1. The TGFβ Receptor Activation Process
Morgan Huse, Tom W. Muir, Lan Xu, Ye-Guang Chen, John Kuriyan, Joan Massagué 
Molecular Cell 
Volume 8, Issue 3, Pages (September 2001)
DOI: /S (01)00332-X

2. Figure 1 Protein Semisynthesis
(A) Schematic describing the semisynthetic protocol. The tetraphosphorylated GS region peptide thioester was synthesized using Fmoc coupling chemistry on a sulfonamide resin. TβR-IΔ was expressed downstream of a histidine tag and Factor Xa recognition sequence. Subsequent cleavage exposed the N-terminal cysteine required for ligation (L196 mutated to Cys).
(B) TβR-I and Smad2 fragments used in these studies. Full-length TβR-I and Smad2 are shown for reference. Green denotes the GS region of TβR-I and blue the kinase domain. Phosphate groups are indicated by lower case p's. The primary sequence for the tetraphosphorylated GS region is shown below the schematic for TβR-I(4P). The cysteine residue that was introduced to enable ligation is colored red. The Smad2 MH2 domain is shown in yellow with the C-terminal tail in single letter code (receptor phosphorylation sites within the tail are colored green). The Smad2 L3 loop is colored cyan and the Smad1 L3 loop red. Primary sequences for both the Smad2 and the Smad1 L3 loops are shown below the schematic for the Smad2 L3 mutant. The two residues that differ between the two proteins in the L3 loop have been highlighted
Molecular Cell 2001 8, DOI: ( /S (01)00332-X)

3. Figure 2
Phosphorylation of the GS Region Activates TβR-I toward the C-Terminal Serines of Smad2
(A) Phosphorylation of the Smad2 MH2 domain by receptor fragments. Each bar represents the average of three experiments. Error bars indicate standard deviation.
(B) Phosphorylation of different Smad2 MH2 domain constructs by TβR-I(4P) and TβR-I(0P). The kinase and substrate used in each reaction are indicated (4P, TβR-I(4P); 0P, TβR-I(0P); WT, wild-type; and Trun, Truncated). Each bar represents the average of three experiments. Error bars indicate standard deviation.
(C) An anti-phospho-Smad2 immunoblot of TβR-I kinase reactions. Samples were subjected to SDS-PAGE, blotted, and probed with anti-phospho-Smad2. The kinase and substrate used in each reaction are indicated (Rec, TβR-I(0P,rec); Δ, TβR-IΔ)
Molecular Cell 2001 8, DOI: ( /S (01)00332-X)

4. Figure 5
A Basic Surface on Smad2 Is Involved in Phosphorylation by TβR-I
(A) The structure of the forkhead associated (FHA) domain of Rad53p in complex with a pThr-containing peptide (Durocher et al., 2000). The entire domain is shown above in green with the core β sandwich colored orange. The peptide is cyan with the pThr sidechain shown in ball and stick representation. The red arrow indicates the viewing angle for the image of the peptide binding site shown below.
(B) The structure of the Smad2 MH2 domain (Wu et al., 2000). The entire domain is shown above in yellow with the core β sandwich colored orange. The Smad2 protein is oriented such that its β sandwich fold is aligned with the corresponding portions of the FHA domain in (A). The L3 loop is colored red and the C terminus of the molecule magenta. The red arrow indicates the viewing angle for the image of the basic surface patch shown below. Residues mutated in this study that affect TβR-I binding are shown in cyan. Residues from the L3 loop previously implicated in receptor interaction are green (Lo et al., 1998). Lys420, a universally conserved residue that forms the base of the positive patch, is shown in blue.
(C) In vitro phosphorylation of the Smad2 basic patch mutants. Kinase reactions containing the indicated Smad2 mutants were subjected to SDS-PAGE, blotted, and probed with anti-phospho-Smad2. The level of phosphorylation has been quantitated for each Smad2 fragment and is shown below (NIH Image).
(D) The Smad2 patch mutants were assessed for TβR-I binding. The indicated GST-Smad2 fusion proteins were used as bait for TβR-I(4P) or TβR-I(0P,rec) in a pull-down assay. The protein retained on the glutathione beads was analyzed by immunoblot using an antibody against TβR-I. The amount of TβR-I coprecipitating with the glutathione beads has been quantitated below (NIH Image).
(E) Flag-tagged expression vectors encoding the indicated Smad2 mutants were transfected into Mv1Lu/L17 cells together with wild-type TβR-I. After TGFβ treatment, the Flag-tagged proteins were immunoprecipitated, and their level of tail phosphorylation was examined by immunoblot using the anti-phospho-Smad2 antibody (upper panel). The expression level of all constructs is comparable (lower panel). The level of phosphorylation has been quantitated for each Smad2 construct after normalization against protein expression and is shown in the bar graph (NIH Image)
Molecular Cell 2001 8, DOI: ( /S (01)00332-X)

5. Figure 3 FKBP12 Inhibition of TβR-I Fragments
The amount of FKBP12 added to each reaction is expressed in equivalents relative to TβR-I kinase. Activity is shown as a percentage of the full activity observed in each experiment. Each data point represents the average of three separate experiments. Error bars indicate standard deviation, and error bars smaller than the symbols are not shown.
(A) An inhibition curve of Smad2 phosphorylation by TβR-I(0P,rec).
(B) An inhibition curve of Smad2 phosphorylation by TβR-I(4P)
Molecular Cell 2001 8, DOI: ( /S (01)00332-X)

6. Figure 4 Phosphorylated TβR-I Binds to Smad2 and Does Not Bind FKBP12
(A) The indicated GST fusion proteins were used as bait for TβR-I(0P,rec), TβR-I(0P), and TβR-I(4P) in a pull-down assay (see Experimental Procedures). The protein retained on the glutathione beads was analyzed by immunoblot using an antibody against TβR-I.
(B) The effect of nucleotides and NPC on the Smad2/TβR-I(4P) and FKBP12/TβR-I(0P,rec) interactions. GST-Smad2 and GST-FKBP12 were used as bait for TβR-I(4P) and TβR-I(0P,rec), respectively. Pull-down assays were supplemented with the indicated small molecules, subjected to SDS-PAGE, and visualized by anti-TβR-I immunoblot
Molecular Cell 2001 8, DOI: ( /S (01)00332-X)

7. Figure 6 The Structure of TβR-I in the Absence of FKBP12
(A) The structure of TβR-I in complex with NPC (left) is compared with the structure of TβR-I in complex with FKBP12 (right) (Huse et al., 1999). The TβR-I protein kinase domain is colored blue with the GS region green and the activation segment magenta. FKBP12 is shown in red. |Fo| − |Fc| difference density for NPC within the nucleotide binding groove is depicted using wire mesh at two contour levels. 3σ electron density is colored cyan, while 6σ density is red. Asp 351 and Arg 372, which form an ion pair in the TβR-I/FKBP12 complex, are illustrated in both images. All ribbon diagrams were generated using RIBBONS (Carson, 1991).
(B) The GS region from the TβR-I/NPC complex (left) is compared to the GS region from the TβR-I/FKBP12 complex (right) (Huse et al., 1999). The kinase N-lobe is shown in blue with the GS region in green. The αGS1 and αGS2 helices are indicated along with selected residues at the FKBP12 interface. On the left, the GS loops from all five TβR-I molecules in the asymmetric unit of the TβR-I/NPC crystals have been overlaid and are colored orange. L195 and L196 are directly engaged by FKBP12 and mark the binding site. Hydrogen bonds are shown in dashed purple.
(C) The TβR-I kinase domain remains in an inhibited conformation in the absence of FKBP12. On the left, the N-lobe from the TβR-I/NPC complex is aligned using its β sheet with TβR-I from the FKBP12 complex (Huse et al., 1999) as well as PKA in active conformation (Zheng et al., 1993). The proteins are viewed from the ATP binding site looking up through the β sheet. The ATP molecule from the PKA structure is shown in ball and stick representation. On the right, the N-lobe from the TβR-I/NPC complex is viewed in isolation. |Fo| − |Fc| electron density for NPC is shown in wire mesh at three different contour levels, 3σ in cyan, 6σ in yellow, and 8σ in magenta. The portion of the inhibitor that extends into the back of the nucleotide binding groove is marked with an asterisk. S280 and L278, which pack against this element, are shown in ball and stick representation along with Y249, which forms the bottom right portion of the ATP binding pocket. The ATP molecule from the PKA structure is shown in thin red lines for comparison
Molecular Cell 2001 8, DOI: ( /S (01)00332-X)

8. Figure 7 A Model for TβR-I Activation
The kinase domain is colored in shades of blue with the N-lobe β sheet, C helix, and C-lobe schematically depicted. The GS region is green, FKBP12 red, and Smad2 yellow. The TβR-I/Smad2 interaction is depicted as having an extended interface that, in addition to incorporating phosphate recognition by the positive surface patch of Smad2, also involves the L45 loop-L3 loop interaction
Molecular Cell 2001 8, DOI: ( /S (01)00332-X)