Structural Basis of MPL Activation by Thrombopoietin

1. High resolution structure of mTPO bound to mMPL shows how ligand induces receptor dimerization and how patient mutations affect this process.

2. MD modeling of JAK2 bound to MPL/TPO complex v. apo-MPL reveals how MPL dimerization or patient mutations can induce JAK2 phosphorylation.

Myeloproliferative leukemia protein (MPL), also known as thrombopoietin receptor, is a class I cytokine receptor that is expressed on hematopoietic progenitors, promoting growth and differentiation toward the megakaryocyte lineage and is critical for normal platelet production. Mutations in MPL, thrombopoietin (TPO), or JAK2 have been implicated in multiple diseases from congenital thrombocytopenias to myeloproliferative neoplasms. The ligand for MPL, TPO, stimulates platelet production by inducing MPL dimerization and results in an active conformation that allows downstream JAK2/STAT5 signaling. Despite the biological importance of this pathway, the molecular signaling mechanism remained unclear. Here we present a 3.39 Å cryo-electron microscopy (cryo-EM) structure of the ectodomain of mouse MPL bound to TPO. The structure revealed both low and high affinity sites between MPL and TPO that contain several pathologic mutations. To better understand TPO-driven MPL signaling, we expanded upon this structure by molecular dynamics (MD) simulations to model the full length (FL) human MPL/TPO complex, and showed that MPL D4-D4 domain interactions are functionally relevant in activity assays. To build on our understanding of downstream activation, we added JAK2 to the MPL/TPO complex by MD simulations. This ternary complex illustrates JAK2 dimerization through the pseudo kinase (PK) domain, illustrates residues important for MPL interactions, and reveals the constitutive activation mechanism of patient mutant V617F. The model also suggests the mechanism of JAK2 tyrosine kinase domain trans-phosphorylation. Overall, our studies illuminate TPO/MPL/JAK2 signaling mechanisms and provide additional insight into the nature of receptor signaling which will further benefit human health.