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SH2-catalytic domain linker heterogeneity influences allosteric coupling across the SFK family.

Register AC, Leonard SE, Maly DJ - Biochemistry (2014)

Bottom Line: Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix.Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases.Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Washington , Seattle, Washington 98195, United States.

ABSTRACT
Src-family kinases (SFKs) make up a family of nine homologous multidomain tyrosine kinases whose misregulation is responsible for human disease (cancer, diabetes, inflammation, etc.). Despite overall sequence homology and identical domain architecture, differences in SH3 and SH2 regulatory domain accessibility and ability to allosterically autoinhibit the ATP-binding site have been observed for the prototypical SFKs Src and Hck. Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix. As a relatively unconserved region between SFK family members, SH2-CD linker sequence variability across the SFK family is likely a source of nonredundant cellular functions between individual SFKs via its effect on the availability of SH3 and SH2 domains for intermolecular interactions and post-translational modification. Using a combination of SFKs engineered with enhanced or weakened regulatory domain intramolecular interactions and conformation-selective inhibitors that report αC helix conformation, this study explores how SH2-CD sequence heterogeneity affects allosteric coupling across the SFK family by examining Lyn, Fyn1, and Fyn2. Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases. Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

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Conformation-selective inhibitors differentiallymodulate the SH3domain accessibilities of Lyn, Fyn1, and Fyn2. (A) Molecular structureof ligand 8 and a crystal structure of 8 bound to the ATP-binding site of Abl (PDB entry 3OXZ). 8 stabilizes the DFG-out inactive conformation and is predicted tostabilize helix αC in an active conformation by forming an electrostaticinteraction with Glu310. (B) Quantification of the SH3 pull-down experimentperformed with LynY527F in the presence of saturating 1, 2, 4, 5, or 8 (mean ± SEM; n = 3). All data arenormalized to the SFKY527F·1 complex(1 does not show a strong preference for SFKAct over SFKSH2eng). (C) Quantification of the SH3 pull-downexperiment performed with Fyn1Y527F and Fyn2Y527F in the presence of saturating 1, 2, 4, 5, or 8 (mean ± SEM; n = 3). All data are normalized to the SFKY527F·1 complex. Representative blots are shown in FigureS6 of the Supporting Information.
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fig7: Conformation-selective inhibitors differentiallymodulate the SH3domain accessibilities of Lyn, Fyn1, and Fyn2. (A) Molecular structureof ligand 8 and a crystal structure of 8 bound to the ATP-binding site of Abl (PDB entry 3OXZ). 8 stabilizes the DFG-out inactive conformation and is predicted tostabilize helix αC in an active conformation by forming an electrostaticinteraction with Glu310. (B) Quantification of the SH3 pull-down experimentperformed with LynY527F in the presence of saturating 1, 2, 4, 5, or 8 (mean ± SEM; n = 3). All data arenormalized to the SFKY527F·1 complex(1 does not show a strong preference for SFKAct over SFKSH2eng). (C) Quantification of the SH3 pull-downexperiment performed with Fyn1Y527F and Fyn2Y527F in the presence of saturating 1, 2, 4, 5, or 8 (mean ± SEM; n = 3). All data are normalized to the SFKY527F·1 complex. Representative blots are shown in FigureS6 of the Supporting Information.

Mentions: Giventhe surprising differences that were observed in how regulatory domainengagement affects ATP-binding site conformation in Fyn1 and Fyn2,we next investigated how αC helix conformation affects intramolecularregulatory domain engagement using our panel of conformation-selectiveligands. SH3 domain accessibility was measured using the pull-downassay described in the legend of Figure 3.Each SFK of interest was incubated with a saturating amount of a conformation-selectiveinhibitor before being exposed to SH3-binding peptide resin. Comparingthe relative amounts of retained Lyn, Fyn1, and Fyn2 when they arebound to active or αC helix-out stabilizing ligands providesa measure of how αC helix conformation influences SH3 domainaccessibility within the context of each SFK. Ligand 8, which stabilizes an inactive activation loop conformation (DFG-out)but an active αC helix conformation (αC helix-in), wasalso tested to investigate the contribution of the activation loopto regulatory domain accessibility. Ligands that stabilize the DFG-outinactive conformation form a hydrogen bond with Glu310 in the αChelix—similar to αC helix-in, active ligands 2–4—and are thus predicted to prefer the ATP-binding sites ofactivated over autoinhibited SFK constructs (Figure 7A). Consistent with this hypothesis, stabilizing the DFG-outconformation of Src and Hck results in increased SH3 domain accessibility.18 SH3 pull-downs were performed with LynY527F, Fyn1Y527F, and Fyn2Y527F in the presenceof a saturating concentration of 1, 2, 5, 6, or 8 (Figure 7B). For each of these experiments, ligand 1,which has a minimal preference for the SFK activation state, was usedas a reference compound. Representative blots for the data in Figure 7B are shown in Figure S6 of the Supporting Information.


SH2-catalytic domain linker heterogeneity influences allosteric coupling across the SFK family.

Register AC, Leonard SE, Maly DJ - Biochemistry (2014)

Conformation-selective inhibitors differentiallymodulate the SH3domain accessibilities of Lyn, Fyn1, and Fyn2. (A) Molecular structureof ligand 8 and a crystal structure of 8 bound to the ATP-binding site of Abl (PDB entry 3OXZ). 8 stabilizes the DFG-out inactive conformation and is predicted tostabilize helix αC in an active conformation by forming an electrostaticinteraction with Glu310. (B) Quantification of the SH3 pull-down experimentperformed with LynY527F in the presence of saturating 1, 2, 4, 5, or 8 (mean ± SEM; n = 3). All data arenormalized to the SFKY527F·1 complex(1 does not show a strong preference for SFKAct over SFKSH2eng). (C) Quantification of the SH3 pull-downexperiment performed with Fyn1Y527F and Fyn2Y527F in the presence of saturating 1, 2, 4, 5, or 8 (mean ± SEM; n = 3). All data are normalized to the SFKY527F·1 complex. Representative blots are shown in FigureS6 of the Supporting Information.
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fig7: Conformation-selective inhibitors differentiallymodulate the SH3domain accessibilities of Lyn, Fyn1, and Fyn2. (A) Molecular structureof ligand 8 and a crystal structure of 8 bound to the ATP-binding site of Abl (PDB entry 3OXZ). 8 stabilizes the DFG-out inactive conformation and is predicted tostabilize helix αC in an active conformation by forming an electrostaticinteraction with Glu310. (B) Quantification of the SH3 pull-down experimentperformed with LynY527F in the presence of saturating 1, 2, 4, 5, or 8 (mean ± SEM; n = 3). All data arenormalized to the SFKY527F·1 complex(1 does not show a strong preference for SFKAct over SFKSH2eng). (C) Quantification of the SH3 pull-downexperiment performed with Fyn1Y527F and Fyn2Y527F in the presence of saturating 1, 2, 4, 5, or 8 (mean ± SEM; n = 3). All data are normalized to the SFKY527F·1 complex. Representative blots are shown in FigureS6 of the Supporting Information.
Mentions: Giventhe surprising differences that were observed in how regulatory domainengagement affects ATP-binding site conformation in Fyn1 and Fyn2,we next investigated how αC helix conformation affects intramolecularregulatory domain engagement using our panel of conformation-selectiveligands. SH3 domain accessibility was measured using the pull-downassay described in the legend of Figure 3.Each SFK of interest was incubated with a saturating amount of a conformation-selectiveinhibitor before being exposed to SH3-binding peptide resin. Comparingthe relative amounts of retained Lyn, Fyn1, and Fyn2 when they arebound to active or αC helix-out stabilizing ligands providesa measure of how αC helix conformation influences SH3 domainaccessibility within the context of each SFK. Ligand 8, which stabilizes an inactive activation loop conformation (DFG-out)but an active αC helix conformation (αC helix-in), wasalso tested to investigate the contribution of the activation loopto regulatory domain accessibility. Ligands that stabilize the DFG-outinactive conformation form a hydrogen bond with Glu310 in the αChelix—similar to αC helix-in, active ligands 2–4—and are thus predicted to prefer the ATP-binding sites ofactivated over autoinhibited SFK constructs (Figure 7A). Consistent with this hypothesis, stabilizing the DFG-outconformation of Src and Hck results in increased SH3 domain accessibility.18 SH3 pull-downs were performed with LynY527F, Fyn1Y527F, and Fyn2Y527F in the presenceof a saturating concentration of 1, 2, 5, 6, or 8 (Figure 7B). For each of these experiments, ligand 1,which has a minimal preference for the SFK activation state, was usedas a reference compound. Representative blots for the data in Figure 7B are shown in Figure S6 of the Supporting Information.

Bottom Line: Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix.Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases.Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Washington , Seattle, Washington 98195, United States.

ABSTRACT
Src-family kinases (SFKs) make up a family of nine homologous multidomain tyrosine kinases whose misregulation is responsible for human disease (cancer, diabetes, inflammation, etc.). Despite overall sequence homology and identical domain architecture, differences in SH3 and SH2 regulatory domain accessibility and ability to allosterically autoinhibit the ATP-binding site have been observed for the prototypical SFKs Src and Hck. Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix. As a relatively unconserved region between SFK family members, SH2-CD linker sequence variability across the SFK family is likely a source of nonredundant cellular functions between individual SFKs via its effect on the availability of SH3 and SH2 domains for intermolecular interactions and post-translational modification. Using a combination of SFKs engineered with enhanced or weakened regulatory domain intramolecular interactions and conformation-selective inhibitors that report αC helix conformation, this study explores how SH2-CD sequence heterogeneity affects allosteric coupling across the SFK family by examining Lyn, Fyn1, and Fyn2. Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases. Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

Show MeSH
Related in: MedlinePlus