Cooperative Assembly of TGF-β Superfamily Signaling Complexes Is Mediated by Two Disparate Mechanisms and Distinct Modes of Receptor Binding

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Cooperative Assembly of TGF-β Superfamily Signaling Complexes Is Mediated by Two Disparate Mechanisms and Distinct Modes of Receptor Binding
  Molecular Cell  Article Cooperative Assembly of TGF- b  Superfamily Signaling Complexes Is Mediated by Two DisparateMechanisms and Distinct Modes of Receptor Binding Jay Groppe, 1,4, * Cynthia S. Hinck, 1 Payman Samavarchi-Tehrani, 2 Chloe Zubieta, 3 Jonathan P. Schuermann, 1  Alexander B. Taylor, 1 Patricia M. Schwarz, 1 Jeffrey L. Wrana, 2, * and Andrew P. Hinck 1, * 1 Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229, USA  2 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada 3 Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CA 94025, USA  4 Present address: Department of Biomedical Sciences, Baylor College of Dentistry, Texas A&M University System Health Science Center,3302 Gaston Ave, Dallas, TX 75246, USA.*Correspondence: (J.G.), (J.L.W.), (A.P.H.) DOI 10.1016/j.molcel.2007.11.039 SUMMARY  Dimeric ligands of the transforming growth factor- b (TGF- b  ) superfamily signal across cell membranesin a distinctive manner by assembling heterotetra-meric complexes of structurally related serine/threo-nine-kinase receptor pairs. Unlike complexes of thebone morphogenetic protein (BMP) branch thatapparently form due to avidity from membrane local-ization, TGF- b  complexes assemble cooperativelythrough recruitment of the low-affinity (type I) recep-tor by the ligand-bound high-affinity (type II) pair.Here we report the crystal structure of TGF- b 3 incomplex with the extracellular domains of both pairsof receptors, revealing that the type I docks and be-comestetheredviauniqueextensionsatacompositeligand-type II interface. Disrupting the receptor-receptor interactions conferred by these extensionsabolishes assembly of the signaling complex andsignal transduction (Smad activation). Althoughstructurally similar, BMP and TGF- b  receptors bindin dramatically different modes, mediating gradedand switch-like assembly mechanisms that mayhave coevolved with branch-specific groups of cyto-plasmic effectors. INTRODUCTION Secretedpolypeptidegrowthfactorsfromdiversefamiliessignalby inducing the assembly of cell-surface transmembrane recep-tors.Inmanycases,identical orsimilarreceptorsbindattwodif-ferentsites,onehighaffinityandonelowaffinity,andassemblyismediated by one of several mechanisms: (1) allosteric, ligand-dependent conformational change in the receptor extracellulardomain that induces receptor-receptor contacts, as exemplifiedby the EGF receptor complex ( Schlessinger, 2002 ), (2) recruit-ment of the second receptor at a lower affinity site by coopera-tive ligand and receptor-receptor interactions as shown for thehuman growth hormone receptor complex ( Wells, 1996 ), and (3)enhanced avidity resulting from membrane localization and re-ceptorpreorientationor‘‘presentation’’( Grasbergeretal.,1986 ).The transforming growth factor- b  (TGF- b  ) superfamily is com-posed of two major branches represented by the prototypicTGF- b s and bone morphogenetic proteins (BMPs) ( Massague´ ,1998; Massague´  et al., 2000 ). TGF- b s are key regulators of cel-lular proliferation, adhesion, extracellular matrix deposition,and the immune system. BMPs, well-known inducers of osteo-genesis, are also considered ‘‘body’’ morphogenetic proteinsdue to their pleiotropic roles in embryonic patterning and mor-phogenesis ( Hogan, 1996; Reddi, 2005 ). TGF- b  superfamilyligands signal by bringing together two pairs of structurally re-lated receptors, designated types I and II ( Wrana et al., 1992;Wrana et al., 1994 ). The extracellular domains share a single‘‘three-finger toxin’’ fold ( Greenwald et al., 1999 ) linked by trans-membrane helices to cytoplasmic serine/threonine kinase do-mains, in contrast to receptors for other growth factors (EGF,FGF, NGF, PDGF, VEGF, and insulin/IGF) often composed of tandem immunoglobulin-like scaffolds linked or associatedwith tyrosine kinase domains.In the TGF- b  paradigm, the type II receptor binds with highaffinity and is responsible for cooperative recruitment and trans-phosphorylation of its low-affinity type I pair ( Wrana et al., 1992;Wrana et al., 1994 ). Either allosteric or direct receptor-receptorinteractions are hypothesized to impart cooperativity to thismacroscopically stepwise mechanism ( Hart et al., 2002; Shiand Massague´ , 2003; Wrana et al., 1994 ). However, cell-basedassays ( Brummel et al., 1994; Koenig et al., 1994; Letsou et al.,1995; Liu et al., 1995; Nohno et al., 1995; Penton et al., 1994;Yamashita et al., 1995 ) and binding studies with extracellulardomains (EDs) ( Greenwald et al., 2003; Hatta et al., 2000; Kirschet al., 2000a; Natsume et al., 1997; Sebald et al., 2004 ) demon-strated that the large assortment of BMP receptors have mixedaffinities for their ligands. For example, ActRII has moderateaffinity for BMP-7 and interacts weakly with BMP-2, whereasBMPRIA binds BMP-2 with high affinity and BMP-7 weakly( Sebald et al., 2004 ). In addition, superposition of the BMP-2:BMPRIA-ED ( Kirsch et al., 2000b ) and BMP-7:ActRII-ED Molecular Cell  29 , 157–168, February 1, 2008 ª 2008 Elsevier Inc.  157  complex structures revealed that the extracellular domainsof the two receptor types neither interact nor induce significantconformational change ( Greenwald et al., 2003 ), confirmed sev-eralyearslaterbydeterminationofcrystalstructuresoftwoBMPternary complexes (  Allendorph et al., 2006; Weber et al., 2007 ).Thus avidity from membrane localization is theorized to promoteassembly of BMP signaling complexes in vivo ( Greenwald et al.,2003; Sebald and Mueller, 2003; Sebald et al., 2004 ).In marked contrast to the autonomous interactions of BMPreceptors, assembly of the TGF- b :T b RII-ED:T b RI-ED complexis sequential and cooperative ( Zu´n˜iga et al., 2005 ). Furthermore,TGF- b s interact with their type II receptor in a manner distinctfrom BMPs, the three-finger toxin scaffold binding with its‘‘knuckles’’ rather than ‘‘fingers’’ at a separate site ( De Cre-scenzo et al., 2006; Hart et al., 2002 ). T b RII also has a uniqueN-terminal extension of 25 disordered residues not required forligand binding ( Boesen et al., 2002a, 2002b; Hart et al., 2002 ).T b RI is predicted to be structurally similar to BMPRIA with theexception of a small loop, the prehelix extension ( Harrisonet al., 2003 ), peculiar to T b RI and ActRIB. Like all type I recep-tors, T b RI has been anticipated to bind in a mode similar toBMPRIA ( Hart et al., 2002; Lin et al., 2006; Shi and Massague´ ,2003; Zu´ n ˜ iga et al., 2005 ). However, in light of the disparatemodes observed for type II receptors, the validity of a commontype I site is questionable ( Sun, 2003 ). Moreover, althoughmembrane attachment is clearly not required for assembly of the TGF- b  ternary complex ( Zu´ n ˜ iga et al., 2005 ), the structuralbasis for the stepwise mechanism has remained uncertain.Here we show that the TGF- b  type I receptor is recruited inahighlycooperativemannerbydirectreceptor-receptor contactatacompositeligand-typeIIinterface.Theligandbindingmodesof T b RI and BMPRIA are distinct, with T b RI interacting largelythrough its hallmark prehelix extension. The disordered N-termi-nal extension of T b RII becomes partially structured in the com-plex, tethering T b RI to its docking site at the composite inter-face. In vitro and cell-based assays show that the T b RII tether,not required for ligand binding, is crucial for ternary complexassembly and signal transduction. Thus two evolutionarily minormodificationsofthree-fingertoxinscaffolds,extensionsofaloopand the N terminus of the type I and type II receptors, respec-tively, conferred a highly cooperative mechanism of assemblyon the TGF- b  signaling complex. RESULTSCooperative Assembly In Vitro Consistent with the stepwise assembly on the cell surface( Wrana et al., 1992, 1994 ) and in solution with the extracellulardomains ( Zu´n˜iga et al., 2005 ), surface plasmon resonance(SPR) binding assays showed that recruitment of T b RI-ED bythe TGF- b 3:T b RII-ED complex is highly cooperative. T b RII-EDbound to TGF- b 3 immobilized on the surface of the sensorchip with an apparent K D  of   0.5  m M, similar to previous studies( De Crescenzo et al., 2006 ) ( Figure 1 A). In contrast, T b RI-ED atconcentrations up to 70  m M produced only a minimal responsedue to its extremely low affinity (   188  m M) for the ligand alone( Figure 1B). However, the affinity of T b RI-ED for the binary com-plex of TGF- b 3:T b RII-ED was  0.6  m M, an enhancement of over300-fold ( Figure 1C). Unlike T b RII-ED, which equilibrated rapidlybased on the sensogram profiles, T b RI-ED associated and dis-sociated more gradually, suggestive of induced fit rather thanrigid body binding.T b RI-EDwasrecruitedinananalogousmannerbybinarycom-plexes of T b RII-ED and TGF- b s 1 and 2 ( Table S1 available on-line). Despite the significantly lower affinity of T b RII for TGF- b 2( Cheifetz et al., 1990; De Crescenzo et al., 2006 ), recruitmentof T b RI-ED by the TGF- b 2:T b RII-ED complex was enabled bythe high concentration of T b RII-ED in the flow, in agreementwith our previous finding that the three ligand isoforms induceassembly in the same general manner ( Zu´ n ˜ iga et al., 2005 ). Toestablish the structural basis for this pronounced cooperative Figure 1. T b RI-ED Is Cooperatively Recruited by the TGF- b 3:T b RII-ED Binary Complex In Vitro Surface plasmon resonance (SPR) sensograms of (A) T b RII-ED binding toTGF- b 3, (B) T b RI-ED binding to TGF- b 3 alone, and (C) T b RI-ED binding toTGF- b 3 in complex with T b RII-ED. Black bars above sensograms mark pe-riod of injection of receptors into the flow at the range of concentrations colorcoded on the right. Molecular Cell TGF- b  Receptor Complex Assembly 158  Molecular Cell  29 , 157–168, February 1, 2008 ª 2008 Elsevier Inc.  effect, we determined the crystal structure of the TGF- b 3:T b RII-ED:T b RI-ED ternary complex. Structure Determination The ternary complex (relative molecular mass 78,000; M r  78K),composed of two T b RI-EDs, two T b RII-EDs, and one TGF- b 3dimer, was isolated by size-exclusion chromatography andcrystallized near neutral pH. The structure was solved at 3.0 A  ˚ resolution by molecular replacement using crystal structures of free TGF- b 3 ( Mittl et al., 1996 ) and TGF- b 3-bound T b RII-ED( Hart et al., 2002 ) successively as search models ( Table 1 ). T b RI-ED model building was facilitated by placing the BMPRIA-ED model ( Kirsch et al., 2000b ) in an MRSAD-phased electrondensity map ( Schuermann and Tanner, 2003 ) and by aligningcysteine sulfur positions with their anomalous signals ( Groppeet al., 2002 ) ( Figure S1 ), which also provided independent verifi- cation of the position of T b RI-ED in the complex. Structure of the TGF- b 3:T b RII-ED:T b RI-EDTernary Complex TheheterotetramericreceptorcomplexcontainsonepairoftypeII receptor extracellular domains bound in a wedge-like fashionbetween the fingertips of the dimeric ligand and one pair of type I receptor extracellular domains docked in clefts of twocomposite ligand-type II receptor interfaces ( Figure 2 A). In con-trast to the open conformation previously observed in complexwith T b RII ( Hart et al., 2002 ), TGF- b 3 adopts the closed confor-mation seen in crystal structures of the free ligand ( Mittl et al.,1996 )( Figure2B).SterichindranceorvanderWaalscontactsbe- tweenboundT b RIandthemajorhelix(  a 3)mayrestricttheligandto the closed state in the ternary complex. Alternatively, theequilibriumbetweenopenandclosedstatesmightbepHdepen-dent, with the open state predominating at pH 4 and below ( Hartet al., 2002; Bocharov et al., 2002 ) and the closed state at pH 5and above ( Mittl et al., 1996 ).T b RIIbinds atthefingertips ofTGF- b 3asintheTGF- b 3:T b RII-ED complex, without any significant change in conformation orinteraction at the type II receptor-ligand interface ( Figure 2B).Relative to the structure of the free ligand, T b RII appears toinduce a small displacement of the long finger of TGF- b 3 uponbinding. The key interactions shown to stabilize the binary com-plex, two hydrogen-bonded ion pairs bordering a hydrophobicarray( DeCrescenzoetal.,2006;Hartetal.,2002 ),arepreservedin the ternary complex. The most striking difference lies withseven residues of the flexible N-terminal extension of T b RIIthatbecomeorderedintheternarycomplex,providingextensivereceptor-receptor contact ( Figure 2 A and Figure S2 ). T b RI shares the three-finger toxin fold shown for the BMPtype I receptor BMPRIA ( Kirsch et al., 2000b ) as well as the typeII receptors T b RII ( Boesen et al., 2002b; Hart et al., 2002 ), ActRII( Greenwald et al., 1999 ), ActRIIB ( Thompson et al., 2003 ), and BMPRII ( Mace et al., 2006 ). Modest differences with respect tothe lengths and conformations of the three fingers are seen be-tween T b RI and BMPRIA (Figures 2C and 3 ). However T b RI con-tains an additional five residues in a loop preceding a single-turnhelix of BMPRIA referred to as the ‘‘prehelix extension’’ (Figures2C and 3, red). These additional residues, which include two highly conserved flanking prolines, form a sharply curved, finger-like projection that plays a central role in docking T b RI at acomposite TGF- b 3:T b RII interface. Ligand Binding Modes of T b RI and BMPRIA Are Distinct Hypothetical models of the TGF- b  ternary complex created bysuperposition of the TGF- b 3:T b RII-ED and BMP-2:BMPRIA-EDcomplexes have been based on the assumption that all TGF- b superfamily type I receptors bind ligand in a similar fashion ata common site ( Hart et al., 2002; Lin et al., 2006; Shi and Mas-sague´ , 2003; Zu´ n ˜ iga et al., 2005 ). A flexible loop preceding the a 3 helix of the ligands, the prehelix loop, has been proposedas the conserved recognition motif for all type I receptors of the superfamily ( Keller et al., 2004 ). However, in keeping withstructure-based sequence alignment of the ligands ( Scheufleret al., 1999 ), superposition of TGF- b 3 and BMP-2 in the BMP-2:BMPRIA-ED complex shows that TGF- b s lack three residuescomprising a key structural element of the prehelix loop of BMPs ( Figure 4 A). Upon binding, this BMP-specific segment Table 1. Crystallographic Data Data Collection StatisticsData collection site SSRL FRD/in-houseSpace group P6 5 22 P6 5 22Unit cell parameters a = b = 66.92 A  ˚ ,c = 254.36 A  ˚ a = b = 66.37 A  ˚ ,c = 257.76 A  ˚  a  =  b  = 90.00  , g  = 120.00  a  =  b  = 90.00  , g  = 120.00  Wavelength (A  ˚  ) 0.979 1.542Resolution range (A  ˚  ) 50.0–3.0 50.0–4.1Number of observations 64,333 107,298Number of unique reflections 6710 5017Completeness (%) 90.3 (100) a 99.9 (100) a  Anomalous completeness (%)    98.8 (98.4) a Mean I/  s  (I) 26.2 (4.2) a 46.32 (17.49) a R sym  on I (%) 7.8 (31.4) a 10.8 (30.6) a Highest resolution shell (A  ˚  ) 3.29–3.0 4.25–4.10Refinement StatisticsResolution range (A  ˚  ) 28.8–3.0R work  /R free  (%) 24.2/29.7Number of protein atoms 2328Number of water atoms 9Mean B factors protein (A  ˚  2  ) 51.7Mean B factors water (A  ˚  2  ) 70.7Rmsd bond lengths (A  ˚  ) 0.006Rmsd bond angles (    ) 0.809Ramachandran Plot Statistics b Most favored regions (%) 86.5 Additionally allowed (%) 13.2Generously allowed (%) c 0.4 a Highest resolution shell. b Non-Gly/Proresidues (Procheck);for all residues, 93.5% adopt favoredconformations, no outliers (MolProbity). c TGF- b 3 Asn42. Similar conformation established for this residue in the2.0 A  ˚  structure of the free ligand ( Mittl et al., 1996 ). Molecular Cell TGF- b  Receptor Complex Assembly Molecular Cell  29 , 157–168, February 1, 2008 ª 2008 Elsevier Inc.  159  Figure 2. Structure of the TGF- b 3:T b RII-ED:T b RI-ED Ternary Complex (A) Ribbon diagram (above) and surface representation (below) portraying the ligand-receptor and receptor-receptor interfaces in the TGF- b  ternary complex.Identical monomers of the homodimeric ligand are distinguished as TGF- b 3  A  , TGF- b 3 B  according to the convention of the BMP-2:BMPRIA-ED complex ( Kirschet al., 2000b ). View is down the shared molecular and crystallographic two-fold symmetry axis (side chains of the TGF- b 3 intermolecular disulfide depicted) withthe membrane-proximal face below, from which the C termini of all four receptors protrude and extend. A principle element mediating assembly, the flexibleN-terminal extension of T b RII, becomes ordered in the ternary complex (outlined with a diffuse green border, above). The prehelix extension of T b RI, dockedat the composite TGF- b 3:T b RII interface, is highlighted (red) as in subsequent figures. These two key elements, both short extensions, contribute substantiallyto the receptor-receptor interface (below).(B) Superpositions of free TGF- b 3 (light blue, light red) and TGF- b 3 of the ternary complex (blue, red) (0.85 A  ˚  rmsd, 111 C a  atoms), and the T b RII extracellulardomain in the binary (light green) and ternary (green) complex crystal structures (0.54 A  ˚  rmsd, 102 C a  atoms). The side chains of key residues required for sta-bilization of the TGF- b 3:T b RII-ED binary complex are depicted. Approximately 920 A  ˚  2 of each TGF- b 3 monomer is buried by T b RII. Molecular Cell TGF- b  Receptor Complex Assembly 160  Molecular Cell  29 , 157–168, February 1, 2008 ª 2008 Elsevier Inc.  (BMP-2Asp53,His54,andLeu55)adoptsan a -helicalconforma-tion complementary to the concave surface of BMPRIA. Theshort prehelix loop of TGF- b  isoforms precludes a similar stabi-lizingligand-typeIreceptorinteraction.Inaddition,smallbutsig-nificantstericclasheswiththetypeIreceptorinthispositionandtheN-terminalhelix(  a 1)uniquetothisbranch(cf.Figure4 A),plusother more conserved elements of TGF- b , necessitate an alter-native mode of binding by T b RI relative to BMPRIA.Indeed, superposition of the ligands in the crystal structures of the TGF- b  ternary and BMP-2:BMPRIA binary complexes showsthatT b RIisrotated  45  relativetoBMPRIAaroundthelongaxisof the ligand, allowing the prehelix extension of T b RI to dockagainst the fingers of TGF- b 3 B  ( Figure 4B). This rotation of T b RI, combined with the compactness of the prehelix loop of TGF- b 3, drastically reduces interaction between TGF- b 3  A   andT b RI,whicharelargelyseparatedbyasolvent-filledchannel( Fig-ure 4C). Contact with TGF- b 3  A   is confined to a 3 and the loopex-iting the helix and limited primarily to van der Waals interactions. Approximately 1338 A  ˚  2 of the TGF- b 3 dimer is buried by T b RI,44% coming from TGF- b 3  A   and 56% from TGF- b 3 B . In compar-ison,1130A  ˚  2 ofBMP-2dimerisburiedbyBMPRIA,68%comingfrom BMP-2  A   and 32% from BMP-2 B  ( Kirsch et al., 2000b ).Interaction of the prehelix extension of T b RI with TGF- b 3 B  ispredominantly hydrophobic and enhanced bythe bordering res-idues Ile54 and Phe60 ( Figure 4D). Due to its two flanking pro-lines (Pro55 and Pro59), the extension curves sharply at eachend, creating a perpendicular bulge that lodges within the widegroove separating the fingers of the ligand. Hydrophobic sidechains of three residues on the receptor side of the interface(T b RI Ile54, Pro55, and Phe60) interdigitate tightly with hydro-phobic side chains of four residues on the ligand (TGF- b 3Trp30, Trp32, Tyr90, and Leu101) that are invariant among theisoforms. Centrally located within the cluster, T b RI Pro55 is inthe  cis  conformation and packs flat against the ligand surface.Interestingly, Phe60 of T b RI does not bind in the hydrophobiccavityatthedimerinterfaceoftheligandina‘‘knob-and-pocket’’fashion like the corresponding residue of BMPRIA (Phe85),a manner hypothesized to be shared by all type I receptors of the superfamily and indicative of a common binding site ( Kirschet al., 2000b ). Instead, the phenyl ring of Phe60 lays along thesurface of the ligand, stacking against the indole ring of TGF- b 3 Trp30.Consistent with this alternative mode of interaction, SPR anal-yses of recruitment of T b RI variants by the TGF- b 3:T b RII-EDbinary complex showed that substitution with bulky hydrophilic(F60Y) or charged (F60E) residues, which would not be accom-modated in the hydrophobic pocket at the dimer interface, onlydiminished binding affinity by about 4- and 8-fold, respectively (C) Superposition of T b RI and BMPRIA extracellular domains (1.28 A  ˚  rmsd, 55 C a  atoms). T b RI is yellow-gold as above, and BMPRIA blue-gray. The prehelixextension of T b RI is highlighted in red, and the three fingers of the receptors are labeled near their tips. Figure 3. Sequence Alignment of Type I Receptors of the TGF- b  Superfamily  The receptor extracellular domains are grouped in two phylogenetic clades, with the secondary structures of T b RI and BMPRIA depicted above their respectivesequences. The ten conservedcysteines arenumbered, boxed in yellow,andconnectedto depict the fivedisulfide linkagesobserved in the crystal structures of T b RI and BMPRIA. The prehelix extension of T b RI is highlighted in red, a glycine common to the BMPRIA clade in magenta, and an invariant asparagine in cyan.ResiduesattheterminiofT b RIandBMPRIAingrayarenotmodeledandpresumablydisordered.TheNterminusofmatureT b RIfollowingsignalpeptidecleavagehas not been experimentally determined. An improved method for prediction (SignalP 3.0 Server) gives high probability to a second cleavage site (A9-L10) moredistal to the NCBI-annotated (NP 004603) site shown. Note thatthe boundaries ofthe prehelix extension defined by alignment (ClustalW) of all seven ALKs are inagreement with the crystal structure yet differ from alignment of T b RI and ActRIB with BMPRIA only ( Harrison et al., 2003 ). Molecular Cell TGF- b  Receptor Complex Assembly Molecular Cell  29 , 157–168, February 1, 2008 ª 2008 Elsevier Inc.  161
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