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Syndecans are single-span membrane proteins playing important roles in cell-cell and cell-matrix interactions. The transmembrane domain of syndecans is critical for signal transduction across the cell membrane. Here, the structure of the transmembrane domain of syndecan-2 in detergent micelles was investigated using solution NMR spectroscopy. Backbone resonance assignment was obtained, and NMR studies show that the transmembrane domain forms a helix in detergent micelles, which is also supported by the hydrogen and deuterium exchange experiment. A study of the dynamics revealed the rigid structure of the transmembrane domain formed in solution, and paramagnetic relaxation enhancement defined the topology of the transmembrane domain in detergent micelles. This structural analysis may facilitate a better understanding of the role of the syndecan-2 transmembrane domain in signal transduction., (© 2019 Federation of European Biochemical Societies.)

The Zika virus (ZIKV) genome encodes a polyprotein that can be post-translationally processed into functional viral proteins. The viral protease is indispensable in the maturation of viral proteins. The Zika protease comprises of two components crucial for catalysis. The N-terminal region of NS3 contains the catalytic triad and approximately 40 amino acids of NS2B are essential for folding and protease activity. NS2B is a membrane protein with transmembrane domains that are critical for the localization of NS3 to the membrane. In this study, we expressed and purified full-length NS2B from ZIKV in E. coli. Purified NS2B was then reconstituted into lyso-myristoyl phosphatidylglycerol (LMPG) micelles. It was found that compared to wild type NS2B, NS2B C11S mutation in LMPG exhibited dispersed cross peaks in the 1 H 15 N-HSQC spectrum, thereby suggesting the feasibility for structural characterization using solution NMR spectroscopy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

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.

The insulin receptor (IR) binds insulin and plays important roles in glucose homeostasis by regulating the tyrosine kinase activity at its C-terminus. Its transmembrane domain (TMD) is shown to be important for transferring conformational changes induced by insulin across the cell membrane to regulate kinase activity. In this study, a construct IR(940-988) containing the TMD was expressed and purified for structural studies. Its solution structure in dodecylphosphocholine (DPC) micelles was determined. The sequence containing residues L962 to Y976 of the TMD of the IR in micelles adopts a well-defined helical structure with a kink formed by glycine and proline residues present at its N-terminus, which might be important for its function. Paramagnetic relaxation enhancement (PRE) and relaxation experimental results suggest that residues following the TMD are flexible and expose to aqueous solution. Although purified IR(940-988) in micelles existed mainly as a monomeric form verified by gel filtration and relaxation analysis, cross-linking study suggests that it may form a dimer or oligomers under micelle conditions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Dengue virus causes serious diseases affecting people in tropical and sub-tropical regions. The nonstructural (NS) protein 2B is an integral membrane protein and important for the regulation of viral protease NS3, which is significant for virus replication. The NS2B-NS3 complex is an important drug target for treating dengue fever. However, little is known about the structure of NS2B in its entirety. Herein, we describe the expression and purification of this integral membrane protein from cell membrane and inclusion bodies of Escherichia coli cells. The initial nuclear magnetic resonance (NMR) and circular dichroism (CD) results indicate that the purified protein adopts alpha-helical structures in LMPG and TDPC micelles., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Dengue virus nonstructural protein 4B (NS4B) is a membrane protein consisting of 248 residues with a crucial role in virus replication and interference with the host innate immunity. The dengue virus serotype 3 NS4B was reconstituted into lyso-myristoyl phosphatidylglycerol (LMPG) micelles. Backbone resonance assignment of NS4B was obtained using conventional solution NMR experiments. Further studies suggested that NS4B contained eleven helices and six of them form five potential transmembrane regions. This study provides atomic level information for an important drug target to control flavivirus infections. [ABSTRACT FROM AUTHOR]

KCNQ1 plays important roles in the cardiac action potential and consists of an N-terminal domain, a voltage-sensor domain, a pore domain and a C-terminal domain. KCNQ1 is a voltage-gated potassium channel and its channel activity is regulated by membrane potentials. The linker between transmembrane helices 4 and 5 (S4–S5 linker) is important for transferring the conformational changes from the voltage-sensor domain to the pore domain. In this study, the structure of the S4–S5 linker of KCNQ1 was investigated by solution NMR, circular dichroism and fluorescence spectroscopic studies. The S4–S5 linker adopted a helical structure in detergent micelles. The W248 may interact with the cell membrane. [ABSTRACT FROM AUTHOR]

Highlights: [•] A protease construct containing full length NS2B and NS3pro of Dengue virus was expressed. [•] Detergent screening was conducted for protein extraction from membrane. [•] Protease was active in the LMPC micelles. [Copyright &y& Elsevier]

Spike protein of SARS-CoV-2 contains a single-span transmembrane (TM) domain and plays roles in receptor binding, viral attachment and viral entry to the host cells. The TM domain of spike protein is critical for viral infectivity. Herein, the TM domain of spike protein of SARS-CoV-2 was reconstituted in detergent micelles and subjected to structural analysis using solution NMR spectroscopy. The results demonstrate that the TM domain of the protein forms a helical structure in detergent micelles. An unstructured linker is identified between the TM helix and heptapeptide repeat 2 region. The linker is due to the proline residue at position 1213. Side chains of the three tryptophan residues preceding to and within the TM helix important for the function of S-protein might adopt multiple conformations which may be critical for their function. The side chain of W1212 was shown to be exposed to solvent and the side chains of residues W1214 and W1217 are buried in micelles. Relaxation study shows that the TM helix is rigid in solution while several residues have exchanges. The secondary structure and dynamics of the TM domain in this study provide insights into the function of the TM domain of spike protein. [ABSTRACT FROM AUTHOR]