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

Please download to get full document.

View again

of 12
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Categories
Published
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: jgroppe@bcd.tamhsc.edu (J.G.), wrana@mshri.on.ca (J.L.W.), hinck@uthscsa.edu (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
Similar documents
View more...
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks