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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.

Show MeSH
Library design and selected Abl SH2-binding monobodies(a) Schematic of the FN3 scaffold. β-Strands are labeled with A–G and loop regions diversified in the combinatorial library are in cyan. Figure generated using PyMOL (www.pymol.org). (b) Library design and loop sequences of Abl SH2-binding monobodies. X refers to a mixture of 30% Tyr (yellow), 15% Ser (red), 10% Gly (Green), 5% each of Trp, Phe and Arg (Green) and 2.5% each of all other amino acids except Cys. Z refers to a mixture of 50% Gly, 25% Tyr and 25% Ser. The numbers indicate positions for HA4. The Tyr87 position, mutated in the HA4Y87A non-binding control, is marked with the asterisk. Because of differences in loop lengths, the numbering does not correspond to previously published monobodies. (c) SPR traces for HA4 binding to immobilized Abl SH2 domain, corrected by subtraction of the sensorgram for a blank run (gray) Parameters for the global Langmuir fit are provided, and the black lines show the best fit. Left, measurements in non-phosphate buffer. Right, measurements in phosphate buffer. (d) Left, fluorescence polarization changes of a rhodamine-labeled pY-peptide as a function of GST-Abl SH2 added to the solution. The concentration of GST-Abl SH2 required to give ∼80% maximum polarization (10 μM, indicated with the arrow) was used for HA4 competition assay shown on the right panel. Right: Fluorescence polarization of the rhodamine-labeled pY-peptide in the presence of GST-Abl SH2 is plotted versus the concentration of monobody added to the solution.
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Figure 1: Library design and selected Abl SH2-binding monobodies(a) Schematic of the FN3 scaffold. β-Strands are labeled with A–G and loop regions diversified in the combinatorial library are in cyan. Figure generated using PyMOL (www.pymol.org). (b) Library design and loop sequences of Abl SH2-binding monobodies. X refers to a mixture of 30% Tyr (yellow), 15% Ser (red), 10% Gly (Green), 5% each of Trp, Phe and Arg (Green) and 2.5% each of all other amino acids except Cys. Z refers to a mixture of 50% Gly, 25% Tyr and 25% Ser. The numbers indicate positions for HA4. The Tyr87 position, mutated in the HA4Y87A non-binding control, is marked with the asterisk. Because of differences in loop lengths, the numbering does not correspond to previously published monobodies. (c) SPR traces for HA4 binding to immobilized Abl SH2 domain, corrected by subtraction of the sensorgram for a blank run (gray) Parameters for the global Langmuir fit are provided, and the black lines show the best fit. Left, measurements in non-phosphate buffer. Right, measurements in phosphate buffer. (d) Left, fluorescence polarization changes of a rhodamine-labeled pY-peptide as a function of GST-Abl SH2 added to the solution. The concentration of GST-Abl SH2 required to give ∼80% maximum polarization (10 μM, indicated with the arrow) was used for HA4 competition assay shown on the right panel. Right: Fluorescence polarization of the rhodamine-labeled pY-peptide in the presence of GST-Abl SH2 is plotted versus the concentration of monobody added to the solution.

Mentions: Protein-based inhibitors are a promising but unproven alternative to small molecule- or phosphopeptide-mediated inhibition of SH2–phosphopeptide interactions. Because protein-protein interactions generally involve larger interfaces than protein-small molecule or protein-peptide interactions, protein-based inhibitors could promote specificity through interactions outside of the conserved phosphopeptide-binding interface. Currently, antibodies are the most common protein-based inhibitors16, but as large, multi-chain, disulfide-stabilized proteins, they are not likely to fold into the functional form in the reducing environment of the cytoplasm. An attractive alternative are engineered single-domain binding proteins. One of the best established systems is based on the tenth human fibronectin type III domain (FN3), a small (10 kDa), highly stable β–sandwich protein with surface loops tolerant to extensive mutations.17 These surface loops form a binding interface analogous to that presented by the complementarity determining regions of antibodies (Fig. 1a). FN3-based binding proteins, called ‘monobodies,’ do not contain disulfides and are thus functional in both oxidizing and reducing environments, making them amenable to in vitro and cellular studies. Moreover, because monobodies tend to recognize binding ‘hot spots’ of the target protein, they often act as competitive inhibitors.18,19


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)

Library design and selected Abl SH2-binding monobodies(a) Schematic of the FN3 scaffold. β-Strands are labeled with A–G and loop regions diversified in the combinatorial library are in cyan. Figure generated using PyMOL (www.pymol.org). (b) Library design and loop sequences of Abl SH2-binding monobodies. X refers to a mixture of 30% Tyr (yellow), 15% Ser (red), 10% Gly (Green), 5% each of Trp, Phe and Arg (Green) and 2.5% each of all other amino acids except Cys. Z refers to a mixture of 50% Gly, 25% Tyr and 25% Ser. The numbers indicate positions for HA4. The Tyr87 position, mutated in the HA4Y87A non-binding control, is marked with the asterisk. Because of differences in loop lengths, the numbering does not correspond to previously published monobodies. (c) SPR traces for HA4 binding to immobilized Abl SH2 domain, corrected by subtraction of the sensorgram for a blank run (gray) Parameters for the global Langmuir fit are provided, and the black lines show the best fit. Left, measurements in non-phosphate buffer. Right, measurements in phosphate buffer. (d) Left, fluorescence polarization changes of a rhodamine-labeled pY-peptide as a function of GST-Abl SH2 added to the solution. The concentration of GST-Abl SH2 required to give ∼80% maximum polarization (10 μM, indicated with the arrow) was used for HA4 competition assay shown on the right panel. Right: Fluorescence polarization of the rhodamine-labeled pY-peptide in the presence of GST-Abl SH2 is plotted versus the concentration of monobody added to the solution.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2926940&req=5

Figure 1: Library design and selected Abl SH2-binding monobodies(a) Schematic of the FN3 scaffold. β-Strands are labeled with A–G and loop regions diversified in the combinatorial library are in cyan. Figure generated using PyMOL (www.pymol.org). (b) Library design and loop sequences of Abl SH2-binding monobodies. X refers to a mixture of 30% Tyr (yellow), 15% Ser (red), 10% Gly (Green), 5% each of Trp, Phe and Arg (Green) and 2.5% each of all other amino acids except Cys. Z refers to a mixture of 50% Gly, 25% Tyr and 25% Ser. The numbers indicate positions for HA4. The Tyr87 position, mutated in the HA4Y87A non-binding control, is marked with the asterisk. Because of differences in loop lengths, the numbering does not correspond to previously published monobodies. (c) SPR traces for HA4 binding to immobilized Abl SH2 domain, corrected by subtraction of the sensorgram for a blank run (gray) Parameters for the global Langmuir fit are provided, and the black lines show the best fit. Left, measurements in non-phosphate buffer. Right, measurements in phosphate buffer. (d) Left, fluorescence polarization changes of a rhodamine-labeled pY-peptide as a function of GST-Abl SH2 added to the solution. The concentration of GST-Abl SH2 required to give ∼80% maximum polarization (10 μM, indicated with the arrow) was used for HA4 competition assay shown on the right panel. Right: Fluorescence polarization of the rhodamine-labeled pY-peptide in the presence of GST-Abl SH2 is plotted versus the concentration of monobody added to the solution.
Mentions: Protein-based inhibitors are a promising but unproven alternative to small molecule- or phosphopeptide-mediated inhibition of SH2–phosphopeptide interactions. Because protein-protein interactions generally involve larger interfaces than protein-small molecule or protein-peptide interactions, protein-based inhibitors could promote specificity through interactions outside of the conserved phosphopeptide-binding interface. Currently, antibodies are the most common protein-based inhibitors16, but as large, multi-chain, disulfide-stabilized proteins, they are not likely to fold into the functional form in the reducing environment of the cytoplasm. An attractive alternative are engineered single-domain binding proteins. One of the best established systems is based on the tenth human fibronectin type III domain (FN3), a small (10 kDa), highly stable β–sandwich protein with surface loops tolerant to extensive mutations.17 These surface loops form a binding interface analogous to that presented by the complementarity determining regions of antibodies (Fig. 1a). FN3-based binding proteins, called ‘monobodies,’ do not contain disulfides and are thus functional in both oxidizing and reducing environments, making them amenable to in vitro and cellular studies. Moreover, because monobodies tend to recognize binding ‘hot spots’ of the target protein, they often act as competitive inhibitors.18,19

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