Structural Basis for the Inhibition of Tyrosine Kinase Activity of ZAP-70
Sebastian Deindl, Theresa A. Kadlecek, Tomas Brdicka, Xiaoxian Cao, Arthur Weiss and John Kuriyan
ZAP-70, a cytoplasmic tyrosine kinase required for T cell antigen receptor signaling, is controlled by a regulatory segment that includes a tandem SH2 unit responsible for binding to immunoreceptor tyrosine-based activation motifs (ITAMs). The crystal structure of autoinhibited ZAP-70 reveals that the inactive kinase domain adopts a conformation similar to that of cyclin-dependent kinases and Src kinases. The autoinhibitory mechanism of ZAP-70 is, however, distinct and involves interactions between the regulatory segment and the hinge region of the kinase domain that reduce its flexibility. Two tyrosine residues in the SH2-kinase linker that activate ZAP-70 when phosphorylated are involved in aromatic-aromatic interactions that connect the linker to the kinase domain. These interactions are inconsistent with ITAM binding, suggesting that destabilization of this autoinhibited ZAP-70 conformation is the first step in kinase activation.
Figure 1. Structural Organization of Inactive ZAP-70 and Comparison with Autoinhibited Src Family Kinases
(A) Domain organization (top) and crystal structure (bottom) of inactive ZAP-70. The kinase domain, SH2-kinase linker, inter-SH2 linker, C-terminal SH2 domain, and N-terminal SH2 domain are shown in blue, red, green, yellow, and orange, respectively. The two phosphotyrosine-binding sites are indicated with magenta spheres. Disordered regions are depicted as dotted lines.
(B) Domain organization (top) and structure (bottom) of autoinhibited Src family kinases. The structure shown here is that of inactive Hck (PDB code 1QCF; Schindler et al., 1999).
Figure 3. Comparison of the Kinase Domain of an Autoinhibited Src Family Kinase with Inactive and Active ZAP-70
Helix αC is highlighted in dark blue, the activation loop is shown in brown, and the short helix in the activation loop following the DFG motif is shown in magenta. The dotted line represents an unordered part of the activation loop. The Src family kinase shown here is Hck (PDB code 1QCF; Schindler et al., 1999). The structure of c-Src is very similar (PDB code 2SRC; Xu et al., 1999). Active ZAP-70: PDB code 1U59 (Jin et al., 2004).
Illustrator file, local only
Figure 4. Apparent Reduction in Flexibility of the Hinge Region of the Kinase Domain
(A) Structures of active (kinase domain only; PDB code 1U59; Jin et al., 2004) and inactive ZAP-70, colored according to the crystallographic temperature factors for each atom. The mean value of the temperature factors for all atoms in helix αF (residues 518 to 534) is approximately the same in both structures, as indicated by the similar colors for atoms in this helix. (B) The hydrogen-bonding network centered on the hinge region of the inactive ZAP-70 kinase domain (left). This network is disrupted in active ZAP-70 (right).
Figure 5. Mutagenesis Identifies Hot Spot Residues in the Linker-Kinase Sandwich
(A) Two schematic views of ZAP-70, rotated by 180° with respect to each other, depict the location of residues in ZAP-70 that are most critical for autoinhibition. These hot spot residues are indicated by yellow stars.
(B) LAT phosphorylation by ZAP-70 mutants. The Tyr315Ala/Tyr319Ala double mutant is referred to as "YYAA" Strongly activating alanine mutations at hot spots of the linker-kinase sandwich are highlighted with yellow stars. Control experiments in the presence of Lck are shown in Figure S4.
Figure 6. Interaction between the Inter-SH2 Linker and the Kinase Domain
Detailed view showing the docking of Pro147 in the inter-SH2 linker into the cleft formed by the side chains of Tyr597 and Tyr598. Helix αI is shown as a light brown surface, and Tyr597 and Tyr598 are shown in magenta surface representation.
Figure 7. A Conformational Change in the Tandem SH2 Unit upon ITAM Binding May Be Coupled to Activation
(A) Left: ITAM-complexed tandem SH2 domains superimposed onto the tandem SH2 unit of inactive ZAP-70, based on helix αL2. The N-terminal SH2 domain of inactive ZAP-70 is not shown. The inter-SH2 linker is green (inactive ZAP-70) or cyan (ITAM-complexed tandem SH2 unit). The doubly phosphorylated ITAM peptide is in purple. Right: A detailed view showing that ITAM binding requires an 10° increase in the elbow angle between helices αL2 and αL3 and leads to the disruption of a hydrogen bond between Gln145 and Ser144.
(B) Model for activation of ZAP-70 by ITAM motifs. Top: Two views, 90° rotated from each other, showing a superimposition of the ITAM-complexed SH2 domains onto inactive ZAP-70 based on the N-terminal SH2 domain. The ITAM-bound tandem SH2 unit is orange (N-terminal SH2 domain), yellow (C-terminal SH2 domain), and green (inter-SH2 linker). The tandem SH2 domains of inactive ZAP-70 are shown in gray. Bottom: Schematic diagram of conformational changes upon ITAM binding.
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