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Abstract
Abstract
The envelope protein from SARS-CoV-2 (E) is a single pass transmembrane protein which accumulates in the endoplasmic reticulum golgi intermediate compartment in host cells, where it acts as a viroporin and an ion channel which plays a critical role in viral budding and assembly. A recent study produced a structure of the pentameric transmembrane domain (TMD) but no structural information is available for the entire protein, and the conformation and dynamics of the C-terminal domain (CTD), which is presumed to be a soluble region, is unknown. Here we present preliminary investigation into the full length of the protein in a native lipid environment utilizing power saturation EPR spectroscopy with site specific introduction of nitroxyl spin-labels as well as Double Electron-Electron Resonance EPR (DEER-EPR) to examine topology of the protein with respect to the lipid bilayer. Our results support a model with moderately buried amphipathic CTD ending in an exposed C terminus.
Background
Background
SARS-CoV-2 Envelope, or S2E, is a transmembrane protein between found between the endoplasmic reticulum and Golgi in cells infected by SARS-CoV-2 (COVID-19).
S2E is believed to be involved in altering the curvature of host membranes to allow nascent virions to bud off of the Golgi as they mature [4, 5].
The C Terminal Domain (CTD) of S2E is responsible for binding and sequestering host factor PALS1, inhibiting formation of tight junction complexes. This leads to pulmonary edema and inflammation, which are characteristic in serve COVID-19 cases [7]
S2E is a homopentameric protein with a single helix passing through the membrane [2] composed of 3 primary domains [1].
Unstructured N-terminal Domain (9 residues)
Transmembrane Domain (TMD) (18 residues)
C-terminal domain (CTD) (37 Residues)
While the structure of the TMD has been determined, there is no structural information is available for the entire protein, and the conformation and dynamics of the C-terminal domain (CTD).
The goal of this project is to present a preliminary investigation into the full length of the protein in a native lipid environment while providing functional mechanisms and potential as a therapeutic target.
Figure 1: S2E causes budding, PALS1 inactivation, and channel activity.
Figure 2: Model of S2E in a membrane environment. The five helical residues form a cation channel.
Methodology
Methodology
Sample Preparation
Sample Preparation
The Liberty Blue Peptide Synthesizer 2.0 was used to generate peptide chains that fold into the S2E protein and allows for testing how specific amino acid changes affect interactions.
Following synthesis, the sample was purified with HPLC and emulsified into a 3:1 POPC:POPG lipid vesicle.
Power Saturation Electron Paramagnetic Resonance (EPR)
Power Saturation Electron Paramagnetic Resonance (EPR)
A biophysical technique that determines orientation and insertion depth of the S2E protein within the lipid bilayer that uses paramagnetic spin labels to distinguish between segments that are buried in the membrane and in the aqueous phase.
Double Electron-Electron Resonance (DEER-EPR)
Double Electron-Electron Resonance (DEER-EPR)
Pulse DEER-EPR is a technique used to investigate spatial relationships between different protein regions and assess conformational changes of S2E in the membrane.
Liposome Calcium Fluorescence Assay
Liposome Calcium Fluorescence Assay
A technique that measures the permeability of liposomes to calcium influx with FURA2, a dye that fluoresces in response to calcium.
Results
Results
EPR Data
EPR Data
Figure 3: Liberty Blue Peptide Synthesizer 2.0.
Figure 4: Electron Paramagnetic Resonance Instrument.
Figure 5: Depth parameter vs labeled residues collected from power saturation. Known TMD and CTD domains colored in green and purple.
Figure 6: EPR data of MTSL Spin Labeled residues of S2E. Sharper peaks indicate less steric constraint.
Liposome Assay
Liposome Assay
Figure 7: Liposome Calcium Fluorescence Assay with FURA2, a dye that fluoresces in response to calcium. Used to determine ion channel activity.
Figure 8: Shifts in FURA2 fluorescence without S2E (blank), with Cysless S2E, and with melittin, a pore forming peptide.
Figure 9: Gradient Depletion as a fraction of melittin vs time of different S2E mutations.
Future Directions
Future Directions
Develop a small molecule drug to inhibit S2E function and compare fluorescence assay results.
Use triangulation of distance pairs obtained by DEER-EPR of double-labeled samples to estimate the angle between CTD and TMD.
Use computational modeling to integrate DEER and CW EPR data to predict the orientation and interactions of the CTD.
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