Lymphoma-associated TIR-domain mutants are located at stable communication hubs in the TIR domain. (A-B) HEK293 cells were transfected with plasmids for designed MyD88 mutants and NF-κB–inducible firefly luciferase and constitutive Renilla luciferase reporter plasmids. Twenty-four hours later, cells were lysed and luciferase activity measured (A) or the lysates were run on SDS-PAGE and analyzed by anti-MyD88 immunoblot (B). (C-D) The MyD88 TIR domain contains highly stable RMSF minima. The 40-nanosecond (C) or 100-nanosecond (D) molecular dynamics simulations were done on WT (black dotted line) and mutant (colored as shown) MyD88 NMR structure 2js7 ensemble (composed of 20 structures each, 20 simulations each); shown are averaged data (C) or the single most representative conformer (D). RMSF over the TIR-domain residues were plotted. RMSF profiles are highly similar and show RMSF minima (black arrows pointing to residues 177, 204, 231/232, 265, and 303/304). (E-F) RMSF minima are shown as green or orange (in case they coincide with lymphoma-associated mutations) spheres and map to a plane describing MyD88 dimer formation (see schematic representation, panel F) as proposed by Bovijn et al.²⁰  Communication hubs are indicated by arrows and coincide with RMSF minima or locate in the interaction plane within the MyD88 dimer. Some lymphoma-associated mutations directly map to hub positions (orange spheres) or locate within this plane (red spheres), with the exception of S143 and T294 (red). Hub positions for which so far no lymphoma-associated mutations have been reported are shown as green spheres. The region of higher flexibility in WT (black dotted line) vs mutated (colored as shown) TIR domains in molecular dynamics simulation (F) is shown in dark blue in panel E. EV, empty vector; IB, immunoblot.