1. Volume 9, Issue 5, Pages 1045-1054 (May 2002)
Structural and Functional Versatility of the FHA Domain in DNA-Damage Signaling by the Tumor Suppressor Kinase Chk2 
Jiejin Li, Brandi L. Williams, Lesley F. Haire, Michal Goldberg, Erik Wilker, Daniel Durocher, Michael B. Yaffe, Stephen P. Jackson, Stephen J. Smerdon 
Molecular Cell 
Volume 9, Issue 5, Pages (May 2002)
DOI: /S (02)

2. Figure 1 Chk2 FHA Structure and Peptide Binding
(A) Oriented peptide library screen for Chk2 FHA phosphothreonine binding specificity. Selectivity ratios (Durocher et al., 2000) for the primary and refinement screens are in parentheses. For the refinement screen, a second phosphopeptide library was synthesized with an isoleucine residue fixed at the pT +3 position. Amino acids are in single-letter code.
(B) Binding of Chk2 FHA to the optimal phosphopeptide measured by isothermal titration calorimetry, essentially as described previously (Durocher et al., 2000).
(C) Structure of the Chk2 FHA domain in complex with the selected phosphopeptide.
Molecular Cell 2002 9, DOI: ( /S (02) )

3. Figure 2 Structural Basis of Binding Specificity
(A) Schematic representation of protein-peptide contacts. Hydrogen bonds are shown as dotted lines with nonpolar/van der Waals interactions indicated by arcs and interacting peptide side chains highlighted in red. Highly conserved residues are highlighted in green.
(B) Least-squares superposition of the Chk2 FHA (white) with Rad53p FHA1 (blue) as a backbone worm shown in stereo. The phosphopeptide from the Chk2 complex is also shown in worm representation with the pThr and Ile +3 side chains highlighted in ball-and-stick. The β4-β5 and β10-β11 loop regions are highlighted in red (Chk2) and green (Rad53p), respectively.
Molecular Cell 2002 9, DOI: ( /S (02) )

4. Figure 3
Chk2 Regulation Is Variably Affected by Mutations in the FHA Domain
(A) Surface plasmon resonance was used to measure the relative binding of approximately equal concentrations of wild-type (WT) and mutated GST-Chk2 FHA fusion proteins to biotinylated peptides immobilized on streptavidin-coated sensor chips. The peptides contain the sequence RHFD(T)YLIRR in which the threonine was either unphosphorylated (T) or phosphorylated (pT).
(B) Extracts were prepared from 293T cells transfected with the indicated plasmid DNA and treated with phleomycin (+) or PBS (−). HA-tagged Chk2 was immunoprecipitated with anti-HA antibody and immunoblotted with anti-Chk2 (pThr68) antibody (upper panel). The blot was stripped and reprobed with anti-HA antibody (lower panel).
(C) 293T cells were transfected with pRc/CMV (Vec) or with pRc/CMV-HAChk2 having wild-type sequence (WT), an inactivating mutation in the kinase domain (KD), or the indicated missense mutation. After exposure to phleomycin (+) or PBS (−), 50 μg of total protein extract was probed with anti-HA antibody. The unphosphorylated (Chk2) and phosphorylated (Chk2-P) forms are indicated by arrows. Nonspecific species are present to a greater or lesser extent in all samples and are indicated by an asterisk.
(D) Both Arg145 and Ile157 are located on the more conserved face of the FHA domain β sandwich, and they are remote from the site of phosphopeptide binding. Arg117 forms a conserved contact with the phosphothreonine.
(E) Arg145 forms an extensive series of van der Waals and hydrogen bonding interactions with a constellation of residues located on β strands 3, 5, and 6.
Molecular Cell 2002 9, DOI: ( /S (02) )

5. Figure 4
The Chk2 FHA Domain Specifically Binds BRCA1 in Phospho-Dependent and -Independent Manners
(A) Wild-type (WT) or mutated GST-Chk2FHA fusion proteins were incubated with extracts from undamaged (C), HU treated (H), or phleomycin treated (P) HeLa cells. Bound proteins were analyzed by Western blot using anti-BRCA1 antibody (BRCA1 Ab-1, Oncogene Research Products).
(B) Extracts from HU-treated HeLa cells (prepared in the absence of phosphatase inhibitors) were treated with 1000 units of λ phosphatase in the absence or presence of 50 mM EDTA for 30 min at 30°C. Extracts were incubated with the GST-Chk2 FHA fusion protein, and bound proteins (lower panel) or 50 μg of total protein (upper panel) was immunoblotted with anti-BRCA1 antibody.
(C) GST-Chk2FHA fusion protein was incubated with HeLa cell extracts in the absence or presence of increasing amounts of peptide with the sequence RHFDpTYLIRR (pT-Y-L-I). Bound BRCA1 was analyzed by Western blot.
(D) Wild-type (WT) or mutant GST-CHK2 FHA fusion proteins were incubated with extracts from γ-irradiated MCF-7 cells, and bound proteins were analyzed by 2-D electrophoresis. One representative section of the resulting gel is shown. Proteins that bound to the wild-type FHA domain but not to either the R117A or I157T mutant FHA domains are highlighted by ovals. Proteins displaying nearly equivalent interactions with wild-type and mutant domains are enclosed in diamond-shaped symbols, and those showing semiconserved interactions are enclosed in squares.
Molecular Cell 2002 9, DOI: ( /S (02) )

6. Figure 5 Comparison of Chk2 FHA and Smad2 MH2 Complexes
(A) The structurally related β sandwich domains are shown in the same orientation, each superimposed with the phosphopeptide (pP) derived from the Chk2 FHA complex structure and the SARA adaptor protein derived from the MH2 domain complex (Wu et al., 2000). The side chains of Ile157 from Chk2 and Arg330 and His331 from Smad2 are shown in space-filling representation. Mutation of Arg330 and His331 severely reduces interaction of Smad2 with phosphorylated TGFβ receptor (Huse et al., 2001).
(B) Structure of a phosphorylated Smad2 MH2 trimer viewed along the 3-fold axis (Wu et al., 2001a). The C-terminal region is highlighted in red. The Chk2 FHA domain (green) is shown superimposed on one MH2 monomer, and the relative position of the phosphopeptide (pP) to that of the Smad C-terminal interacting region is shown.
Molecular Cell 2002 9, DOI: ( /S (02) )