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. 2014 Nov 18;107(10):2325-36.
doi: 10.1016/j.bpj.2014.10.013.

Structural Insight Into the Transmembrane Domain and the Juxtamembrane Region of the Erythropoietin Receptor in Micelles

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Free PMC article

Structural Insight Into the Transmembrane Domain and the Juxtamembrane Region of the Erythropoietin Receptor in Micelles

Qingxin Li et al. Biophys J. .
Free PMC article

Abstract

Erythropoietin receptor (EpoR) dimerization is an important step in erythrocyte formation. Its transmembrane domain (TMD) and juxtamembrane (JM) region are essential for signal transduction across the membrane. A construct compassing residues S212-P259 and containing the TMD and JM region of the human EpoR was purified and reconstituted in detergent micelles. The solution structure of the construct was determined in dodecylphosphocholine (DPC) micelles by solution NMR spectroscopy. Structural and dynamic studies demonstrated that the TMD and JM region are an ?-helix in DPC micelles, whereas residues S212-D224 at the N-terminus of the construct are not structured. The JM region is a helix that contains a hydrophobic patch formed by conserved hydrophobic residues (L253, I257, and W258). Nuclear Overhauser effect analysis, fluorescence spectroscopy, and paramagnetic relaxation enhancement experiments suggested that the JM region is exposed to the solvent. The structures of the TMD and JM region of the mouse EpoR were similar to those of the human EpoR.

Figures

Figure 1
Figure 1
Topology of the EpoR. (A) Diagram of the EpoR. The extracellular region, TMD and JM region, and cytoplasmic region are shown in blue, gray, and green, respectively. The membrane is shown as a brown box. The TMD contains residues L216–L247. (B) Topology of the hEpoR construct used in this study. The hEpoR sequence was obtained from the UniProt Knowledgebase (http://www.uniprot.org; accession number P19235). Sequence numbering does not include the N-terminal 25-residue signal peptide. (C) Sequence alignment of hEpoR and mEpoR. The TMD is highlighted with solid lines and the JM region is highlighted with blue and dashed lines. The different amino acids are indicated with triangles. The conserved hydrophobic residues in the JM region are indicated with black circles. To see this figure in color, go online.
Figure 2
Figure 2
Cross-linking of hEpoR in different membrane systems. Protein was cross-linked using GA. Samples were separated by SDS-PAGE and followed by western blot using an anti-his antibody. The bands above the dimer are trimer or oligomers of the hEpoR. D, dimer; M, monomer.
Figure 3
Figure 3
1H-15N-HSQC spectra of the hEpoR in different membrane systems. (A) 1H-15N-HSQC spectrum of 1.2 mM of hEpoR in 30 mM LMPC micelles. (B) 1H-15N-HSQC spectrum of 1.5 mM of hEpoR in 30 mM LMPG micelles. (C) 1H-15N-HSQC spectrum of 0.6 mM hEpoR in 20% DHPC/DMPC bicelles (q = 0.33). (D) 1H-15N-HSQC spectrum of 1.5 mM of hEpoR in 80 mM SDS micelles. All data were acquired at 40°C. The 1H-15N-HSQC spectrum of hEpoR in DPC micelles is shown in Fig. 4A.
Figure 4
Figure 4
Assignment and secondary structure analysis of hEpoR in DPC micelles. (A) Assigned 1H-15N-HSQC spectrum of hEpoR in DPC micelles. (B) Deviations of the observed Cα chemical-shift values from the corresponding random-coil chemical-shift values. (C) Secondary structure analysis of hEpoR in DPC micelles by TALOS+. The positive value is the possibility (0–1) of the corresponding amino acid being a helix. (D) NOE connections of the hEpoR in DPC micelles. (E) H-D exchange experiment. The 1H-15N-HSQC spectrum of the hEpoR in 90% D2O and assignment of the crosspeaks are shown with the residue name and sequence number.
Figure 5
Figure 5
Relaxation measurements (600 MHz) of the hEpoR in DPC micelles. 15N R1, R2, and hetNOE values of the hEpoR were obtained at 313 K. R1, R2, R2/R1, and hetNOE values are shown as a function of residue number.
Figure 6
Figure 6
Structure of the hEpoR in DPC micelles. (A) Twenty structures of the hEpoR are superimposed. The side chains of the amino acids are shown in sticks. Carbon, nitrogen, and oxygen atoms are shown in green, blue, and red, respectively. The structures have been deposited in the PDB under accession number 2MV6. (B) The 20 superimposed structures are shown with backbone atoms. (C) Ribbon representation of the hEpoR for the lowest-energy conformer. The N-terminal residues are flexible. The TMD and JM region form one helix. (D) Color-coded electrostatic surface potential for the hEpoR. Positive and negative potentials are shown in blue and red, respectively. Positively charged residues and the three conserved hydrophobic residues in the JM region are labeled with the residue name and sequence number. (E) Ribbon representation of the TMD and JM region of the hEpoR. The TMD and JM region are shown in purple and green, respectively. The heights of the TMD and JM region are shown. The calculation and all of the figures were made using PyMOL (http://www.pymol.org). (F) Helix-wheel representation of the JM region. (G) Ribbon presentation of the JM region. The three hydrophobic residues are shown in sticks.
Figure 7
Figure 7
Model of the hEpoR in a micelle. (A) NOEs between water protons and amide protons of residues at the C-terminus of the hEpoR. Slices from a 3D 1H-15N-HSQC-NOESY experiment are shown. The signal from water protons is indicated with a dotted line. (B) PRE experiment for the hEpoR. The ratio of peak intensities of a residue in the presence (Ip) and absence (I0) of 2 mM Gd-DTPA is plotted against the residue number. (C) Fluorescence spectroscopy of the hEpoR in DPC micelles. (D) Model of the hEpoR in a micelle. The micelle is drawn as a sphere with a diameter of 32 Å. The N-terminus of the construct is shown in cyan. The TMD is shown as a purple helix and the JM region is shown as a green helix. This figure was made using PyMOL (http://www.pymol.org). To see this figure in color, go online.
Figure 8
Figure 8
NMR study of the mEpoR. (A) Superimposed 1H-15N-HSQC spectra of hEpoR (black) and mEpoR (red). (B) Assigned 1H-15N-HSQC spectrum of mEpoR in DPC micelles. (C) Deviations of the observed mEpoR Cα chemical-shift values from the corresponding random-coil chemical-shift values. (D) 13Cα chemical-shift difference between the mEpoR (Cαm) and hEpoR (Cαh). Residues that differ between the hEpoR and mEpoR are shown as open circles. To see this figure in color, go online.
Figure 9
Figure 9
Model of the EpoR extracellular domain, TMD, and JM region. Only a single molecule is shown. The model was generated by manually linking two structures (PDB ID 1EBP and the structure determined in this study) in PyMOL. The two proline residues are shown in sticks. The extracellular domain, TMD, and JM region are shown in different colors. The membrane is indicated as two lines. To see this figure in color, go online.

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