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Solution structure of the transmembrane domain of the mouse erythropoietin receptor in detergent micelles.

Li Q, Wong YL, Yueqi Lee M, Li Y, Kang C - Sci Rep (2015)

Bottom Line: Residues from S216 to T219 in mEpoR form a short helix.Fluorescence spectroscopy and sequence analysis indicate that the C-terminal JM region is exposed to the solvent.Helix wheel result shows that there is hydrophilic patch in the TMD of the mEpoR formed by residues S231, S238 and T242, and these residues might be important for the receptor dimerization.

View Article: PubMed Central - PubMed

Affiliation: Institute of Chemical &Engineering Sciences, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

ABSTRACT
Erythropoiesis is regulated by the erythropoietin receptor (EpoR) binding to its ligand. The transmembrane domain (TMD) and the juxtamembrane (JM) regions of the EpoR are important for signal transduction across the cell membrane. We report a solution NMR study of the mouse erythropoietin receptor (mEpoR) comprising the TMD and the JM regions reconstituted in dodecylphosphocholine (DPC) micelles. The TMD and the C-terminal JM region of the mEpoR are mainly α-helical, adopting a similar structure to those of the human EpoR. Residues from S216 to T219 in mEpoR form a short helix. Relaxation study demonstrates that the TMD of the mEpoR is rigid whilst the N-terminal region preceding the TMD is flexible. Fluorescence spectroscopy and sequence analysis indicate that the C-terminal JM region is exposed to the solvent. Helix wheel result shows that there is hydrophilic patch in the TMD of the mEpoR formed by residues S231, S238 and T242, and these residues might be important for the receptor dimerization.

No MeSH data available.


Related in: MedlinePlus

Exposure of the C-terminal JM region to the solvent.(A) NOE between residues in the JM region and water. The slice from a 3D-HSQC-NOESY was plotted. The NOE between amide proton and water protons is indicated as a solid line. The assignments of the Hα atoms are labeled with sequence number. Sequential connection of amide protons are shown with solid lines. (B) Solvent exposure analysis using protein sequence. HSE analysis was carried out and the resulting HSE values and CN are plotted against residue number. (C) Fluorescence spectroscopy of the mEpoR in DPC micelles. The experiment was conducted as described in Materials and Methods.
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f4: Exposure of the C-terminal JM region to the solvent.(A) NOE between residues in the JM region and water. The slice from a 3D-HSQC-NOESY was plotted. The NOE between amide proton and water protons is indicated as a solid line. The assignments of the Hα atoms are labeled with sequence number. Sequential connection of amide protons are shown with solid lines. (B) Solvent exposure analysis using protein sequence. HSE analysis was carried out and the resulting HSE values and CN are plotted against residue number. (C) Fluorescence spectroscopy of the mEpoR in DPC micelles. The experiment was conducted as described in Materials and Methods.

Mentions: To further understand whether the C-terminal JM region is exposed to the solvent, we analyzed our NOE data. It is clear that the amide protons from hydrophilic residues compassing 250 to 257 exhibited NOEs with water protons, demonstrating that these residues were not buried in DPC micelles (Fig. 4A). Interestingly, amide protons of the three conserved hydrophobic residues including L253, I257 and W258 showed no NOE or weak NOEs with water protons, which indicated that these residues may interact with micelles or having exchanges. We then performed bioinformatics analysis of the construct using a half-sphere exposure (HSE) algorithm14. In the HSE analysis, a residue is considered as a sphere with two half parts. HES-up corresponds to the direction of the chain side of a residue and HES-down corresponds to the direction of the opposite side. This method also predicts the contact number (CN) that is a measurement of a residue burial in proteins. The HSE-up showed that most residues in the TMD and few residues in both N- and C-termini were protected from exposure. Taking the HES-down and CN results together, the N- and C-termini of the constructs are exposed to the solvent (Fig. 4B). Although this bioinformatics study might not suitable for mEpoR, the information obtained suggested that the C-terminal JM region is exposed to the solvent. There is only one tryptophan residue present (W258) in the protein sequence. Tryptophan fluorescence experiment was carried out. In the fluorescence spectrum, the emission maximum of the mEpoR in DPC micelles was 350 nm (Fig. 4C), indicating that W258 is exposed to the solvent because the emission maximum will be shifted to a lower wavelength if the tryptophan residue is buried in a hydrophobic environment.


Solution structure of the transmembrane domain of the mouse erythropoietin receptor in detergent micelles.

Li Q, Wong YL, Yueqi Lee M, Li Y, Kang C - Sci Rep (2015)

Exposure of the C-terminal JM region to the solvent.(A) NOE between residues in the JM region and water. The slice from a 3D-HSQC-NOESY was plotted. The NOE between amide proton and water protons is indicated as a solid line. The assignments of the Hα atoms are labeled with sequence number. Sequential connection of amide protons are shown with solid lines. (B) Solvent exposure analysis using protein sequence. HSE analysis was carried out and the resulting HSE values and CN are plotted against residue number. (C) Fluorescence spectroscopy of the mEpoR in DPC micelles. The experiment was conducted as described in Materials and Methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4551963&req=5

f4: Exposure of the C-terminal JM region to the solvent.(A) NOE between residues in the JM region and water. The slice from a 3D-HSQC-NOESY was plotted. The NOE between amide proton and water protons is indicated as a solid line. The assignments of the Hα atoms are labeled with sequence number. Sequential connection of amide protons are shown with solid lines. (B) Solvent exposure analysis using protein sequence. HSE analysis was carried out and the resulting HSE values and CN are plotted against residue number. (C) Fluorescence spectroscopy of the mEpoR in DPC micelles. The experiment was conducted as described in Materials and Methods.
Mentions: To further understand whether the C-terminal JM region is exposed to the solvent, we analyzed our NOE data. It is clear that the amide protons from hydrophilic residues compassing 250 to 257 exhibited NOEs with water protons, demonstrating that these residues were not buried in DPC micelles (Fig. 4A). Interestingly, amide protons of the three conserved hydrophobic residues including L253, I257 and W258 showed no NOE or weak NOEs with water protons, which indicated that these residues may interact with micelles or having exchanges. We then performed bioinformatics analysis of the construct using a half-sphere exposure (HSE) algorithm14. In the HSE analysis, a residue is considered as a sphere with two half parts. HES-up corresponds to the direction of the chain side of a residue and HES-down corresponds to the direction of the opposite side. This method also predicts the contact number (CN) that is a measurement of a residue burial in proteins. The HSE-up showed that most residues in the TMD and few residues in both N- and C-termini were protected from exposure. Taking the HES-down and CN results together, the N- and C-termini of the constructs are exposed to the solvent (Fig. 4B). Although this bioinformatics study might not suitable for mEpoR, the information obtained suggested that the C-terminal JM region is exposed to the solvent. There is only one tryptophan residue present (W258) in the protein sequence. Tryptophan fluorescence experiment was carried out. In the fluorescence spectrum, the emission maximum of the mEpoR in DPC micelles was 350 nm (Fig. 4C), indicating that W258 is exposed to the solvent because the emission maximum will be shifted to a lower wavelength if the tryptophan residue is buried in a hydrophobic environment.

Bottom Line: Residues from S216 to T219 in mEpoR form a short helix.Fluorescence spectroscopy and sequence analysis indicate that the C-terminal JM region is exposed to the solvent.Helix wheel result shows that there is hydrophilic patch in the TMD of the mEpoR formed by residues S231, S238 and T242, and these residues might be important for the receptor dimerization.

View Article: PubMed Central - PubMed

Affiliation: Institute of Chemical &Engineering Sciences, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

ABSTRACT
Erythropoiesis is regulated by the erythropoietin receptor (EpoR) binding to its ligand. The transmembrane domain (TMD) and the juxtamembrane (JM) regions of the EpoR are important for signal transduction across the cell membrane. We report a solution NMR study of the mouse erythropoietin receptor (mEpoR) comprising the TMD and the JM regions reconstituted in dodecylphosphocholine (DPC) micelles. The TMD and the C-terminal JM region of the mEpoR are mainly α-helical, adopting a similar structure to those of the human EpoR. Residues from S216 to T219 in mEpoR form a short helix. Relaxation study demonstrates that the TMD of the mEpoR is rigid whilst the N-terminal region preceding the TMD is flexible. Fluorescence spectroscopy and sequence analysis indicate that the C-terminal JM region is exposed to the solvent. Helix wheel result shows that there is hydrophilic patch in the TMD of the mEpoR formed by residues S231, S238 and T242, and these residues might be important for the receptor dimerization.

No MeSH data available.


Related in: MedlinePlus