Structural Basis for Activation of the Thrombopoietin Receptor (MPL) By Its Ligand (TPO)

Myeloproliferative leukemia protein (MPL), also known as thrombopoietin receptor, is a class I cytokine receptor expressed on hematopoietic progenitors that is critical for normal platelet production by promoting myeloid progenitor growth and differentiation toward the megakaryocyte lineage. The ligand for MPL, thrombopoietin (TPO), stimulates platelet production through binding MPL via two sequential sites, a high- and then a low-affinity interface that together induce MPL dimerization and result in an active conformation allowing downstream JAK2/STAT5 signaling. Despite intense interest in characterizing the molecular mechanism of the interaction between MPL and TPO over the last several decades, no experimental structural evidence has been reported. Here we present a 3.39 Å cryo-EM structure of the ectodomain of mouse MPL bound to TPO. TPO features both low and high affinity sites that each bind to one MPL monomer in agreement with previous functional studies. Our structure reveals that several patient mutations associated with congenital amegakaryocytic thrombocytopenia (CAMT) and aplastic anemia are found at MPL-TPO interaction sites. The structure further explains additional patient mutations, as well as published experimental mutations that have previously been the basis of structure-function studies. Building on this structure, we generated a molecular dynamics simulation model of the full length human MPL-TPO complex, revealing that TPO binding to full length MPL results in unexpected MPL-MPL intramolecular interactions that stabilize the dimer to allow for efficient signaling. Functional characterization of deleterious mutations predicted by our structure and modelling have revealed additional residues critical to MPL-TPO and MPL-MPL interactions, providing further validation of our proposed structure. Overall, our studies reveal new insight into a receptor-cytokine interaction critical for normal hematopoiesis and platelet production that are frequently mutated in disease, provide the structural basis underlying existing experimental findings, and provide additional insight into the nature of hematopoietic growth factor signaling which could benefit future therapeutic research.