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A potent and highly specific FN3 monobody inhibitor of the Abl SH2 domain.

Wojcik J, Hantschel O, Grebien F, Kaupe I, Bennett KL, Barkinge J, Jones RB, Koide A, Superti-Furga G, Koide S - Nat. Struct. Mol. Biol. (2010)

Bottom Line: HA4 disrupted intramolecular interactions of Abl involving the SH2 domain and potently activated the kinase in vitro.Within cells, HA4 inhibited processive phosphorylation activity of Abl and also inhibited STAT5 activation.This work provides a design guideline for highly specific and potent inhibitors of a protein interaction domain and shows their utility in mechanistic and cellular investigations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.

ABSTRACT
Interactions between Src homology 2 (SH2) domains and phosphotyrosine sites regulate tyrosine kinase signaling networks. Selective perturbation of these interactions is challenging due to the high homology among the 120 human SH2 domains. Using an improved phage-display selection system, we generated a small antibody mimic (or 'monobody'), termed HA4, that bound to the Abelson (Abl) kinase SH2 domain with low nanomolar affinity. SH2 protein microarray analysis and MS of intracellular HA4 interactors showed HA4's specificity, and a crystal structure revealed how this specificity is achieved. HA4 disrupted intramolecular interactions of Abl involving the SH2 domain and potently activated the kinase in vitro. Within cells, HA4 inhibited processive phosphorylation activity of Abl and also inhibited STAT5 activation. This work provides a design guideline for highly specific and potent inhibitors of a protein interaction domain and shows their utility in mechanistic and cellular investigations.

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HA4 activates autoinhibited Abl(a) Left, an overlay of the HA4/Abl SH2 complex (blue; only HA4 is shown for clarity) and autoinhibited Abl (1OPL)36 highlighting steric clash (boxed) between the HA4 FG loop (black mesh) and the Abl αI-αI′ helix (red mesh). The SH3, SH2 and kinase domains of Abl are colored in red, yellow and green, respectively. In the blow-up, SH2 is omitted for clarity. (b) Mutually exclusive interactions of two SH2 residues (Arg153 and Arg189) between the two structures. Residues in the SH2 and kinase domains of the autoinhibited Abl structure are shown with the carbon atoms in cyan and green, respectively. Residues in the SH2 domain and HA4 in the HA4/SH2 complex are shown with the carbon atoms in yellow and white, respectively. Polar interactions involving Arg153 and Arg189 of the SH2 domain are shown with dashed lines. SH2 residues are labeled in red. (c) Effects of HA4 on Abl activation. Left, the activity of the Abl kinase is plotted as a function of HA4 (solid black), phosphopeptide (red), and HA4Y87A (open squares) concentrations. The activity was normalized relative to that in the absence of monobody or peptide. Right, the normalized activity of an autoinhibition-defective mutant of Abl kinase (G2APP: open boxes, dashed line) and the activity of the highly activated Bcr-Abl fusion protein (solid boxes, solid line) plotted as a function of HA4 concentration.
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Figure 5: HA4 activates autoinhibited Abl(a) Left, an overlay of the HA4/Abl SH2 complex (blue; only HA4 is shown for clarity) and autoinhibited Abl (1OPL)36 highlighting steric clash (boxed) between the HA4 FG loop (black mesh) and the Abl αI-αI′ helix (red mesh). The SH3, SH2 and kinase domains of Abl are colored in red, yellow and green, respectively. In the blow-up, SH2 is omitted for clarity. (b) Mutually exclusive interactions of two SH2 residues (Arg153 and Arg189) between the two structures. Residues in the SH2 and kinase domains of the autoinhibited Abl structure are shown with the carbon atoms in cyan and green, respectively. Residues in the SH2 domain and HA4 in the HA4/SH2 complex are shown with the carbon atoms in yellow and white, respectively. Polar interactions involving Arg153 and Arg189 of the SH2 domain are shown with dashed lines. SH2 residues are labeled in red. (c) Effects of HA4 on Abl activation. Left, the activity of the Abl kinase is plotted as a function of HA4 (solid black), phosphopeptide (red), and HA4Y87A (open squares) concentrations. The activity was normalized relative to that in the absence of monobody or peptide. Right, the normalized activity of an autoinhibition-defective mutant of Abl kinase (G2APP: open boxes, dashed line) and the activity of the highly activated Bcr-Abl fusion protein (solid boxes, solid line) plotted as a function of HA4 concentration.

Mentions: The SH2 domain plays an important role in Abl autoinhibition by interacting with the C-terminal lobe of the kinase domain.36 The binding of Abl SH2 to phosphopeptide ligands activates Abl in vitro, and is thought to mimic the activation of Abl by cellular substrates.23,41 Superposition of the HA4/Abl SH2 structure on the autoinhibited structure of Abl indicates steric clashes between HA4 and the kinase domain (Fig. 5a). In addition, HA4 directly interacts with SH2 residues that form salt bridges and hydrogen bonds with the C-lobe of the kinase in autoinhibited Abl (Fig. 5b). These observations suggest that the binding of HA4 to the SH2 domain should disrupt the autoinhibited conformation, thereby activating the kinase. Indeed, in in vitro Abl kinase assays HA4 and a phosphopeptide derived from c-Jun (Kd = ∼7 μM)41 caused a dose-dependent increase in Abl activity but HA4Y87A did not (Fig. 5c, left). HA4 was a substantially more potent activator than the phosphopeptide, in line with its ∼1000× higher affinity for the SH2 domain.


A potent and highly specific FN3 monobody inhibitor of the Abl SH2 domain.

Wojcik J, Hantschel O, Grebien F, Kaupe I, Bennett KL, Barkinge J, Jones RB, Koide A, Superti-Furga G, Koide S - Nat. Struct. Mol. Biol. (2010)

HA4 activates autoinhibited Abl(a) Left, an overlay of the HA4/Abl SH2 complex (blue; only HA4 is shown for clarity) and autoinhibited Abl (1OPL)36 highlighting steric clash (boxed) between the HA4 FG loop (black mesh) and the Abl αI-αI′ helix (red mesh). The SH3, SH2 and kinase domains of Abl are colored in red, yellow and green, respectively. In the blow-up, SH2 is omitted for clarity. (b) Mutually exclusive interactions of two SH2 residues (Arg153 and Arg189) between the two structures. Residues in the SH2 and kinase domains of the autoinhibited Abl structure are shown with the carbon atoms in cyan and green, respectively. Residues in the SH2 domain and HA4 in the HA4/SH2 complex are shown with the carbon atoms in yellow and white, respectively. Polar interactions involving Arg153 and Arg189 of the SH2 domain are shown with dashed lines. SH2 residues are labeled in red. (c) Effects of HA4 on Abl activation. Left, the activity of the Abl kinase is plotted as a function of HA4 (solid black), phosphopeptide (red), and HA4Y87A (open squares) concentrations. The activity was normalized relative to that in the absence of monobody or peptide. Right, the normalized activity of an autoinhibition-defective mutant of Abl kinase (G2APP: open boxes, dashed line) and the activity of the highly activated Bcr-Abl fusion protein (solid boxes, solid line) plotted as a function of HA4 concentration.
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Figure 5: HA4 activates autoinhibited Abl(a) Left, an overlay of the HA4/Abl SH2 complex (blue; only HA4 is shown for clarity) and autoinhibited Abl (1OPL)36 highlighting steric clash (boxed) between the HA4 FG loop (black mesh) and the Abl αI-αI′ helix (red mesh). The SH3, SH2 and kinase domains of Abl are colored in red, yellow and green, respectively. In the blow-up, SH2 is omitted for clarity. (b) Mutually exclusive interactions of two SH2 residues (Arg153 and Arg189) between the two structures. Residues in the SH2 and kinase domains of the autoinhibited Abl structure are shown with the carbon atoms in cyan and green, respectively. Residues in the SH2 domain and HA4 in the HA4/SH2 complex are shown with the carbon atoms in yellow and white, respectively. Polar interactions involving Arg153 and Arg189 of the SH2 domain are shown with dashed lines. SH2 residues are labeled in red. (c) Effects of HA4 on Abl activation. Left, the activity of the Abl kinase is plotted as a function of HA4 (solid black), phosphopeptide (red), and HA4Y87A (open squares) concentrations. The activity was normalized relative to that in the absence of monobody or peptide. Right, the normalized activity of an autoinhibition-defective mutant of Abl kinase (G2APP: open boxes, dashed line) and the activity of the highly activated Bcr-Abl fusion protein (solid boxes, solid line) plotted as a function of HA4 concentration.
Mentions: The SH2 domain plays an important role in Abl autoinhibition by interacting with the C-terminal lobe of the kinase domain.36 The binding of Abl SH2 to phosphopeptide ligands activates Abl in vitro, and is thought to mimic the activation of Abl by cellular substrates.23,41 Superposition of the HA4/Abl SH2 structure on the autoinhibited structure of Abl indicates steric clashes between HA4 and the kinase domain (Fig. 5a). In addition, HA4 directly interacts with SH2 residues that form salt bridges and hydrogen bonds with the C-lobe of the kinase in autoinhibited Abl (Fig. 5b). These observations suggest that the binding of HA4 to the SH2 domain should disrupt the autoinhibited conformation, thereby activating the kinase. Indeed, in in vitro Abl kinase assays HA4 and a phosphopeptide derived from c-Jun (Kd = ∼7 μM)41 caused a dose-dependent increase in Abl activity but HA4Y87A did not (Fig. 5c, left). HA4 was a substantially more potent activator than the phosphopeptide, in line with its ∼1000× higher affinity for the SH2 domain.

Bottom Line: HA4 disrupted intramolecular interactions of Abl involving the SH2 domain and potently activated the kinase in vitro.Within cells, HA4 inhibited processive phosphorylation activity of Abl and also inhibited STAT5 activation.This work provides a design guideline for highly specific and potent inhibitors of a protein interaction domain and shows their utility in mechanistic and cellular investigations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.

ABSTRACT
Interactions between Src homology 2 (SH2) domains and phosphotyrosine sites regulate tyrosine kinase signaling networks. Selective perturbation of these interactions is challenging due to the high homology among the 120 human SH2 domains. Using an improved phage-display selection system, we generated a small antibody mimic (or 'monobody'), termed HA4, that bound to the Abelson (Abl) kinase SH2 domain with low nanomolar affinity. SH2 protein microarray analysis and MS of intracellular HA4 interactors showed HA4's specificity, and a crystal structure revealed how this specificity is achieved. HA4 disrupted intramolecular interactions of Abl involving the SH2 domain and potently activated the kinase in vitro. Within cells, HA4 inhibited processive phosphorylation activity of Abl and also inhibited STAT5 activation. This work provides a design guideline for highly specific and potent inhibitors of a protein interaction domain and shows their utility in mechanistic and cellular investigations.

Show MeSH
Related in: MedlinePlus