1. Structure and Activation Mechanism of the CHK2 DNA Damage Checkpoint Kinase 
Zhenjian Cai, Nabil H. Chehab, Nikola P. Pavletich 
Molecular Cell 
Volume 35, Issue 6, Pages (September 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1 Structure of the CHK2K249R Dimer
(A) Linear representation of CHK2 depicting the SCD, FHA, and kinase domain (KD) boundaries and the two truncated CHK2K249R of the P1 and P crystal forms.
(B) Schematic representation of the CHK2K249R dimer in the P crystal form with the two protomers colored in red and blue. The FHA domains and the N and C lobes of the bilobal kinase domain are labeled. The secondary structure elements of one protomer are labeled by numbers for β strands and letters for α helices, with the FHA labels followed by the symbol (′) (Figure S1). The yellow spheres indicate the approximate positions of the phosphoThr phosphate group based on the structure of the isolated FHA-phosphopeptide complex (Li et al., 2002). Dotted lines indicate disordered regions, including the activation loops.
(C) View of the CHK2K249R dimer looking down the vertical axis of (A).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
The CHK2K249R Dimer Forms through Intermolecular FHA-KD, FHA-FHA, and FHA-Linker Interfaces
(A) Close-up view of the intermolecular interface between the FHA (red) and kinase (blue) domains of the two protomers. The interacting amino acids of the FHA are shown in pink, and those of the kinase N lobe are shown in blue. Green dotted lines indicate groups within hydrogen bonding distance, and asterisks indicate residues mutated in cancer. For clarity, only a subset of the secondary structure elements is labeled.
(B) Close-up view of a portion of the dimer interface that involves FHA-FHA and FHA-linker intermolecular contacts, colored as in (A).
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4. Figure 3
The Dimeric CHK2 Structure Is Consistent with the SCD-FHA Association Being Intermolecular
Molecular surface representation of the CHK2 dimer showing the position of a high-affinity peptide cocrystallized with the isolated FHA domain of CHK2 (Li et al., 2002). The peptides, one for each FHA domain, are positioned by superimposing the FHA domains. Assuming the peptide's phosphoThr is equivalent to Thr68 and the same direction of the polypeptide chain, the peptide would correspond to residues Thr65–Tyr72 of the SCD. Dotted lines highlight the 34 Å line of sight between the C terminus of the peptide and the N terminus of the FHA domain. Orientation as in Figure 1C.
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5. Figure 4 SCD-Independent CHK2K249R Dimerization in Solution
(A) Size-exclusion chromatography of CHK2K249R (residues 70–512) at increasing concentrations (blue profile at 120 μM; red, 220 μM; black, 480 μM; green, 710 μM). The retention volumes of proteins used as molecular weight standards are indicated on top.
(B) Quantitation of the size-exclusion chromatography profiles of (A), showing that the fraction of the protein that elutes in the dimer peak increases with increasing protein concentration. Quantitation is approximate due to the partial overlap of the two peaks at the higher protein concentrations.
(C) Native gel analysis of CHK2K249R (residues 70–512) at the indicated concentrations, showing the appearance of a faster-migrating second band starting at a protein concentration of 50 μM.
(D) Size-exclusion chromatography profiles of SCD-containing CHK2K249R (residues 40–512), showing that the phospho-mimetic T68E mutation (green profile) enhances dimerization of CHK2K249R, whereas the I157T (red) and W97A (purple) mutations at the FHA-KD and FHA-FHA interfaces, respectively, reduce dimerization. All proteins were at 300 μM.
(E) Dimer fraction of the proteins shown in (D), calculated based on size-exclusion chromatography analyses at multiple protein concentrations for each protein. The dimer fractions of the elution profiles in (D) are indicated by an asterisk (∗).
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6. Figure 5 CHK2 Kinase Conformation
(A) The activation loop of the P form (red) adopts the inactive conformation characterized by the formation of the αT1 helix and disorder (dotted line) of the subsequent 12 residues. In the P1 crystal form (green), part of the activation loop adopts a conformation similar to the active state, which, as seen in the structure of the active PKA (gray) (Zheng et al., 1993), is characterized by the formation of the β9 strand instead of the αT1 helix. The autophosphorylation sites of CHK2 (Thr378) and PKA (phosphoThr197) are also shown. Residue numbers of the CHK2 secondary structure elements are labeled.
(B) The quaternary structure of the CHK2 dimer (red and blue) differs from the previously reported crystallographic symmetry-related dimer of the isolated CHK2 kinase domain (magenta and yellow) (Oliver et al., 2006). The two dimers were superimposed by aligning one kinase domain in each dimer. The Asp347 catalytic residue of each kinase domain is shown in black, and the intradimer distances between the Asp347 side chains in each of the two dimers are indicated by black dotted lines.
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7. Figure 6 Dissociation of the CHK2 Dimer after Activation
(A) Size-exclusion chromatography profiles of active CHK2 either lacking the SCD (residues 70–512, 470 μM, red) or containing the SCD (residues 40–502, 310 μM, blue) exhibit singe peaks coincident with the monomer peaks of the corresponding kinase-dead CHK2K249R proteins (thinner magenta and cyan lines, respectively).
(B) Model of a hypothetical, active CHK2 dimer (yellow and green) superimposed on the inactive dimer structure (red and blue) by aligning the N lobes. The αB, αEF, and αG secondary structure elements that could result in intermolecular steric clashes when the kinase is activated are labeled.
(C) Comparison of the kinase C lobe positions in the inactive structure (red and blue) and in the model of the active dimer (yellow and green), looking up the vertical direction in (B). The disordered segment (residues 393–404) between the αEF and αF is indicated by a dotted line. For clarity, the disordered activation loop between the start of αEF (residue 388) and the end of β8 is not indicated.
Molecular Cell  , DOI: ( /j.molcel )
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