Sonja Lorenz
Wang Stamp


Crystal structure of an SH2-kinase construct of c-Abl and effect of the SH2 domain on kinase activity

Sonja Lorenz, Patricia Deng, Oliver Hantschel, Giulio Superti-Furga, and John Kuriyan

Biochem J. 2015 Jun 1;468(2):283-91     (local copy)

Abstract

Constitutive activation of the non-receptor tyrosine kinase c-Abl (Abl1) in the Bcr-Abl1 fusion oncoprotein is the molecular cause of chronic myeloid leukemia. Recent studies have indicated that an interaction between the SH2 domain and the N-lobe of the c-Abl kinase domain has a critical role in leukemogenesis. To dissect the structural basis of this phenomenon we studied c-Abl constructs comprising the SH2 and kinase domains in vitro. We present a crystal structure of an SH2-kinase domain construct bound to dasatinib, which contains the relevant interface between the SH2 domain and the N-lobe of the kinase domain. We show that the presence of the SH2 domain enhances kinase activity moderately and that this effect depends on contacts in the SH2-N-lobe interface and is abrogated by specific mutations. Consistently, formation of the interface decreases slightly the association rate of imatinib with the kinase domain. That the effects are small compared to the dramatic in vivo consequences suggests an important function of the SH2-N-lobe interaction might be to help disassemble the autoinhibited conformation of c-Abl and promote processive phosphorylation, rather than substantially stimulate kinase activity.

Figures from the paper

(Click on the small image to get a higher-resolution version.)
Figure 1A from paper

Figure 1A - Conformational states of c-Abl


Schematic depicting two distinct domain arrangements in the autoinhibited and active states of c-Abl. In the autoinhibited, assembled state the N-terminal myristoyl group binds to the kinase domain and allows the SH2 and SH3 domains to dock onto it [2,40]. Open autoinhibited conformations also exist [5]. In the extended, active conformation, the SH2 domain forms an interface with the N-lobe of the kinase domain, involving the αC patch and Tyr 412 in the activation loop undergoes rapid autophosphorylation

Figure 1B from paper

Figure 1B - Conformational states of c-Abl


Schematic representation of critical components of the active site in various states of the Abl kinase domain: the inactive conformation as seen in the complex with imatinib, the Src-like inactive conformation, and the active conformation. The DFG motif at the base of the activation loop and αC helix are shown. Ionic interactions are indicated as dotted lines.

Figure 2 from paper

Figure 2 - Effect of the SH2-N-lobe interaction on kinase activity


Kinase activities, as measured by the Vmax values, of the SH2-KD construct and two mutanted variants thereof referenced to the activity of the isolated kinase domain. Perturbation of the SH2-N-lobe interface by the mutation I164E nullifies the stimulating effect of the SH2 domain, while the mutation T231R enhances it.

Figure 3 from paper

Figure 3 - Effect of the SH2-N-lobe interaction on imatinib binding


Imatinib binding rates (kon) of the SH2-KD construct and two mutants thereof referenced to that of the isolated kinase domain. Formation of the native SH2-N-lobe interface decreases the rate of drug binding and is reduced further by the mutation T231R. Mutational perturbation of the interface (I164E), however, enhances the binding rate compared to the wildtype.

Figure 4A from paper

Figure 4A - Crystal structure of the Abl SH2-KD construct


Cartoon representation of the crystal structure of the SH2-KD construct (chain B) in complex with dasatinib (cyan, ball-and-stick representation). The SH2 domain is colored yellow, the kinase domain blue. The side chains of two residues at the SH2-N-lobe interface, Ile 164 and Thr 231, whose roles were studied by mutagenesis are highlighted.

Figure 4B from paper

Figure 4B - Crystal structure of the Abl SH2-KD construct


Detailed view of the active site in the SH2-KD structure (top) compared to the published structure of the isolated kinase domain in complex with dasatinib, pdb ID: 2GQG (bottom) [24]. The activation loop is highlighted in orange and helix αC in blue. In contrast to the active state of the isolated kinase domain (DFG-Asp In/αC-Glu In), the SH2-KD construct adopts an intermediate state (DFG-Asp Out/αC-Glu Out).


Supplemental figures from the paper

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Supplemental Figure 1 from paper

Supplemental Figure 1 - Effect of the SH2-N-lobe interaction on Abl activity after pre-phoshorylation by Hck


Kinase activities of the pre-phosphorylated KD, SH2-KD, and two mutated variants thereof referenced to the activity of the isolated, unphosphorylated kinase domain (see Figure 2). Pre-incubation with Hck does not significantly enhance the activity of the isolated kinase domain, but slightly increases the activities of the three SH2-KD constructs (by a maximum factor of ~ 1.7). The relative activities of the pre-phosphorylated constructs are similar to what was seen without pre-phosphorylation (Figure 2). Perturbation of the SH2-N-lobe interface by the mutation I164E reduces the stimulating effect of the SH2 domain, while the mutation T231R enhances it.

FSupplemental igure 2 from paper

Supplemental Figure 2 - Detail of the active site in the crystal structure of the SH2-KD construct with dasatinib


Cartoon representation of the active site in the crystal structure of the SH2-KD construct (chain B, grey) in complex with dasatinib (cyan, ball-and-stick representation). The DFG motif is colored orange and shown in ball-and–stick representation. The Fo-Fc omit electron density (contoured at 3.0 σ) for the DFG motif and dasatinib is shown.

Supplemental Figure 3A from paper

Supplemental Figure 3A - Model of how Abl dimerization might promote processive substrate phosphorylation


Two peptides were modeled onto the structure of the Abl dimer observed in our crystal: A phosphotyrosine-containing peptide was modeled onto the SH2 domain of Abl subunit II based on the crystal structure of a peptide-bound SH2 domain of Src (PDB ID: 1SPS) [1]. The substrate peptide bound to the active site of Abl subunit I was modeled based on a crystal structure of the kinase domain with an ATP-peptide conjugate (PDB ID: 2G2I) [2]. In our model the two peptides have the same directionality and could, in principle, represent two parts of a substrate with multiple tyrosine residues that spanning across the dimer interface.

Supplemental Figure 3B from paper

Supplemental Figure 3B - Model of how Abl dimerization might promote processive substrate phosphorylation


Detail of the crystallographic dimer interface shown in (A). The interface is centered on a key aromatic stacking interaction between Tyr 158 of the SH2 domain of subunit II (yellow) and Tyr 468 in helix αC of the C-lobe of subunit I (light blue).