Structure of mMPL ECD bound to mTPO core domain. (A) Domain schematics for MPL and TPO. Normal numbering refers to residues in mouse proteins whereas numbers in parentheses indicate residues in human proteins. (B) Cryo-EM map of the mMPL ECD/mTPO core domain complex with proteins color-coded. mTPO is colored in orange and 2 copies of mMPL ECD are in shades of blue. (C) Cartoon representation of the mMPL ECD/mTPO core domain complex with mMPL domains color-coded (mTPO in orange, D1 in magenta, D2 in cyan, and D3 in light green). The locations of the high (cyan) and low affinity (dark blue) sites are indicated by ovals. (D) Zoomed-in view of interactions in the high affinity site. (E) Zoomed-in view of interactions in the low affinity site. In panels D-E, hydrogen bonds are represented by black, dashed lines, numbering in italics refers to residue numbering in human proteins, loss of function residues mutated in CAMT are in red, and gain-of-function mutated residues in purple. (F) STAT5-nanoluciferase/STAT5b–based reporter assay with hTPO. A HEK293 STAT5-NanoLuc/STAT5b expressing stable cell line was used as a source of STAT5b and STAT5 response element upstream of a NanoLuc reporter element. Cells were transfected with FL WT hMPL plasmid and recombinant WT or mutant hTPO core domain protein was added to induce response. Please see the Methods and supplemental Methods sections for further details. The hTPO R38H CAMT mutation was used for comparison purposes with other mutants. (G) Reporter assay using a similar strategy as in panel F but with transfected WT or mutant FL hMPL plasmid and with recombinant WT hTPO core domain protein added to induce response. In panels F-G, a representative result is shown with error bars representing the standard deviation of technical replicates. The 50% effective concentration (EC50) values represent half maximal signal activation as measured by NanoLuc activity. The table shows the mean value ± standard deviation from 3 independent experiments. Mutated residues are color-coded to indicate whether they make key interactions in the high affinity site (cyan), the low affinity site (dark blue), or both sites (green).

Structure of mMPL ECD bound to mTPO core domain. (A) Domain schematics for MPL and TPO. Normal numbering refers to residues in mouse proteins whereas numbers in parentheses indicate residues in human proteins. (B) Cryo-EM map of the mMPL ECD/mTPO core domain complex with proteins color-coded. mTPO is colored in orange and 2 copies of mMPL ECD are in shades of blue. (C) Cartoon representation of the mMPL ECD/mTPO core domain complex with mMPL domains color-coded (mTPO in orange, D1 in magenta, D2 in cyan, and D3 in light green). The locations of the high (cyan) and low affinity (dark blue) sites are indicated by ovals. (D) Zoomed-in view of interactions in the high affinity site. (E) Zoomed-in view of interactions in the low affinity site. In panels D-E, hydrogen bonds are represented by black, dashed lines, numbering in italics refers to residue numbering in human proteins, loss of function residues mutated in CAMT are in red, and gain-of-function mutated residues in purple. (F) STAT5-nanoluciferase/STAT5b–based reporter assay with hTPO. A HEK293 STAT5-NanoLuc/STAT5b expressing stable cell line was used as a source of STAT5b and STAT5 response element upstream of a NanoLuc reporter element. Cells were transfected with FL WT hMPL plasmid and recombinant WT or mutant hTPO core domain protein was added to induce response. Please see the Methods and supplemental Methods sections for further details. The hTPO R38H CAMT mutation was used for comparison purposes with other mutants. (G) Reporter assay using a similar strategy as in panel F but with transfected WT or mutant FL hMPL plasmid and with recombinant WT hTPO core domain protein added to induce response. In panels F-G, a representative result is shown with error bars representing the standard deviation of technical replicates. The 50% effective concentration (EC50) values represent half maximal signal activation as measured by NanoLuc activity. The table shows the mean value ± standard deviation from 3 independent experiments. Mutated residues are color-coded to indicate whether they make key interactions in the high affinity site (cyan), the low affinity site (dark blue), or both sites (green).

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