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. 2010 Dec;6(12):2392-402.
doi: 10.1039/c0mb00115e. Epub 2010 Oct 4.

Peptide ligands that use a novel binding site to _target both TGF-β receptors

Lingyin Li  1 , Brendan P Orner, Tao Huang, Andrew P Hinck, Laura L Kiessling
Affiliations

Peptide ligands that use a novel binding site to _target both TGF-β receptors

Lingyin Li et al. Mol Biosyst. 2010 Dec.

Abstract

The transforming growth factor beta (TGF-β) signaling pathway plays myriad roles in development and disease. TGF-β isoforms initiate signaling by organizing their cell surface receptors TβRI and TβRII. Exploration and exploitation of the versatility of TGF-β signaling requires an enhanced understanding of structure-function relationships in this pathway. To this end, small molecule, peptide, and antibody effectors that bind key signaling components would serve as valuable probes. We focused on the extracellular domain of TβR1 (TβRI-ED) as a _target for effector screening. The observation that TβRI-ED can bind to a TGF-β coreceptor (endoglin) suggests that the TβRI-ED may have multiple interaction sites. Using phage display, we identified two peptides LTGKNFPMFHRN (Pep1) and MHRMPSFLPTTL (Pep2) that bind the TβRI-ED (K(d)≈ 10(-5) M). Although our screen focused on TβRI-ED, the hit peptides interact with the TβRII-ED with similar affinities. The peptide ligands occupy the same binding sites on TβRI and TβRII, as demonstrated by their ability to compete with each other for receptor binding. Moreover, neither interferes with TGF-β binding. These results indicate that both TβRI and TβRII possess hot spots for protein-protein interactions that are distinct from those used by their known ligand TGF-β. To convert these compounds into high affinity probes, we exploited the observation that TβRI and TβRII exist as dimers on the cell surface; therefore, we assembled a multivalent ligand. Specifically, we displayed one of our receptor-binding peptides on a dendrimer scaffold. We anticipate that the potent multivalent ligand that resulted can be used to probe the role of receptor assembly in TGF-β function.

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Figures

FIG. 1
FIG. 1
(A) Schematic depiction of the TGF-β signaling pathway. The covalently linked TGF-β homodimer (orange) binds to two copies of TβRII (green) which forms non-covalent homodimers as well as higher order oligomers. The TGF-β/TβRII complex then recruits two copies of TβRI (purple). This quinary complex enables the constitutively active TβRII to catalyze the phosphorylation of the serine residues in the juxtamembrane GS domain of TβRI. Upon GS domain phosphorylation, the adjacent kinase domain catalyzes the phosphorylation and activation of receptor-regulated Smad proteins (R-Smad), Smad2 and Smad3 (blue) with the help of an adaptor protein SARA (Smad anchor for receptor activation, light brown). Phosphorylated Smad2 and Smad3 dissociate from SARA and bind to common Smad (co-Smad), Smad4 (teal), which facilitates the translocation of this complex into the nucleus. Once in the nucleus, the Smads bind to different DNA binding partners to control gene expression. Structures used in the creation of this Fig. were determined by X-ray crystallographic analysis and rendered using PyMOL molecular graphics. PDB files used to construct this scheme follow: PDB ID 3KFD (for TGF-β1:TβRI-ED:TβRII-ED ternary structure), 2QLU (for activin receptor type IIB cytoplasmic domain residues 188-483, which is homolous to TβRII residues 267-592), 1IAS (for TβRI cytoplasmic domain residue 171-503), 1DEV and 1U7F (for unphosphorylated Smad3 bound to the Smad binding-domain of SARA), 1U7F (for phosphorylated Smad3 and Smad3:Smad3:Smad4 trimeric complex). For extracellular and intracellular segments whose structures have not been determined by X-ray crystallography, online software NetSurfP (http://www.cbs.dtu.dk/services/NetSurfP/) was used to predict secondary structures, and α-helices and transmembrane domains are represented by cylinders. (B) Structures of the extracellular domains of TβRI (purple, PDB ID 2PJY: C), TβRII (dark green, PDB ID 2PJY: B), BMPR-IA (magenta, PDB ID 2GOO: B) and ActRII (light green, PDB ID 2GOO: C), indicate these proteins share a common three-finger toxin fold stabilized by four disulfide bonds.
FIG. 2
FIG. 2
Phage display against TβRI yields peptides that bind TβRI-ED and TβRII-ED indistinguishably. The binding of (A) phage clone 1 and (B) phage clone 2 to immobilized TβRI-ED and TβRII-ED was assessed using a phage-based ELISA. (C) ELISA-based competition binding assay. Pep1 derived from phage clone 1 and (D) Pep2 derived from clone 2 were tested for inhibition of phage clone binding to immobilized receptors (550 pM of clone 1 and 39 pM of clone 2 were used). (E) An assay with Pep1competing with phage clone 2 (39 pM) for binding to either immobilized TβRI-ED or TβRII-ED. The IC50 value for Pep1 with phage clone 2 and TβRI-ED is 110 μM; the corresponding value for TβRII-ED is 156 μM. (F) An assay with Pep2 competing with phage clone 1 (550 pM) for binding to either immobilized TβRI-ED or TβRII-ED. The IC50 value for Pep2 inhibiting phage clone 1 binding to TβRI-ED is 256 μM; the corresponding value for TβRII-ED is 274 μM. Error bars represent the mean ± the standard deviation in (A) to (F).
FIG. 3
FIG. 3
Pep1 and Pep2 do not compete with TGF-β in binding to either TβRI-ED or TβRII-ED. (A) Binding of TGF-β1 (41 pM to 30 nM) to TβRII-ED was tested using SPR. TβRI-ED and TβRII-ED were immobilized through their lysine residues. A protein-free flow cell was used as control. TGF-β binds to TβRII-ED with a saturating concentration of 10 nM. At the concentrations tested, TGF-β1 has no observable affinity to TβRI-ED (data not shown). (B) Pep1 does not compete with TGF-β in binding to TβRII-ED. (C) TGF-β1 initiated luciferase gene expression in an Mv1Lu reporter cell line stably transfected with a SBE(CAGA)12-luciferase reporter gene. TβRI kinase inhibitor SB-431542 inhibited TGF-β regulated luciferase gene expression. Pep1 and (D) Pep2 do not alter the cellular response to TGF-β1. In this competition assay, 10 pM TGF-β1 was used.
FIG. 4
FIG. 4
Multivalent display of Pep1 on G3 dendrimer. (A) Peptides identified from phage display can be displayed on multivalent scaffolds to afford ligands with increased avidity. (B) Synthetic scheme for conjugating Pep1 to G3 PAMAM dendrimer.
FIG. 5
FIG. 5
(A) Binding affinities of the dendrimer to TβRI-ED, (B) TβRII-ED, (C) BMPR-IA-ED, (D) ActRII-ED, (E) endoglin-ED and (F) β-glycan-ED were assessed by SPR. All proteins were immobilized through their lysine residues. A protein-free flow cell was used as control. The dendrimer binds to TβRI-ED and TβRII-ED, but not to Endoglin-ED at concentrations ranging from 2.93 nM to 1.5 μM. In a separate experiment, the dendrimer did not interact with BMPR-IA-ED, ActRII-ED or β-glycan-ED at concentrations ranging from 47 nM to 6 μM.
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