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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
Structural basis for HA4's specificity toward Abl and Abl2(a) Amino-acid sequence alignment of Abl, Abl2 and Src family SH2 domains. Residues within 5Å of the HA4 interface are colored as follows: gray, residues where the consensus amino acid among the Src family members is identical to that of Abl; yellow, conservative substitutions; and red, non-conservative substitutions. Residues in the SH2 CD loop are in the blue box. Residues in the peptide-binding interface, inferred from the Lck structure39 are indicated with asterisks. (b) Cartoon model of the Lck SH2 domain structure with a bound peptide (1LCJ).39 The phosphopetide (sticks) lies across the central strand (βD). The pY-binding pocket and the Y+3 pocket are on either side of βD. (c) Conservation of HA4-interacting residues shown on the surface of the Lck SH2 domain. A phosphopeptide (sticks) highlights the overlap between phosphopeptide-binding and HA4-binding interfaces. (d) HA4/Abl complex (left) and hypothetical HA4/Lck complex (right, modeled by aligning Lck SH2 with Abl SH2 in the HA4/Abl SH2 complex), emphasizing SH2 CD-loop residues (blue). The two SH2 domains are viewed from an equivalent direction. HA4 is shown as a cartoon model, with the DE loop shown as orange sticks and gray mesh. The CD loop of the Lck SH2 domain that creates a protruding knob and is predicted to clash with the DE loop of HA4 is enclosed in the solid circle. The equivalent region in the Abl SH2 domain is marked with the dotted circle.
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Figure 4: Structural basis for HA4's specificity toward Abl and Abl2(a) Amino-acid sequence alignment of Abl, Abl2 and Src family SH2 domains. Residues within 5Å of the HA4 interface are colored as follows: gray, residues where the consensus amino acid among the Src family members is identical to that of Abl; yellow, conservative substitutions; and red, non-conservative substitutions. Residues in the SH2 CD loop are in the blue box. Residues in the peptide-binding interface, inferred from the Lck structure39 are indicated with asterisks. (b) Cartoon model of the Lck SH2 domain structure with a bound peptide (1LCJ).39 The phosphopetide (sticks) lies across the central strand (βD). The pY-binding pocket and the Y+3 pocket are on either side of βD. (c) Conservation of HA4-interacting residues shown on the surface of the Lck SH2 domain. A phosphopeptide (sticks) highlights the overlap between phosphopeptide-binding and HA4-binding interfaces. (d) HA4/Abl complex (left) and hypothetical HA4/Lck complex (right, modeled by aligning Lck SH2 with Abl SH2 in the HA4/Abl SH2 complex), emphasizing SH2 CD-loop residues (blue). The two SH2 domains are viewed from an equivalent direction. HA4 is shown as a cartoon model, with the DE loop shown as orange sticks and gray mesh. The CD loop of the Lck SH2 domain that creates a protruding knob and is predicted to clash with the DE loop of HA4 is enclosed in the solid circle. The equivalent region in the Abl SH2 domain is marked with the dotted circle.

Mentions: The HA4 structure resembles a cupped hand consisting of the ‘palm’ and ‘fingers’ upon which the Abl SH2 domain rests (Fig. 3b). The palm consists of one β-sheet of the FN3 scaffold and the C-terminal residue of the BC loop (Tyr35). The contacts made by HA4 ‘palm’ residues, contributing ∼40% of the total interface area, are at the periphery of the phosphopeptide-binding interface. A β-hairpin formed by a long FG loop of HA4 (residues 79–90) corresponds to the fingers that recognize the center of the phosphopeptide-binding interface, which make up ∼60% of the interface. The finger residues in the FG loop closely mimic the canonical backbone conformation of an SH2-bound phosphopeptide (Fig. 3c). In the stereotypical mode of SH2-phosphopeptide interaction, the phosphopeptide lies perpendicular to the SH2 central β-sheet, and interacts with surface cavities on either side of the central β-strand (βD) (Fig. 4b).38,39 One of these cavities accommodates pY, while the other forms a pocket for the amino acid three-residues C-terminal to pY (termed “Y+3”). In the HA4 FG loop, Tyr87 is inserted into the pY pocket where it forms cation-π interactions with two arginines along the sides of the pocket (Arg194 and Arg153; note that two-digit numbers refer to HA4 residues and three-digit numbers to SH2 residues), and polar contacts with Ser181 at the base (Fig. 3d). Residues Ser84–Gly86 form a network of polar interactions with SH2 residues at the periphery of the pY pocket. Although Asp83 does not contact the SH2 domain, it forms intramolecular hydrogen bonds with Gly86 and Tyr87, which may be important for supporting the FG loop conformation. Met88 makes hydrophobic contacts (Fig. 3b) and forms a main-chain H-bond with His192 of the SH2 domain (Fig. 3d). C-terminal to Met88, the main chain bends away from the surface such that the ‘Y+3 pocket’ of the SH2 domain is not engaged by the amino acid three residues C-terminal to Tyr87 (Fig. 3c). Instead, distinct from the canonical mode of peptide-SH2 interactions, Trp80, at the N-terminus of the FG loop, forms a large hydrophobic surface that blankets the Y+3 binding pocket.


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)

Structural basis for HA4's specificity toward Abl and Abl2(a) Amino-acid sequence alignment of Abl, Abl2 and Src family SH2 domains. Residues within 5Å of the HA4 interface are colored as follows: gray, residues where the consensus amino acid among the Src family members is identical to that of Abl; yellow, conservative substitutions; and red, non-conservative substitutions. Residues in the SH2 CD loop are in the blue box. Residues in the peptide-binding interface, inferred from the Lck structure39 are indicated with asterisks. (b) Cartoon model of the Lck SH2 domain structure with a bound peptide (1LCJ).39 The phosphopetide (sticks) lies across the central strand (βD). The pY-binding pocket and the Y+3 pocket are on either side of βD. (c) Conservation of HA4-interacting residues shown on the surface of the Lck SH2 domain. A phosphopeptide (sticks) highlights the overlap between phosphopeptide-binding and HA4-binding interfaces. (d) HA4/Abl complex (left) and hypothetical HA4/Lck complex (right, modeled by aligning Lck SH2 with Abl SH2 in the HA4/Abl SH2 complex), emphasizing SH2 CD-loop residues (blue). The two SH2 domains are viewed from an equivalent direction. HA4 is shown as a cartoon model, with the DE loop shown as orange sticks and gray mesh. The CD loop of the Lck SH2 domain that creates a protruding knob and is predicted to clash with the DE loop of HA4 is enclosed in the solid circle. The equivalent region in the Abl SH2 domain is marked with the dotted circle.
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Related In: Results  -  Collection

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Figure 4: Structural basis for HA4's specificity toward Abl and Abl2(a) Amino-acid sequence alignment of Abl, Abl2 and Src family SH2 domains. Residues within 5Å of the HA4 interface are colored as follows: gray, residues where the consensus amino acid among the Src family members is identical to that of Abl; yellow, conservative substitutions; and red, non-conservative substitutions. Residues in the SH2 CD loop are in the blue box. Residues in the peptide-binding interface, inferred from the Lck structure39 are indicated with asterisks. (b) Cartoon model of the Lck SH2 domain structure with a bound peptide (1LCJ).39 The phosphopetide (sticks) lies across the central strand (βD). The pY-binding pocket and the Y+3 pocket are on either side of βD. (c) Conservation of HA4-interacting residues shown on the surface of the Lck SH2 domain. A phosphopeptide (sticks) highlights the overlap between phosphopeptide-binding and HA4-binding interfaces. (d) HA4/Abl complex (left) and hypothetical HA4/Lck complex (right, modeled by aligning Lck SH2 with Abl SH2 in the HA4/Abl SH2 complex), emphasizing SH2 CD-loop residues (blue). The two SH2 domains are viewed from an equivalent direction. HA4 is shown as a cartoon model, with the DE loop shown as orange sticks and gray mesh. The CD loop of the Lck SH2 domain that creates a protruding knob and is predicted to clash with the DE loop of HA4 is enclosed in the solid circle. The equivalent region in the Abl SH2 domain is marked with the dotted circle.
Mentions: The HA4 structure resembles a cupped hand consisting of the ‘palm’ and ‘fingers’ upon which the Abl SH2 domain rests (Fig. 3b). The palm consists of one β-sheet of the FN3 scaffold and the C-terminal residue of the BC loop (Tyr35). The contacts made by HA4 ‘palm’ residues, contributing ∼40% of the total interface area, are at the periphery of the phosphopeptide-binding interface. A β-hairpin formed by a long FG loop of HA4 (residues 79–90) corresponds to the fingers that recognize the center of the phosphopeptide-binding interface, which make up ∼60% of the interface. The finger residues in the FG loop closely mimic the canonical backbone conformation of an SH2-bound phosphopeptide (Fig. 3c). In the stereotypical mode of SH2-phosphopeptide interaction, the phosphopeptide lies perpendicular to the SH2 central β-sheet, and interacts with surface cavities on either side of the central β-strand (βD) (Fig. 4b).38,39 One of these cavities accommodates pY, while the other forms a pocket for the amino acid three-residues C-terminal to pY (termed “Y+3”). In the HA4 FG loop, Tyr87 is inserted into the pY pocket where it forms cation-π interactions with two arginines along the sides of the pocket (Arg194 and Arg153; note that two-digit numbers refer to HA4 residues and three-digit numbers to SH2 residues), and polar contacts with Ser181 at the base (Fig. 3d). Residues Ser84–Gly86 form a network of polar interactions with SH2 residues at the periphery of the pY pocket. Although Asp83 does not contact the SH2 domain, it forms intramolecular hydrogen bonds with Gly86 and Tyr87, which may be important for supporting the FG loop conformation. Met88 makes hydrophobic contacts (Fig. 3b) and forms a main-chain H-bond with His192 of the SH2 domain (Fig. 3d). C-terminal to Met88, the main chain bends away from the surface such that the ‘Y+3 pocket’ of the SH2 domain is not engaged by the amino acid three residues C-terminal to Tyr87 (Fig. 3c). Instead, distinct from the canonical mode of peptide-SH2 interactions, Trp80, at the N-terminus of the FG loop, forms a large hydrophobic surface that blankets the Y+3 binding pocket.

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