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Organization of the SH3-SH2 Unit in Active and Inactive Forms of the c-Abl Tyrosine Kinase

Bhushan Nagar, Oliver Hantschel, Markus Seeliger, Jason M. Davies, William I. Weis, Giulio Superti-Furga and John Kuriyan

Molecular Cell (2006) Vol. 21: 787-798   Local Copy

Summary / Figures / Tables / Supplemental Data / PDB coordinates

Summary: The tyrosine kinase c-Abl is inactivated by interactions made by its SH3 and SH2 domains with the distal surface of the kinase domain. We present a crystal structure of a fragment of c-Abl which reveals that a critical N-terminal cap segment, not visualized in previous structures, buttresses the SH3-SH2 substructure in the autoinhibited state and locks it onto the distal surface of the kinase domain. Surprisingly, the N-terminal cap is phosphorylated on a serine residue that interacts with the connector between the SH3 and SH2 domains. Small-angle X-ray scattering (SAXS) analysis shows that a mutated form of c-Abl, in which the N-terminal cap and two other key contacts in the autoinhibited state are deleted, exists in an extended array of the SH3, SH2, and kinase domains. This alternative conformation of Abl is likely to prolong the active state of the kinase by preventing it from returning to the autoinhibited state.

Figures (Click on the small image to view the bigger one):

Figure 1. Schematic Diagram of the c-Abl Constructs Used and the Structure of the Cap Region; Table 1

  1. AblN-cap was used for both the crystal structure and SAXS analyses. Residues from the N-terminal cap in AblN-cap that were deleted are indicated with gray shading, and residues that were included are highlighted in pink.
  2. Surface representation of AblN-cap with the cap region shown as a backbone model in pink. Residues that connect the cap to the myristoyl but could not be modeled are shown as pink spheres. Helix αI of the kinase domain is colored purple. A black box indicates the region magnified in (C).
  3. Hydrophobic surface rendition of AblN-cap showing cap interactions with the SH2 domain and SH3-SH2 connector. Increasing hydrophobicity of the surface is indicated with darker shades of green. The cap is shown as sticks, where carbon, nitrogen, and oxygen atoms are colored orange, blue, and red, respectively. Labeled are residues that are well ordered and make direct interactions with the protein. The water molecule hydrogen bonded to Lys70 is shown as a blue sphere. Molecular figures were generated with PyMOL.

Figure 2. The Phosphoserine Binding Site

  1. Closeup of the phosphoserine interactions with the SH3-SH2 connector. Difference electron density contoured at 4σ before inclusion of the phosphate group into the refinement model is shown. A water molecule is indicated with a blue sphere, and green dashes depict hydrogen bonds. Carbon atoms from the main body of the protein are colored gray, and those from the cap are colored light orange. Nitrogen, oxygen, and phosphorus atoms are colored blue, red, and orange, respectively. Only selected side chains are shown.
  2. Surface and ribbon rendition of the SH2 domain of c-Abl depicting the locations that the phosphoserine from the N-terminal cap and a modeled phosphotyrosine ligand (from Hck pdbcode 1QCF) bind. In red is the SH3-SH2 connector extending from the SH2 domain.
  3. (Left) Charged surface rendition of AblN-cap depicting the positively charged region on Abl with which the N-terminal cap interacts. Shown in sticks are residues of the cap, which were omitted from the calculation. (Right) The corresponding calculation for c-Src (PDB code 2SRC) shows a relatively neutral surface.
  4. Kinase assays assessing the effects of mutating Ser69. Abl immunoprecipitates were probed with anti-Abl and anti-phosphotyrosine antibodies (lower panels) and assayed for catalytic activity by in vitro kinase assays using an optimal substrate peptide. The histograph shows the in vitro kinase activity (mean with standard deviation of two experiments done in duplicate, black bars) and levels of autophosphorylation (mean with standard deviation of two immunoprecipitations, gray bars) of the mutated Abl constructs relative to c-Abl and corrected for endogenous c-Abl levels.

Figure 3. SAXS Scattering Data; Table 2

  1. Log of scattered intensity versus Q. The curves have been vertically offset to aid visualization.
  2. Guinier plots. The linear regions of the low Q section are shown as black lines. Lines have been vertically offset.
  3. Plots of the interatomic distances (P[r] function).

Figure 4. Shape Reconstructions

  1. Kinase domain. The backbone of the crystal structure of the c-Abl kinase domain (blue; PDB code 1OPJ) is superimposed onto the shape reconstruction (shown as green mesh).
  2. AblN-cap. Superimposed is the crystal structure of AblN-cap shown as a green backbone onto the shape reconstruction (gray mesh).
  3. Ablactivated. The kinase domain and SH2 and SH3 domains are colored red, green, and blue, respectively. Indicated on the right view is the part of the model that may correspond to the crystal structure of disassembled c-Abl from the original crystallographic analysis of c-Abl 1b. The SH3 domain was placed by visual inspection, ensuring that its C terminus was in close proximity to the N terminus of the SH2 domain.

Figure 5. Potential Role for the SH2-N Lobe Interface in Ablactivated

  1. Ribbon and surface representations of the disassembled molecule of c-Abl from the original c-Abl 1b crystallographic analysis. A possible location for the SH3 domain based on crystal symmetry constraints and weak electron density is indicated. The SH2-kinase linker that could not be modeled is indicated by pink spheres. Helix αI, which has straightened out in the disassembled molecule of Abl, is colored purple.
  2. In vitro kinase activities of the Abl constructs used in the SAXS analysis. The histograph shows the mean (black bars) with errors from the least squares fit of Km and Vmax from two experiments (error bars). The active proteins used for kinetic analysis are produced in bacteria and therefore do not get myristoylated. Therefore, to test a construct corresponding to AblN-cap (which was produced in insect cells and autoinhibited by myristoylation at the N terminus), we used bacterially expressed Abl83-534, which represents a partially autoinhibited state.
  3. Cartoon diagram depicting the relationship between the two states of c-Abl, AblN-cap and Ablactivated, and the structural parameters determined from the SAXS data. The intermediate position of AblSH2-mutant is indicated.

Tables:   Table 1 Table 2

Supplemental Data

Figure S1.  Shape reconstruction of c-Abl lb in which the internal docking site of the Sh3 domain has been disrupted and the myristoylation site has been mustated but the N-terminal cap region is intact.