRALRR 59 Inh-α ------CQGLELARELVLAKVRALFLDAL---GPPAVTREGGD------PG------VRR 39 BMP-3 ------ERPKPPFPELRKAVPGDRTAGGGPDSELQ------PQ------DKVSEHMLR 40 GDF-10 ------SHRAPAWSALPAAADGLQGDRDLQRHPGDAAATLG------PS------AQDMVAVHMHR 48 BMP-15 ------MEHRAQMAEGGQSSIALLAEAPTLPLIEEL-LEESPGEQPRK------PR------LLGHSLRYMLE 54 GDF-9 ------SQASGGEAQIAASAELESGAMPWSLLQHIDERDRAGLLPALFKVLSVGRGGSPRLQ------PD------SRALHYMKK 67 GDF-1 ------PVPPGPAAALLQALGLRDEPQGA------PR------LRPVPPVMWR 35 GDF-3 ------QEYVFLQFLGLDKAPS------PQ------KFQPVPYILKK 29 BMP-9 ------KPLQSWGRGSAGGNAHSPLGVPGGGLPEHTFNLKMFLENVKVDFLRSLNLSGVPSQDKTRVE------P------PQYMID 69 BMP-10 ------SPIMNLEQSPLEEDMSLFGDVFSEQDGVDFNTLLQSMKDEFLKTLNLSDIPTQDSAKVD------P------PEYMLE 66 Nodal ------TVATALLRTRGQPSSPS------PL------AYMLS 24 BMP-2 ------LVPELGRRKFAAASSGRPSSQPSDEVLSEFELRLLSMFGLKQRPT------PS------RDAVVPPYMLD 58 BMP-4 ------GASHASLIPETGKKKVAEIQGHAGGRRSGQ-SHELLRDFEATLLQMFGLRRRPQ------PS------KSAVIPDYMRD 66 Dpp_Dr KNKSKQLVNKPNHNKMAVKEQRSHHKKSHHHRSHQPKQASASTESHQSSS I ES I FVEEPTLVLDREVAS I NVPANAKA I I AEQGPSTYSKEAL I KDKLKPD - - - - - PSTLVE I EKSLLS 209 Scw_Dr ------TTYVTTNNHIEMPIYQKRPLSEQMEMIDILDLGDRPRRQAEPN------LHNSASKFLLE 54 Gbb_Dr ------TQSGIYIDNGKDQTIMHRVLSEDDKLDVSYEILEFLGIAERPTHLSSHQLSLR------KSAPKFLLD 62 BMP-5 ------DNHVHSSFIYRRLRNHERREIQREILSILGLPHRPR------PF---SPGKQASSAPLFMLD 53 BMP-7 ------DFSLDNEVHSSFIHRRLRSQERREMQREILSILGLPHRPR------PH---LQGKH-NSAPMFMLD 56 BMP-6 CCGPPPLRPPLPAAAAAAAGGQLLGDGGSPGRTEQPPPSPQSSSGFLYRRLKTQEKREMQKE I LSVLGLPHRPRPLHGLQQPQPPALRQQEEQQQQQQLPRGEPPPGRL - KSAPLFMLD 118 BMP-8 ------GGGPGLRPPPGCPQRRLGARERRDVQREILAVLGLPGRPR------PRAPPAASRLPASAP L FMLD 60 GDF-5 TGGLTQPKKDEPKKLPPRPGGPEPKPGHPPQTRQATARTVTPKGQLPGGKAPPKAGSVPSSFLLKKAREPGPPREPKEPFRPPP I T ------PH ------EYMLS 148 GDF-6 ------FQQASISSSSSSAELGSTKGMRSRKEGKMQRAPRDSDAGREGQEPQPRPQDEPRAQQPRAQEPPGRGPRVV------PH------EYMLS 78 GDF-7 ------RDGLEAAAVLRAAGAGPVRSPGGGGGGGGGGRTLAQAAGAAAVPAAAVPRARAARRAAGSGFRNGSVV--PH------HFMMS 75 GDF-15 ------LSLAEASRASFPG------PS------ELHSEDSRFRE 26 AMH EQAFLGAVQRARWGPRDLATFGVCNTGDRQAALPSLRRLGAWLRDPGGQRLVVLHLEEVTWEPTPSLRFQEPP ------PG------GAGPPELALLV 147 LEFTY1 ------LTGEQLLGSLLRQLQLKEVPTLDRADMEELVI------PT------HVRAQYVAL 43 LEFTY2 ------LTEEQLLGSLLRQLQLSEVPVLDRADMEKLVI------PA------HVRAQYVVL 43 α2 Fastener β1 β1′ β2 α3

TGF-β1 LYNSTRDRVAGESAEPE------PEPEADYYAKEVTRVLMVETHNEIYDKFKQS------THSIYMFFNTSE 110 TGF-β2 IYNSTRDLLQEKASRRA------AAC---ERERSDEEYYAKEVYKIDMPPFFPSENAIPPTFYR------PYFRIVRFDVSA 117 TGF-β3 LYNSTRELLEEMHGERE------EGC---TQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKG------ITSKVFRFNVSS 118 myosta QYDVQRDD-SSDGSLED------DDYHATTETI ITMPTESDFLMQVD------GKPKCCFFKFSS 121 GDF-11 LHDFQGDALQPEDFLEE------DEYHATTETV I SMAQETDPAVQTD ------GSPLCCHFHFSP 144 Inh-βA LHVGKVGENGYVEI------EDDIGRRAEMNELMEQTSEIITFAESG------TARKTLHFEISK 122 Inh-βB LHAGKVREDGRVEI ------PHLDGHASPGADGQERVSEIISFAETDGLA------SSRVRLYFFISN 137 Inh-βC LHGVPQGALLEDNR------EQECEIISFAETGLST------IN QTRLDFHFSS 102 Inh-βE LQPGSVAPGN------GEEVISFATVTDSTS------AYSSLLTFHLST 96 Inh-α LPRRHALGGFTHRGSEP------EEEEDVSQA I LFPATDASCEDKSAARGLAQEA - - - - - E - EGLFRYMFRPSQ 101 BMP-3 LYDRYSTVQAARTPGSL------EGGSQPWRPRLLREGNTVRSFRAAAAETL------ERKGLYIFNL TS 98 GDF-10 LYEKYSRQGARPGG------GNTVRSFRARLEVV------DQKAVYFFNL TS 88 BMP-15 LYRRSADSHGHPRENRT------IGATMVRLVKPLTSVARPHRGTWHIQILG-----FPLRPNRGLYQLVR 114 GDF-9 LYKTYATKEGI PKSNRS------HLYNTVRLFTPCTRHKQAPGDQVTGI------LPSVELLFNLDR 122 GDF-1 LFRRRDPQETRSGSRRT------SPGVTLQPCHVEELGVAGNIVRHIPDRGAPTRASEPASAAGH------CPEWTVVFDLSA 106 GDF-3 IFQDREAAATTGVSRDL------CYVKELGVRGNVLRFLPDQGFFLYPKKISQASS------CLQKLLYFNLSA 91 BMP-9 LYNRYTSDKSTT------PASNIVRSFSMEDAISITATEDFP------FQKHILLFN I S- 116 BMP-10 LYNKFATDRTS------MPSANIIRSFKNEDLFSQPVSFNG------LRKYPLLFNVS- 112 Nodal LYR------DPLPRADIIRSLQAEDVAVD------GQNWT FAFDFSF 59 BMP-2 LYRRHSGQPGSPAPDHR------LERAASRANTVRSFHHEESLEELPETSG------KTTRRFFFNLSS 115 BMP-4 LYRLQSGEEEEEQIHST------GLEYPERPASRANTVRSFHHEEHLEN I PGTSE ------NSAFRFLFNLSS 127 Dpp_Dr LFNMKRPPKIDRSKIIIPEPMKKLYAEIMG------HELDSVNIPKPGLLTKSANTVRSFTHKDSKIDDRFP------HHHRFRLHFDVKS 288 Scw_Dr VYNEISEDQEPKEVLHQRHKR------SLDDDILISNEDRQEIASCNSILTFSSRLKPEQLDN------ELDMHITFNTND 123 Gbb_Dr VYHR I TAEEGLSDQDEDDDYERG------HRSRRSADLEEDEGEQQKNFITDLDKRAIDESDIIMTFLNKRHHNVDELRHEHGRRLWFDVSN 148 BMP-5 LYNAMTN - - - EENPEESEYSVRASLAEETRGARKGYPASPNGYPRR I QLSRTTPLTTQSPPLASLHDTNFLNDADMVMSFVNLVERDKDFSHQR------RHYKEFRFDLTQ 156 BMP-7 LYNAMAV---EEGGGPGGQGFSYPYKAVFS------TQGPPLASLQDSHFLTDADMVMSFVNLVEHDKEFFHPR------YHHREFRFDLSK 133 BMP-6 LYNALSADNDEDGASEGERQQSWPHEAASSSQRRQPPPGAAHPLNRKSLLAPGSGSGGASPLTSAQDSAFLNDADMVMSFVNLVEYDKEFSPRQ------RHHKEFKFNLSQ 224 BMP-8 LYHAMAGDDDEDGAPAE------QRLGRADLVMSFVNMVERDRALGHQE ------PHWKEFRFDLTQ 115 GDF-5 LYRTLSDADRKGGNSSV------KLEAGLANT I TSF I DKGQDDRGPV ------VRKQRYVFD I SA 201 GDF-6 IYRTYSIAEKLGINASF------FQSSKSANTITSFVDRGLDDLSHTP------LRRQKYL FDVSM 132 GDF-7 LYRSLAGRAPAGAAAVS------ASGHGRADT I TGFTDQATQDESAA ------ETGQSFLFDVSS 128 GDF-15 LRKRYEDLLTRLRANQS------WEDSNTDLVPAPAVR I LT ------PEV - - - - - RLGSGGHLHLR I SR 78 AMH LYPGPGPEVTVTRAGLP--GAQSLCPSRD------TRYLVLAVDRPAGAWRGSGLALTLQPRGEDSRLST------ARLQALLFGDDH 221 LEFTY1 LQRSHGDRSRGKRFSQS ------FREVAGRFLALE------ASTHLLVFGMEQ 84 LEFTY2 LRRSHGDRSRGKRFSQS------FREVAGRFLASE------ASTHLLVFGMEQ 84 α3 β3 α3′ β4 β5

TGF-β1 LREAVPEP--VLLSRAELRLLRLK------LKVEQHVELYQKYSNN------SWRYLSNRLLA----PSDSPEWLSFDVTGVVRQWLSRGG--EIE 186 TGF-β2 MEKNA - - - - - SNLVKAEFRVFRLQ ------NPKARVPEQR I ELYQ I LKSKDLT ------SPTQRY I DSKVVK - - - - TRAEGEWLSFDVTDAVHEWLHHKD - - RNL 199 TGF-β3 VEKNR - - - - - TNLFRAEFRVLRVP ------NPSSKRNEQR I ELFQ I LRPDEH I ------AKQRY I GGKNLP - - - - TRGTAEWLSFDVTDTVREWLLRRE - - SNL 199 myosta KIQY------NKVVKAQLWIYL------RPVETPTTVFVQILRLIKPMKDG------TRYTGIRSLKLD-MNPGTGIWQSIDVKTVLQNWLKQPE- -SNL 200 GDF-11 KVMF------TKVLKAQLWVYL------RPVPRPATVYLQILRLKPLTGEGTA------GGGGGGRRHIRIRSLKIE-LHSRSGHWQSIDFKQVLHSWFRQPQ - - SNW 231 Inh-βA EGSDL-----SVVERAEVWLFL------KVPKANRTRTKVT I RLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVD - - - - - ARKSTWHVFPVSSS I QRLLDQGK - - SSL 218 Inh-βB EGNQN-----LFVVQASLWLYL------KLLPYVLEKGSRRKVRVKVYFQEQGH------GDRWNMVEKRVD-----LKRSGWHTFPLTEAIQALFERGE--RRL 218 Inh-βC DRTAGD----REVQQASLMFFV------QLPSNTTWTLKVRVLVLGPHN------TNL T LATQY L LE - - - - - VDASGWHQLP LGPEAQAACSQGH - - L TL 179 Inh-βE PRSH------HLYHARLWLHV------LPTLPGTLCLRIFRWGPRRRRQ------GSRTLLAEHHI------TNLGWHTLTL---PSSGLRGEKS-GVL 167 Inh-α HTRS ------RQVTSAQLWFHT ------GLDRQGTAASNSSEPLLGLLALSPG------GPVAVPMSLG- - - - - HAPPHWAVLHLATSALSLLTHPV - - LVL 178 BMP-3 LTKS------ENI LSATLYFCIGELGN ISLSCPVSGGCSHHAQRKHIQIDLSAWTLK------FSRNQSQLLGHLSVDMAKSHRD I MSWLSKD I TQLLRKAKENEE - - FLI 195 GDF-10 MQDS------EMILTATFHFYS-EPPRWPRALEVLCKPRAKNASGRPLPLGPPTRQHLLFR------SLSQNTATQGLLRGAMAL - -APPPRGLWQAKD I SP I VKAARRDGE - - LLL 188 BMP-15 ATV------VYRHHLQLTR------FNLSCHVEPWVQKNPTN------HFPSSEGDSSKPSLMSNAWKEMDI TQLVQQRFWNNKGHRIL 185 GDF-9 ITTV------EHLLKSVLLYNI------NNSVSFSSAVKCVCNLMIKEPKSSSR------TLGRAPYSFTFNSQFEF-----GKKHKWIQIDVTSLLQPLVASNK--RSI 207 GDF-1 VEPA------ERPSRARLELRFAAA------AAAAPEGGWELSVAQAGQ------GAGADPGPVLLRQLV--P------ALGPPVRAELLGAAWARNASWPRSL 184 GDF-3 IKER------EQLTLAQLGLDL------GPNSYYNLGPELELALFLVQEPHVWGQ------TTPKPGKMFVLRSVPWPQ-----GAVHFNLLDV---AKDWNDNPR--KNF 174 BMP-9 IPRH------EQITRAELRLYVSCQ------NHVDPSHDLKGSVVIYDVLDGTDAW------DSATETKTFLVSQDIQD------EGWETLEVSSAVKRWVRSDSTKSKN 202 BMP-10 IPHH------EEVIMAELRLYTLVQ------RDRMIYDGVDRKITIFEVLESKGDN------EGERNMLVLVSGEIYG-----TNSEWETFDVTDAIRRWQKSGS--STH 197 Nodal LSQQ------EDLAWAELRLQLS ------SPVDLPTEGSLA I E I FHQPKPDTEQ------ASDSCLERFQMDLFTVTLSQVTFSLGSMVLEVTRPLSKWLKR- - - - - PG 145 BMP-2 IPTE------EFITSAELQVFR------EQMQDALGNNSSFHHRINIYEIIKPATAN------SKFPVTRLLDTRLVN-----QNASRWES FDV TPAVMRWTAQGH - - ANH 201 BMP-4 I PEN ------EV I SSAELRLFR ------EQVDQGPDWERGF - HR I N I YEVMKPPAEV ------VPGHL I TRLLDTRLVH - - - - - HNVTRWET FDVSPAV LRWTREKQ - - PNY 213 Dpp_Dr I PAD ------EKLKAAELQLTRDALS - - QQVVASRSSANRTRYQVLVYDITRVGVRG------QREPSYLLLDTKTVR-----LNSTDTVS LDVQPAVDRWLASPQ - - RNY 378 Scw_Dr VPVD ------LSLVQAMLR I YK ------QPSLVDRRANFTVSVYRKLDNRQDFS------YRILGSVNTTS- - - - -SQRGWLEFNL TDT LRYWLHNKGLQRRN 203 Gbb_Dr VPND------NYLVMAELRIYQNAN------EGKWLTANREFTITVYAIGTGTLGQ------HTMEPLSSVNTTG- - - - -DYVGWLELNV TEGLHEWLVKSK - - DNH 230 BMP-5 IPHG------EAVTAAEFRIYK------DRSNNRFENETIKISIYQI IKEYTNR------DADLFLLDTRKAQ- - - - -ALDVGWLVFDITVTSNHWVINPQ- -NNL 237 BMP-7 IPEG------EAVTAAEFRIYK------DYIRERFDNETFRISVYQVLQEHLGR------ESDLFLLDSRTLW- - - - -ASEEGWLVFDITATSNHWVVNPR- -HNL 214 BMP-6 IPEG------EVVTAAEFRIYK------DCVMGSFKNQTFLISIYQVLQEHQHR------DSDLFLLDTRVVW-----ASEEGWLEFDITATSNLWVVTPQ--HNM 305 BMP-8 IPAG------EAVTAAEFRIYK------VPSIHLLNRTLHVSMFQVVQEQSNR------ESDLFFLDLQTLR- - - - -AGDEGWLVLDVTAASDCWLLKRH- -KDL 195 GDF-5 LEKD------G-LLGAELRILRKKPSDTAK-PAAPGGGRAAQLKLSSCPSGR------QPASLLDVRSVPG----LDGSGWEVFDIWKLFRNFKNS------A 280 GDF-6 LSDK------EELVGAELRLFR------QAPSAPWGPPAGPLHVQLFPCLSP------LLLDARTLDPQ--GAPPAGWEVFDVWQGLRHQPWK------205 GDF-7 LNDA------DEVVGAELRVLRRGSPE----SGPGSWTSPPLLLLSTCPGAA------RAPRLLYSRAAEP----LVGQRWEAFDVADAMRRHRREPR--PPR 209 GDF-15 AALPEGLPEASRLHRALFRLS------PTASRSWDVTRP--LRRQLSLARPQAPAL 126 AMH RCFTR------MTPALLLLPR------SEPAPLPAHGQLDTVPFPPPR------PSAELEESPPS------ADPFLETL-TRLVRALRVPPARASAP 293 LEFTY1 RLPPNS- - - - -ELVQAVLRLFQEPVPKAALHRHGRLSPRSARARVTVEWLRVRDDGS------NRTSL I DSRLVS - - - - - VHESGWKAFDVTEAVNFWQQLSRPRQPL 176 LEFTY2 RLPPNS- - - - -ELVQAVLRLFQEPVPKAALHRHGRLSPRSAQARVTVEWLRVRDDGS------NRTSL I DSRLVS - - - - - VHESGWKAFDVTEAVNFWQQLSRPRQPL 176

Figure 1. Sequence alignment of the transforming growth factor b (TGF-b) family. Sequences were aligned with MAFFT (Katoh and Standley 2013), as described (Shi et al. 2011), with slight structural realignment of TGF-b1 and bone morphogenetic protein (BMP)-9 procomplexes (Mi et al. 2015). Structure elements of pro-TGF-b1 (Shi et al. 2011) and pro-BMP-9 (Mi et al. 2015) are labeled and shown as colored lines (upper and lower lines, respectively). (Legend continues on following page.)

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a022103 7 Downloaded from http://cshperspectives.cshlp.org/ at Harvard University Library on October 5, 2016 - Published by Cold Spring Harbor Laboratory Press

A.P. Hinck et al.

β7 β9′ β10 α5 α5 TGF-β1 GFRLSAHCSCDSRD------NTLQVD I NGFT ------TGRRGDLAT I HGMNRPFLLLMATPLERAQ------240 TGF-β2 GFK I SLHCPCCTFVPSNNY I I PNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLE------273 TGF-β3 GLEISIHCPCHTFQP-NGDILENIHEVMEIKFKGVDNED---DHGRGDLGRLKKQKDHHNPHLILMMIPPHRLD------269 myosta GIEIKALDENGHDLAVTFPG------PGEDGLNPFLEVKVTDT------237 GDF-11 GIEINAFDPSGTDLAVTSLG------PGAEGLHPFMELRVLEN------268 Inh-βA DVRIACEQCQESGASLVLLGKKKKKEE------EGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSEDHP------284 Inh-βB NLDVQCDSCQELAVVPVFV------DPGEESHRPFVVVQARLGDSR------258 Inh-βC ELVLEGQVAQSSVIL------GGAAHRPFVAARVRVGGK------212 Inh-βE KLQLDCRPLEGNSTV------TGQPRRLLDTAGHQQPFLELKIRANEPGA------211 Inh-α LLRCPLCTCSA------RPEATPFLVAHTRTRPPSG------208 BMP-3 GFN ITSK------GRQLPKRRLPFPEPYILVYANDAAISEPESVVSSLQGHRNFPTGTVPKWDSHIRAALSIERRKKRSTGVLLP 274 GDF-10 SAQLDSE------ERDPGVPRPSPYAPYILVYANDLAISEPNSVAVTLQRYDPFPA---GDPEPRAAPNNSADPRVRRAAQATGP 264 BMP-15 RLRFMCQQQKD------SGGLELWHGTSSLDIAFLLLYFNDTHKSIRKAK------229 GDF-9 HMS I NFTCMKDQLEHP------SAQNGLFNMTLVSPSL I LYLNDTSAQAYHSW------YSLHYKRRPSQ 265 GDF-1 RLALALR------PRAPAACARLAEASLLLVTLDPRLCHP------218 GDF-3 GLFLEILVKEDRDSGVNFQPEDT------CARL RCSLHASLLVVTLNPDQCH------220 BMP-9 KLEVTVE------SHRKGCDTLD I SVP------PGSRNLPFFVVFSNDHSSG------TKETRL ELREMISHEQESVLKKLSK 267 BMP-10 QLEVHIESKHDEAEDAS------SGRLEIDTS------AQNKHNPLLIVFSDDQSSD------KERKE ELNEMISHEQLPELDNLGL 266 Nodal ALEKQMS------RVAGECWPRPP------TPPATNVLLMLYSNLSQEQRQLG------186 BMP-2 GFVVEVAHLEEKQGVSK----RHVR I SRSLHQDEH------SWSQIRPLLVTFGHDGKGHP------252 BMP-4 GLA I EVTHLHQTRTHQG - - - -QHVR I SRSLPQGSG------NWAQLRPL LVT FGHDGRGHA ------264 Dpp_Dr GLLVEVRTVRSLKPAPHHHVR----LRRSADEAHE------RWQHKQPLLFTYTDDGRHKARSIR------433 Scw_Dr ELR I S IGDSQLSTFAAG------LVTPQA------SRTSLEPFIVGYFNGPELLVKIQKLR------252 Gbb_Dr GIYIGAHAVNRPDREVKLDDIG------LIHRKVDDEFQPFMIGFFRGPELIKATAHS------282 BMP-5 GLQLCAETGDGRSINVKSAGL------VGRQGPQSKQPFMVAFFKASEVLLRSV------285 BMP-7 GLQLSVETLDGQSINPKLAGL------IGRHGPQNKQPFMVAFFKATEVHFRSI------262 BMP-6 GLQLSVVTRDGVHVHPRAAGL------VGRDGPYDKQPFMVAFFKVSEVHVRTT------353 BMP-8 GLRLYVETEDGHSVDPGLAGL------LGQRAPRSQQPFVVTFFRASPSPIRTP------243 GDF-5 QLCLELEAWERGRAV ------DLRGLGF - - - DRAARQVHEKALFLVFGRTKKRDLFFN ------E I KARSGQDDK 340 GDF-6 QLCLELRAAWGELDAGEAEARARGPQQPPPP------DLRSLGF---GRRVRPPQERALLVVFTRSQRKNLFAEMREQL------GSAEAAGPGAGAEGSWPPPSGAPDA 300 GDF-7 AFCLLLRAVAGPVPSPL------ALRRLGFGWPGGGGSAAEERAVLVVSSRTQRKESLFREIRAQ------ARALGAALASEPLPDPGTGTA 289 GDF-15 HLRLSPPPSQSDQLL------AESSSARPQLELHLRPQAARG------162 AMH RLALDPDALAGFPQGLVNLSDPAALERLLDG------EEPLLLLLRPTAATTGDPAPLHDP------TSAPWATALARRVAAELQAAAAELRS 374 LEFTY1 LLQVSVQREHLGPLASGAHKLV------RFASQGAPAGLGEPQLELHTLDLGD------223 LEFTY2 LLQVSVQREHLGPLASGAHKLV------RFASQGAPAGLGEPQLELHTLDLRD------223 Growth factor domain α1 β1 β2 TGF-β1 ------HLQSSRHRR------ALDTNYCFSSTEKNCCVRQLYIDFRKD 276 TGF-β2 ------SQQTNRRKKR------ALDAAYCFRNVQDNCCLRPLYIDFKRD 310 TGF-β3 ------NPGQGGQRKKR------ALDTNYCFRNLEENCCVRPLYIDFRQD 307 myosta ------PKRSRR------DFGLDCDEHSTESRCCRYPLTVDFE-A 269 GDF-11 ------TKRSRR------NLGLDCDEHSSESRCCRYPLTVDFE-A 300 Inh-βA ------HRRRRR------GLECDGKVNICCKKQFFVSFK-D 312 Inh-βB ------HRIRKR------GLECDGRTNLCCRQQFFIDFR-L 286 Inh-βC ------HQIHRR------GIDCQGGSRMCCRQEFFVDFR-E 240 Inh-βE ------GRARRR------TPTCEPATPLCCRRDHYVDFQ-E 239 Inh-α ------GERARR----STPLMSWPWSPSALRLLQRPPEEPAAHANCHRVALN ISFQ-E 255 BMP-3 LQNNELPGAE------YQYKKDEVWEERKPYKTLQAQAPEKSKNKKKQRKGPHRKSQTLQFDEQTLKKARR------KQWIEPRNCARRYLKVDFA-D 359 GDF-10 LQDNELPGLDERPPRAHAQHFHKHQLWP--SPFRALKPRPGRKDRRKKGQEV-FMAASQVLDFDEKTMQKARR------KQWDEPRVCSRRYLKVDFA-D 354 BMP-15 ------FLPRGMEEFMERESLLRRTRQADGISAEVTA ------SSSKHSGPENNQCSLHPFQ I SFR - Q 284 GDF-9 GPDQERSLSA------YPVGEEAAEDGRSSHHRHRRGQETVSSELKKPLGPASFNLSEYFRQFLLPQNECELHDFRLSFS - Q 340 GDF-1 ------LARPRR------DAEPVLGGGPGGACRARRLYVSFR-E 249 GDF-3 ------PSRKRR------AAIPVPKLSCKNLCHRHQLFINFR-D 251 BMP-9 DGSTEAGESS------HEEDTDGHVAAGSTLARRKR------SAGAGSHCQKTSLRVNFE-D 316 BMP-10 DSFSSGPGEE------ALLQMRSNIIYDSTARIRR------NAKGNYCKRTPLYIDFK-E 313 Nodal -GSTLLWEAE------SSWRAQEGQLSWEWGKRHRR------HHLPDRSQLCRKVKFQVDFN-L 236 BMP-2 ------LHKREKR------QAKHKQRKRLKSSCKRHPLYVDFS-D 284 BMP-4 ------LTRRRRAKR------SPKHHSQRARKKNKNCRRHSLYVDFS-D 300 Dpp_Dr ------DVSGGEGGGKGGRNKRQP------RRPTRRKNHDDTCRRHSLYVDFS-D 475 Scw_Dr ------FKRDLEKRRAGGGS------PPPPPPPPVDLYRPPQSCERLNFTVDFK- E 295 Gbb_Dr ------SHHRSKRSASHPRKRKKSVS------PNNVPLLEPMESTRSCQMQTLYIDFK-D 329 BMP-5 ------R-AANKRKNQNRNK ------SSSHQDSSRMSS - VGDYNTSEQKQACKKHELYVSFR- D 334 BMP-7 ------RSTGSKQRSQNRSK ------TPKNQEALRMAN - VAENSSSDQRQACKKHELYVSFR - D 312 BMP-6 ------RSASSRRRQQSRNR------STQSQDVARVSS-ASDYNSSELKTACRKHELYVSFQ - D 403 BMP-8 ------RAVRPLRRRQPK-K------SNELPQANRLPGIFDDVRGSHGRQVCRRHELYVSFQ-D 293 GDF-5 TVYEYLFSQR------RKRRAPLATRQGKRPSKNLK------ARCSRKALHVNFK-D 384 GDF-6 RPWLPSPGRR------RRRTAFASRHGKRHGKKSR------LRCSKKPLHVNFK-E 343 GDF-7 SPRAVIGGRR------RRRTALAG-TRTAQGSGGGAGRGHGRRGR------SRCSRKPLHVDFK-E 341 GDF-15 ------RRRARAR ------NGDHCPLGPGRCCRLHTVRASLE - D 193 AMH LPGLPPATAP------LLARLLALCP------GGPGGLGDPLRALLLLKALQGLRVEWRGRDPRGPGRAQR------SA--GATAADGPCALRELSVDLR-A 455 LEFTY1 ------YGAQGD------CDPEAPMTEGTRCCRQEMYIDLQ-G 253 LEFTY2 ------YGAQGD------CDPEAPMTEGTRCCRQEMYIDLQ-G 253 α2 β3 β4 α3′ α3 β5 β6 β7 β8 TGF-β1 LGW--KWIHEPKGYHANFCLGPCPYIWSLD-----T----QYSKVLALYNQH--N-PGASAAPC--CVPQALEPLPIVYYVG----RKPKVEQLSNMIVRSCKCS------361 TGF-β2 LGW--KWIHEPKGYNANFCAGACPYLWSSD-----T----QHSRVLSLYNTI--N-PEASASPC--CVSQDLEPLTILYYIG----KTPKIEQLSNMIVKSCKCS------395 TGF-β3 LGW--KWVHEPKGYYANFCSGPCPYLRSAD-----T----THSTVLGLYNTL - -N- PEASASPC- -CVPQDLEPLT I LYYVG- - - -RTPKVEQLSNMVVKSCKCS ------392 myosta FGW- -DWI I APKRYKANYCSGECEFVFLQK- - YPHT ------HLVHQA- -N- PRGSAGPC- -CTPTKMSP I NMLYFNGK- - - EQI I YGKI PAMVVDRCGCS ------352 GDF-11 FGW--DWIIAPKRYKANYCSGQCEYMFMQK--YPHT------HLVQQA--N-PRGSAGPC--CTPTKMSPINMLYFNDK---QQIIYGKIPGMVVDRCGCS------383 Inh-βA IGW-NDWIIAPSGYHANYCEGECPSHIAGTSGSSLS----FHSTVINHYRMRGHS-PFANLKSC--CVPTKLRPMSMLYYDDG---QNIIKKDIQNMIVEECGCS------406 Inh-βB IGW-NDWIIAPTGYYGNYCEGSCPAYLAGVPGSASS----FHTAVVNQYRMRGLN-P-GTVNSC--CIPTKLSTMSMLYFDDE---YNIVKRDVPNMIVEECGCA------379 Inh-βC IGW-HDWIIQPEGYAMNFCIGQCPLHIAGMPGIAAS----FHTAVLNLLKANTAA-GTTGGGSC--CVPTARRPLSLLYYDRD---SNIVKTDIPDMVVEACGCS------334 Inh-βE LGW-RDWI LQPEGYQLNYCSGQCPPHLAGSPGI AAS - - - - FHSAVFSLLKAN- -N- PWPASTSC- -CVPTARRPLSLLYLDHN- - -GNVVKTDVPDMVVEACGCS ------331 Inh-α LGW- ERWI VYPPSF I FHYCHGGCGLH I PPN - - LSLP - - - - VPGAPPTPAQPY - - S - LLPGAQPCCAALPGTMRPLHVRTTSDGG- - YSFKYETVPNLLTQHCACI ------348 BMP-3 I GW- SEWI I SPKSFDAYYCSGACQFPMPKS - - LKPS - - - - NHAT I QS I VRAV - -GVVPG I PEPC - - CVPEKMSSLS I LFFDEN - - - KNVVLKVYPNMTVESCACR------450 GDF-10 I GW- NEWI I SPKSFDAYYCAGACEFPMPK I - - VRPS - - - - NHAT I QS I VRAV - -G I I PG I PEPC - - CVPDKMNSLGVLFLDEN - - - RNVVLKVYPNMSVDTCACR------445 BMP-15 LGW- DHWI I APPFYTPNYCKGTCLRVLRDG - - LNSP - - - - NHA I I QNL I NQL - - V - DQSVPRPS - - CVPYKYVP I SVLM I EAN - - -GS I LYKEYEGMI AESCTCR ------374 GDF-9 LKW-DNWIVAPHRYNPRYCKGDCPRAVGHR--YGSP----VHTMVQNIIYEK--L-DSSVPRPS--CVPAKYSPLSVLTIEPD---GSIAYKEYEDMIATKCTCR------430 GDF-1 VGW-HRWVIAPRGFLANYCQGQCALPVALS--GSGGPPALNHAVLRALMHAA--A-PGAADLPC--CVPARLSPISVLFFDNS---DNVVLRQYEDMVVDECGCR------343 GDF-3 LGW- HKWI I APKGFMANYCHGECPFSLT I S - - LNSS----NYAFMQALMHAV--D-P-EIPQAV--CIPTKLSPISMLYQDNN---DNVILRHYEDMVVDECGCG------340 BMP-9 IGW-DSWIIAPKEYEAYECKGGCFFPLADD--VTPT----KHAIVQTLVHLK--F-PTKVGKAC--CVPTKLSPISVLYKDDM--GVPTLKYHYEGMSVAECGCR------407 BMP-10 IGW-DSWIIAPPGYEAYECRGVCNYPLAEH--LTPT----KHAIIQALVHLK--N-SQKASKAC--CVPTKLEPISILYLDKG---VVTYKFKYEGMAVSECGCR------403 Nodal IGW-GSWIIYPKQYNAYRCEGECPNPVGEE--FHPT----NHAYIQSLLKRY--Q-PHRVPSTC--CAPVKTKPLSMLYVDN----GRVLLDHHKDMIVEECGCL------325 BMP-2 VGW-NDWI VAPPGYHAFYCHGECPFPLADH- - LNST- - - -NHAIVQTLVNSV- - - -NSKIPKAC- -CVPTELSAISMLYLDE- - -NEKVVLKNYQDMVVEGCGCR------373 BMP-4 VGW- NDWI VAPPGYQAFYCHGDCPFPLADH - - LNST----NHAIVQTLVNSV----NSS I PKAC - - CVPTELSA I SMLYLDE - - - YDKVVLKNYQEMVVEGCGCR ------389 Dpp_Dr VGW- DDWI VAPLGYDAYYCHGKCPFPLADH - - FNST - - - - NHAVVQTLVNNMN - - - PGKVPKAC - - CVPTQLDSVAMLYLNDQ - - - STVVLKNYQEMTVVGCGCR ------565 Scw_Dr LHM- HNWV I APKKFEAYFCGGGCNFPLGTK - -MNAT----NHAIVQTLMHLK----QPHLPKPC--CVPTVLGAITILRYLN---EDIIDLTKYQKAVAKECGCH------384 Gbb_Dr LGW-HDWI I APEGYGAFYCSGECNFPLNAH- -MNAT----NHAIVQTLVHLLE---PKKVPKPC--CAPTRLGALPVLYHLND---ENVNLKKYRNMIVKSCGCH------419 BMP-5 LGW-QDWI I APEGYAAFYCDGECSFPLNAH- -MNAT----NHAIVQTLVHLM--F-PDHVPKPC--CAPTKLNAISVLYFDD---SSNVILKKYRNMVVRSCGCH------424 BMP-7 LGW-QDWI I APEGYAAYYCEGECAFPLNSY - -MNAT----NHAIVQTLVHFI--N-PETVPKPC--CAPTQLNAISVLYFDD---SSNVILKKYRNMVVRACGCH------402 BMP-6 LGW- QDWI I APKGYAANYCDGECSFPLNAH - - MNAT - - - - NHA I VQTLVHLM- - N - PEYVPKPC - - CAPTKLNA I SVLYFDD - - - NSNV I LKKYRNMVVRACGCH ------493 BMP-8 LGW- LDWVI APQGYSAYYCEGECSFPLDSC - -MNAT----NHAILQSLVHLM--K-PNAVPKAC--CAPTKLSATSVLYYDS---SNNVILRKHRNMVVKACGCH------383 GDF-5 MGW- DDWI I APLEYEAFHCEGLCEFPLRSH - - LEPT - - - - NHAV I QTLMNSM- - D - PESTPPTC - - CVPTRLSP I S I LF I DS - - - ANNVVYKQYEDMVVESCGCR ------474 GDF-6 LGW- DDWI I APLEYEAYHCEGVCDFPLRSH - - LEPT - - - - NHA I I QTLMNSM- - D - PGSTPPSC - - CVPTKLTP I S I LY I DA - - -GNNVVYKQYEDMVVESCGCR ------433 GDF-7 LGW-DDWIIAPLDYEAYHCEGLCDFPLRSH--LEPT----NHAIIQTLLNSM--A-PDAAPASC--CVPARLSPISILYIDA---ANNVVYKQYEDMVVEACGCR------431 GDF-15 LGW-ADWVLSPREVQVTMCIGACPSQFRAA-----N----MHAQIKTSLHRL--K-PDTVPAPC--CVPASYNPMVLIQKTD----TGVSLQTYDDLLAKDCHCI------279 AMH ----ERSVLIPETYQANNCQGVCGWPQSDR-----NPRYGNHVVLLLKMQVR--G-AALARPPC--CVPTAY-AGKLLISLSE---ERISAHHVPNMVATECGCR------542 LEFTY1 MKWAENWVLEPPGFLAYECVGTCRQPPEAL------AFKWPFLGPRQC---IASETDSLPMIVSIKEGGRTRPQVVSLPNMRVQKCSCASDGALVPRRLQP 345 LEFTY2 MKWAKNWVLEPPGFLAYECVGTCQQPPEAL------AFNWPFLGPRQC---IASETASLPMIVSIKEGGRTRPQVVSLPNMRVQKCSCASDGALVPRRLQP 345

Figure 1. (Continued) Potential proprotein convertase (PC) and tolloid cleavage sites are marked with green and red circles, respectively. Potentially N-glycosylated asparagines in all family members and known phosphory- lated serines in mature BMP-15 and growth and differentiation factor 9 (GDF-9) (Tibaldi et al. 2010) are shown in bold red. All 33 human TGF-b family polypeptides are shown, except BMP-8B; the almost identical BMP-8A is shown as BMP-8. Three well-characterized Drosophila melanogaster BMPs are included: Decapentaplegic (Dpp), Screw (Scw), and Glass bottom boat (Gbb). Closely related family members appear in adjacent rows.

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Structural Biology and Evolution of the TGF-b Family

antagonist binding (TGF-b1 homodimer and in part a result of its large and complex pro- GF homodimer, Box 1). domain, which enables complex regulation of The common focus in the TGF-b family biological function during signaling in extra- field on the structures and activities of the ma- cellular environments that can be layered onto ture proteins might suggest that the prodomain cell-surface and intracellular signaling in con- functions primarily in proper folding and dime- trol of agonism and antagonism. The following rization of GF domains during biosynthesis; section highlights the important functional role however, evolution suggests otherwise. Among of the prodomains in the TGF-b family. the six CKGF groups described above, the GPHs, bursicons, and BMP antagonists have no prodomains and many are disulfide-linked STRUCTURES AND FUNCTIONS OF TGF-b dimers. Thus, prodomains are not essential FAMILY PROCOMPLEXES for folding or dimerization of proteins in the Biosynthesis and Latency CKGF superfamily. Moreover, the glial-derived neurotrophic factors (GDNFs), including neur- TGF-b family members are synthesized with turin, artemin, and persephin, have GF domains large, 250 residue amino-terminal prodo- that are very similar in sequence to those of the mains that are required for proper folding and TGF-b family, dimerize through the equivalent dimerization of the smaller, 110 residue car- cysteine, and have similar structures (Wanget al. boxy-terminal GF domains (Fig. 1) (Gentry and 2006). Yet,GDNF prodomains are much shorter Nash 1990; Gray and Mason 1990). During pro- (41–75 residues) than TGF-b family prodo- domain and GF domain folding and disulfide mains (250 residues), and have no detectable bond formation in the endoplasmic reticulum sequence homology (Shi et al. 2011). Two other (ER), TGF-bs 1–3 also become disulfide-linked CKGF families, the nerve growth factor (NGF) to LTBPs. Studies on TGF-b1, activin, and and PDGF families, have prodomains of 100 AMH suggest that the carboxy-terminal GF do- residues that are functionally important and main folds either concomitantly with, or sub- structurally characterized. The PDGF prodo- sequently to, the amino-terminal prodomain main remains bound after cleavage and shields (Gray and Mason 1990; Belville et al. 2004; Wal- the receptor binding site (Shim et al. 2010). In ton et al. 2009). contrast, the NGF prodomain is poorly ordered Processing by PCs (Miyazono et al. 1991) and dissociates from the GF after cleavage; occurs following transit out of the ER. All nonetheless, the prodomain has an important TGF-b family members have one or more PC regulatory role because it alters the stoichiom- cleavage sites (green dots, Fig. 1) (Constam etry and symmetry of NGF binding to its recep- 2014). PCs are a family of secreted, or more tor p75NTR (Feng et al. 2010). commonly membrane-bound, Golgi serine These evolutionary comparisons show that proteases. Seven PCs cleave after a basic residue CKGF domains do not inherently require pro- (Arg or Lys) (Seidah and Prat 2012). These in- domains for biosynthesis or dimerization and clude PC1, PC2, furin, PC4, PC5, PACE4, and that prodomains of even smaller size than those PC7 (Seidah and Prat 2012). Most PC family of TGF-b have important functional roles in the members reside in the Golgi and most TGF-b PDGF and NGF families. Among the six CKGF family members are cleaved between the prodo- protein families (see section on CKGF Domain main and GF domain in the Golgi prior to se- and Its Families), the TGF-b gene family has cretion. However, some PC family members are both the largest prodomain and the largest secreted, and some TGF-b family members, in- number of members in vertebrates (33 in hu- cluding nodal and myostatin, are cleaved extra- mans), whereas bursicons lack prodomains and cellularly (Anderson et al. 2008; Blanchet et al. are extinct in deuterostomes. It is thus tempting 2008; Constam 2014). to propose a prodomain-centric view that the PC cleavage is regulated by many fac- evolutionary success of the TGF-b family is tors, including association with other proteins

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A.P. Hinck et al.

(Blanchet et al. 2008), but most PCs cleave after mains have two b-sheets that partially over- the motif (R/K)-Xn-(R/K), in which Xn is a 0, 2, lap in the hydrophobic core. Long meandering 4, or 6 amino acid spacer, and R (Arg) is highly loops and a substantial a4-helix cover the re- favored over K (Lys) immediately before the mainder of the core (Fig. 2C,D). The b1-strand cleavage site (Seidah and Prat 2012). Often, of the arm domain hydrogen bonds to the b7- multiple PC cleavage motifs are present (Fig. strand in the GF finger to form a super-b-sheet 1) and are used (Israel et al. 1992; Akiyama (Fig. 2E,F). This super-b-sheet knits the pro- et al. 2012; Tilak et al. 2014). domain and GF together. Substantial twisting The tendency of the prodomains and GFs to of the arm b1-strand between the crossed-arm dissociate after secretion varies greatly among and open-arm conformations enables the arm the TGF-b family. TGF-b, GDF-8 (myostatin), domain to reorient by 90˚, while nonetheless and GDF-11 procomplexes are so stable that maintaining equivalent super-b-sheet hydro- their GFs are kept latent by the associating pro- gen bonds in TGF-b and BMP-9 procomplexes domain, whereas the BMP-2 prodomain readily (Fig. 2A,B,E,F). dissociates during GF purification (Hammonds A long loop at the opposite end of the arm et al. 1991; Israel et al. 1992). On the other hand, domain special to pro-TGF-b1, b2, and b3 con- the prodomains and GFs of BMP-7 and BMP-9 tains cysteines that disulfide link the two arm largely remain associated during purification domains together to stabilize the crossed-arm (Brown et al. 2005; Gregory et al. 2005), and procomplex conformation (Fig. 2A). The disul- many BMP prodomains bind their GFs with fide-linked region is termed the bowtie knot; the high affinity (Sengle et al. 2008). region that follows the knot and includes the RGD motif is termed the bowtie tail. The bowtie tail greatly changes in conformation when Structures of Prodomain–GF Complexes bound to integrins (Dong et al. 2014; X Dong, Structures of TGF-b1 (Shi et al. 2011) and BMP- B Zhao, and TA Springer, unpubl.). 9 (Mi et al. 2015) procomplexes, which share Elements amino terminal to the arm do- only 11% prodomain sequence identity, illumi- main surround the GF dimer in the crossed- nate TGF-b family diversity (Fig. 2A,B). The arm conformation of pro-TGF-b and form a prodomain contains an arm domain flanked straitjacket (Fig. 2A,E) (Shi et al. 2011). The by shorter, partially a-helical, amino- and car- a1-helix is buried between the two GF mono- boxy-terminal straitjacket elements (Fig. 2C,D). mers in an extensive interface that includes TGF-b1 and BMP-9 procomplexes reveal over- the amphipathic, carboxy-terminal end of the all crossed-arm and open-arm conformations, a1-helix (Fig. 2A,E). Hydrophobic residues in respectively, with markedly different orienta- three turns of the a1-helix interface with aro- tions between arm domain monomers (Fig. matic residues on one GF monomer. a1-helix 2A,B). In the open-arm conformation of pro- burial helps stabilize the crossed-arm confor- BMP-9, the two arm domains point away from mation with its unique arm domain orientation, one another and are not in contact (Fig. 2B). In and displaces the GF a3-helix from its usual the crossed-arm conformation of pro-TGF-b1, position in the interface between the two mono- the arm domains point toward one another and mers. The latency lasso loosely surrounds the dimerize at the bowtie (Fig. 2A). In contrast to GF finger (Fig. 2A,E). The fastener, a short seg- the arm domain, the segments amino-terminal ment just before the arm domain, binds to the (a1-helix, latency lasso, and fastener) and car- a1-helix and completes GF encirclement (TGF- boxy-terminal (a5-helix) to the arm domain b1 homodimer, Box 1, Fig. 2A,C,E). These ele- interact differently with the GF domain in the ments form a straitjacket that prevents receptor two conformations (Fig. 2A–F). binding (Fig. 2G) and maintains TGF-b latency, Despite differences in orientation, the arm as verified by mutation (Shi et al. 2011). domains of TGF-b1 and BMP-9 show overall In native pro-TGF-b complexes with LTBP similar folds. TGF-b1 and BMP-9 arm do- and the cell-surface leucine-rich repeat protein

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Structural Biology and Evolution of the TGF-b Family

A Pro- Bowtie Pro- B domain 1 domain 2 Pro- Prodomain 1 Prodomain 2 RGD RGD BMP-9 Arm Arm Arm Arm domain domain domain domain Pro- α2 TGF-β1 α 2 α2 α5 α5 α2 Fastener GF 1 GF 2 Finger α5 Growth Growth Strait- factor 2 factor 1 jacket Latency Latency α α lasso lasso 1 1 Association region Cys β linkage G Pro-TGF- 1, receptors

R type I Pro- domain β C TGF- 1 D BMP-9 Arm prodomain prodomain domain GF α Bowtie 2 Latency RGD β4 β9′ α1 lasso β R type II Arm β3 4 β5 domain β5 β2 β2 β7 Arm β7 GF domain β1′ β α 6 α4 β6 4 β3 H Pro-BMP-9, receptors β3 α3 β10 β10 R type I β α2 β1 1 Arm α2 Fastener domain α5 α5 Latency lasso α1 GF Pro- α2 domain α5

R type II GF E Pro-TGF-β1 F Pro-BMP-9 β Pro-BMP-9, inhibitor 10 Pro- Pro- I β1 β10 α domain 1 domain 1 2 β1 Arm α5 α2 domain Finger β7 β7 α5 Fastener α5 β6 β6 Clip GF Pro- Latency domain lasso α3 Growth CV2 factor 2 α α2 Growth 1 factor 2 Growth Growth GF VWC factor 1 factor 1 domain

Figure 2. Procomplexes of transforming growth factor (TGF)-b1 and bone morphogenetic protein 9 (BMP-9) have crossed-arm and open-arm conformations, respectively. Crystal structures are shown in cartoon represen- tation. Some labels are shown in the colorof the corresponding proteins or protein segment. Cysteine side chains are shown as yellow sticks and in some cases with sulfur atom spheres. (A,B) Overall structures of pro-TGF-b1 (Shi et al. 2011) (A), and pro-BMP-9 (Mi et al. 2015) (B) in identical orientations with superimposition on GF dimers. Yellow spheres in A show Cys residues that disulfide-bond to latent TGF-b binding protein (LTBP) or GARP.(C,D) The prodomains in identical orientations after superimposition on the arm domains. Arm domain secondary structural elements common or unique to TGF-b1 and BMP-9 are labeled in black and red, respec- tively. Amino-terminal and carboxy-terminal appendages to the arm domain are labeled and are, in order, the a1-helix, latency lasso, a2-helix, and fastener (all amino-terminal) and the a5-helix (carboxy-terminal). (E,F) Interacting regions of the prodomain and GF shown in identical orientations after superimposition on GF dimers. (G–I) Competition of the prodomain with receptor and inhibitor binding. Complexes are shown in identical orientations after superimposition on GF dimers. For clarity, GFs in receptor and inhibitor complexes are omitted. (G) TGF-b1 procomplex (Shi et al. 2011) superimposed on TGF-b1 complex with ALK5 (R type I) and TbRII (R type II) (Radaev et al. 2010). (H,I) BMP-9 procomplex (Mi et al. 2015) superimposed on (H ) BMP-9 complex with ALK1 (R type I) and ActRIIB (R type II) (Townson et al. 2012) or superimposed on (I) BMP-2 complex with a crossveinless 2(CV2)VWC domain fragment (Zhang et al. 2008).

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A.P. Hinck et al.

32 (LRRC32 or GARP), cysteines in an “associ- (Fig. 1). The a2-helix has a unique role at the ation region” that comprises the most amino- interface between the arm and GF domains. terminal portion of each prodomain (Figs.1 and The a2-helix is amphipathic, and its hydropho- 2A) link to two cysteines in a single LTBP or bic face shields hydrophobic residues in the GARP molecule in a 2:1 complex (Shi et al. GF fingers from solvent. Between the crossed- 2011; Wang et al. 2012). Thus, the prodomains arm and open-arm conformations, the a2-helix are covalently linked together, directly through maintains its position relative to the GF fingers the bowtie knot at one end of the wreath-shaped (Fig. 2A,B,E,F). In contrast, the a2-helix moves procomplex, and indirectly through an inter- 90˚ with respect to the arm domain between mediate protein on the opposite side of the crossed- and open-arm conformations (Fig. procomplex (Fig. 2A). Unpublished pro-TGF- 2C,D). Reorientation of the a2-helix in pro- b crystal structures show that, in the absence of BMP-9 allows it to interact with the arm do- an LTBP or GARP partner, the association re- main in a way not seen in pro-TGF-b1(Mi gion of 10 residues is unstructured or varies et al. 2015). in structure depending on the crystal lattice The a5-helix in pro-BMP-9 is much longer (X Dong, B Zhao, and TA Springer, unpubl.). than in pro-TGF-b1 (Fig. 1), orients differently Proper disulfide bond formation is guided by relative to the arm domain, and binds to a sim- noncovalent interactions surrounding the cys- ilar region of the GF domain as the a1-helix in teines. Because the TGF-b binding-like (TB3) pro-TGF-b1 (Fig. 2A,B,E,F) (Mi et al. 2015). domain in LTBP and the LRRC domain in The BMP-9 prodomain a5 helix inserts into GARP that disulfide-link to pro-TGF-b have the hydrophobic groove formed by the fingers no structural similarity, the association region of one GF monomer and a3-helix of the other must adopt different conformations when GF monomer (Fig. 2F). Thus, instead of dis- linked to these different partners. Furthermore, placing the GF a3-helix as the prodomain a1- because the complex is 2:1, each prodomain helix does in pro-TGF-b1, the a5-helix in pro- monomer must associate with a different cys- BMP-9 binds adjacent to the GF a3-helix (Fig. teine and surrounding region in the binding 2F), enabling a relaxed, mature-like GF confor- partner, resulting in a different structure in the mation in pro-BMP-9. In pro-TGF-b1 the a5- association region of each monomer (shown in helix also binds the GF; it fills a small gap be- Fig. 3A,B). tween the arm domain and a1-helix (Fig. 2E) In the open-arm conformation of pro-BMP- (Shi et al. 2011). 9, the prodomain a1-helix and latency lasso are A remarkable feature of the pro-TGF-b not visualized (Fig. 2B) (Mi et al. 2015). Provoc- dimer is a swap in the GF monomer that each atively, the a1-helix sequence and especially its prodomain monomer embraces. The swap was amphipathic signature are well conserved be- suggested based on the structure of PC-cleaved tween BMP-9 and TGF-b procomplexes and pro-TGF-b1, in which prodomain residues pre- among the majority of TGF-b family members ceding the PC cleavage site are disordered (Shi (Fig. 1). This suggests that pro-BMP-9 may as- et al. 2011). In a structure with the PC site mu- sume an alternative, crossed-arm conforma- tated (B Zhao, X Dong, and TA Springer, un- tion with an ordered a1-helix that resembles publ.), the linkage is also flexible, as expected pro-TGF-b. Models show that such a confor- for a polypeptide that is susceptible to PC cleav- mation is plausible for pro-BMP-9 (schematized age. However, the disordered polypeptide is in Fig. 3D), and movies show a pathway for in- only long enough to span one of the two possi- terconversion between open-arm and crossed- ble prodomain–GF connections (TGF-b1 arm conformations of pro-BMP-9 (see online monomer, Box 1). This establishes that the Movie 1 at cshperspectives.cshlp.org) (Mi et al. most intimate contacts are between pro- and 2015). GF domains of different polypeptide chain The sequence of the prodomain a2-helix is monomers in the precursor in the ER. Thus, conserved in all 33 TGF-b family polypeptides the straitjacket, a2-helix, arm domain, and

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Structural Biology and Evolution of the TGF-b Family

ABLatent TGF-β Latent TGF-β D RG D RG RG D RG D Prodomain Arm Arm Arm Arm domain domain TGF-β domain domain

GF Strait- GF GF jacket

Association S S S S S S region S S LTBP 70 residues ...... TB3 600 residues GARP

Extracellular matrix Plasma membrane

C TGF-β activation by cytoskeletal force transmitted by integrins D RG RG D αvβ6 αvβ6 Active TGF-β Arm Arm R GD GD domain domain Prodomain R Arm Arm domain domain GF

GF Strait- GF jacket Cytoskeletal force

S S S S S S S S

LTBP or GARP LTBP or GARP

D Model for exchange between crossed and open-arm conformations ECM? Pro-BMP + matrix ? (2) Cell α (b) 5 α Pro-BMP 1 Prodomains (c) (a) (1) α1 Pro-BMP

α5 α1 (3) (4) GF α5 +

P P

Type I Type II

Figure 3. Models for growth factor (GF) latency, activation, and binding to receptors. (A,B) Schematic repre- sentation of how anchoring molecules present pro-transforming growth factor b1 (TGF-b1) in the extracellular matrix (ECM) or on cell surfaces, and how the association region at the amino terminus of the prodomain must take on different conformations to bind distinct anchoring molecules and two different sites within each anchor molecule. (C) Schematic representation of pro-TGF-b1 activation (Shi et al. 2011). Integrins, such as avb6, attached to the actin cytoskeleton exert traction force against RGDLXXL/I motifs in pro-TGF-b1 and -b3, which are covalently linked to anchor molecules in the ECM or on the cell surface. The most force-susceptible structural region is the straitjacket and its latency lasso, which will be elongated. Consequently, TGF-b1is released, and shifts conformation to its free form. (D) Models for conformational change between crossed- arm, latent, and open-arm nonlatent conformations of pro-BMPs (Mi et al. 2015). (1) Open-arm, nonlatent conformation; (2) putative crossed-arm conformation of pro-BMPs stabilized by association with ECM com- ponents; (3,4) step-wise binding to type I (3) and type II (4) receptors. Putative pathways for secretion (a,b) and conformational interconversion (c) are marked.

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a5-helix from prodomain monomer 1 sur- electron density in certain crystallization con- round the GF from monomer 2 (Fig. 2A). ditions (Mi et al. 2015). These structural results Prodomain–GF swapping has important together with receptor competition experi- biological implications, because it can provide ments suggest that, among BMPs, type I recep- a mechanism for preferential formation of het- tors may displace the prodomain a5-helix from erodimers over homodimers when a cell syn- the GF, whereas the prodomain a2-helix and thesizes monomers for two different TGF-b arm domain remain bound to the GF and in- family members. In zebrafish embryos, BMP- hibit type II receptor binding (Sengle et al. 2008; 2/7 heterodimers pattern the dorsoventral Mi et al. 2015). axis, whereas the respective BMP-2 and BMP-7 Chordin and chordin-related BMP inhib- homodimers are insufficient (Little and Mullins itors contain one or several von Willebrand 2009). Overexpression of recombinant human factor C (VWC) domains as BMP-inhibiting BMPs also suggests greater activity of BMP-2/5, moieties. Most interestingly, the binding of 2/6, 2/7, and 4/7 heterodimers than the corre- such a domain of the chordin-member cross- sponding BMP homodimers (Israel et al. 1996). veinless 2 to BMP-2 (Zhang et al. 2008) is rem- Complementary interactions between swapped iniscent of prodomain binding to BMP-9 prodomains and GF domains must also be (Fig. 2I). The VWC domain binds to the GF important among inhibin a- and b-subunits, fingers similarly to the a2-helix and arm do- which form both activating activin b homo- main, whereas an amino-terminal appendage dimers and inhibitory inhibin ab heterodimers called clip binds to the same site as the prodo- (Walton et al. 2009). main carboxy-terminal appendage, the a5-helix (Fig. 2I, further described in section on Chordin Family Members). It is, therefore, theoretically Competition between Prodomains, possible that prodomains could help deliver Receptors, and Antagonists for GF Binding GFs to receptors by shielding GFs from inhibi- Prodomains encoded by the 33 TGF-b family tors; a similar mechanism has been shown for genes have important yet poorly understood chordins (Ashe and Levine 1999; De Robertis roles, including extending the range of BMP et al. 2000; Harland 2001). signaling in vivo (Cui et al. 2001; Harrison et al. 2011; Akiyama et al. 2012; Constam 2014). Integrin- and Force-Dependent Release Many prodomains form strong complexes of TGF-b1 from the Prodomain with their GFs (Brown et al. 2005; Sengle et al. 2008), and some of these bind tightly TGF-bs evolved in deuterostomes with a unique enough to compete for binding of the GFs to integrin-dependent mechanism of activation. their cognate receptors (Sengle et al. 2008; Wal- In the crossed-arm conformation of pro-TGF- ton et al. 2009; Mi et al. 2015). Interestingly, the b1, binding to both type I and type II receptors prodomains seem to compete mainly with type is completely blocked (Fig. 2G) (Shi et al. 2011). II receptors but rarely with type I receptors An integrin-binding RGDLXX(L/I) motif in (Sengle et al. 2008; Hauburger et al. 2009; Mi the prodomains of TGF-b1 and 3 locates to et al. 2015). the shoulder region of their arm domains, be- The strong effect of BMP prodomains on tween the b7- and b10-strands of the arm do- type II receptor binding can be explained by main (Fig. 1). Integrins avb6 and avb8 bind the pro-BMP-9 structure (Fig. 2H). The prodo- this motif with 10 nM affinity, which is ex- main arm domain and a2-helix occupy the type tremely high for integrins (Dong et al. 2014). II receptor-binding site. In contrast, only the Integrin binding is not sufficient to release and prodomain a5-helix blocks the type I receptor thus to activate TGF-b (Shi et al. 2011). An- binding site (Fig. 2H). The a5-helix binds choring pro-TGF-b covalently to LTBP in the weakly as suggested by the relatively small sur- extracellular matrix (ECM) or covalently to face on the GF that it buries, and by its weak GARP on cell surfaces is required for activation

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Structural Biology and Evolution of the TGF-b Family

(Munger et al. 1999; Annes et al. 2004; Yoshi- drophobic face of the amphipathic a1-helix naga et al. 2008; Wanget al. 2012). Furthermore, (Walton et al. 2009). Thus, multiple family activation requires the cytoplasmic domain of members besides pro-TGF-b1 may adopt an the integrin b-subunit, which connects to the a1-helix–associated, putative crossed-arm actin cytoskeleton and exerts traction (Munger conformation. et al. 1999). These and other experiments Conformational changes between latent, (Wipff et al. 2007; Wipff and Hinz 2008) suggest crossed-arm conformations and nonlatent, that traction force exerted by integrins on the open-arm conformations may regulate whether RGDLXX(L/I) motif in the arm domain, and procomplexes are stored in the ECM in a latent its resistance on the opposite side of the pro- form or released in a nonlatent form for sig- domain by association region cysteines that link naling (Fig. 3D) (Mi et al. 2015). Because the to LTBP and GARP, is required for activation crossed-arm conformation is tensed with more (Fig. 3C). The results suggest a model in which surface area buried between the GF and prodo- applied traction force distorts the straitjacket main, and with the GF surrounded by a strait- region and results in release of TGF-b (Shi jacket, it may correspond to a latent conforma- et al. 2011). tion. In contrast, the open-arm conformation may correspond to a nonlatent conformation. This idea is consistent with latency of crossed- Conformational Change in Procomplexes arm pro-TGF-b1 (Shi et al. 2011) and nonla- as a Mechanism for Regulating Activity tency of open-arm pro-BMP-7 and -9 (Mi et al. in the TGF-b Family 2015). Although most members of the TGF-b Some members of the TGF-b family may be family are nonlatent when overexpressed as re- able to access both open-arm and crossed-arm combinant proteins (Fig. 3D, pathway a, struc- procomplex conformations (Fig. 3D). BMP-9 ture 1) (Brown et al. 2005; Sengle et al. 2011; Mi may be one of these; its procomplex structure et al. 2015), latency may differ in vivo, in which shows an open-arm conformation (Fig. 3D, cells cosynthesize TGF-b family members with structure 1), whereas strong conservation of its components of the ECM (Fig. 3D, pathway b). a1-helix sequence suggests the presence of a Prodomains can bind to ECM components, distinct, putative crossed-arm conformation including heparin, proteoglycans, LTBP, and (Fig. 3D, structure 2) (Mi et al. 2015). Modeling fibrillin (Gregory et al. 2005; Anderson et al. of a crossed-arm BMP-9 procomplex shows that 2008; Sengle et al. 2008, 2011; Li et al. 2010); the prodomain a1-helix and GF can associate in one or more of these may bind to procomplexes, a crossed-arm conformation that recapitulates and thereby stabilize the crossed-arm con- the hydrophobic interface in pro-TGF-b1 (see formation (Fig. 3D, structure 2) and enable online Movie 1 at cshperspectives.cshlp.org) the GF domain, which is very short-lived in (Mi et al. 2015). BMP-9 regulates vascular de- vivo, to reach storage concentrations as high velopment. Mutations in its prodomain can as 100 ng/g in demineralized bone (Pietrzak cause syndromes resembling hereditary hemor- et al. 2006). The short lifetimes of free BMPs, rhagic telangiectasia (HHT) caused by muta- together with significant storage depots of tions in receptors for BMP-9 (David et al. BMPs in bone, and colocalization of GFs with 2009; Castonguay et al. 2011; Wooderchak- prodomains (Sampath and Reddi 1981, 1984; Donahue et al. 2013). The location of mutations Gregory et al. 2005; Pietrzak et al. 2006), suggest associated with an HHT-like condition in the latency in vivo. Furthermore, purification of BMP-9 prodomain a1-helix and in the sub- BMPs showed a 60-fold increase in total activity sequent loop supports the hypothesis of a in the first two steps, consistent with possible crossed-arm conformation. A similar embrace purification away from an inhibitor such as the between the prodomain a1-helix and mature prodomain (Luyten et al. 1989). Release from domains in inhibin a and bA is shown by storage depots in vivo may be associated with mapping of disruptive mutations to the hy- conformational change to the open-arm con-

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formation, which is ready for receptor or inhib- third of the family contains substantial se- itor binding (Fig. 3D, pathway c). quence carboxy-terminal to the arm domain Among different family members, differ- (Fig. 1) that might fulfill a function similar to ences in structural elements, including those the a5-helix in competing with the a1-helix corresponding to the straitjacket and fastener, for GF binding. may regulate whether crossed-arm or open- Prodomain sequences corresponding to the arm conformations predominate. The prodo- association region in TGF-b1 reveal interesting main a1 and a5-helices are likely to compete peculiarities (Fig. 1). An abundance of Arg res- for overlapping binding sites on the GF in idues in BMP-5, 6, 7, and 8 suggests possible pro-BMP-9 (Mi et al. 2015). Therefore, the binding to heparan sulfate proteoglycans. relative strengths of a1- and a5-helix bind- Many members have long, hydrophilic associa- ing among different TGF-b family mem- tion regions that are likely unfolded, unless they bers may regulate which conformation is pre- associate with another macromolecule. Unusu- ferred (Fig. 3D, pathway c; see online Movie 1 at al features include continuous repeats of 7 to 11 cshperspectives.cshlp.org). In a single family mem- Ala residues (GDF-11 and BMP-6), 7 Pro resi- ber, binding to ECM components, and proteo- dues (inhibin bB) or 10 Gly residues (GDF-7). lytic removal by PC or tolloid proteases of In similar positions to the LTBPor GARP-linked helical segments amino- or carboxy-terminal Cys of TGF-b1, one or two Cys are present in the to the arm domain may regulate which state association regions of myostatin, GDF-11, in- predominates. Many family members contain hibin-bA, bB, bC, and bE, inhibin-a, BMP-6, known or putative PC or tolloid/BMP protease and BMP-8. These unexplained features high- cleavage sites in these segments (Fig. 1). The light how much remains to be learned about the open-arm conformation of BMP-9 can readily prodomains of the TGF-b family, both as re- bind to type I receptors, with displacement of combinant proteins, and in their native context the a5-helix (Fig. 3D, structure 3) (Mi et al. in tissues in which our understanding of their 2015). The final step in signaling could then binding partners remains incomplete. The GF be complete prodomain dissociation followed domains in the family also show great variation by binding to type II receptors (Fig. 3D, struc- in sequence and structure and can bind to ECM ture 4). components as well as to receptors, as described in the next section.

Implications of Prodomain Diversity for Structure and Function of the TGF-b SPECIFICITY AND PROMISCUITY Family IN GF-RECEPTOR INTERACTIONS What can we deduce from sequence (Shi et al. GF Structures 2011) and structural alignment (Mi et al. 2015) of all 33 human TGF-b family polypeptides Three-dimensional structures have been re- (Fig. 1)? The minimum TGF-b family prodo- ported for more than ten different TGF-b fam- main, exemplified by the short (169 residues) ily GFs (Table 1, Fig. 4). The GFs consist of two prodomain of GDF-15, contains an a2-helix extended monomers joined together in most, and an arm domain core containing the b1, but not all cases, by a single interchain disulfide b2, b3, b6, b7, and b10-strands and the a4- bond. The monomers adopt the shape of a helix (Figs. 1 and 2C,D). The b4 and b5- curled left hand, with the heel of one hand pack- strands on the edge of the arm domain b-sand- ing into the palm of the other (GF homodimer, wich closest to the arm–arm interface in pro- Box 1). The heel is formed by a 3-4 turn a-helix TGF-b1 are dispensable, as they are absent in (GF monomer, Box 1). The fingers are formed GDF-15 (Fig. 1). All members except GDF-6, by two loops that extend from the CK and adopt GDF-15, and possibly AMH contain an a1-he- a b-strand conformation in most family mem- lix with an amphipathic signature (Fig. 1). A bers (GF monomer, Box 1). Finger nomencla-

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ture differs in the family. For TGF-bseach or by exchange between preformed homo- b-strand is counted as one finger, and thus dimers (Liao et al. 2003; McNatty et al. 2005). TGF-bs are described as having four fingers; Their heterodimers have more pronounced bi- whereas for BMP, GDF, and activin GFs, and ological activities than the homodimers (Mot- DAN family antagonists (see section on The tershead et al. 2013, 2015; Peng et al. 2013). Dan Family), each two-stranded b-ribbon is Curiously, some GFs including BMP-9 contain counted as one finger, and thus these CKGF the cysteine for the interchain disulfide, yet their domains are described as having two fingers. homodimers fail to be quantitatively disulfide- The thumb, when present, is formed by a short linked (Wei et al. 2014). amino-terminal a-helix. TGF-bs, AMH and ac- GF homodimers within the same subfamily tivins have a fourth disulfide that tethers the have similar backbone conformations, as illus- one-turn amino-terminal thumb a-helix to trated with TGF-b2 overlayed on TGF-b3, or the amino-terminal end of b-strand 1. This di- BMP-2 overlayed on BMP-9 (Fig. 4B). GFs be- sulfide causes the amino-terminal a-helix to longing to different subfamilies also share sim- extend away from the fingers to form a pro- ilarity, especially in the region of the CK and the nounced thumb (Fig. 4A, TGF-b3). The thumb fingers extending to the knuckle, but tend to region of BMP-2 has basic, heparin-binding res- have significant differences in the heel a3-helix, idues and is disordered in crystal structures the “prehelix extension,” and the fingertips (Scheufler et al. 1999); GDF-5 lacks sequence (Fig. 4B, overlay of TGF-b3 and BMP-2). Such for the thumb (Figs. 1 and 4A). differences correlate with sequence differences; TGF-b family proteins can be expressed as for example, in the prehelix extension before both homodimers and heterodimers. When a the heel a3-helix, inhibin-b s are five residues cell synthesizes two polypeptide subunits that longer, and BMPs and GDFs are three residues then heterodimerize, prodomain-GF swapping longer than TGF-bs (Fig. 1). Furthermore, the (see section on Structures of Prodomain–GF structural differences in the heel, prehelix ex- Complexes) provides a mechanism for facilitat- tension, and fingertips are functionally impor- ing heterodimer formation (Israel et al. 1996). tant, because these regions comprise the prima- Inhibin subunits are interesting because dime- ry binding sites, not only for the ectodomains rization of their b-subunits generates activins, of the signaling receptors, but also for prodo- whereas b-subunit heterodimerization with the mains and modulator proteins, which have dis- inhibin a-subunit forms inhibins. Recombi- tinct preferences for specific subclasses of GFs, nant BMP heterodimers, including BMP-4/7 or, in some cases, even for single GFs. (Aono et al. 1995; Suzuki et al. 1997; Nishi- Structural studies have shown that not all matsu and Thomsen 1998) and BMP-2/7 (Buijs family members are stable as “closed” dimers et al. 2012; Zheng et al. 2012), are more active (Fig. 4A,B) in which the heel region of one than their homodimers both in cultured cells monomer extensively packs onto the palm re- and in animals (Aono et al. 1995; Israel et al. gion of the opposing monomer. NMR shows 1996; Suzuki et al. 1997; Nishimatsu and Thom- that TGF-b3 is very dynamic (“open”), with sen 1998; Zhu et al. 2006; Buijs et al. 2012; little order in the regions including the heel Zheng et al. 2012). However, we know little a3-helix that are important in the dimer inter- about the relative abundance of BMP homo- face (Bocharov et al. 2000, 2002; Huang et al. dimers and heterodimers in vivo, and structures 2014). Although extensive NMR data have for biologically relevant TGF-b family hetero- been collected on both TGF-b1 and TGF-b3, dimers are currently lacking. an NMR model of the dimer could only be The GF domains of GDF-3, GDF-9, and constructed for TGF-b1 (Hinck et al. 1996). BMP-15 lack the cysteine residue that forms In agreement, the TGF-b1 solution structure the interchain disulfide, but they can still form is closed, whereas TGF-b3 can crystallize in an stable homodimers. BMP-15 and GDF-9 can open conformation, with little contact between form heterodimers either during biosynthesis monomers except near the interchain disulfide

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Table 1. Structures that illuminate transforming growth factor b (TGF-b) family signaling NMR or X-ray Molecules (resolution, A˚ ) PDB entry References Growth factors TGF-b1 NMR 1KLC Hinck et al. 1996 TGF-b2 X-ray (2.0, 1.95) 2TGI, 1TFG Daopin et al. 1992; Schlunegger and Grutter 1992; Daopin et al. 1993 TGF-b3 X-ray (3.3, 2.0) 1TGK, 1TGJ Mittl et al. 1996 BMP-2 X-ray (2.7, 2.65) 3BMP, 1REU Scheufler et al. 1999; Keller et al. 2004 BMP-3 X-ray (2.21) 2QCQ Allendorph et al. 2007 BMP-6 X-ray (2.49, 2.50, 2.1) 2QCW, 2R52, Allendorph et al. 2007; Saremba et al. 2R53 2008 BMP-7 X-ray (2.8, 2.0) 1BMP, 1LXI Griffith et al. 1996; Greenwald et al. 2003 BMP-9 X-ray (2.33) 1ZKZ, 4MPL Brown et al. 2005; Wei et al. 2014 Activin A X-ray (2.0) 2ARV Harrington et al. 2006 GDF-5 X-ray (2.28, 2.40) 1WAQ, 2BH5 Nickel et al. 2005; Schreuder et al. 2005 Nodal/BMP-2 chimera X-ray (1.91) 4N1D Esquivies et al. 2014 Activin A/BMP-2 X-ray (2.14) 4MID Yoon et al. 2014 chimera Procomplexes Pro-TGF-b1 X-ray (3.05 A˚ ) 3RJR Shi et al. 2011 Pro-BMP-9 X-ray (3.3 A˚ , 3.25 A˚ ) 4YCG, 4YCI Mi et al. 2015 Receptors ActRII X-ray (1.5) 1BTE Greenwald et al. 1999 TbRII NMR, X-ray (1.05) 1PLO, 1KS6, Boesen et al. 2002; Deep et al. 2003; 1M9Z Marlow et al. 2003 BMPRII X-ray (1.2, 1.45) 2HLR, 2HLQ Mace et al. 2006 ALK1 NMR 2LCR Mahlawat et al. 2012 ALK3 NMR, X-ray (2.7) 3K3G, 3NH7 Klages et al. 2008; Harth et al. 2010 ALK5 NMR 2L5S Zuniga et al. 2011 Antagonists Sclerostin NMR 2K8P, 2KD3 Veverka et al. 2009; Weidauer et al. 2009 PRDC X-ray (2.25) 4JPH Nolan et al. 2013 Nbl1/Dan X-ray (2.5, 1.8) 4X1J, 4YU8a Nolan et al. 2013 CV2 fragment NMR 2MBK Fiebig et al. 2013 Coreceptors Betaglycan fragment X-ray (2.0, 2.7) 3QW9, 4AJV Lin et al. 2011; Diestel et al. 2013 Cripto fragment NMR 2J5H Calvanese et al. 2006 Complexes BMP-2:ALK3 X-ray (2.90, 1.86) 1ES7, 1REW Kirsch et al. 2000; Keller et al. 2004 BMP-2:ALK3/ALK6 X-ray (2.5, 2.4, 2.6) 2QJB, 2QJ9, Kotzsch et al. 2008 chimera 2QJA TGF-b3:TbRII X-ray (2.15) 1KTZ Hart et al. 2002 Activin A:ActRIIB X-ray (3.1, 2.3) 1NYU, 1S4Y Thompson et al. 2003; Greenwald et al. 2004 BMP-7:ActRII X-ray (3.3) 1LX5 Greenwald et al. 2003 GDF-5:ALK6 X-ray (2.1) 3EVS Kotzsch et al. 2009 GDF-5:ALK3 X-ray (2.28) 3QB4 Klammert et al. 2015 BMP-2:ActRII:ALK3 X-ray (2.2) 2GOO Allendorph et al. 2006 Continued

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Structural Biology and Evolution of the TGF-b Family

Table 1. Continued NMR or X-ray Molecules (resolution, A˚ ) PDB entry References BMP-2:ActRIIB:ALK3 X-ray (1.85, 1.92) 2H62, 2H64 Weber et al. 2007 TGF-b3:TbRII:ALK5 X-ray (3.0) 2PJY Groppe et al. 2008 TGF-b3:Antibody Fab X-ray (3.1) 3EO1 Gru¨tter et al. 2008 TGF-b1:TbRII:ALK5 X-ray (3.0) 3KFD Radaev et al. 2010 BMP-9:ActRIIB:ALK1 X-ray (3.36) 4FAO Townson et al. 2012 BMP-2:CV2 fragment X-ray (2.7 A˚ ) 3BK3 Zhang et al. 2008 BMP-7:Noggin X-ray (2.42) 1M4U Groppe et al. 2002 GDF-8:Follistatin 288 X-ray (2.15 A˚ ) 3HH2 Cash et al. 2009 Activin A:Follistatin 288 X-ray (2.8 A˚ ) 2B0U Thompson et al. 2005 Activin A:Follistatin- X-ray (2.48 A˚ ) 3B4V Stamler et al. 2008 like 3 Activin A:Follistatin Fs12 X-ray (2.0 A˚ ) 2ARP Harrington et al. 2006 Activin A:Follistatin 315 X-ray (3.40) 2P6A Lerch et al. 2007 GDF-8:Follistatin-like 3 X-ray (2.4 A˚ ) 3SEK Cash et al. 2012 RGMa:BMP-2 X-ray (3.2) 4UHY Healey et al. 2015 RGMb:BMP-2 X-ray (2.85, 2.8) 4UHZ, 4UIO Healey et al. 2015 RGMc:BMP-2 X-ray (2.35) 4UI1 Healey et al. 2015 RGMb:BMP-2:Neogenin X-ray (3.15) 4UI2 Healey et al. 2015 RGMb:Neogenin X-ray (2.3, 6.6, 2.8) 4BQ6, 4BQ7, Bell et al. 2013 4BQ8 Norrin:Frizzled4 X-ray (3.0) 5BQC Chang et al. 2015 NMR, Nuclear magnetic resonance; BMP, bone morhphogenetic factor; GDF, growth and differentiation factor. aR.J. Owens, Oxford Protein Production Facility.

bond (Fig. 4C) (Hart et al. 2002). However, Open forms may represent the preferred TGF-b3 also crystallizes in the closed confor- conformation for several family TGF-b family mation (Mittl et al. 1996). Similarly, activin A proteins, and might impart them with their has been captured in alternative conformations distinctive activities, for example by altering that show large variation and asymmetry in the kinetics and/or affinities of binding to the monomer–monomer orientation (Fig. 4D) signaling receptors or by exposing residues that (Thompson et al. 2003; Greenwald et al. allow recognition by a secreted antagonist. The 2004). For both TGF-b3 and activin A, the latter was suggested by a study of TGF-b chime- less ordered, “open” conformation shows disor- ras, in which cultured dermal fibroblasts mi- der at the dimerization-proximal end of the grate in response to TGF-b3 or a TGF-b3-b1- monomer. The other end of the monomer, b3 chimera that adopted the open form, but with its b-sheets and CK is more stable, and not in response to TGF-b1 or a TGF-b1-b3- makes the overall shapes of closed and open b1 chimera that adopted the closed form monomers similar (Fig. 4C,D). TGF-b3in (Huang et al. 2014). complex with type I and type II receptors shows a closed structure, in agreement with binding of type I receptors near the monomer–mono- Structures of Type I and Type II Receptor mer interface, requiring ordering of this region Ectodomains (Groppe et al. 2008). Similarly, when activin TGF-b family GF dimers initiate signaling by A is surrounded by two follistatin monomers, binding to type I and type II receptors. The it assumes a symmetric, closed conformation receptor ectodomains of 100 residues are cys- (see section on follistatin) (Thompson et al. teine-rich. Homology in sequence and structure 2005; Harrington et al. 2006). is detectable between type I and type II recep-

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A Closed GF homodimers B Closed GF homodimer overlays β β β β TGF- 3/TGF- 2 TGF- 3/TGF- 2 BMP-2/BMP-9 BMP-2/BMP-9 http://cshperspectives.cshlp.org/ ..Hnke al. et Hinck A.P. 20 KnuckleKnuckl KnuckleKnuck TGF-β3 BMPBMP-22 KnuckleKnuckle Knuckle

FingersFing Fingers FingersFingers Fourth disulfide Prehelix FingersFingers Prehelix Prehelix αN Cystine extension Thumb extensionextension extension

dacdOln ril.Ct hsatceas article this Cite Article. Online Advanced Prehelix knot Thumb extension Knuckle TGF-β3/BMP-2 TGF-β3/BMP-2 GDF-5 KnuckleKnuckle atHarvardUniversityLibraryonOctober5,2016-PublishedbyColdSpring Fingers Harbor LaboratoryPress FingersFing Prehelix PrehelixPrehelix extension extensionti

C Open TGF-β3 homodimer Closed and open TGF-β3 monomers D Open ActA homodimer Closed and open ActA monomers N Monomer 2 Monomer 2 Monomer 1 Monomer 1 Fingers Heel Fingers Heel Helix Helix Fingers odSrn abPrpc Biol Perspect Harb Spring Cold Knuckle KnKnuckleuckle Cystine knot CyCystinestine knotkno Knuckle Thumb N ThumbThumb N N ActA homodimers N N TGF-β3 homodimers closed and open closed and open N Open Open Open Open monomer 1 monomer 2 monomer 1 monomer 2 Closed Closed monomer 1 monomer 2 o:10.1101 doi: Closed Closed monomer 1 monomer 2 N N N / cshperspect.a022103 Figure 4. Structures of transforming growth factor b (TGF-b) family growth factors (GFs). All structures are of disulfide- linked GF homodimers. Ribbon diagrams are color-coded by monomer (with names in black) or color-coded by monomer or GF (with corresponding color-coded names). Cysteine side chains are shown as yellow sticks. (A) Closed GF structures. (B) Superposition of closed GFs. (C) Comparison of open (1KTZ) and closed (1TGK) TGF-b3 dimers and monomers from homodimer crystal structures. (D) Comparison of open (1NYU) and closed (2B0U) activin A dimers and monomers from homodimer crystal structures. Structures are of TGF-b2 (Daopin et al. 1992), TGF-b3 (Mittl et al. 1996; Hart et al. 2002), growth and differentiation factor 5 (GDF-5) (Nickel et al. 2005), bone morphogenetic protein 2 (BMP-2) (Scheufler et al. 1999), BMP-9 (Brown et al. 2005), and activin A (Thompson et al. 2003). Downloaded from http://cshperspectives.cshlp.org/ at Harvard University Library on October 5, 2016 - Published by Cold Spring Harbor Laboratory Press

Structural Biology and Evolution of the TGF-b Family

tors in both their ectodomains and kinase ternative interface is promoted by two impor- domains, and thus they arose from a common tant structural differences in TbRII compared ancestor (Box 1, Fig. 5). Four of the seven mam- with ActRII, ActRIIB, and BMPRII. First, the malian type I receptors and four of the five b4-b5 loop is extended by seven to eight resi- mammalian type II receptors are structurally dues in TbRII relative to other type II receptors characterized (Table 1, Fig. 6). Because the and lacks the f disulfide (Figs. 5 and 6H). The receptor ectodomain fold has three fingers absence of this disulfide allows the extended b4- (RI and RII, Box 1) and the superfamily includes b5 loop to fold onto the concave surface of neuro- and cardiotoxins, it is sometimes termed the b4-b3-b5 sheet, thus blocking GF binding the three-finger toxin fold (Greenwald et al. through this interface. Second, the b1-b2 loop 1999). The fold comprises a central three-strand- in TbRII is extended by more than 10 residues ed b4-b3-b5 antiparallel b-sheet flanked on its compared with all other type I and type II re- convex surface by a smaller two-stranded b1-b2 ceptors of the family, except AMHRII (Fig. 5). antiparallel b-sheet, and along the edge of b- The b1-b2 loop is further stabilized by both the strand 5 by an extended loop of variable struc- e disulfide at its base, as in type I receptors, and ture that connects b-strands 4 and 5 (Fig. 6). the TbRII-specific g disulfide near its tip (Figs. 5 Type I and type II receptors all share a com- and 6H). The extended loop wraps onto the mon set of four disulfide bonds termed a, b, convex surface of the b4-b3-b5 sheet and packs c, and d (Figs. 5 and 6). The e–g disulfides, tightly against it. This unusual structural feature which are present in only some receptors is likely important for proper positioning and (Figs. 5 and 6), alter structures to enable distinc- bracing of the short b2-strand, which forms the tive GF binding modalities. Thus, the f disul- primary structural element used by TbRII to fide, which is present in ActRII, ActRIIB, and bind the three TGF-b isoforms. BMPRII (Figs. 5 and 6E–G), braces the amino- The AMHRII type II receptor is unusual in terminal portion of the b4-b5 loop so that it that it includes only one of the two cysteines that points away from the convex surface of the b4- form the f disulfide found in other family type b3-b5 sheet. This is important for the function II receptors, including ActRII, ActRIIB, and of ActRII (Greenwald et al. 2003; Allendorph BMPRII (Fig. 5). However, this cysteine is pre- et al. 2006) and ActRIIB (Thompson et al. dicted to form a disulfide unique to AMHRII 2003; Weber et al. 2007), and likely BMPRII as with another cysteine that immediately pre- well (Yin et al. 2008), as these receptors use the cedes the carboxy-terminal cysteine of the con- exposed face of the b4-b3-b5 sheet, along with served a disulfide (Fig. 5). This putative h disul- the b4-b5 loop, to bind their cognate GFs. The e fide is predicted by the structure and sequence- disulfide is present in all type I receptors, as well based alignment in Figure 5, because aligned as TbRII (Figs. 5 and 6A–D,H). The e disulfide residues in homologous type II receptors have in type I receptors fulfills an analogous role as Ca atom separations and side chain orienta- the f disulfide in type II receptors, as it precludes tions consistent with disulfide bond formation. the b1-b2 loop from occluding the convex sur- face of the b4-b3-b5 sheet, which along with Structures of GF Complexes with Receptors the critical b4-b 5 loop, binds cognate GFs (Kirsch et al. 2000). TGF-b family GF dimers greatly outnumber the The TGF-b type II receptor, TbRII, is type I and type II receptors, and thus, multiple unique among the type I and type II receptors GFs must share the same type I or II receptors, of the family in that it binds its cognate GFs or in some cases, share both type I and type II through a distinct interface, namely, an edge receptors. Nonetheless, sequence and structure b-strand on the smaller b1-b2-sheet, the b2- provide a basis for understanding this sharing; strand, and the flanking b1 and b10 strands particular subfamilies of type I and type II re- (Figs. 6H and 7F) (Hart et al. 2002; Groppe ceptors (TGF-b receptors, Box 1) preferentially et al. 2008; Radaev et al. 2010). Use of this al- bind and transduce signals for particular sub-

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a e b c d β1 β2 β3 β4 β5 atHarvardUniversityLibraryonOctober5,2016-PublishedbyColdSpring

ALK1 4FAO 10 LVTCTCESPHC ------K ------GPTCRG------AWCTVVLVREEGRHPQE-HRGCGNL-H------REL------CRGR----P--T-E---FVNHYCCD-SHLCNHNVSL 80 Harbor LaboratoryPress ALK2 12 LYMCVCEGLSC------G-----NEDHCEG------QQCFSSLSINDGFHVY--QKGCFQV-Y-EQG--KMT------CKTP----P--S----PGQAVECCQ-GDWCNRNITA85 ALK3 2H62 35 FLKCYCSG-HC- - -PD- - - - -DA- - - - - INNTCITN------GHCFAI IEEDDQGETTL-ASGCMKY- - - -EG- -SDFQ- - - - -CK- -D- - -SP- -KA-QLRRTIECCR-TNLCNQYLQP 113 ALK6 3EVS 16 I LRCKCHH - HC - - - PE - - - - - DS - - - - - VNNICSTD------GYCFTMIEEDDSGMPVV-TSGCLGL----EG--SDFQ-----CR------D-TP-IPHQRRSIECCTERNECNKDLHP 95 ALK4 8 ALLCACTS--C------L---Q-ANYTCETD------GACMVSIFNLD-GMEHH-VRTCIPKVELV---PAGKP---FYCL------S-SE----DLRNTHCCY-TDYCNRIDLR84 ALK5 3KFD 9 ALQCFCHL--C------T----KDNFTCVTD------GLCFVSVTETT-DKVIH-NSMCIAEIDLI---PRDRP---FVCA------P-SS-KTGSVTTTYCCN-QDHCNKIELP 88 ALK7 5 GLKCVCLL--C------D----SSNFTCQTE------GACWASVMLTN-GKEQV-IKSCVSLPEL ------NAQ- - - VFCH------S - SN- - - -NVTKTECCF - TDFCNNI TLH 78 Align. RMSD 3100001237*557-66- - - -45- - - -7-5111112305103100000124- - - -642101010123------446610010**------5525---3521110000-1001112233 TBRII 1KTZ 25 PQLCKFCDVRF- - - -STCDNQKSCMSNC-SITSICEK-P- -QEVCVAVWRKND-ENITL-ETVCHDPKLPYHDFILEDAA-SPKCIMK- - - -EK-K-KPGETFFMCSCS-SDECNDNI IF 126 BMPRII 2HLQ 4 ERLCAFKDPYQQDL - G I GESR I S - - - - HENGT I LCSK - - - - GSTCYGLWEKSKG - D I NLVKQGCWSH - - - - I GDPQE - CH - YEECVVTTTPPS I - Q - N - - GTYRFCCCS - TDLCNVNFTE 112 ActRII 2GOO 8 TQECLFFNANWERD-RT-----N----Q-TGVEPCYGDKDKRRHCFATWKNISG-SIEIVKQGCWLD------DINCYDRTDCVEK----KD-S-P---EVYFCCCE -GNMCNEKFSY 97 ActRIIB 2H62 7 TREC I YYNANWELE - RT - - - - - N - - - - Q - SGLERCEGEQDKRLHCYASWRNSSG - T I ELVKKGCWLD ------DFNCYDRQECVAT - - - - EE - N - P - - - QVYFCCCE - GNFCNERFTH 96

odSrn abPrpc Biol Perspect Harb Spring Cold AMHRII 4 RRTCVFFEAPGVRGSTKTLGELLDTGTELPRAI RC- - - - LYSRCCFGIWNLTQD-RAQVEMQGCRDSD------EPGCE - SLHCDPS - - - - PRAHPSPGSTLFTCSCG- TDFCNANYSH 105 g h c e b f

Figure 5. Alignment of type I and type II receptor ectodomains by structure and sequence. Type I and type II receptors with structures (labeled with PDB codes) were aligned to one another structurally using DeepAlign (Wang et al. 2013); that is, only residues that are in equivalent positions in three-dimensional structures are aligned in the same column in the sequence alignment. Ca atom root mean square deviation (RMSD, A˚ ) of each type I and type II receptor structure to their average position in the structural superposition is shown at each position in the alignment. Gaps were closed to minimize the length of the alignment; corresponding positions lack an RMSD value. Asterisks in the RMSD row show o:10.1101 doi: positions in which type I receptor cysteines are poorly aligned with one another structurally and were manually aligned. Human type I and type II receptors were also aligned separately from one another by sequence using MAFFT (Katoh and Standley 2013). Sequences not represented by structures were then aligned to those in the structural alignment. Color code is modified from ClustalX. Known disulfide bonds are color coded and lettered, except for the “h” disulfide in AMHRII, / cshperspect.a022103 which is predicted by the structural alignment (see section on Structures of Type I and Type II Receptor Ectodomains). b- strand arrows above the alignment shown with dark or light shading show positions where all or some structures, respectively, have b-structure. Downloaded from http://cshperspectives.cshlp.org/ at Harvard University Library on October 5, 2016 - Published by Cold Spring Harbor Laboratory Press

Structural Biology and Evolution of the TGF-b Family

Type I receptors A ALK6 (BMPRIB) B ALK3 (BMPRIA) CDALK1 (AcvRL1) ALK5 (TβRI) β1 Prehelix extension β1 β1 d β β β β β β β β β bc 4- 5 1 bc 4- 5 4- 5 4- 5 a d b c d β β loop β b d loop a loop 1 d loop 2 2 a ca a bc β2 β β5 β β β5 2 e 2 5 e β e e 5 e β5 β β 3 3 β3 β3 β3 β β4 β4 4 β4 β4

Type II receptors E ActRII F ActRIIB GHBMPRII TβRII

β 1 β4-β5 β β β β β β f 1 a β4-β5 4- 5 1 4- 5 loop c f a loop I533 b c loop a b loop b c f c β d b d 2 d a d β β2' β5 β5 β β 2 D32D 2 β5 β2 β 1 5 β3 2 β3 e g β3 β1' β3 β4 β 4 β4 β4

Figure 6. Type I and type II receptors of the transforming growth factor b (TGF-b) family. Structures of type I (A–D) and II (E–H ) receptor ectodomains are shown as ribbon diagrams, with disulfide bonds depicted as sticks with yellow sulfur atoms. Disulfide bond letters and b-strand numbers correspond to those in Fig. 5. A loop without reliable electron density in ALK3 is shown as a dashed line. Structures are for ALK6 (Kotzsch et al. 2009), ALK3 (Kirsch et al. 2000), ALK1 (Townson et al. 2012), ALK5 (Groppe et al. 2008), ActRII (Greenwald et al. 1999), ActRIIB (Thompson et al. 2003), BMPRII (Mace et al. 2006), and TbRII (Boesen et al. 2002).

families of GFs (TGF-b GFs, Box 1). Thus, the (Liu et al. 1995). In contrast, TbRII and AMH- closely related type I receptors ALK3 and ALK6 RII are highly specific or exclusive for binding bind and transduce signals for the BMP clade and transducing signals for the TGF-bs and comprising the BMP-5, 6, and 7 subfamily, the AMH, respectively (Baarends et al. 1994; BMP-2 and 4 subfamily, or the GDF-5, 6, and 7 Liu et al. 1995). TbRII does not ally with any subfamily, yet not for the activin or the myosta- other type II receptors in the family tree and lies tin and GDF-11 subfamilies in the activin clade closest to the type I receptors; however, AMH- or for TGF-b (ten Dijke et al. 1994b). The type I RII is the most divergent from the consensus receptors ALK4 and ALK5, which diverge from type II receptor sequence (TGF-b receptors, ALK3 and ALK6 (TGF-b receptors, Box 1), Box 1). show the opposite GF binding properties; they Among receptor ectodomain:GF complexes bind and transduce signals for the activin sub- (Table 1), binary type I or II receptor:GF com- family and the myostatin and GDF-11 subfam- plexes and ternary type I receptor:type II recep- ily in the activin clade, and for TGF-b, but not tor:GF complexes show nearly identical binding for BMP-5, 6, and 7, BMP-2 and 4, or GDF-5, 6, positions for type I and type II receptors (Fig. and 7 in the BMP clade (ten Dijke et al. 1994a). 7). Type II receptors bind the distal ends of the The three type II receptors ActRII, ActRIIB, and GF dimer, at the knuckle (ActRII and ActRIIB) BMPRII bind and transduce signals for BMP-5, or fingertips (TbRII) of the GF. Thus, residues 6, and 7, BMP-2 and 4, GDF-5, 6, and 7. Fur- within a single monomer determine type II thermore, the receptors ActRII and ActRIIB also receptor binding specificity. In contrast, type I interact with activin A and B (receptor binding receptors bind across the dimer interface at the data for activin C and E is unclear) as well as wrist, and thus can show selectivity for open myostatin, and thus act across clades, yet do versus closed homodimers or homodimer ver- not bind or signal for the TGF-bsorAMH sus heterodimer (Fig. 7). The GF:receptor struc-

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A.P. Hinck et al.

ABALK6 - GDF-5 ActRII - BMP-7 CActRIIB - ActA

ALK6 ActRIIB ActA ActRII BMP-7 GDF-5 GDF-5

ALK6 BMP-7 ActRII ActA ActRIIB 90° ALK6 90° 90° ActA ActA BMP-7 BMP-7

GDF-5 GDF-5

ActRII ActRII ALK6 ActRIIB ActRIIB

D ALK3 - ActRIIB - BMP-2 E ALK1 - ActRIIB - BMP-9 F ALK5 - TβRII - TGF-β3 ALK5 ALK1 TβRII ALK3 TGF-β3 ActRIIBRIIB TβRII N-term ActRIIBRIIB BMP-9BMP-9

BMP-2BMP-2 K97 BBMP-2MP-2 BMP-9BMP-9 D57 P55 TβRII ActRIIBActRII TGF-β3 AALK3LK3 ActRIIBAct TβRII N-term ALK1ALK1 R58 ALK5 P59 D118 ALK3 90° 90° F24 90° ALK3 BMP-9 BMP-9 TβRII N-term ALK1 TGF-β3 TGF-β3 ALK1 TβRII TβRII

BMP-2 BMP-2

ActRIIB ActRIIB ALK5 ALK5 ActRIIB ActRIIB

Figure 7. Representative growth factor (GF) complexes with type I and type II receptors. Structures are shown for representative binary (A–C) or ternary (D–F) complexes as ribbon diagrams with pink and light blue GF monomers, and type I and type II receptor ectodomains in orange-red and green, respectively. Orthogonal views are shown above and below one another in each panel. The ActRIIB:activin A structure model with a closed activin GF is made by superimposing open monomer complexes from the ActRIIB:activin A structure (Thomp- son et al. 2003) on the closed activin dimer from the activin A:Fst288 structure (Thompson et al. 2005). Panel F (inset) shows the interactions of Pro55, Asp57, Arg58, and Pro59 in the ALK5 pre-helix extension with Lys97 on TGF-b3 and Asp118 in TbRII, and of ALK5 with Phe24 in the amino-terminal tail of TbRII. Structures are for ALK6:GDF-5 (Kotzsch et al. 2009), ActRII:BMP-7 (Greenwald et al. 2003), ActRIIB:activin A (Thompson et al. 2003), ActRIIB:ALK3:BMP-2 (Weber et al. 2007), ActRIIB:ALK1:BMP-9 (Townson et al. 2012), and TbRII:ALK5:TGF-b3 (Groppe et al. 2008) complexes. N-term, amino terminal.

tures provide insight into many patterns of re- and ActRIIB binding to two different GFs fur- ceptor:GF binding that have been reported. ther show essentially identical orientations in all Promiscuous binding of many GFs from the cases (Fig. 8E–G). Promiscuity of ActRII and BMP and activin clades by ActRII and ActRIIB, ActRIIB for binding a large number of GFs for example, is evident from their identical ori- agrees with the similarity in the receptor struc- entations for binding BMP-7, activin A, BMP-2, tures and knuckle regions of the GFs, which are and BMP-9 dimers (Fig. 7B–E) (Greenwald among their most structurally conserved fea- et al. 2003, 2004; Thompson et al. 2003; Allen- tures. Promiscuity is further promoted by the dorph et al. 2006; Weber et al. 2007; Townson predominantly hydrophobic composition of et al. 2012). Consistent with their similar b4- the interface (Fig. 9E–G). Among 23 residues b3-b5 sheet concave surfaces and b4-b5 loop in BMP-2 that directly contact ActRIIB in the occluding f disulfide (Fig. 6E,F), ActRII and structure of the BMP-2:ActRIIB:ALK3 complex, ActRIIB complement the convex knuckle of substitution with alanine of only six residues the GF in a manner that is nearly indistinguish- perturbed binding (Kirsch et al. 2000; Weber able (Fig. 7B–E). Superpositions of ActRIIB et al. 2007). Among these, only a Leu is con- binding to three different GFs and of ActRII served among the diverse GFs bound by these

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ABCDALK3 ALK3 ALK3 ALK6 ALK3 ALK1 ALK3 ALK5 BMP-2 GDF-5 BMP-2 GDF-5 BMP-2 BMP-9 BMP-2 TGF-β3 atHarvardUniversityLibraryonOctober5,2016-PublishedbyColdSpring Harbor LaboratoryPress odSrn abPrpc Biol Perspect Harb Spring Cold

Type II receptor binding modes

β EFGActRIIB ActRIIB ActRIIB ActRIIB ActRIIB ActRII HActRIIB T RII BMP-2 ActA BMP-2 BMP-9 BMP-2 BMP-7 BMP-2 TGF-β3 tutrlBooyadEouino h TGF- the of Evolution and Biology Structural o:10.1101 doi: / cshperspect.a022103

Figure 8. Relative positioning of type I and type II receptor ectodomains on growth factor (GF) dimers. Positions of type I receptors on GF dimers in complexes are compared by superimposition using GF dimers on the ALK3:BMP-2 complex (A– D); similarly, positions of type II receptors on GF dimers in complexes are compared by superimposition using GF dimers on the ActRIIB:BMP-2 complex (E–H ). Receptor names are color-coded as in their ribbon diagrams. GFs used for superposition are shown with black names below names of their bound receptors. GF dimers are shown as surfaces, with monomers shaded pink and light blue. Overlaid type I and type II receptors are in yellow, whereas ALK3 and ActRIIB are in orange–red and green, respectively. Complex structures are for ALK3:GDF-5 (Klammert et al. 2015), ALK6:GDF-5 b

(Kotzsch et al. 2009), ALK1:ActRIIB:BMP-9 (Townson et al. 2012), ALK5:TbRII:TGF-b3 (Groppe et al. 2008), ALK3: Family BMP-2 (Kirsch et al. 2000), ActRIIB:ActA (Thompson et al. 2003), ActRII:BMP-7 (Greenwald et al. 2003), and ALK3: 25 ActRIIB:BMP-2 (Weber et al. 2007). Downloaded from http://cshperspectives.cshlp.org/ ..Hnke al. et Hinck A.P. 26

Type I receptor interfaces

A ALK3 - BMP-2 B ALK6 - GDF-5 C ALK1 - BMP-9 D ALK5 - TGF-β3

dacdOln ril.Ct hsatceas article this Cite Article. Online Advanced M106 D23 W36 Y103 K97 TGF-β3 Y103 I68 R78 F85 W33 Y90 BMP-2 BMP-2 GDF-5 F66 Y99 V92 W31 D89 W25 P55 E75 W22 BMP-9 W28 BMP-9 D57 ALK3 ALK6 F54 K53 TGF-β3 L76 R58 atHarvardUniversityLibraryonOctober5,2016-PublishedbyColdSpring ALK1 H73 Harbor LaboratoryPress L29 F43 D118 F24 F49 R57 V22 ALK3 GDF-5 TβRII

odSrn abPrpc Biol Perspect Harb Spring Cold Type II receptor interfaces

E ActRIIB - ActA F ActRIIB - BMP-2 G ActRII - BMP-7 H TβRII - TGF-β3

Finger 4 Y42 D32 R94 F83 F42 TβRII F83 W60 V92 V81 Y42 K102 W60 V81 W60 F83 L92 ActRIIB L90 ActRIIB ActRII β I30 A34 S88 L61 A34 S88 L61 L125 1’ H39 V98 V81 Y90 Finger 3 I100 H39 L115 A58 S113 L61 I53 D104 W32 A63 β2 Finger 2 L27 L27 o:10.1101 doi: β1 W30 ActA BMP-7 BMP-7 E119 R25 Finger 1 BMP-7 ActA BMP-7 TGF-β3 / cshperspect.a022103

Figure 9. Type I and type II receptor–GF interfaces. Interfaces show growth factors (GFs) bound to type I receptors (A–D) or type II receptors (E–H ). Cartoon diagrams show GFs as pink and light blue monomers, and type I and type II receptors in red–orange and green, respectively, with key side chains in stick with carbons in the same colors, red oxygens and blue nitrogens. Structures correspond to those in the Figure 8 legend. Downloaded from http://cshperspectives.cshlp.org/ at Harvard University Library on October 5, 2016 - Published by Cold Spring Harbor Laboratory Press

Structural Biology and Evolution of the TGF-b Family

receptors (Leu residues 92, 90, and 125 in acti- and differ in the length and sequence of the loop vin A, BMP-2, and BMP-7, respectively, in Fig. bearing Arg94 (Fig. 1), correlating with the dif- 9E–G). Thus, the similar overall shape and hy- ferent shapes of fingertip loops (Fig. 4B, lower) drophobicity of the knuckle in GFs that bind and creating high specificity for TbRII. ActRII and ActRIIB enables the promiscuity Type I receptor ectodomain:GF complexes that is possible for predominantly hydrophobic also provide insights into specificity, but the interfaces (Weber et al. 2007). differences are more subtle. Thus, complexes How TbRII specifically binds TGF-b1–3 containing ALK1, 3, 5, and 6 with GFs as diverse is evident from the structure of the TGF- as GDF-5, BMP-2, BMP-9 and TGF-b3, show b3:TbRII:ALK5 ternary complex (Figs. 6H, 7F, that the type I receptors are positioned similarly, and 9H) (Groppe et al. 2008). TbRII binds in although not identically, at the wrists of the GFs the cleft between the fingers of the GF, using (Fig. 7A,D–F) (Kirsch et al. 2000; Groppe et al. primarily the b2, but also the flanking b1 and 2008; Kotzsch et al. 2009; Townson et al. 2012). b10 strands. Remarkably, the TbRII binding site The closely related type I receptors ALK3 and on the GF does not overlap with those of ActRII ALK6 (TGF-b receptors, Box 1) bind similarly and ActRIIB (Fig. 8H). The ability of TbRII to at the wrist and contact each GF monomer bind a distinct site on the GF is driven by dif- roughly equally (Kirsch et al. 2000; Keller et al. ferences in receptor structure that change the 2004; Kotzsch et al. 2009; Klammert et al. 2015). overall shape of the binding surface (see section ALK1 and ALK5 belong to subfamilies distinct on structures of type I and type II receptor ec- from that containing ALK3 and ALK6 (Box 1). todomains); sequence and loop shape differ- The structural features that cause ALK1 and ences in the finger region of TGF-bs relative to ALK5 binding positions to shift relative to the other GFs enable specificity (Fig. 4B, lower) (De ALK3/6 subfamily (Fig. 8A–D) are discussed in Crescenzo et al. 2006; Baardsnes et al. 2009). the next two paragraphs. The interface that stabilizes TbRII in the cleft The type I receptor ALK1 is divergent in between the TGF-b fingertips is unique relative sequence from ALK3 and ALK6 (TGF-b recep- to that used by ActRII and ActRIIB, and is char- tors, Box 1) and binds and transduces signals acterized by an inner hydrophobic portion, for BMP-9 and BMP-10, but not other BMPs with Ile53 of TbRII packing into a shallow and GDFs (David et al. 2007). ALK1 binds both pocket in TGF-b formed by Trp32, Tyr90, and monomers at the wrist, but the directionality of Val92 (Fig. 9H) (Groppe et al. 2002; Radaev the strands that comprise its central b4-b3-b5 et al. 2010). The inner hydrophobic portion is sheet is rotated relative to that of ALK3 and flanked on either edge by hydrogen-bonded ALK6 (Mahlawat et al. 2012; Townson et al. ion pairs between the side chain carboxylate 2012), as shown by the dashed lines in Figure groups of Asp32 and Glu119 on TbRII and 7D,E. Repositioning ALK1 relative to ALK3 and the side chain guanidinium groups of Arg25 ALK6 is primarily driven by a two-residue and Arg94 on the fingertip loops of TGF-b shortening in the segment between the Cys of (Fig. 9H). The two arginine residues contribute the c disulfide at the carboxy-terminal end of b- .30% of the total binding energy and are in- strand 4 and the Cys of the d disulfide in the 310 variant in TGF-b1 and 3, which bind TbRII helix of the b4-b5 loop (Figs. 5 and 6). Short- with high affinity (De Crescenzo et al. 2006; ening causes this segment of ALK1 to be more Baardsnes et al. 2009). The two arginine resi- rigid and shifted closer to b-strand 5 than in dues are conservatively replaced by lysine in ALK3 and ALK6 (Fig. 6A–C) (Mahlawat et al. TGF-b2, which binds TbRII with low affinity, 2012; Townson et al. 2012). Loop shortening in and substitution of these with arginine increases ALK1, together with conformational changes in the affinity of TGF-b2 to that of TGF-b1 and 3 the prehelix extension and other loops in the (De Crescenzo et al. 2006; Baardsnes et al. wrist region of BMP-9 (Fig. 4B), leads to steric 2009). Relative to all other GFs, TGF-bshavea clashes when ALK1 is positioned onto BMP-9 one-residue insertion in the loop bearing Arg25, in the same manner as ALK3 on BMP-2 (Mah-

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A.P. Hinck et al.

lawat et al. 2012; Townson et al. 2012). The shifts toward the fingertips in which it binds in structure of the BMP-9:ActRIIB:ALK1 complex the cleft between TGF-b and TbRII. Reposi- shows that these clashes are alleviated when tioning orchestrates a number of interactions ALK1 binds BMP-9 by a rotation of 20˚ rela- with TGF-b and TbRII that are required for tive to ALK3 bound to BMP-2 (Fig. 7D,E) cooperative assembly of the TGF-b signaling (Townson et al. 2012). complex (Fig. 7F, inset) (Groppe et al. 2008; ALK5 (also known as TbRI) is in a type I Radaev et al. 2010; Zuniga et al. 2011). In sum- receptor subfamily distinct from the ALK1 and mary, alternative positioning of ALK1 and ALK 3/6 subfamilies (TGF-b receptors, Box 1). ALK5 provide distinct interfaces and stabilizing Compared with these receptors, ALK5 binds interactions that enable segregation of GF spec- more toward the TGF-b fingertips, which en- ificities of the ALK1 and ALK5 subfamilies from ables it to additionally contact TbRII (Fig. 7F, the ALK3 and ALK6 subfamily. upper panel) (Groppe et al. 2008). TbRII has a Further details on amino acid sidechain longer amino-terminal segment than other type and structural features that underlie the selec- II receptors (Fig. 5), and a portion of this seg- tivities displayed by type I and type II recep- ment becomes ordered by this contact, with tors, despite promiscuity in binding multiple Val22 and Phe24 in TbRII binding to a hydro- GFs, are summarized in Table 2. These details phobic pocket in ALK5 (Fig. 7F, inset). The extend to differences among ActRII, ActRIIB, repositioning of ALK5 relative to ALK3 and and BMPRII, and between ALK3 and ALK6 ALK6 is, like ALK1 repositioning, driven by (Table 2). a modification of the segment between the c disulfide cysteine in the carboxy-terminal end of b-strand 4 and the d disulfide cysteine Coreceptors in the 310 helix within the b4-b5 loop, but in this case with a five-residue extension rather Several families of coreceptors regulate GF sig- than a two-residue shortening (Figs. 5 and 6). naling. One subfamily comprises betaglycan The PRDRP sequence motif in the prehelix ex- and endoglin, which show sequence homology tension, between the c and d disulfide in Alk5, with one another. They contain amino-terminal is rigid and forms a tight turn at its amino- orphan and carboxy-terminal zona pellucida terminal cis Pro-55 (Figs. 5, 6D, and 7F, inset) (ZP) domains (Gougos and Letarte 1990; (Zuniga et al. 2011). The extension precludes Bork and Sander 1992; Esparza-Lopez et al. ALK5 from binding similarly to ALK3 due to 2001), single-pass transmembrane domains, clashes with hydrophobic residues in the wrist. and short cytoplasmic domains of ,50 amino These clashes cause ALK5 to reposition so that it acid residues. Both coreceptors can be shed

Table 2. Structural basis of GF binding preferences by ActRII, ActRIIB, ALK3, and ALK6 Preference Interactions that promote binding to preferred GF References ActRII and ActRIIB bind Intramolecular salt bridge between Lys102 and Asp104 on Thompson et al. 2003; activins 100- to 1000- finger 4 of activin A promotes hydrophobic and Allendorph et al. fold more avidly than H-bond interactions with ActRIIB; ion pairs of Glu111 2007; Weber et al. BMPs and GDFs and Arg87 of activin Awith Lys37 and Asp62 of ActRIIB 2007 BMPRII binds BMPs and BMPRII Tyr113 promotes binding to BMP-2, BMP-7, and Greenwald et al. 2003; GDFs, but does not GDF-5, but inhibits binding to activin A Yin et al. 2008 bind activins ALK6 binds GDF-5 10- Rotation of ALK6 by about 9˚ brings its b1-b2 loop close Keller et al. 2004; to 20-fold more avidly to Arg57, which protrudes from the pre-helical loop of Kotzsch et al. 2009 than ALK3 GDF-5; the equivalent loop of ALK3 has a different conformation that cannot accommodate Arg57 GDF, Growth differentiation factor; BMP, bone morphogenetic protein.

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Structural Biology and Evolution of the TGF-b Family

from the cell surface as soluble fragments Alt et al. 2012). BMP-9, BMP-10, endoglin, and (Lamarre et al. 1994; Hawinkels et al. 2010). ALK1 may form a regulatory circuit to control Betaglycan interacts with TGF-bs, BMP-2 angiogenesis (Tillet and Bailly 2014). and 4, GDF-5, and inhibins (Lewis et al. 2000; Cripto is the eponymous member of a small Esparza-Lopez et al. 2001; Kirkbride et al. 2008). protein family (Ciardiello et al. 1991) and serves Most importantly, betaglycan binds the three as a coreceptor for several TGF-b family GF di- TGF-bs and, hence, was identified as the TGF- mers (Yeoand Whitman 2001; Cheng et al. 2003, b type III receptor. It was initially thought to 2004; Gray et al. 2003; Chen et al. 2006). Cripto predominantly function by presenting TGF-b2 and its family members are attached to the cell to TbRII, and thereby compensate for TGF-b2’s membrane by a glycosylphosphatidylinositol low intrinsic affinity for TbRII (Lopez-Casillas (GPI)-moiety (Minchiotti et al. 2000). Like be- et al. 1993). In agreement, betaglycan knockout taglycan and endoglin, cripto can be shed, but by and TGF-b2 knockout mice each show perinatal GPI phospholipase D rather than by proteolysis lethality and display many phenotypic charac- (Watanabe et al. 2007). Cripto functions in teristics in common (Stenvers et al. 2003). Be- switching between nodal and activin signaling, cause the cytoplasmic domain of betaglycan can which use the same type I and type II receptors interact with b-arrestin and mediate internali- (Rosa 2002). As nodal requires cripto for bind- zation of TGF-bs and their receptors, betagly- ing to ALK4, whereas activins do not, nodal sig- can can also directly modulate TGF-b signaling naling is repressed in cells lacking cripto, where- (Chen et al. 2003; McLean et al. 2013). as activins can signal via ALK4 and ActRII/IIB Betaglycan’s ZP domain comprises amino- (Yeo and Whitman 2001). Structurally, cripto terminal (ZP-N) and larger carboxy-terminal comprises two consecutive small cysteine-rich (ZP-C) immunoglobulin-like subdomains. Of domains, an amino-terminal EGF-like domain these, only ZP-C interacts with TGF-b (Wiater and a CFC-domain. Both domains contain three et al. 2006). Structural and functional analyses of disulfides required for folding. Although the the betaglycan ZP-C domain suggest that a short EGF domain is involved in binding to nodal, peptide segment, named EHP,harbors the main the CFC domain interacts with the type I recep- binding determinants of the betaglycan ZP-C tor ALK4 (Adkins et al. 2003; Calvanese et al. subdomain for TGF-b (Fig. 10A) (Lin et al. 2006). A structure for the CFC domain in its 2011; Diestel et al. 2013). Understanding how unbound form shows that two residues impli- betaglycan interacts with TGF-b GFs to provide cated in interaction with ALK4, His104 and high affinity (Mendoza et al. 2009) will require Trp107, form a patch on the protein surface structures of its orphan domain, and GF com- (Fig. 10B) (Calvanese et al. 2006). However, fur- plexes with the orphan and ZP-C domains. ther insights into how cripto bridges nodal to Endoglin has unique biological functions ALK4 require a structure of the nodal:ALK4:- compared with betaglycan (Nassiri et al. cripto ternary complex. 2011). Its importance in vascular development RGMs, initially identified for their func- is shown by loss-of-function mutations in the tions in guiding retinal axons and neural tube vascular disorder HHT type 1 (HHT1) (Arde- closure (Muller et al. 1996; Monnier et al. 2002) lean and Letarte 2015). BMP-9 and -10 are sim- are BMP-specific coreceptors (Babitt et al. 2005; ilarly important in vasculogenesis (Chen et al. Samad et al. 2005). Mammals have three RGM 2013), and mutations in the BMP-9 prodomain family members, RGMa, RGMb/DRAGON and ALK1 cause similar HHTsyndromes (Tillet and RGMc/hemojuvelin (HJV) (Camus and and Bailly 2014). Within the TGF-b family, en- Lambert 2007). They are highly conserved, doglin has high affinity and specificity for BMP- with 40%–50% amino acid sequence identity, 9 and -10 (Castonguay et al. 2011). It binds both and thus have identical protein architectures. BMPs through its orphan domain, does not RGMs both interact with the type I transmem- compete or synergize with ALK1, but competes brane protein neogenin, a netrin family recep- with type II receptors (Castonguay et al. 2011; tor, to mediate neuronal functions (Matsunaga

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A.P. Hinck et al.

Betaglycan ABCZP-C domain N N Cripto BMP-2 CFC-domain

N α1 α 3 α2 C

C RGMbND EHP segment Disulfide bonds C Neogenin E RGMB D BMP-2 ND BMP-2

Leu103 Trp31

His106 -GDPH- Phe49 RGMb RGMbCD RGMb CD Neogenin ND Pro50 90°

BMP-2 F RGMbCD Neogenin RGMBND

Lys88 BMP-2 Phe49 Phe85 Pro50 Neogenin ALK3 RGMbCD RGMbND

Figure 10. Coreceptors for transforming growth factor b (TGF-b) family members. (A) The carboxy-terminal zona pellucida (ZP-C) domain of betaglycan (3QW9) (Lin et al. 2011). (B) The coreceptor cripto comprises small epidermal growth factor (EGF)-like and CFC domains. The CFC domain of cripto adopts an extended architecture lacking secondary structure and is stabilized by three disulfide bonds (yellow sticks) (2J5H, Calva- nese et al. 2006). Two residues involved in binding of the CFC-domain to ALK4, His104, and Trp107, are shown with green side chains. (C) The amino-terminal domain (ND) of RGMb (RGMbND) in cartoon (green), bound to a BMP-2 dimer (surface, with type I and type II receptor binding sites in light green and pink, respectively) (PDB 4UI0 [Healey et al. 2015]). (D) Similar structural elements are used in the interaction of BMP-2 with RGMbND (upper panel) and ALK3 (lower panel). Leu103, conserved in all three repulsive guidance molecules (RGMs), engages in a hydrophobic knob-into-hole interaction resembling that of the phenylalanine residue conserved among BMP type I receptors (Phe85 in ALK3 and Phe66 in ALK6). Moreover, the Phe-Pro motif (surface and side chains in magenta) found in Smad1-, 5-, and/or 8-activating BMPs forms a hydrophobic patch between the 1st and 3rd helix of the RGM three-helix bundle to mimic interaction with ALK3. His106 in RGMb, conserved in all three RGMs, makes a p-stacking interaction with a conserved Trp in bone morphogenetic protein (BMP) and renders BMP-binding by RGMs pH-sensitive. (E) RGM binds via RGMND (green cartoon) to BMP (gray and black monomer surfaces with the type I and type II receptor sites in light green and pink, respectively) and via RGMBCD (cyan cartoon) to neogenin (dark blue cartoon) (PDB 4UI2 [Healey et al. 2015]). RGMbCD links to RGMND with a flexible linker (dashed lines) that is only partially observed in the crystal structure (green cartoon with disulfides to RGMbCD in gold stick). The acid-labile Asp-Pro bond (marked with GDPH) is at the carboxy-terminal end of b-strand 1 of the b-sandwich of RGMbCD. This b-strand and two disulfide bonds maintain covalent linkage between RGMbND and RGMbCD despite cleavage at the Asp-Pro bond. (F)AsinE, but viewed along the dyad of the BMP GF dimer.

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Structural Biology and Evolution of the TGF-b Family

et al. 2004; Rajagopalan et al. 2004), and bind coreceptor would be released from its pH-de- BMPs to modulate BMP signaling. The rela- pendent complex with BMP and the type II re- tionship between these two functions is poorly ceptor, enabling the type I receptor to replace understood, but both neogenin and BMP bind- RGM in the complex, as type I receptor interac- ing are required for the proper function of HJV tion with BMPs is not pH-sensitive (Heinecke in regulating systemic iron stores (Kuns-Hashi- et al. 2009; Healey et al. 2015). Signaling would moto et al. 2008; Zhang et al. 2009). thusoccurinthecytoplasmicenvironmentasfor RGMs contain an amino-terminal domain receptors on the cell surface, but on the cytoplas- (ND) with three disulfide-linked a-helices that mic face of a distinct membrane compartment binds BMPs, a flexible linker, a carboxy-termi- where other colocalizing molecules might differ nal domain (CD) with two tightly packed b- (Healeyet al. 2015). This might alter the shape of sheets that contains an acid-labile Asp-Pro pep- signaling as shown for some G protein-coupled tide bond and binds to neogenin, a PC cleavage receptors, in which a second wave of signaling site, and a carboxy-terminal GPI membrane an- occurs on endosomes that moves the site of sec- chor (Bell et al. 2013; Healeyet al. 2015). Soluble ond messenger production relative to effectors forms of RGMa and RGMc result from process- (Tsvetanova et al. 2015). ing by the PCs furin and/or SKI-1 (subtilisin And how does neogenin influence the BMP- kexin isozyme-1) (Tassew et al. 2012). RGM interaction? The ternary complex struc- How do RGMs stimulate BMP signaling? ture of BMP-2:RGMb:neogenin not only shows Structural and microscopy studies suggest that that RGMs can simultaneously bind BMPs and endocytosis of BMPs by RGMs and neogenin neogenin (Fig. 10E,F), but also revealsthat bind- promotes type I and type II receptor signaling ing of type II receptors to this complex is again in an intracellular compartment (Healey et al. not blocked. Thus, formation of quaternary 2015). The first 120 amino-terminal residues of complexes consisting of RGMs, neogenin, RGMs, RGMND, fold into an antiparallel three- BMP, and type II receptors is possible (Healey helical bundle that binds BMPs at the receptor et al. 2015). Although RGMCD binding to neo- type Isite (wrist epitope)(Fig.10C). When com- genin brings neogenin into close proximity to plexes of RGMs bound to BMP-2 are compared BMP bound to RGMND, no direct interactions with ALK3 bound to BMP-2, the third helix of between BMP and neogenin are observed (Fig. RGMND mimics the interaction of the short a- 10E,F) (Healey et al. 2015). The modular archi- helix of ALK3 with BMP-2 (Fig. 10D). One key tecture of RGMs with their two domains inde- element for the RGM–BMP interaction is a his- pendently binding two different partners, BMPs tidine residue conserved in all RGM proteins, and neogenin, of which BMP is a dimer and His106 in RGMb, which forms a p-stacking in- neogenin is dimerized on RGM binding (Bell teraction with the second Trp of the W-X-X-W et al. 2013), suggest that RGMs could bridge motif in BMPs (Fig. 10D). Binding of RGMs individual complexes and facilitate formation to BMPs is inhibited at pH ,6.5, presumably of supramolecularclusters. Indeed, high-resolu- because of histidine protonation. This feature tion microscopy shows that BMP-2 mediates points toward a mechanism by which RGMs clustering of RGM–neogenin complexes into might stimulate BMP signaling. Because RGMs large assemblies at the cell surface (Healey et al. block BMP binding to type I but not type II 2015). The physiological significance of supra- receptors, it is proposed that RGM–BMP– molecular cluster formation is unknown, al- BMPRII complexes form at the cell surface, though this may enable BMPs to modulate neo- and that these are then endocytosed in a clath- genin signaling, and neogenin to modulate BMP rin-dependent manner. As type I receptors, signaling, adding more complexity to BMP re- including ALK3, are constitutivelyendocytosed, ceptor activation (Mueller 2015). endosomes might form that contain RGM– In summary, TGF-b and BMP coreceptors BMP–type RII complexes as well as free ALK3. are functionally and structurally diverse, and Because of endosomal acidification, the RGM the presence or absence of a coreceptor can

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A.P. Hinck et al.

have large qualitative (changing gene expres- side of noggin, opposite the concave BMP bind- sion) and quantitative effects (sensitizing cells ing site, and support noggin’s C-clamp-like cur- for a GF) on downstream signaling. vature (Fig. 12A,B). Thus, whereas the TGF-b CKGF dimer is linked by monomers that over- REGULATION OF TGF-b/BMP SIGNALING lap in the palm and heel (GF homodimer, Box BY MODULATOR PROTEINS—BMPs AS 1), and dimerize through cysteine 6, the noggin SIGNALING HUBS dimer links the two CKGF domain monomers more distally toward their carboxy-terminal Structural Diversity of TGF-b/BMP end at cysteine 11 (Fig. 11F,G), leading to a lon- Antagonists ger and more curved dimer that straddles and A hallmark of the TGF-b/BMP family is the clamps the BMP dimer (Fig. 12A,B). plethora of extracellular modulator proteins Distinct structural elements in noggin block that bind GFs and regulate signaling. More the BMP type I (wrist) and type II (knuckle) than 25 modulator proteins are known. receptor epitopes (Fig. 12A–D). A 25-residue Attempts have been made to classify TGF-b/ segment within the amino-terminal region of BMP modulator proteins structurally. A com- noggin is spatially separated from the CKGF mon hallmark is the large number of cysteine and a-helical domains in a complex with residues. Although the diversity of these mod- BMP, fastens to more than one BMP monomer ulators is stunning, they can be grouped, based in a U-shape, and is therefore termed the “clip” on structural features and sequence homology, (Fig. 12A) (Groppe et al. 2002). In this segment, into six distinct families: noggin, members of residues Pro35 to Ser38 snugly fold into the the Dan family (Fig. 11), the follistatin family, center of the wrist epitope, and block binding the CCN family, the chordin family, and twisted to type I receptors. The clip then turns over the gastrulation (Tsg). The Dan and CCN families fingers of the BMP and runs over the periphery as well as noggin have CKGF domains. Notably, of the knuckle epitope. In contrast, the noggin the diversity of BMP and activin antagonists fingers at the end of its C-clamp extend over the contrasts with the paucity or absence of modu- fingertips of the BMP and shield a large part of lators of TGF-b itself, perhaps correlating with the knuckle epitope, thereby blocking access the distinct integrin-dependent mechanism of and binding of type II receptors. Hydrophobic TGF-b activation. interactions dominate both the fingertip and clip interfaces, similarly to type I and type II receptor interfaces with BMPs. Moreover, Noggin—The BMP Antagonist Paradigm Pro35 in the noggin clip, which is highly con- Noggin, a component of the Spemann organiz- served among different species, extends into a er (Smith and Harland 1992), antagonizes the hydrophobic pocket in the wrist epitope of ventralizing activity of BMPs in dorsoventral BMP-7, mimicking the knob-into-hole interac- patterning and regulates skeletogenesis (Zim- tion found in various BMP-type I receptor in- merman et al. 1996; Brunet et al. 1998). Noggin teractions (Fig. 12A–D) (Hatta et al. 2000; binds to the same sites on BMP-7 as the BMP Kirsch et al. 2000; Groppe et al. 2002; Kotzsch type I and type II receptors, and thereby im- et al. 2009). The importance of Pro35 for the pedes BMP signaling (Fig. 12A–D) (Groppe BMP-neutralizing activity of noggin is high- et al. 2002). Noggin contains a CKGF domain lighted by its mutation in skeletal malformation like TGF-b GF domains, but has a 10- rather diseases (Gong et al. 1999; Mangino et al. 2002). than 8-membered CK ring (Fig. 12A). Further- The mechanism of noggin binding to BMPs more, its monomers dimerize through a Cys preserves much of the chemistry of receptor residue in a position (cysteine 11 in Fig. binding to BMPs, including shared hydropho- 11G,H) that differs from that in TGF-b family bic interfaces and the knob-into-hole packing proteins, and through an additional a-helical seen with most type I receptors; however, details domain (Fig. 12A). The helices lie on the convex of the distribution and arrangement of the

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Structural Biology and Evolution of the TGF-b Family

A E PRDC Frizzled4 1 Loop 1 CRD 4 10 9 7 3 Loop 3 Norrin 5 Loop 2 8 1 10 9 Loop 1 2 4 1 Loop 1 3 Glycan B Nbl1 10 7 9 6-5’ 5 Loop 3 4 -6′ 7 3 11 –11′ Loop 3 8 5 11 2 Loop 2 8 Loop 2 TGF-β2 1 Loop 1 2 4 9 1 Loop 1 F C Sclerostin 4 10 10 9 7 3 3 6–6′ 7 Loop 2 Loop 3 Loop 3 G Noggin Loop 2 8 3 2

4 1 Loop 1 9 D HCGβ 1 Loop 1 Loop 2 10 4 9 7 3 10 ′ Loop 3 3 11 –11 7 Loop 3 Loop 2 Clip

H Loop 1 Loop 2 142 3 PRDC 4JPH71 DWCKTQPLRQTVS- - - -E-EGCRS-RT- - ILNRFCYG-Q-C-NS-FYI -PRHVKKE-E Nbl1 4X1J33 AWCEAKNITQIVG-H- -S-G-CEA-KS- - IQNRACLG-Q-C-FS-YSV-PN-T- -FPQ Sclerostin 2KD353 YSCRELHYTRFLT-D- -G-P-C-R-SAKPVTELVCSG-Q-C-GP-ARL -L- - -PNAIG Norrin 5BQC37 RRCMRHHYVDSIS-HPLY-K-CSS-KM- -VLLARCEG-H-CSQA-SRS-EP-LSFSTV Noggin 1M4U153 T FCPV L YAWNDLG - S ------RFWPRY--VKVGSCFSKRSC-SV-P------TGF-β2 1TFG13 DNCCLRPLY I DFKRDLGWKW- I HEPKG - - YNANFCAG - A - C - PYLWSSDTQHSRVLSL HCGβ 1QFW7 PRCRPINATLAVE-K- -EGC-PVC- IT- -VNTTICAG-Y-C-PT-MTRVLQ-G- -V- - Alignment RMSD 5321211112323------3-445222- -22211112-2-1-3------CK CK CK Loop 2 Loop 3 51076 8 9 11 PRDC 4JPH 115 DSF-Q- --- SCAFCKPQRVTSV I VELECPGLDPP - - - FR I - K - K I - QKVKHCRCMS - V Nbl1 4X1J 73 - - STESLV - HCDSCMPAQSMWE I VTLECPGHEE - - VPRVD - K - LV - EK I LHCSCQACG Sclerostin 2KD3 96 RVKWWRPNGPDFRC I PDRYRAQRVQLLCGG- - - - AAP - RS - R - KV - RLVASCKCKR - - Norrin 5BQC 85 LKQPFR - S - SCHCCRPQTSKLKALRLRCS - - - - GGMR - LT - A - TY - RY I LSCHCEECN Noggin 1M4U 188 ------EGMVCKPSKSVHLTVLRWRCQRRGGQRCGWIPI-QY-PIISECKCSC-- TGF-β2 1TFG 65 YNT I NPEASASPCCVSQDLEPLT I LYY I G - - - - KTP - - K I - EQLSNMI VKSCKCS - - - HCGβ 1QFW 49 ----LPAL-PQVVCNYRDVRFESIRLPGCP---R-GVNPV-V-SY-AVALSCQCALCR Alignment RMSD ------432111122232221346------633-3-23-21111111133-

Figure 11. Structural alignment of Dan family monomers to cystine knot (CK) growth factor (CKGF) super- family representatives with similar monomer–monomer orientations. (A–G) CKGF monomers in cartoon with a-helices in cyan and b-strands in magenta. Disulfides are shown in stick and are color-coded with their cysteines numbered as in panel H. Cysteines in intermolecular disulfides are indicated by showing the partner Cys in the other monomer with its number preceded by a dash and followed by a hyphen. All monomers but sclerostin are from dimeric structures. A frizzled domain and glycan cocrystallized with norrin are shown in cartoon and stick, respectively. Monomers are in identical orientations, and aligned in two columns. (H ) Structure-based sequence alignment. Cysteines disulfide-bonded to one another are in identical colors, except those that participate in interchain disulfides in some families and intrachain disulfides in others (positions 5, 6, and 11) are shown in red. Disulfides unique to particular families are shown in thinner silver stick and colored silver in panel H. Other residues are colored according to ClustalX. CK disulfides are labeled between the two rows of the alignment and link Cys residues distant in sequence; the three long loops between CK disulfides are also marked. (Legend continues on following page.)

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structural elements differ. Remarkably, the nog- ily antagonists (Nolan and Thompson 2014). gin–BMP complex partially shares its building They include neuroblastoma suppressor of tu- principle with the TGF-b1 proprotein complex; morigenicity 1 (Nbl1, Dan, DAND1), gremlin/ the folded noggin CKGF domain, like the pro- Drm (DAND2), protein related to Dan and cer- domain arm domain, blocks the type II recep- berus (PRDC, DAND3, gremlin2), cerberus tor site, and the single element clip in noggin, (DAND4), and coco/dante (DAND5) (Fig. like the prodomain straitjacket, blocks type I 12E). These five members are the smallest pro- receptor access to the GF (Fig. 12A; Box 1). In teins known to modulate BMP signaling. Scle- both noggin and the TGF-b1 prodomain, a cen- rostin and USAG1 (also known as wise, ectodin, tral domain together with intermolecular disul- and SOSD) also belong to the Dan modulator fide bonds forms a covalent homodimer, which family (BMP antagonists, Box 1, Figs. 11 and explains the extremely tight binding to the 12E), but they antagonize Wnt and not BMP GF. Moreover, both interact with the ECM; the signaling. Dan family members share a central TGF-b1 prodomain is covalently linked to CKGF domain of 90 residues that may be sub- LTBP, and noggin has heparin binding sites to stantially elaborated with amino- and carboxy- interact with the ECM (Groppe et al. 2002; terminal extensions that add another 100 res- Paine-Saunders et al. 2002) that can locally re- idues (Fig. 11A–C, 12E, lower). Extensions in- tain a bound GF to limit its radius of action. clude a-helices in PRDC (Nolan et al. 2013) and Noggin binds in vitro to most BMPs with unstructured regions in sclerostin (Veverka et al. no apparent dissociation, which suggests that 2009; Weidauer et al. 2009). These might be- the concentration of active ligand would be come structured when bound to GFs or ECM effectively decreased for all BMPs that it tar- components and might resemble in this respect gets (Zimmerman et al. 1996; Seemann et al. the long amino-terminal extensions in many 2009; Song et al. 2010; Schwaerzer et al. 2012). TGF-b family prodomains (Fig. 1). However, in cell-based assays some BMPs, for All Dan and sclerostin family proteins con- example, BMP-6, have been described as nog- tain an 8-membered CK ring with the same gin-resistant, although their affinity to noggin sequence motif as in TGF-b family CKGF do- is in the same range as for nonresistant ligands mains (C-X-G-X-C and C-X-C) (Figs. 11 and (Song et al. 2010). Furthermore, mutations that 12E). Besides the six CK cysteines, Dan proteins render BMPs noggin-resistant only marginally contain two additional cysteines (designated C2 decrease their affinity to noggin (Song et al. and C8 in Fig. 11) that disulfide-link the two b- 2010; Schwaerzer et al. 2012), possibly indicat- ribbon fingers at the fingertips (Figs. 11 and ing that the inhibition mechanism is more com- 12F,G). The same eight Cys residues are shared plex in vivo. with carboxy-terminal CK (CTCK) domains, which are closely related to CKGF domains. CTCK domains have been structurally charac- The Dan Family—A Heterogeneous Group of terized in von Willebrand factor (Zhou and / Antagonists for BMPs and or Wnt Factors Springer 2014) and norrin (Ashe and Levine With seven known members in mammals, Dan 1999; Chang et al. 2015). Three long loops in modulators are the largest family of TGF-b fam- CKGF and CTCK domains link the CK residues

Figure 11. (Continued) For alignments to CKGF families with monomers dimerized in different orientations (neurotrophin in the nerve growth factor [NGF] family, and platelet-derived growth factor [PDGF] in the PDGF family) (see Zhou and Springer 2014). CKGF and CTCK monomers (PDB codes are shown in panel H ) were aligned to one another structurally using DeepAlign (Wang et al. 2013). Structurally equivalent residues are shown aligned by sequence as described in Figure 5 legend. Ca atom root mean square deviation (RMSD, A˚ )of monomers in each position relative to the average at each position are shown below the alignment. Gaps were closed to minimize the length of the alignment; corresponding positions lack an RMSD value.

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Structural Biology and Evolution of the TGF-b Family

A Dimerization Noggin X6 C3 domain C4 10-Membered C5 ring C2 C6 C1 X1 X5 X2 Fingers X4 X3 B * Clip Fingers

90°

Fingers Clip BMP-7 Clip C D Type II receptor Type II receptor Type I Type II receptor receptor GF

90° GF

Type I Type I Type I receptor Type II receptor receptor receptor

E F Sclerostin Cerberus (DAND4) Finger 2, loop 3 Loop 2 PRDC (DAND3) Gremlin (DAND2) Coco (DAND5) Finger 1, loop 1 Nbl1/Dan (DAND1) Sclerostin USAG1 CKGF domain NC

Loop 1 2 3 G PRDC H Nbl1/Dan Finger 2, loop 3 Finger 2, Loop 3 Amino terminus Additional Loop 2 disulfide Finger 1, Loop 2 loop 1 90° Finger 1, loop 1

Amino terminusB PRDCB Monomer 2

Monomer 1

PRDCA Amino terminusA

Figure 12. The bone morphogenetic protein (BMP) modulator noggin, members of the Dan family, and sclerostin. (A) Noggin binds BMPs (PDB 1M4U [Groppe et al. 2002]) in a clamp-like manner with the fingers of its cystine knot (CK) growth factor (CKGF) domain (red) blocking type II receptor binding, and a peptide segment termed clip (green) shielding the type I receptor epitopes. Hydrophobic Pro35 of noggin, engaging in the knob-into-hole interaction motif is shown as a gold sphere. The noggin dimer is formed by interactions between the a-helical dimerization domains and stabilized by an intermolecular disulfide bond (asterisk). Disulfides are shown with gold sticks and the CK motif (box) is shown in detail. (B)AsinA but viewed down the dyad axis of the noggin and GF dimers. (C and D) A prototypical BMP ligand-receptor complex, with type I and type II receptors as green and red ribbons, respectively, and the GF dimer as gray surface (PDB 2H64 [Weber et al. 2007]) shown in similar orientations as in A and B.(Legend continues on following page.)

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A.P. Hinck et al.

(GF monomer, Box 1, Fig. 11). The Dan family Furthermore, although PRDC and Nbl1 con- (Nolan et al. 2013; Nolan et al. 2015), sclerostin tain one or two Cys residues equivalent in se- (Veverka et al. 2009; Weidauer et al. 2009), and quence and structure to those in positions CTCK domains have similarly curved, long C5 and C11 in CTCK domains, they do not b-ribbon fingers (loops 1 and 3) stabilized by form equivalent disulfides. PRDC has a C5 the fingertip C2-C8 disulfide (Figs. 11A–C,E cysteine that curiously has no disulfide bond and 12F–H). Human chorionic gonadotrophin partner, whereas in Nbl1 C5 and C11 form an (HCG) in the hormone CKGF family and nog- intramolecular disulfide that contrasts with in- gin have similarly long, disulfide-stabilized termolecular disulfides in CTCK domains (Fig. fingers, but the disulfides are between non- 11A,B,E). equivalent cysteines (Fig. 11D,G,H). Loop 2 of In contrast to Dan proteins, which are TGF-b family GF domains forms the heel a- shown or considered to be dimers (Stanley et helix; in contrast, loop 2 of PRDC, Nbl1, and al. 1998; Pearce et al. 1999; Kattamuri et al. CTCK domains extends far from the CK, forms 2012), sclerostin and USAG1 are both mono- a b-ribbon in some members, and continues meric (Kusu et al. 2003; Winkler et al. 2003; the curvature of loops 1 and 3 (Figs. 11 and Lintern et al. 2009). In the absence of a dimer 12G,H). interface, loop 2 of sclerostin is not stabilized by Different loop 2 structures correlate with the van der Waals interactions and is highly flexible amount of overlap between monomers when and disordered (Fig. 12F). Although sclerostin they dimerize. TGF-b family CKGF domains was initially described as a BMP antagonist dimerize through the cysteine in position C6 (Kusu et al. 2003; Winkler et al. 2003), it func- (C6-C60 disulfide), whereas CTCK domains di- tions as an antagonist of the canonical Wnt merize through reciprocal C5-C60 and C6-C50 pathway and binds and blocks the Wnt co- disulfides and an additional C11-C110 disulfide receptor LRP5/6 (van Bezooijen et al. 2004, (Fig. 11). Because C5 and C6 are two residues 2007; Krause et al. 2010; Boschert et al. 2013). apart in a C5-X-C6-C7 sequence that includes Similarly, the close sclerostin relative USAG1 C7 of the CK, the dimer interface slides two inhibits Wnt signaling (Itasaki et al. 2003). residues in CTCK dimers so that their knots Structure–function studies show that the NXI are two b-ribbon positions further apart than motif, present in loop 2 of sclerostin and in TGF-b GF dimers. The dimer interface in the USAG1 but not the Dan clade (Figs. 11H and Dan family proteins PRDC and Nbl1 and in 12F), binds to the first b-propeller domain in HCG slides similarly to that in CTCK domains, the extracellular domain of LRP6 and thereby correlating with their similarly shaped loop 2 competes with binding of Wnt GFs (Bourhis (Fig. 11). However, these Dan proteins and et al. 2011; Holdsworth et al. 2012; Boschert HCG form dimers that are not disulfide-linked. et al. 2013).

Figure 12. (Continued)(E) Phylogenetic tree of the Dan family (upper panel) and schematic of domain organization (lower panel) with the CKGF domain boxed, its eight disulfides indicated by black lines, and the segments forming three loops colored as in panels F-H. The highly diverse segments amino- and carboxyl termini to the CKGF domain are shown as horizontal lines. (F) Sclerostin NMR structure (PDB 2KD3 [Wei- dauer et al. 2009]). The cartoon representation (upper) shows the NXI motif (red spheres) and the Ca atom trace (lower) shows the ensemble of 15 structures highlighting the flexibility of loop 2. Loops are colored as in E (lower). (G) PRDC (PDB 4JPH [Nolan et al. 2013]). The cartoon representation (upper, with one monomer colored as in E, lower) shows cysteines as gold sticks, with the unpaired Cys boxed. The lower panel shows PRDC as a surface, with the a-helical amino-terminal segments as blue Ca atom traces. Hydrophobic residues, which on exchange for alanine attenuate binding to BMP binding, are colored red. (H ) Two Nbl1/Dan crystal structures (PDB 4X1J [Nolan et al. 2015], upper) and (PDB 4YU8, Table 1, lower). One monomer is colored as in E (lower). The other monomer(s) associates as in PRDC (gray, upper) or in a different manner, with two nearby monomers in the crystal lattice shown in two shades of gray (lower).

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Structural Biology and Evolution of the TGF-b Family

In contrast to the monomeric Wnt antago- with CTCK domains and HCG (Fig. 12G) (Ke nists sclerostin and USAG1, the CTCK family et al. 2013; Zhou and Springer 2014; Chang et al. member norrin is a dimeric Wnt agonist. Nor- 2015). Additionally, fingers 1 and 2 form two rin stimulates canonical Wnt signaling by bind- b-ribbons that extend from the central four- ing frizzled4, LRP6, and glycosaminoglycans stranded b-sheet, reminiscent of the wrist in (GAGs). A synthetic heparin glycan mimetic TGF-b GFs (Nolan et al. 2013). However, in- binds to both Frizzled and the norrin fingertips, stead of an overall butterfly-shaped architecture providing a model for how ECM components like TGF-b dimers, the PRDC monomers (Fig. may contribute to the formation of signaling 11A) assemble to form a longer arc-like struc- complexes (Fig. 11E) (Ke et al. 2013; Chang ture (Fig. 12G). et al. 2015). As described in the section on the The structure of the CKGF domain of brief history of TGF-b family evolution, the Nbl1 is very similar to that of PRDC, despite TGF-b and Wnt signaling pathways coevolved only 35% sequence identity (Figs. 11A,B and in primitive metazoans. Remarkably, CKGF/ 12G,H) (Nolan et al. 2015). Like PRDC, Nbl1 CTCK domains evolved to serve as both ago- fails to form disulfide-linked dimers, with the nists and antagonists in each of these pathways. C5 and C11 cysteines forming an intramolec- The ECM has an important organizing role in ular disulfide (Fig. 11B). This is remarkable both families. Binding sites for GAGs are at the because the C5 Cys in one Nbl1 monomer is amino termini of some GFs (Ruppert et al. positioned very close to the C11 Cys of the 1996), in the prodomains of activin A and my- other monomer in the dimer, and thus only ostatin (Li et al. 2010; Sengle et al. 2011), in the slight structural adjustment would be required dimerization domain in noggin (Groppe et al. to form intermolecular disulfides similar to 2002), and along one side of the extended scle- those in the CTCK domains of VWF and nor- rostin molecule harboring residues from all rin (Fig. 11E) (Ke et al. 2013; Zhou and three loops (Veverka et al. 2009). Therefore, Springer 2014; Chang et al. 2015). Dimeriza- such sites must have evolved independently tion in all of these structures is over essentially multiple times. identical interfaces, and gives the dimers an Although all dimeric Dan proteins are effec- arc-like shape, although the Nbl1 dimer is tive BMP antagonists, coco and cerberus also more curved and shows an S-like shape com- block nodal and Wnt signaling (Glinka et al. pared with PRDC (Fig. 12G,H, upper) (Nolan 1997; Piccolo et al. 1999; Bell et al. 2003). Cer- et al. 2015). Both PRDC and Nbl1 form non- berus binds and inhibits Wnt, nodal and BMPs covalent dimers in solution (Kattamuri et al. at mutually noncompetitive sites. Furthermore, 2012). However, another structure of Nbl1 the amino-terminal portion of cerberus is re- (PDB entry 4YU8, Table 1) fails to show the quired for BMP binding and Wnt antagonism same dimerization interface in crystal lattices whereas the carboxy-terminal portion includ- (Fig. 12H, lower). ing the CKGF domain is sufficient for nodal Without structural data on a Dan protein antagonism (Piccolo et al. 1999). bound to BMP or nodal, it is unclear whether The prototypical Dan protein PRDC forms Dan proteins inhibit GF signaling by impeding a homodimer that is not disulfide-linked, de- binding of both receptors like noggin, or of only spite its odd number of Cys residues (Fig. one receptor type, as shown for Tsg (Zhang et al. 12G) (Nolan et al. 2013). Dimerization of 2007). Furthermore, the architecture of PRDC PRDC is between extremely long b-strands in and Nbl1 differs from other structurally charac- each monomer that begin in loop 1 and extend terized BMP modulators and lacks C-clamp- through the CK into loop 2 (Figs. 11 and 12G). or clip-like structures. However, a contiguous These join with antiparallel b-strands in the patch of hydrophobic residues on the convex other halves of loops 1 and 2 to form an ex- surface of PRDC is required for binding and tensive “intermolecular” four-stranded b-sheet, inhibition of BMP signaling in vitro (Fig. 12G, and build a large dimer interface in common lower) (Nolan et al. 2013). Several of these

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residues are conserved in Nbl1, which binds or 315, respectively, thus creating further com- BMP-2 and 7 and GDF-5 with different affini- plexity. They bind activin with different af- ties compared with PRDC. The importance of finities, and longer isoforms partially lack the these residues in encoding BMP selectivity was ability to bind to heparin and proteoglycans shown by exchange of patch residues from (Kumar 2005). PRDC into Nbl1, which yielded an Nbl1 variant Structures of follistatin and FSTL-3 bound mimicking PRDC (Nolan et al. 2015). Interest- to activin A or myostatin provide insights ingly, in PRDC the hydrophobic patch respon- into inhibitory mechanisms and specificity sible for BMP binding seems to be covered by a (Thompson et al. 2005; Harrington et al. dynamic a-helix in the amino-terminal seg- 2006; Cash et al. 2009, 2012). Although follista- ment (Fig. 12G, lower). A similar helix is not tin and FSTL-3 block type I and type II receptor observed in the Nbl1 structure (Fig. 12H) (No- binding sites, they bind the GF domain vastly lan et al. 2015). differently from noggin (Figs. 12A and 13A). Follistatin wraps around activin on the perim- eter of the dyad (two-fold rotation axis) of the Follistatin Antagonizes Activins as Well GF dimer (Fig. 13B). In contrast, noggin binds as BMPs on one side of the dyad axis. Viewed normal to Follistatin (Fst) was initially described as a pro- this axis (as in Fig. 12A), noggin resembles a tein that antagonizes activin signaling. How- C-shaped clamp with the ends of the clamp ever, follistatin also binds BMPs and GDFs, in- gripping the GF fingers. In the case of follistatin, cluding BMP-2, 4, 6, and 7 as well as myostatin/ two modulator molecules embrace the GF such GDF-11, albeit with lower affinities (Iemura that almost its entire surface seems covered (Fig. et al. 1998; Amthor et al. 2002, 2004; Glister 13A,B). In contrast to noggin, follistatin is a et al. 2004; Schneyer et al. 2008). Fst is a mod- monomer when not bound to the GF. However, ular protein comprising a unique 65-residue on binding, two follistatin monomers (struc- amino-terminal domain (Fs0) followed by three tures of follistatin 288 are shown) tightly inter- follistatin domains (Fs1, Fs2, Fs3) of 70 to 85 act in a head-to-tail fashion and form a stable, residues each (Fig. 13A,B). Follistatin domains noncovalent dimer (Thompson et al. 2005). are in turn each composed of EGF-like and The type I receptor epitope of activin is blocked Kazal subdomains (Hohenester et al. 1997; by the unique amino-terminal domain (Fs0) of Schneyer et al. 2004). follistatin (Fig. 13A). Here, the key element is Fs domains together with other domains are Phe52 located in the second a-helix of the Fs0 present in follistatin-like (FSTL) proteins. Al- domain, which makes a knob-into-hole inter- though FSTL-1 and 3 are known to regulate action with the wrist epitope of activin A simi- activin and BMP signaling, FSTL-4, FSTL-5, larly to type I receptors that bind BMPs (Hatta SMOC1, and SMOC2 are not yet characterized et al. 2000; Keller et al. 2004). Fs1, and primarily for effects on activin and BMP signaling. Fur- Fs2, shield the type II receptor epitope by main- thermore, SMOC1 and SMOC2 contain addi- ly using hydrophobic interactions as found in tional domains characteristic of the SPARC complexes of activins and BMPs bound to their family (Schneyer et al. 2004; Bradshaw 2012). type II receptors (Fig. 13A,B). The final domain, The follistatin family thus includes modulators Fs3, wraps around the fingertips of activin A with similar mechanisms of action (binding to and engages in predominantly polar contacts GFs and blocking receptor binding) and similar with the amino-terminal Fs0 domain of the specificities for activins and BMPs, yet its mem- other follistatin monomer, thus stabilizing en- bers are built from several types of domains circlement of the ligand (Fig. 13A,B). that are assembled into distinctive architectures. The selectivity of follistatin for activins and Three isoforms of follistatin, Fst288, Fst303, and BMPs is explained by a central core of GF amino Fst315 have identical amino-terminal regions, acids in the binding sites for the Fs0 and Fs1/2 but end at carboxy-terminal residues 288, 303, domains that are conserved in activins and

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Structural Biology and Evolution of the TGF-b Family

A B Fs3 Fs1 Fs3 Follistatin domain B Fs0 domain Fs3 Fs1 90° domain

Fs2 Myostatin Fs2

Fs0 Fs2 domain domain Fs3 Myostatin Fs0 FollistatinA Fs1

Follistatin Fs1-2 CDEFSLT3 3

2 1

Activin A Activin A

F G Subdomain 1 β1 β3 Chordin VWC CHRD CHRD CHRD CHRD VWC VWC VWC β1′ β β1′′ Clip 2 β β CHL-1/CHL-2 VWC VWC VWC β3 2 1 TIL Kielin VWC VWC VWC VWC VWC VWC VWC VWC VWC VWD CCN1-6 IGFBP VWC TSP CKGF TIL crossveinless 2 VWC VWC VWC VWC VWC VWD

Collagen IIA CV2 VWC1 VWC H I

CV2 VWC1 CV2 VWC1 CV2 VWC1bound CV2 VWC1free Subdomain Clip 1 Subdomain 1 BMP-2 Clip

Subdomain 2

Clip Subdomain 2

Figure 13. Modulators of the follistatin and chordin families. (A,B) Two follistatin-288 molecules shown in ribbon cartoon wrap around myostatin shown as a surface (PDB 3HH2 [Cash et al. 2009]). Fs0 (green) blocks access to the type I receptor epitope (light green) with a knob-into-hole interaction indicated by a gold sphere; Fs1 (red) and Fs2 (magenta) block the type II receptor-binding site (pink). (C–E) Open and closed confor- mations of activin A. (C,D) Shows activin monomers as light and dark gray surfaces in different orientations (dashed lines) in an open complex with an Fs1-2 fragment of follistatin (C, PDB 2ARP [Harrington et al. 2006]) and in a closed conformation with FSTL3 (D, PDB 3B4V [Stamler et al. 2008]). (E) Compares monomer– monomer orientations in activin A and myostatin dimers by superimposing on one monomer (gray) and showing the orientation of the other monomer (colored and numbered). The colored and numbered monomers are from dimeric structures of myostatin bound to follistatin-288 (red, 1), activin A bound to FSTL3 (cyan, 2) and activin-A bound to the follistatin Fs1-2 fragment (blue, 3). (Legend continues on following page.)

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A.P. Hinck et al.

BMPs but not in TGF-bs (Thompson et al. Chordin Family Members Exert a Dual 2005). The contribution of individual follistatin Modulator Function with Both Pro- and domains to binding differs depending on the Anti-BMP Activities GF (Cash et al. 2009). For example, when bound to myostatin compared with activin A, the Members of the chordin family are modular Fs0 domain shifts more into the GF wrist epi- proteins with VWC domains (Fig. 13F). VWC tope to better interact. To avoid a clash with domains (Bork 1993), also confusingly known Fs0, Fs3 in the other monomer shifts away as cysteine-rich domains in chordin (Francois from myostatin. Additionally, the ability of et al. 1994; Sasai et al. 1994), are 60 to 75 residues activin A to show both closed and open con- long and are found, often in multiple tandem formations (see section on GF structures) is re- repeats, in .70 human proteins, including the flected in differences in monomer–monomer TGF-b family modulator proteins chordin, ven- orientation in complexes with different follista- troptin, chordin-like 2, kielin, and crossveinless tin fragments (Fig. 13C–E) (Thompson et al. 2 (CV2, also known as BMP endothelial regu- 2003, 2005; Greenwald et al. 2004; Harrington lator or BMPER) (Fig. 13F) (Garcia Abreu et al. et al. 2006). 2002). Chordin is a large multidomain pro- The Fs0 domain in follistatin and FSTLs may tein that contains chordin (CHRD) domains play a role in discriminating between high-affin- in addition to VWC domains (Fig. 13F). Expres- ity ligands (i.e., activins, myostatin, and GDF- sion of chordin fragments in embryos showed 11), and low-affinity binders i.e., BMP-2, 4, 7, that BMP’s dorsalizing activity and BMP bind- and others). The type I receptor ALK3 still in- ing reside in its VWC domains (Larrain et al. teracts with BMP-2 when the latter is bound to 2000). Structures of VWC domains in collagen follistatin, suggesting that the Fs0 domain does IIA and CV2 show two subdomains (Fig. not bind to or cannot block the wrist epitope in 13G,H) (O’Leary et al. 2004; Zhang et al. BMPs (Iemura et al. 1998). Lacking this interac- 2008). Subdomain 2 is highly dynamic (Fig. tion might also explain the lower affinities of 13I, right) (O’Leary et al. 2004), which may be follistatin binding to BMPs and indicate that related to the finding that six tandem VWC do- the inhibition of BMPs by follistatin is function- mains in von Willebrand factor give rise to a allydifferent from that of activins (see also Stam- region with an extended, highly flexible struc- leret al. 2008). A role for the Fs3 domain in BMP ture (Springer 2014). binding is suggested by the finding that FSTL-3, An amino-terminal fragment of CV2 bound which lacks Fs3 and thus cannot fully encircle to BMP-2 exemplifies how a chordin family activin A and myostatin (Fig. 13D) (Stamler member binds and inhibits BMPs (Fig. et al. 2008; Cash et al. 2012), binds activin A 13G,H) (Zhang et al. 2008). The CV2 fragment and myostatin with high affinity, but cannot comprises an amino-terminal segment (termed bind BMPs (Sidis et al. 2006). clip, as in the structurally distinct noggin) and

Figure 13. (Continued)(F) Chordin family members are mosaics of tandem domains. Although von Willebrand factor C (VWC) domain(s) mediate bone morphogenetic protein (BMP) binding, other domains present include chordin (CHRD), von Willebrand type D (VWD), trypsin inhibitor-like (TIL), insulin-like growth factor binding protein (IGFBP), thrombospondin (TSP) and cystine knot growth factor (CKGF). (G)VWC domain of collagen IIA (left) and VWC domain 1 of CV2 (right). Amino-terminal peptide segments and VWC subdomains 1 and 2 are green, red and blue, respectively. A structurally variable segment of VWC subdomain 1 is shown in yellow. (H ) The VWC1 domain of CV2 shown in ribbon with knob-into-hole residue shown as gold sphere bound to BMP-2 shown as surface with type I and type II receptor sites colored green and pink, respectively (PDB 3BK3 [Zhang et al. 2008]). (I) Similarity in architecture of VWC1 domain of CV2 bound to BMP-2 in crystals (left) and free in solution (right panel, NMR ensemble of 10 structures, PDB 2MBK [Fiebig et al. 2013]).

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Structural Biology and Evolution of the TGF-b Family

VWC domain 1. In the 2:2 complex, the clip tic (anti-BMP) (Francois et al. 1994) as well as folds into the type I receptor epitope, whereas agonistic (pro-BMP) (Ross et al. 2001) roles the amino-terminal subdomain of VWC1 binds depending on context (Larrain et al. 2001). the type II receptor site, showing how CV2 in- For instance, very strong expression of CV2 in hibits BMP binding to both types of receptors Drosophila occurs during formation of the wing (Zhang et al. 2007, 2008). The interaction mode crossvein, which requires high BMP signaling resembles attachment of a paperclip (CV2) to a activity (Conley et al. 2000). This dual activity sheet of paper (BMP) with the amino-terminal might be explained for chordin by formation peptide and VWC subdomain 1 folding over of ternary complexes with BMPs and Tsg, opposite sides of the GF finger (Fig. 13H), and which by itself is a modulator protein that can shares some similarities with the noggin–BMP block BMP signaling (Oelgeschlager et al. 2000; interaction (Fig. 12A). In both cases, a linear Chang et al. 2001; Ross et al. 2001; Scott et al. peptide in an amino-terminal segment, clip, 2001). Ternary complexes might be transported efficiently interferes with type I receptor bind- to locations with low BMP expression, in which ing, and a more globular domain occupies the the pro-BMP effect is then mediated when the type II receptor binding site. If the clip were GF is released from the complex (Ashe and Le- highly flexible in the unbound state, it would vine 1999; De Robertis et al. 2000; Harland be puzzling how this short motif could contrib- 2001). Localized release is achieved when chor- ute significantly to BMP binding. However, din is proteolytically cleaved by zinc metallo- NMR analysis of the VWC1 domain of CV2 in proteases (Piccolo et al. 1997). In this context, its unbound state reveals that the clip segment Tsg binds and neutralizes the resulting chordin is pre-oriented to form a hook that facilitates fragments, which otherwise would block BMP cooperative binding to BMP-2 (Fig. 13I) (Fiebig signaling (Larrain et al. 2001). Thus, in vivo Tsg et al. 2013). can toggle chordin between a BMP-antagonistic In contrast to Dan family members, most and a BMP-agonistic function, and therefore VWC domains and most mosaic proteins with functions as a switch rather than inhibitor as VWC domains do not act as BMP antagonists. observed in vitro. CV2 can similarly interact Furthermore, the various VWC domains in with Tsg to yield a stronger BMP antagonist chordin, chordin-like 2 and CV2 (Fig. 13F) (Zakin et al. 2008). However, its pro-BMP role bind BMPs with varying affinity, and some do is due to its ability to directly interact with chor- not bind BMPs at all (Zhang et al. 2007). Among din and chordin-Tsg-BMP complexes (Ambro- the five VWC domains of CV2, only VWC1 sio et al. 2008). By binding to the ECM via its binds BMPs, and among the four VWC domains heparin-binding site, CV2 does not diffuse far of chordin, only the first, third and the fourth from its site of expression and can thus seques- interact with BMPs (Zhang et al. 2007). Further- ter and locally concentrate chordin–Tsg–BMP more, some VWC domains only bind to the type complexes, which when destroyed by tolloid, I receptor site, and some interact with only the lead to a highly focused release of BMP ligand type II receptor site, whereas others, like the first (Ambrosio et al. 2008; Zakin et al. 2010; for VWC domain of CV2, block both sites (Zhang review, see O’Connor et al. 2006). Binding of et al. 2007, 2008). Moreover, binding architec- Tsg and CV2 to chordin fragments blunts vir- tures differ. For instance, CV2 and chordin-like tually all residual BMP-antagonistic activities of 2 bind BMPs symmetrically in 2:2 complexes, these three modulator proteins. Notably, CV2 whereas chordin interacts with BMPs in a 1:2 binds to chordin’s second VWC domain, which asymmetric complex (Zhang et al. 2007). itself does not bind BMPs. In CV2 a complex Additionally, chordin family members reg- epitope comprising the first four VWC domains ulate BMP activity by a mechanism far more is responsible for binding to chordin (Zhang complex than for any other modulator family et al. 2010). These findings highlight the multi- (Ashe and Levine 1999). Chordin and family faceted binding properties of the VWC domains members exert dual roles, displaying antagonis- within the chordin family.

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This section has shown the high structural advance in this area shows how RGM family diversity of BMP modulators. Binding to a members bind GFs, yet lacks a bound type II shared interaction partner, in this case BMPs, receptor in the structure, and raises ques- does not require identical protein structures, tions by suggesting that type I receptors do can be achieved by different domains and not bind until reaching an intracellular, mem- architectures, and has evolved independently brane-enveloped compartment. This finding multiple times. The diversity of interaction blurs the line between extracellular and intra- partners also highlights the hub-like character cellular signaling. Work on modulator proteins of the TGF-b/BMP GFs, which they recognize. has revealed how structurally diverse antago- As the structures of many modulator pro- nists including follistatins, noggin, and chordin teins including Tsg and members of the multi- fragments can bind GFs, although complexes domain CCN family have not yet been deter- with the Dan family remain elusive. We still mined, even greater structural diversity among lack ternary complexes with other ECM com- BMP binding partners is likely to be discovered ponents, and structural understanding of how in the future. chordin members can both antagonize and ag- onize BMP signaling. One of the greatest strengths in the field, PERSPECTIVE the use of recombinant proteins, also led to This review summarized our knowledge of one of its greatest weaknesses, the current pau- the structures of proteins that regulate TGF-b city of work on physiological protein complex- family signaling in extracellular environments, es from tissues. Many TGF-b family and mod- and illustrated how the CKGF domain evolved ulator proteins were discovered by molecular to both agonize and antagonize TGF-b family cloning or genetics as cDNAs or genes, and and Wnt signaling. Structures of prodomain the glycoproteins they encode have not been complexes with GF dimers suggest a new para- characterized in vivo. Weaknesses in structural digm for regulation of release of GFs by conver- characterization include a lack of complex sion from crossed-arm to open-arm procom- structures showing how procomplexes, GFs, plex conformations. TGF-b family and other and modulator proteins bind ECM compo- pathway components evolved in the earliest nents to form signaling hubs and regulate ex- metazoans, and integrin- and force-dependent tracellular signaling. Additionally, all structural release of the TGF-b GF from the procomplex studies to date on receptor complexes used ho- evolved in deuterostomes. However, how GF re- modimers, even though heterodimers are im- lease from other procomplexes occurs in vivo portant, and structural biology shows that the remains unclear. The conformational change type I receptor binds the interface between the model requires extension beyond the two exem- two GF monomers. The two such type I sites plary procomplexes reported, and hypotheses on a heterodimer would be distinct from one that binding to ECM components or prodo- another, as well as from those in homodimers. main cleavage by proteases regulate procomplex In vivo, the type I and type II receptors in the conformational changes and GF release need to receptor–ligand complex need not be the same be tested. The final extracellular step in signaling at the two sites on a GF dimer, even for homo- activation (i.e., binding of TGF-b family GFs dimers, creating further complexity in signal- to type I and type II receptors at the cell surface) ing. Structures of such asymmetric complexes is well characterized structurally with ternary with homo- and heterodimeric ligands are par- complexes. These studies enable insight into ticularly challenging. Furthermore, an under- the specificity and promiscuity in extracellular standing of signaling hubs in the extracellular signaling, but await characterization of recep- environment will require new tools and tech- tor complexes with biological GF heterodimers. niques for isolating hub proteins from, and Furthermore, structural understanding of how characterizing them in, their native environ- coreceptors bind GFs remains incomplete. One ments.

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Structural Biology and Evolution of the TGF-β Family

Andrew P. Hinck, Thomas D. Mueller and Timothy A. Springer

Cold Spring Harb Perspect Biol published online September 16, 2016

Subject Collection The Biology of the TGF-β Family

Transcriptional Control by the SMADs Structural Biology and Evolution of the TGF-β Caroline S. Hill Family Andrew P. Hinck, Thomas D. Mueller and Timothy A. Springer TGF-β Signaling from Receptors to Smads Structural Basis of Intracellular TGF-β Signaling: Akiko Hata and Ye-Guang Chen Receptors and Smads Apirat Chaikuad and Alex N. Bullock Agonists and Antagonists of TGF-β Family Signaling Receptors for TGF-β Family Members Ligands Carl-Henrik Heldin and Aristidis Moustakas Chenbei Chang Activins and Inhibins: Roles in Development, The Discovery and Early Days of TGF-β: A Physiology, and Disease Historical Perspective Maria Namwanje and Chester W. Brown Harold L. Moses, Anita B. Roberts and Rik Derynck Regulation of the Bioavailability of TGF-β and Bone Morphogenetic Proteins TGF- β-Related Proteins Takenobu Katagiri and Tetsuro Watabe Ian B. Robertson and Daniel B. Rifkin TGF-β and the TGF-β Family: Context-Dependent Roles in Cell and Tissue Physiology Masato Morikawa, Rik Derynck and Kohei Miyazono

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