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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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SH2 protein microarray assays of HA4 specificity(a) Fluorescence images of SH2 chips following incubation with fluorophore-labeled HA4. Each SH2 domain is spotted in duplicate. A listing of spotted SH2 domains is provided in Supplementary Table 1. Spots for the SH2 domains shown in (b) are enclosed in the boxes. (b) The fluorescence signal strengths of individual SH2 domain spots are plotted as a function of HA4 concentration. The curves show the best fit of a 1:1 binding model, and the estimated dissociation constants are provided.
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Figure 2: SH2 protein microarray assays of HA4 specificity(a) Fluorescence images of SH2 chips following incubation with fluorophore-labeled HA4. Each SH2 domain is spotted in duplicate. A listing of spotted SH2 domains is provided in Supplementary Table 1. Spots for the SH2 domains shown in (b) are enclosed in the boxes. (b) The fluorescence signal strengths of individual SH2 domain spots are plotted as a function of HA4 concentration. The curves show the best fit of a 1:1 binding model, and the estimated dissociation constants are provided.

Mentions: We sought to rigorously characterize the specificity of HA4 using SH2 domain protein microarrays.4 Critically, these protein microarrays retain most of the spotted SH2 domains in their folded, functional state. Of the of 84 SH2 domains present on the chip (Supplementary Table 1), HA4 interacted most strongly with the Abl and Abl2 (Arg) SH2 domains (Fig. 2a, left panel), indicating a high level of specificity. Because the Abl and Abl2 SH2 domains share >90% sequence identity, this cross reactivity was expected. At higher concentrations of HA4, binding to a small number of additional SH2 domains was also detected (Fig. 2a, right panel). Due to differences in the amount and quality of SH2 domains at individual spots, signal intensity does not directly correlate with the strength of interaction. Therefore, we analyzed the microarray data for each spot as a function of HA4 concentration. The apparent Kd values for the Abl and Abl2 SH2 domains were nearly identical (22 nM and 18 nM, respectively; Fig. 2b), and were consistent with those obtained by SPR. The majority of the spotted SH2 domains (63 domains) show no signal above background levels even with 500 nM HA4, the highest concentration used (data not shown). Furthermore, 15 randomly selected SH2 domains had no detectable interactions with HA4 as tested with SPR, indicating Kd > 5 μM and confirming the microarray results (data not shown).


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)

SH2 protein microarray assays of HA4 specificity(a) Fluorescence images of SH2 chips following incubation with fluorophore-labeled HA4. Each SH2 domain is spotted in duplicate. A listing of spotted SH2 domains is provided in Supplementary Table 1. Spots for the SH2 domains shown in (b) are enclosed in the boxes. (b) The fluorescence signal strengths of individual SH2 domain spots are plotted as a function of HA4 concentration. The curves show the best fit of a 1:1 binding model, and the estimated dissociation constants are provided.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2926940&req=5

Figure 2: SH2 protein microarray assays of HA4 specificity(a) Fluorescence images of SH2 chips following incubation with fluorophore-labeled HA4. Each SH2 domain is spotted in duplicate. A listing of spotted SH2 domains is provided in Supplementary Table 1. Spots for the SH2 domains shown in (b) are enclosed in the boxes. (b) The fluorescence signal strengths of individual SH2 domain spots are plotted as a function of HA4 concentration. The curves show the best fit of a 1:1 binding model, and the estimated dissociation constants are provided.
Mentions: We sought to rigorously characterize the specificity of HA4 using SH2 domain protein microarrays.4 Critically, these protein microarrays retain most of the spotted SH2 domains in their folded, functional state. Of the of 84 SH2 domains present on the chip (Supplementary Table 1), HA4 interacted most strongly with the Abl and Abl2 (Arg) SH2 domains (Fig. 2a, left panel), indicating a high level of specificity. Because the Abl and Abl2 SH2 domains share >90% sequence identity, this cross reactivity was expected. At higher concentrations of HA4, binding to a small number of additional SH2 domains was also detected (Fig. 2a, right panel). Due to differences in the amount and quality of SH2 domains at individual spots, signal intensity does not directly correlate with the strength of interaction. Therefore, we analyzed the microarray data for each spot as a function of HA4 concentration. The apparent Kd values for the Abl and Abl2 SH2 domains were nearly identical (22 nM and 18 nM, respectively; Fig. 2b), and were consistent with those obtained by SPR. The majority of the spotted SH2 domains (63 domains) show no signal above background levels even with 500 nM HA4, the highest concentration used (data not shown). Furthermore, 15 randomly selected SH2 domains had no detectable interactions with HA4 as tested with SPR, indicating Kd > 5 μM and confirming the microarray results (data not shown).

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