Limits...
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.

Show MeSH

Related in: MedlinePlus

Binding preferences of conformation-selective inhibitors revealdifferences in allosteric coupling among Lyn, Fyn1, and Fyn2. (A)Conformation-selective inhibitor panel. 2–4 favorthe ATP-binding site of active (αC helix-in) SFKs, while 5–7 stabilize the αC helix-out, inactive ATP-bindingsite conformation. These ligands allow systematic analysis of theATP-binding site conformation in response to domain engagement. (B)Ligands that stabilize active and αC helix-out conformationsmake different contacts with the ATP-binding sites of SFKs. The leftpanel shows Src bound to 2 with the ATP-binding sitein an active conformation (PDB entry 3EN4). An electrostatic interaction betweenthe inhibitor and E310 in the αC helix is shown. The right panelshows Src bound to 6 with the ATP-binding site in theαC helix-out inactive conformation (PDB entry 4DGG). Helix αCis rotated out of the active site, disrupting the interaction betweenK295 and E310. (C) Quantitative comparison of the fold differencesin Ki values between activated SFKs (SFKAct) and their respective autoinhibited constructs (SFKSH2eng) for 1–4. Previously reported datafor Src and Hck are plotted for reference.18 (D) Quantitative comparison of the fold differences in Ki values between activated SFKs (SFKAct) andtheir respective autoinhibited constructs (SFKSH2eng) for 6 and 7. Previously reported data for Src andHck are plotted for reference. The left-most column, marked with adagger symbol, shows that the absolute fold difference in Ki value could not be determined because inhibitoraffinity is lower than the enzyme concentration used in the assay.Ligand 5, which is marked with an asterisk in panel A,is too potent for Ki determination. Allvalues are the average of assays performed in triplicate; the SEMfor each value is less than 20% of the average Ki value. Ki values are listed inFigure S3 of the Supporting Information.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230323&req=5

fig4: Binding preferences of conformation-selective inhibitors revealdifferences in allosteric coupling among Lyn, Fyn1, and Fyn2. (A)Conformation-selective inhibitor panel. 2–4 favorthe ATP-binding site of active (αC helix-in) SFKs, while 5–7 stabilize the αC helix-out, inactive ATP-bindingsite conformation. These ligands allow systematic analysis of theATP-binding site conformation in response to domain engagement. (B)Ligands that stabilize active and αC helix-out conformationsmake different contacts with the ATP-binding sites of SFKs. The leftpanel shows Src bound to 2 with the ATP-binding sitein an active conformation (PDB entry 3EN4). An electrostatic interaction betweenthe inhibitor and E310 in the αC helix is shown. The right panelshows Src bound to 6 with the ATP-binding site in theαC helix-out inactive conformation (PDB entry 4DGG). Helix αCis rotated out of the active site, disrupting the interaction betweenK295 and E310. (C) Quantitative comparison of the fold differencesin Ki values between activated SFKs (SFKAct) and their respective autoinhibited constructs (SFKSH2eng) for 1–4. Previously reported datafor Src and Hck are plotted for reference.18 (D) Quantitative comparison of the fold differences in Ki values between activated SFKs (SFKAct) andtheir respective autoinhibited constructs (SFKSH2eng) for 6 and 7. Previously reported data for Src andHck are plotted for reference. The left-most column, marked with adagger symbol, shows that the absolute fold difference in Ki value could not be determined because inhibitoraffinity is lower than the enzyme concentration used in the assay.Ligand 5, which is marked with an asterisk in panel A,is too potent for Ki determination. Allvalues are the average of assays performed in triplicate; the SEMfor each value is less than 20% of the average Ki value. Ki values are listed inFigure S3 of the Supporting Information.

Mentions: The data in Figure 2 do not provide informationabout the overall conformation of the ATP-binding site for each SFKregulatory state mutant. To thoroughly explore how domain engagementinfluences SFK ATP-binding sites, a method for sensing ATP-bindingsite conformation is required. To provide more information about thisparameter, the sensitivities of activated and autoinhibited Lyn, Fyn1,and Fyn2 constructs to conformation-selective, ATP-competitive inhibitorswere determined. A representative panel of ATP-competitive ligandsthat are known or predicted to stabilize distinct SFK ATP-bindingsite conformations was employed (Figure 4A).By determining affinities of these ligands for various regulatorystate SFK mutants, one can determine the influence of intramolecularinteractions on ATP-binding site conformation. All inhibitors testedare pyrazolopyrimidine-based compounds with variable substituentsat the C3 position. 1 (PP2) contains a 4-chlorophenylgroup at the C3 position and has previously been found to have a minimalactivation state preference for Src and Hck, making it a useful controlfor these studies.122–4 display small aryl moieties containing hydrogen bond donors and/oracceptors from their C3 positions and have been found to be selectivefor activated Src and Hck constructs over their autoinhibited forms.12,18 These inhibitors are predicted to stabilize an active ATP-bindingsite conformation by forming electrostatic interactions with catalyticresidues that are aligned for catalysis. In contrast, 5–7 contain extended hydrophobic substituents at the C3 position, whichhave been shown to stabilize the ATP-binding sites of SFKs in an inactiveconformation that involves rotation of the αC helix out of acatalytically competent alignment, the αC helix-out inactiveconformation. Inhibitors of this class have been found to have a higheraffinity for autoinhibited Src and Hck constructs than their activatedcounterparts. Unlike 2–4, 5–7 are not compatible with the αC helix being in an active conformation(see Figure 4B). Profiling active compatible(αC helix-in) and active incompatible (αC helix-out) ligandsagainst Lyn and Fyn regulatory state mutants provides insight intohow the allosteric relationship between αC helix conformationand regulatory domains for Fyn and Lyn compares to that for Src andHck.


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

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

Binding preferences of conformation-selective inhibitors revealdifferences in allosteric coupling among Lyn, Fyn1, and Fyn2. (A)Conformation-selective inhibitor panel. 2–4 favorthe ATP-binding site of active (αC helix-in) SFKs, while 5–7 stabilize the αC helix-out, inactive ATP-bindingsite conformation. These ligands allow systematic analysis of theATP-binding site conformation in response to domain engagement. (B)Ligands that stabilize active and αC helix-out conformationsmake different contacts with the ATP-binding sites of SFKs. The leftpanel shows Src bound to 2 with the ATP-binding sitein an active conformation (PDB entry 3EN4). An electrostatic interaction betweenthe inhibitor and E310 in the αC helix is shown. The right panelshows Src bound to 6 with the ATP-binding site in theαC helix-out inactive conformation (PDB entry 4DGG). Helix αCis rotated out of the active site, disrupting the interaction betweenK295 and E310. (C) Quantitative comparison of the fold differencesin Ki values between activated SFKs (SFKAct) and their respective autoinhibited constructs (SFKSH2eng) for 1–4. Previously reported datafor Src and Hck are plotted for reference.18 (D) Quantitative comparison of the fold differences in Ki values between activated SFKs (SFKAct) andtheir respective autoinhibited constructs (SFKSH2eng) for 6 and 7. Previously reported data for Src andHck are plotted for reference. The left-most column, marked with adagger symbol, shows that the absolute fold difference in Ki value could not be determined because inhibitoraffinity is lower than the enzyme concentration used in the assay.Ligand 5, which is marked with an asterisk in panel A,is too potent for Ki determination. Allvalues are the average of assays performed in triplicate; the SEMfor each value is less than 20% of the average Ki value. Ki values are listed inFigure S3 of the Supporting Information.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230323&req=5

fig4: Binding preferences of conformation-selective inhibitors revealdifferences in allosteric coupling among Lyn, Fyn1, and Fyn2. (A)Conformation-selective inhibitor panel. 2–4 favorthe ATP-binding site of active (αC helix-in) SFKs, while 5–7 stabilize the αC helix-out, inactive ATP-bindingsite conformation. These ligands allow systematic analysis of theATP-binding site conformation in response to domain engagement. (B)Ligands that stabilize active and αC helix-out conformationsmake different contacts with the ATP-binding sites of SFKs. The leftpanel shows Src bound to 2 with the ATP-binding sitein an active conformation (PDB entry 3EN4). An electrostatic interaction betweenthe inhibitor and E310 in the αC helix is shown. The right panelshows Src bound to 6 with the ATP-binding site in theαC helix-out inactive conformation (PDB entry 4DGG). Helix αCis rotated out of the active site, disrupting the interaction betweenK295 and E310. (C) Quantitative comparison of the fold differencesin Ki values between activated SFKs (SFKAct) and their respective autoinhibited constructs (SFKSH2eng) for 1–4. Previously reported datafor Src and Hck are plotted for reference.18 (D) Quantitative comparison of the fold differences in Ki values between activated SFKs (SFKAct) andtheir respective autoinhibited constructs (SFKSH2eng) for 6 and 7. Previously reported data for Src andHck are plotted for reference. The left-most column, marked with adagger symbol, shows that the absolute fold difference in Ki value could not be determined because inhibitoraffinity is lower than the enzyme concentration used in the assay.Ligand 5, which is marked with an asterisk in panel A,is too potent for Ki determination. Allvalues are the average of assays performed in triplicate; the SEMfor each value is less than 20% of the average Ki value. Ki values are listed inFigure S3 of the Supporting Information.
Mentions: The data in Figure 2 do not provide informationabout the overall conformation of the ATP-binding site for each SFKregulatory state mutant. To thoroughly explore how domain engagementinfluences SFK ATP-binding sites, a method for sensing ATP-bindingsite conformation is required. To provide more information about thisparameter, the sensitivities of activated and autoinhibited Lyn, Fyn1,and Fyn2 constructs to conformation-selective, ATP-competitive inhibitorswere determined. A representative panel of ATP-competitive ligandsthat are known or predicted to stabilize distinct SFK ATP-bindingsite conformations was employed (Figure 4A).By determining affinities of these ligands for various regulatorystate SFK mutants, one can determine the influence of intramolecularinteractions on ATP-binding site conformation. All inhibitors testedare pyrazolopyrimidine-based compounds with variable substituentsat the C3 position. 1 (PP2) contains a 4-chlorophenylgroup at the C3 position and has previously been found to have a minimalactivation state preference for Src and Hck, making it a useful controlfor these studies.122–4 display small aryl moieties containing hydrogen bond donors and/oracceptors from their C3 positions and have been found to be selectivefor activated Src and Hck constructs over their autoinhibited forms.12,18 These inhibitors are predicted to stabilize an active ATP-bindingsite conformation by forming electrostatic interactions with catalyticresidues that are aligned for catalysis. In contrast, 5–7 contain extended hydrophobic substituents at the C3 position, whichhave been shown to stabilize the ATP-binding sites of SFKs in an inactiveconformation that involves rotation of the αC helix out of acatalytically competent alignment, the αC helix-out inactiveconformation. Inhibitors of this class have been found to have a higheraffinity for autoinhibited Src and Hck constructs than their activatedcounterparts. Unlike 2–4, 5–7 are not compatible with the αC helix being in an active conformation(see Figure 4B). Profiling active compatible(αC helix-in) and active incompatible (αC helix-out) ligandsagainst Lyn and Fyn regulatory state mutants provides insight intohow the allosteric relationship between αC helix conformationand regulatory domains for Fyn and Lyn compares to that for Src andHck.

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