Asia on aiemmin ajateltu:Viime vuoden tammikuulta on artikkeli netissä. Siinä on isoloidusti tutkittu yhden C-terminaalisen monomeerin ominaisuudet- jolloin se osoitautuu IDP-tyyppiseksi mitä suurimamssa määrin eli fysiologisissa oloissa saattaa muodostaa minkä tahansa struktuurin interaktioproteiininsa kanssa. siis siinä piilee monia resursseja toimia viruksen elinsyklin eduksi. Otan aika laajasti sitaattia.
https://www.sciencedirect.com/science/article/pii/S0042682221002300
Microsecond simulations and CD spectroscopy reveals the intrinsically disordered nature of SARS-CoV-2 spike-C-terminal cytoplasmic tail (residues 1242–1273) in isolation
The importance of coronavirus spike protein is apparent from it surface-exposed location, suggesting it is a prime target after viral infection for cell-mediated and humoral immune responses as well as artificially designed vaccines and antiviral therapeutics. The SARS-CoV-2 homo-trimeric spike glycoprotein consists of an extracellular unit anchored by a transmembrane (TM) domain in viral membrane and a cytoplasmic domain (Walls et al., 2020). It is secreted as monomeric 1273 amino acid long protein from endoplasmic reticulum (ER) shortly after which it trimerizes to facilitate the transport to the Golgi complex (Duan et al., 2020; Walls et al., 2020). Moreover, N-linked high mannose oligosaccharide side chains that are added to spike monomer in ER are further modified in Golgi compartments (Duan et al., 2020).
Spike is one of the most extensively studied protein among all of SARS-CoV-2 proteome. So far, based on Uniprot database, approximately two hundred structures have been reported using X-ray crystallography and cryo-electron microscopy techniques. However, these structures consist of S1 subunit of spike but lacks the transmembrane and cytoplasmic C-terminal regions present in S2 subunit or with missing electron densities in cytoplasmic region.
The distal S1 subunit (residues 14–685) contains a N-terminal domain, a C-terminal domain, and two subdomains (Fig. 1). The C-terminal domain of S1 is the receptor-binding domain or RBD, has a receptor-binding motif (RBM) which interacts with human angiotensin converting enzyme 2 (ACE2), chief target receptor of SARS-CoV-2 on human cells (Lan et al., 2020). RBM is present as an extended loop insertion which binds to bottom side of the small lobe of ACE2 receptor.
The S2 subunit (residues 686–1273) has a hydrophobic fusion peptide, two heptad repeats, a transmembrane domain, and a cytoplasmic C-terminal tail (Fig. 1).
Fig. 1. Domain architecture of spike Glycoprotein: depiction of available structures in open and closed states, transmembrane domain, and cytoplasmic C-terminal tail. Based on prediction of transmembrane region in the spike protein by CCTOP, a consensus-based predictor, the boundaries of all domains have been defined. As per CCTOP prediction, the transmembrane region of spike lies within the residues 1216–1241, and so, the cytoplasmic region of spike has been used in this study with the residues 1242–1273.
As of yet, cytoplasmic domain of spike protein is the least explored region despite of such extensive research in pandemic times. It is of particular importance as it contains a conserved ER retrieval signal (KKXX) (Lontok et al., 2004).
In SARS-CoV and SARS-CoV-2 spike proteins, a novel dibasic KLHYT (KXHXX) motif present at extreme ends of the C-terminus plays a crucial role in its subcellular localization (Giri et al., 2020; McBride et al., 2007; Sadasivan et al., 2017).
Also, deletions in cytoplasmic domain of coronavirus spike are implicated in viral infection in recent reports (Bosch et al., 2005; Dieterle et al., 2020; Ou et al., 2020; Ujike et al., 2016). SARS-CoV and SARS-CoV-2 spike having a deletion of last ∼20 residues displayed increased infectivity of single-cycle vesicular stomatitis virus (VSV)–S pseudotypes (Dieterle et al., 2020; Ou et al., 2020).
Contrarily, short truncations of cytoplasmic domain of Mouse Hepatitis Virus (MHV) spike protein (△12 and △25) had limited effect on viral infectivity while the long truncation of 35 residues interfered with both viral-host cell membrane fusion and assembly.
Importantly, it is also shown to interact with the membrane protein inside host cells (Bosch et al., 2005). In our previous report, the cytoplasmic tail is predicted to be a MoRF (Molecular Recognition Feature) region (residues 1265–1273) by a predictor MoRFchibi (Giri et al., 2020). The MoRF regions in proteins are disorder-based binding regions that contribute the binding to DNA, RNA, and other proteins. In the same report, it is also found to contain many DNA and RNA binding residues (Giri et al., 2020).
Despite of availability of several structures of spike protein using advanced techniques like cryo-EM, the structure of cytoplasmic domain is not yet clear due to its ‘missing electron density’. Generally, intrinsically disordered proteins show such characteristic of missing electron density and lacks a well-defined three-dimensional structure (Uversky, 2020).
Additionally, the consensus-based disorder prediction by MobiDB has shown this region to be disordered (Piovesan et al., 2021). Considering these arguments, we aimed to understand the cytoplasmic domain of the SARS-CoV-2 spike protein to gain further insights. To this end, we computationally analyzed its behavioural dynamics using molecular dynamic (MD) simulations up to 1 microsecond (μs) and validated it with CD spectroscopy based experiments. This report's outcomes will help to understand this domain's structure and function and provide knowledge to explore the interaction of spike protein with other viral and host proteins.
2. Material and methods
Fig. 2. Transmembrane region prediction from five web servers: A. CCTOP, B. PSIPRED, C. SPLIT, D. TMHMM, and E. TMPred. F. Table showing predicted transmembrane residues. All predictors work as standalone except CCTOP which works based on consensus from multiple predictors. Considering this, the cytoplasmic region of spike is chosen from residues 1242–1273 as CCTOP has predicted residues 1216–1241 in transmembrane region.
3.1.1. Disorder prediction
In our recent study, we have identified the disordered and disorder-based binding regions in SARS-CoV-2 where the cytoplasmic domain at C-terminal of spike protein is found to be disordered (Giri et al., 2020). Again, we analyzed the disorderedness in selected cytoplasmic region using multiple predictors, including PONDR family, IUPred2A (redox state), and PrDOS predictors. Out of six predictors, three predictors from PONDR family have predicted it as highly disordered, PrDOS has predicted it as moderately disordered, and PONDR FIT has predicted it as least disordered. Additionally, IUPred2A has been used with its redox-state calculation function due to high number of cysteine residues present in the peptide (Fig. 3). As per calculations, the redox minus (where all cysteines are replaced by serine) state has shown high disorder propensity while redox plus has shown least propensity.
Fig. 4. Structure models of spike full-length and C-terminal cytoplasmic domain (1242–1273 residues): A. Full-length spike protein model using AlphaFold2, B. Modelled structure through PEP-FOLD web server, visualized in Maestro, and C. Secondary structure analysis using PSIPRED web server.
3.1.3. Simulation with OPLS 2005
In the last three decades, many advancements have been made in forcefields and hardware related to MD simulation to match the experimental events. Long MD simulations up to microseconds or milliseconds are incredibly insightful to study structural conformations occurring at the nanoscale level. We have recently explored various regions of different SARS-CoV-2 proteins through computational simulations and experimental techniques that are very well correlated (Gadhave et al., 2020a, 2020b, 2020a). This study performed 1 μs MD simulations of C-terminal cytoplasmic domain of spike protein (1242–1273 residues) to understand its dynamic nature. As obtained from structure modelling through PEP-FOLD, the model contains one small helix at C-terminal with residues 1265LKGV1268 (Fig. 4B). According to 2struc webserver (Klose et al., 2010), the total helix propensity contribute approximately 12.5% of total secondary structure while rest of the region is constituted by turns and extended coils. The secondary structure prediction of spike C-terminal tail region contains a β-strand of five residues 1262EPVLK1266, as predicted by PSIPRED webserver (Buchan and Jones, 2019) (Fig. 4C).
After analyzing the disorder propensity and secondary structure composition, we performed a rigorous simulation of cytoplasmic region (residues 1242–1273) to understand its atomic movement and structural integrity. A total of 1 μs simulation was done after 50,000 steps of steepest descent method-based energy minimization. It has been observed that the structure of spike C-terminal cytoplasmic region remains to be unstructured throughout the simulation. Based on mean distance analysis, the peptide simulation setup showed massive deviations up to 7.5 Å which does not attain any stable state (Fig. 5A). As shown in Fig. 5B, mean fluctuation in residues is observed to be within the range of 1.6–6.4 Å. The intramolecular hydrogen bond analysis demonstrates the highly fluctuating trend portraying no stable helical or beta sheet conformation adoption by the residues (Fig. 5E). The secondary structure timeline (Fig. 5C & D) also reveals the disordered state of spike C-terminal cytoplasmic region during the 1 μs simulation time (none of the frames captured α-helix or β-sheets) which is further depicted in the snapshot of 1 μs frame in Fig. 5F and the trajectory movie up to 1 μs (supplementary movie 1).
We have also modelled the cytosolic part of Spike protein from 1235 to 1273 residues as defined in Uniprot database and two predictors (TMPred: 1216–1235, and TMHMM: 1214–1236) used in this study. In modelled structure, the helical propensity in cytosolic region was shown by 1237–1245 residues. Using above described OPLS 2005 forcefield parameters, the all-atoms explicit solvent MD simulation was carried out for 1 μs. The trajectory analysis has been shown in Supplementary Fig. 1, the cytosolic region has revealed majorly unstructured region along with a small β-strand of two residues 1258FD1259 after 1 μs. The upward trend of RMSD values illustrates the highly deviating atomic positions and fluctuating RMSF shows the change in structural property of residues (Supplementary Figs. 1A and 1B). Also, the decreasing number of hydrogen bonds demonstrates the breaking of helices in the structure (Supplementary Fig. 1C). The time-dependent secondary structure element analysis illustrates that a total of 15% secondary structure was formed that includes mainly alpha helix and small percentage of beta strands (Supplementary Figs. 1D and 1E; red: alpha helix and blue: beta strands). After huge structural transitions, the structural composition of last frame of simulation is shown with a small beta strand of two residues and other regions to be disordered (Supplementary Fig. 1F). The snapshots at every 100 ns till 1 μs show the structural transitions in Spike cytoplasmic region (Supplementary Fig. 2).
Fig. 6. Depiction of representative from top 10 clusters from 1 μs simulation trajectory. Size of each cluster is shown which represent the total number of frames in the trajectory based on RMSD calculated with reference structure (frame 1). The protein backbone of all frames is shown as superimposition. The N- to C-terminal protein structures are colored from red to blue, respectively.
-----. However, we also tried to get the synthesized peptide of residues 1235–1273 of spike but due to multiple cysteine residues it was not feasible.
Further, it was of utmost importance to validate MD simulation outcomes using experimental techniques. The water-soluble peptide of spike residues 1242–1273 at 25 μM concentration exhibits a prominent negative peak at approximately 198 nm in far-UV CD spectra which defines the unstructured nature of a protein. Infact, we have also checked the secondary structure state in presence of a reducing agent, DTT, then also, the peptide is observed to be disordered with significant negative ellipticity. Further, in presence of helix inducer solvent, TFE, the peptide adopts helical structure. However, SDS micelles in surroundings of peptide generates little changes in the peptide structure which may signify its inability to gain structure. Also, in presence of sucrose, the CD spectra of peptide corresponds to the disordered conformation. Under the influence of crowding agents like Dextran-70 and PEG (8000), conservation of disordered structure indicates that no -intra chain forces are acting in between the residues. Based on this combination of facts, we have interpreted that spike C-terminal cytosolic tail (residues 1242–1273) as an intrinsically disordered region. Generally, an IDPR gains any structure upon interacting with its interacting partner or in physiological conditions (Wright and Dyson, 1999). In its unstructured state, it may function as a MoRF to bind with COP1 coated transporting vesicles which localizes the Spike protein into ER. As described earlier, the interaction of C-terminal domain of Spike protein is reported with other structural proteins like M which is highly likely to occur in its disordered form with extended radius.
5. Conclusion
The cytoplasmic region of spike glycoprotein of SARS-CoV-2 has not been studied yet. Given its extreme importance in functioning of spike protein, the structure and its dynamics has been investigated here. The advancement in computational powers and excessive improvements in forcefields have empowered structural biology. Newly developed algorithms and their user-friendly approach allow correlating the outcomes with experimental observations. In this article, we have identified the transmembrane region in spike protein by employing distinguished web predictors. This cleared the composition of amino acids forming cytoplasmic domain. Further, the secondary structure and disorder predisposition analysis demonstrated it to be highly disordered. We have demonstrated the structural conformation of cytoplasmic domain (1242–1273 residues) of spike protein at a microsecond timescale using computational simulations. As revealed, this domain is purely unstructured or disordered after 1 μs and have not gained any structural conformation throughout the simulation period. Experimental outcomes also confirm the intrinsic disordered state of cytoplasmic domain of spike. The intrinsic disordered nature of peptide is shown in presence of macromolecular crowders. Based on our previous study (Giri et al., 2020), cytoplasmic tail of spike glycoprotein has molecular recognition features therein which needs to be explored further. The disordered nature of cytosolic region may possibly have implications to interact with other viral proteins during virion assembly as well as host proteins and transporting vesicles during localization in ERs. In this study, the multiple conformations during the simulation process adds up to even more interesting speculations.
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