Fig. 3.

PIN1 binds to and isomerizes the pS110-P111 motif, located C-terminal to the IRAK-M DD. a Binding of the PIN1 WW domain to IRAK-M-pS110 peptide is demonstrated by overlaid regions extracted from ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled PIN1 WW domain that show progressive peak shifts with increasing peptide concentration (apo = red, purple = highest concentration). b Changes in chemical shift (∆ω) with increasing IRAK-M-pS110 peptide concentration (filled circles) were fit to a simple bimolecular interaction model (solid lines) to yield the apparent dissociation constant, K_D^App = 60.7 ± 11.5 µM (mean ± s.d.). c PIN1 catalysis of IRAK-M-pS110 and IRAK-M-S110E peptides is demonstrated by ROESY spectra of each peptide in the presence or absence of PIN1. In the presence of PIN1 ( + PIN1, top two spectra), cross peaks between cis and trans appear for both IRAK-M-pS110 and IRAK-M-S110E peptides. In the absence of PIN1 (-PIN1, bottom two spectra), no cross peaks were observed for either peptide. d ¹H-¹⁵N HSQC spectrum of cleaved ¹⁵N-IRAK-M[1–119:R56D,Y61E]. e Ribbon diagram of the IRAK-M DD structure determined by NMR, showing the six alpha helices and highlighting the Y105-L20 interaction that anchors the C-terminal tail to the structure. f A ClusPro generated model of IRAK-M DD docked into a modified Myddosome oligomer containing three IRAK1 DD subunits in the L, M and N positions in the 3MOP structure. Blue: six MyD88 DD subunits, Green: Four IRAK4 DD subunits, Cyan: three IRAK1 DD homology model subunits, Yellow: docked NMR structure of the IRAK-M DD. g–j Proposed model for IRAK-M and PIN1 regulation of IRAK1 mediated immunity signaling. g IRAK1 homotetramer assembled on the Myddosome is able to achieve full hyperphosphorylation through each IRAK1 undefined domain (UD) being phosphorylated by neighboring IRAK1 kinase domains (KDs). h In the presence of IRAK-M, heterotetrameric IRAK-M/IRAK1 assembles on the Myddosome where IRAK-M DD replaces every-other IRAK1 DD subunit, preventing further IRAK1 hyper-phosphorylation and inhibiting IRAK1 release from the Myddosome thereby suppressing IRAK1-mediated inflammation. i IRAK1 KD phosphorylates S110 in next-neighbor IRAK-M. j Binding of PIN1 to IRAK-M pS110P (IRAK-M phosphorylated at residue S110) and subsequent isomerization induces release of IRAK-M from the Myddosome for downstream PIN1- and IRAK-M dependent signaling