Fig. 6
SOCS1 binds to the activation loop peptides from all four JAKs. a Table showing the affinity and enthalpy of each SOCS1/JAK activation loop peptide interaction as determined by isothermal titration calorimetry. Human SOCS1 binds to phosphorylated peptides representing all four JAK activation loops. The first phosphotyrosine of the JAK1 activation loop (pTyr1034) is the key residue for SOCS1 binding. Errors represent standard error of the mean from three independent experiments. b Representative ITC binding data for the four JAK activation loop peptides (doubly-phosphorylated) binding to SOCS1 used to generate a. c NMR analysis of phosphopeptide binding. 1H–15N SOFAST HMQC spectra of ggSOCS1 in the presence (blue) and absence (black) of the JAK1 activation loop are shown overlaid. Both spectra were assigned, the labels indicate the positions of amide resonances from the bound form of the protein. d NMR analysis of JAK binding in the presence of exogenous phosphopeptide. 1H–15N SOFAST HMQC spectra of ggSOCS1/activation loop complex in the presence (red) and absence (blue) of the JAK1 kinase domain are shown overlaid. The sidechain epsilon NH resonance of Arg127 from both JAK-bound and unbound forms are indicated. These are of opposite phase to backbone amide resonances. The presence of the Arg127 resonance in that section of spectra indicates that the phosphopeptide-binding groove is occupied in both complexes. e NMR analysis of JAK binding in the absence of exogenous phosphopeptide. 1H–15N SOFAST HMQC spectra of ggSOCS1 in the presence (green) and absence (black) of the JAK1 kinase domain are shown overlaid. The line-broadening in the green spectra indicated the formation of a SOCS1/JAK complex and the lack of the Arg127 sidechain resonance in either spectra indicates the phosphotyrosine-binding groove is unoccupied
