hMPL D4-D4 interaction stabilizes the hMPL dimer upon hTPO binding. (A) The optimized computational modeling structure of the FL hMPL dimer bound to hTPO, embedded in a lipid bilayer membrane, as derived from a ∼1.5 μs MD simulation. (B) Detailed view of the intermolecular interactions formed at the identified D4-D4 interface in the activated MPL dimer. Hydrogen bonds are represented by cyan dashed lines. The stability analysis of interaction at the D4-D4 interface measured as: (C) the interdomain distance between D4-D4, considering the center of mass of the Cα atoms within residues 435-440. These residues delineate the antiparallel β-sheet, formed at the interface between the 2 hMPL D4 domains; and (D) the RMSD analysis, within residues 435-440 for both hMPL chains that define the antiparallel β-sheet at the interface. The RMSD was computed for all heavy atoms (nonhydrogen atoms), including side chains. For this analysis, each simulation snapshot was compared against the final configuration derived from the ∼1.5-μs MD simulation. (E) Reporter assay using transfected human FL hMPL WT and D4-D4 mutant with recombinant WT hTPO core domain. A representative result is shown with error bars representing the standard deviation of technical replicates. The table shows the mean ± standard deviation from 3 independent experiments. (F) The WSXWS motif in the D4 domain (gray and light violet) contributes to preserving the structural stability of the established D4-D4 interface.