【病毒外文文獻】2019 Membrane Cholesterol Modulates Oligomeric Status and Peptide-membrane Interaction of Severe Acute Respiratory Syndr

Subscriber access provided by Karolinska Institutet University Library is published by the American Chemical Society 1155 Sixteenth Street N W Washington DC 20036 Published by American Chemical Society Copyright American Chemical Society However no copyright claim is made to original U S Government works or works produced by employees of any Commonwealth realm Crown government in the course of their duties B Biophysics Physical Chemistry of Biological Systems and Biomolecules Membrane Cholesterol Modulates Oligomeric Status and Peptide membrane Interaction of Severe Acute Respiratory Syndrome Coronavirus Fusion Peptide Geetanjali Meher Surajit Bhattacharjya and Hirak Chakraborty J Phys Chem B Just Accepted Manuscript DOI 10 1021 acs jpcb 9b08455 Publication Date Web 19 Nov 2019 Downloaded from pubs acs org on November 20 2019 Just Accepted Just Accepted manuscripts have been peer reviewed and accepted for publication They are posted online prior to technical editing formatting for publication and author proofing The American Chemical Society provides Just Accepted as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance Just Accepted manuscripts appear in full in PDF format accompanied by an HTML abstract Just Accepted manuscripts have been fully peer reviewed but should not be considered the official version of record They are citable by the Digital Object Identifier DOI Just Accepted is an optional service offered to authors Therefore the Just Accepted Web site may not include all articles that will be published in the journal After a manuscript is technically edited and formatted it will be removed from the Just Accepted Web site and published as an ASAP article Note that technical editing may introduce minor changes to the manuscript text and or graphics which could affect content and all legal disclaimers and ethical guidelines that apply to the journal pertain ACS cannot be held responsible for errors or consequences arising from the use of information contained in these Just Accepted manuscripts 1 Membrane Cholesterol Modulates Oligomeric Status and Peptide Membrane Interaction of Severe Acute Respiratory Syndrome Coronavirus Fusion Peptide Geetanjali Meher Surajit Bhattacharjya and Hirak Chakraborty School of Chemistry Sambalpur University Jyoti Vihar Burla Odisha 768 019 India Centre of Excellence in Natural Products and Therapeutics Sambalpur University Jyoti Vihar Burla Odisha 768 019 India School of Biological Sciences Nanyang Technological University 60 Nanyang Drive Singapore 637551 Address correspondence to Hirak Chakraborty E mail hirak suniv ac in or hirakchakraborty Phone 91 8008716419 or Surajit Bhattacharjya Email surajit ntu edu sg Fax 65 6791 3856 Page 1 of 33 ACS Paragon Plus Environment The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 ABSTRACT The N terminal fusion peptide residues 770 788 of S2 glycoprotein of the severe acute respiratory syndrome corona virus SARS CoV exposed upon receptor binding is crucial for virus entry into the host cell The fusion peptide alters the membrane organization and dynamics of the host membrane to facilitate membrane fusion Generally the effect of fusion peptide on the membrane is sensitive to the lipid composition of target membranes In this present work we have utilized steady state and time resolved fluorescence spectroscopy in tandem with circular dichroism spectroscopy to elucidate the binding oligomeric status secondary structure of the fusion peptide and its impact on the depth dependent membrane organization and dynamics We have used depth dependent fluorescence probes 1 6 diphenyl 1 3 5 hexatriene DPH and its trimethylammonium derivative TMA DPH to evaluate the effect of the peptide binding along the bilayer normal We have exploited the energy transfer efficiency of tryptophan between TMA DPH and DPH to determine the relative location of the solitary tryptophan present in the membrane bound fusion peptide We have further evaluated the effect of membrane cholesterol on the binding and organization of the peptide and the impact of peptide binding on the depth dependent physical properties of the membrane at various cholesterol concentrations Our results clearly demonstrate that the membrane cholesterol alters the oligomeric status of the membrane bound peptide and the effect of peptide binding on the depth dependent membrane organization and dynamics The role of cholesterol is important as the eukaryotic host cells contain good amount of cholesterol that might be important for the entry of pathogenic viruses Page 2 of 33 ACS Paragon Plus Environment The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 3 INTRODUCTION Severe Acute Respiratory Syndrome SARS is an emerging form of pneumonia caused by SARS CoVs 1 3 Coronaviruses are enveloped positive stranded RNA viruses with the largest genome and are characterized by 3 4 envelope proteins embedded on their surface 4 Like other enveloped viruses coronavirus enters the host cell via fusion of the lipid bilayer of viral envelope with the host cell membrane The surface glycoprotein S of SARS CoV binds to the host cell receptors angiotensin converting enzyme ACE2 and CD209L to induce membrane fusion 5 6 Recent studies have shown SARS CoV to enter the cell via receptor mediated endocytosis 2 7 The binding of S1 subunit of S protein with the receptor leads to cleavage of the protein thereby initiating conformational changes in the other subunit S2 8 While the S1 subunit binds to the cell surface receptor S2 subunit induces fusion between the virus and target cell membranes 8 10 The conformational change in S2 subunit exposes the N terminal fusion peptide which plays an instrumental role in the fusion process 9 The S2 protein contains heptad repeats HR1 and HR2 and a transmembrane region at the C terminus in addition to the membrane active fusion peptide FP internal fusion peptide IFP and pre transmembrane peptide PTM HR1 and HR2 regions are known to form antiparallel oligomers 11 14 Atomic resolution structure of HR1 and HR2 complex had shown the formation of a six helix bundle which is an important characteristic of class I fusion peptide 15 18 However there has been no information yet regarding the oligomeric status of fusion peptide and its implication in membrane fusion Generally fusion peptide is a stretch of 20 25 amino acids located at the N terminus of class I viral fusion protein Its interaction with the host cell has been extensively shown to be the first step of fusion between virus and host cells 19 An alternative mechanism suggests the requirement of both receptor mediated endocytosis and pH driven conformational change for the fusion 20 21 In that case SARS CoV has been proposed to be Page 3 of 33 ACS Paragon Plus Environment The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 4 internalized in the cell through endocytosis followed by its exposure to a low pH environment thus leading to the proteolytic cleavage of S1 and S2 domains Moreover cholesterol and sphingolipid rich raft microdomains have been also shown to be involved in virus entry 21 22 In general membrane composition plays a key role in the behavior of fusion protein and its effect on membrane fusion by modulating the organization and dynamics of both the membrane as well as the fusion protein 23 24 The fusion peptide of SARS CoV has been shown to preferentially bind to membrane containing negatively charged lipids owing to 2 formal charges on the peptide at physiological pH 3 Insertion of fusion peptide into the membrane reduces the dipole potential of negatively charged membranes the effect being more pronounced in presence of cholesterol 25 In addition SARS CoV fusion peptide promotes water penetration in the hydrophobic region of DMPC and DMPG membranes remarkably more in the latter 3 Lipid composition also has significant impact on the rate of lipid mixing Large unilamellar vesicles LUVs containing cholesterol undergo faster lipid mixing than the membranes devoid of cholesterol 3 25 Cholesterol is known to have unique effect on the fusion peptide structure membrane interaction and fusion The SARS CoV fusion peptide has been recently shown to assume a bent helical conformation V shaped around residues 5 18 and largely non helical or extended conformations around the N terminal residues W2 T4 in DPC micelles 26 27 However it has a propensity to adopt sheet structure upon interaction with lipid membranes 28 FTIR spectra of SARS CoV fusion peptide had shown extended strands with strong intermolecular interactions in presence of phospholipids 3 In the current study we have investigated the structure and oligomeric status of SARS CoV fusion peptide and its effect on the organization and dynamics of POPC POPG membranes with varying amounts of cholesterol Our results revealed the effect of cholesterol on structure and oligomeric status of the peptide We have further evaluated the effect of fusion peptide on membrane Page 4 of 33 ACS Paragon Plus Environment The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 5 organization and dynamics at various membrane compositions We have extensively utilized steady state and time resolved fluorescence properties of tryptophan in the SARS CoV fusion peptide and of two depth dependent extrinsic fluorophores DPH and TMA DPH in order to elucidate the oligomeric status of the peptide and its effect on depth dependent membrane organization and dynamics We have determined secondary structure of the fusion peptide in different membranes using circular dichroism spectroscopy Our results demonstrated the binding affinity of SARS CoV fusion peptide to increase with increasing membrane cholesterol Moreover the fusion peptide demonstrated concentration dependent oligomerization in cholesterol containing membrane with high oligomerization propensity in the membrane containing 20 mol of cholesterol The impact of peptide binding on membrane properties depends on the lipid composition of the membrane and oligomeric status of the peptide The peptide assumes majorly unstructured conformation with approximately 20 sheet and 10 helical conformation in the membrane of different lipid compositions The fusion peptide of SARS CoV mainly partitions in the interfacial region of POPC POPG 80 20 mol and POPC POPG Chol 60 20 20 mol membranes and penetrates in the hydrophobic region of the membrane in POPC POPG Chol 70 20 10 mol membranes Taken together our present work provides a detailed overview of the structure oligomeric status and penetration depth of the SARS CoV fusion peptide in membranes containing varying cholesterol concentration and its effect on the organization and dynamics of the membrane MATERIALS AND METHODS Materials 1 Paimitoyl 2 oleoyl sn glycero 3 phosphocholine POPC cholesterol Chol and 1 paimitoyl 2 oleoyl sn glycero 3 phosphoglycerol sodium salt POPG were purchased from Avanti Polar Lipids Alabaster AL 1 6 Diphenyl 1 3 5 hexatriene DPH and Page 5 of 33 ACS Paragon Plus Environment The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 6 trimethylammonium derivative of DPH TMA DPH were purchased from Molecular Probes Invitrogen Eugene OR Sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate were obtained from Merck India Spectroscopic grade DMSO was purchased from Spectrochem India All other chemicals used in the study were of the highest available purity Water was purified in a Millipore Bedford MA Milli Q water purification system Peptide Synthesis The fusion peptide of SARS CoV was purchased commercially from GL Biochem China with purity of 98 The peptide sequence was MWKTPTLKYFGGFNFSQIL without any modification in the N and C terminals Small aliquots of peptide stock solutions prepared in DMSO were added to the vesicle suspensions The amount of DMSO was always less than 1 v v such that it had no detectable effect on either fusion or membrane structure Preparation of Vesicles Large unilamellar vesicles LUVs diameter approximately 100 nm were prepared from a mixture of POPC POPG 80 20 mol or POPC POPG Chol 70 20 10 mol and 60 20 20 mol the concentration of lipid was kept constant at 200 M in all experiments We used DPH to probe the hydrophobic region of the membrane and TMA DPH to probe the interfacial region The concentration of DPH or TMA DPH was kept constant at 1 M 1 mol with respect to lipid concentration to minimize the probe induced alteration of membrane structure The lipid was dissolved in chloroform and air dried to make a thin film The film was kept overnight in a vacuum desiccator to ensure complete removal of chloroform The lipid film was hydrated swelled by adding 10 mM phosphate buffer pH 7 4 The sample was vortexed for 1h for uniform dispersion of lipids LUVs with a diameter of 100 nm were prepared by extrusion technique using Avanti Mini Extruder Alabester AL as described previously 29 Background samples were prepared the same way except that the peptides were omitted Page 6 of 33 ACS Paragon Plus Environment The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 7 Small aliquots of peptides and probes were added from their respective stock solutions prepared in DMSO to prepare the working solutions DMSO content was always less than 1 v v since such a small quantity of DMSO had no detectable effect on membrane structure and its interaction with peptide 30 Steady State Fluorescence Measurements Steady state fluorescence measurements were carried out in Hitachi F 7000 Japan spectrofluorometer using quartz cuvettes of 1 cm path length Tryptophan was excited at 295 nm and its fluorescence monitored from 310 to 450 nm Excitation and emission slits with a nominal band pass of 5 nm were used for all measurements Background peptide free intensities of samples were subtracted from each sample spectrum to eliminate the contribution of solvent Raman peak and other scattering artefacts Fluorescence anisotropy measurements of DPH and TMA DPH were performed using the same instrument fixing excitation wavelength at 360 nm and monitoring emission at 428 nm Excitation and emission slits with a nominal band pass of 5 nm were used for this set of experiments Fluorescence anisotropy measurement of Tryptophan was performed similarly with excitation wavelength at 295 nm and emission at 350 nm Excitation and emission slits with a nominal band pass of 10 nm were used for the measurement of tryptophan fluorescence anisotropy Background peptide free intensities of samples were subtracted from each sample spectrum to eliminate the contribution of solvent Raman peak and other scattering artefacts in case of fluorescence anisotropy of Tryptophan Anisotropy values were calculated using the following equation 31 2V V V HV V V HI G Ir I G I 1 Page 7 of 33 ACS Paragon Plus Environment The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 8 where G IHV IHH grating correction or G factor IVV and IVH are the measured fluorescence intensities with excitation polarizer vertically oriented and emission polarizer vertically and horizontally oriented respectively Time resolved Fluorescence Measurements Fluorescence lifetimes were calculated from time resolved fluorescence intensity decays using the IBH 5000F Nano LED equipment Horiba Jobin Yvon Edison NJ and Data Station software in the time correlated single photon counting TCSPC mode A pulsed light emitting diode LED was used as the excitation source This LED generates optical pulse at 290 338 nm with pulse duration 1 2 ns and is run at 1 MHz repetition rate The LED profile instrument response function was measured at the excitation wavelength using Ludox colloidal silica as the scatterer To optimize the signal to noise ratio 10 000 photon counts were collected in the peak channel All experiments were performed using emission slits of band pass 16 nm The sample and the scatterer were alternated after every 10 acquisition to ensure compensation for shape and timing drifts occurring during data collection This arrangement also prevents prolonged exposure of the sample to the excitation beam thereby avoiding any possible photo damage of the fluorophore Data were stored and analyzed using DAS 6 2 software Horiba Jobin Yvon Edison NJ Fluorescence intensity decay curves were deconvoluted with the instrument response function and analyzed as a sum of exponential terms A considerable plot was obtained with random deviation about zero with a minimum 2 value of 1 2 or less Intensity averaged mean lifetimes avg for tri exponential decays of fluorescence were calculated from the decay times and pre exponential factors using the following equation 31 2 2 Page 8 of 33 ACS Paragon Plus Environment The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 9 where i is the fraction that shows i lifetime Circular Dichroic Spectroscopy Circular Dichroic CD spectra of SARS CoV fusion peptide 7 5 M in three different membranes were measured in a Jasco 1500 Japan Spectrophotometer The spectra were recorded in the wavelength range of 190 260 nm using cylindrical quartz cuvette of 4 mm path length The spectra were scanned in 0 5 nm wavelength increments with band width of 2 nm and scan rate of 50 nm min All circular dichroic spectra were averages of at least 3 consecutive scans The background spectrum without protein was recorded with the same parameters and was subtracted from each sample spectrum All CD measurements were carried out in 5 mM phosphate buffer pH 7 4 The ellipticity values obtained from the instrument were converted into molar ellipticity using the following equation 10 7 where is the ellipticity measured by CD spectroscopy b is the path length in cm n is the number of amino acid residues present in the fluorophore and c is the concentration of protein solution in moles litre 1 The lipid and peptide concentrations used in the CD measurements were 100 M and 7 5 M respectively The CD spectra were analyzed using Dichroweb for the evaluation of secondary structural components 32 33 Fluorescence Resonance Energy Transfer Efficiency Measurements The penetration depth of SARS CoV fusion peptide was measured in three different membranes by monitoring the ratio of fluorescence resonance energy transfer FRET efficiency of tryptophan between TMA DPH and DPH DPH and TMA DPH are known to locate at two different regions of the bilayer DPH being at an average distance of approximately 7 8 from the centre of the bilayer while TMA DPH locates at the interfacial region owing to its polar trimethylammonium group with an average distance of approximately 10 9 from the centre of the bilayer 34 We have utilized the distance Page 9 of 33 ACS Paragon Plus Environment The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12