1. Volume 8, Issue 6, Pages 1303-1312 (December 2001)
Structural Basis of Smad1 Activation by Receptor Kinase Phosphorylation 
Bin Y Qin, Benoy M Chacko, Suvana S Lam, Mark P de Caestecker, John J Correia, Kai Lin 
Molecular Cell 
Volume 8, Issue 6, Pages (December 2001)
DOI: /S (01)

2. Figure 1
Smad1 Trimerizes and Is Activated upon C-Terminal Tail Pseudophosphorylation
(A) Sedimentation velocity analysis was performed on S1LC (6 to 49 μM) (upper panel) and S1LC(2D) (2 to 26 μM) (lower panel) and presented as g(s*20,w) versus s*20,w plots. The data were converted to weight average S20,w values and best fit to a monomer-dimer-trimer model. The fits are shown in the insert, where the weight average S20,w values of S1LC and S1LC(2D) at different concentrations are shown by triangles and squares, respectively. The monomer S value (vertical line in the upper panel), 2.72 s20,w, was determined by extrapolation of the S1LC data, and the trimer S value (vertical line in the lower panel), 5.67 s20,w, was determined as described (Correia et al., 2001). These values were used in the fitting and agree with results from sedimentation equilibrium experiments (Table 1). The substitution of two aspartic acids at the end of the sequence (SDVD) dramatically enhances trimerization potential of this Smad1 construct, increasing the overall trimerization constant K3 from 1.11 × 107 M−2 to 2.29 × 109 M−2.
(B) Smad1(2D) super-activates Smad1/4-dependent transcriptional responses. NMuMg cells were transfected with SBE-Lux along with full-length wild-type (WT) Smad1 or the Smad1(2D) mutant, with or without full-length WT Smad4. The expression levels of Smad1 and Smad4 constructs are similar. Lysates from NMuMg cells transfected with Flag-tagged Smad1 and Smad4-Myc were separated by SDS-PAGE and immunoblotted using anti-Flag M2 (Sigma) or anti-Myc 9E10 monoclonal antibodies, as indicated.
Molecular Cell 2001 8, DOI: ( /S (01) )

3. Figure 2
Structural Basis of Phosphorylation-Induced Smad1 Trimerization
(A) Crystal structure of S1LCS showing the subunit packing arrangement in the asymmetric unit. The four subunits in the asymmetric unit are shown by the ribbon representation. The 3-fold NCS axis is perpendicular to the page and indicated by a cross. The 3-fold crystallographic axis is indicated by a horizontal line. The regions where the C-terminal tail interacts with the L3 loop are circled. The L3 loops are colored black.
(B) Stereo view of the Fo-Fc omit map of the L3 loop/tail region contoured at 2.5 σ. The residues lining the tail binding pocket are labeled in black, while the tail residues are labeled in red.
(C) Surface electrostatic potential presentation of the L3 loop/tail interaction. The residues lining the tail binding pocket are labeled in black, while the tail residues are labeled in red.
(D) Stereo view of the L3 loop/tail interaction, where the L3 loop is in the closed conformation. The L3 loop main chain and side chain are colored in cyan and green, respectively. The C-terminal tail is colored in pink. The location of Gly419 is shown by a sphere. H-bond interactions are shown by red dotted line.
(E) Stereo view of the unliganded L3 loop in subunit B, where the L3 loop is in the open conformation. The L3 loop main chain and side chain are colored in cyan and green, respectively. The location of Gly419 is shown by a sphere.
(F) Smad1 trimerization induces tilting of the three-helix bundle structure. The Smad2-SARA complex structure is superimposed on subunit B of the Smad1 trimer over the β sandwich core (Smad1 residues used are 273–302, 309–359, 371–387, 413–417, and 434–442; Smad2 residues used are 270–299, 307–357, 369–385, 411–415, and 432–440). The β sandwich core was used for superposition because it is conserved in all Smad proteins, and is likely to be structurally rigid. After superposition, the rms deviation of the β sandwich core Cα trace is 0.7 Å. However, the rms deviation of the Cα trace when superimposed over the entire structure (β sandwich core and the three-helix bundle structure) is 1.6 Å, indicating that there is relative movement between the β sandwich core and the three-helix bundle structure. A similar result is obtained when the Smad2-SARA structure is superimposed on subunit A or C, but is not shown for clarity. The Smad2 MH2 domain is colored in red. The SARA SBD is colored in pink. The arrows indicate the direction of helix 4 and 5 movement.
Molecular Cell 2001 8, DOI: ( /S (01) )

4. Figure 3
Biochemical Analysis of Smad1 Homomeric Interaction and Smad1-Smad4 Heteromeric Interaction by Size-Exclusion Chromatography
(A) Trimerization of Smad1 requires C-terminal pseudophosphorylation, the conserved trimer interface, and the L3 loop phosphoserine binding pocket. The concentration of the protein samples at loading was 150 μM. The SDS-PAGE of the eluted fractions stained with Coomassie blue is shown. The elution positions of the molecular weight standards are marked above the first gel panel.
(B) The Smad1-Smad4 heteromeric complex is a trimer containing two Smad1 and one Smad4 subunits. The heteromeric interaction requires both the conserved trimer interface and the L3 loop phosphoserine binding pockets. The SDS-PAGE of the eluted fractions stained with Coomassie blue is shown. The total relative mole ratio refers to the ratio of S1LC(2D) to S4AF before loading to the column. The complex relative mole ratio refers to the ratio of S1LC(2D) to S4AF in fraction 16 and 17 (elution peak of the complex), as determined by analysis of Coomassie-stained bands with the Fluor-S MultiImager and MultiAnalyst software (Bio-Rad). The standard deviations were obtained through multiple measurements of background at different regions of the gel. The ratio of S1LC(2D) to the S4AF mutants was 1:1 before loading to the column.
(C) Sedimentation velocity analysis of S1LCS(2D) (X line; 9.77 μM) titrated with increasing amounts of S4AF (solid lines; 2.59, 5.79, 10.16, 15.15, μM). Experiment was performed at 24.7°C, 42,000 rpm, + 2 mm TCEP, and presented as a plot of g(s*20,w) versus S*20,w to demonstrate the formation of heterotrimer. The vertical dashed line corresponds to trimer (4.682 S20,w). At higher S4AF concentrations, the excess S4AF sediments as a monomer, indicated by the vertical dotted line at S20,w. Similar results were obtained with the S1LC(2D) construct.
Molecular Cell 2001 8, DOI: ( /S (01) )

5. Figure 4
Basis of Preferential Interaction between Smad4 and R-Smad As Revealed by Structural Modeling of the Smad4-Smad1 Heterotrimeric Complex
(A) The three-helix bundle structure of Smad4 is tilted more toward the subunit interface (indicated by a black circle) to promote heterotrimeric assembly between Smad1 and Smad4. The Smad1 subunits are colored in green. The Smad4 subunit is shown in red. The modeling was performed by superimposing the Smad4 subunit on top of the Smad1 subunit C over the β sandwich core (Smad4 residues used for the superimposition are 322–341, 346–356, 360–391, 397–411, 424–441, 499–510, and 517–541; Smad1 residues used are 270–289, 292–302, 307–338, 343–357, 369–386, 410–421, and 428–452). The model was then examined for subunit interactions. The model created this way maintains all key trimeric contacts observed in the Smad1 trimer and contains additional favorable interactions. The black arrows show the direction of the helix movement.
(B) Closeup view of the Smad1 homotrimeric interface. The side chains of helix 1 and 4 are colored in green and pink, respectively.
(C) Closeup view of the hypothetical model of the Smad1-Smad4 heterotrimeric interface. The side chains of Smad1 helix 1 are colored in green. The side chains of Smad4 helix 4 are colored in pink. Potential H-bonds are shown by red dotted lines.
(D) Sequence alignment of helix 1 and 4 in Smad proteins. The residues are color-coded according to (B) and (C). Conserved residues are marked with an asterisk (*).
Molecular Cell 2001 8, DOI: ( /S (01) )

6. Figure 5 An Allosteric Model for Smad Activation
At the basal state, R-Smad is monomeric. Upon ligand binding and receptor kinase activation, the R-Smad is recruited to the receptor complex by both the receptor kinase cytoplasmic domain (via the L45 loop) and the anchoring protein. Phosphorylation of the C-terminal SXS sequence of the R-Smad energetically favors subunit trimerization, and that formation of trimers is sterically incompatible with receptor association. The R-Smad homotrimer is energetically less stable than the R-Smad/Smad4 heterotrimer due to the presence of unique favorable interactions in the R-Smad/Smad4 subunit interface.
Molecular Cell 2001 8, DOI: ( /S (01) )