key: cord-0818475-ju95s37p authors: Castin, Jesu E.; Gideon, Daniel A.; Sudarsha, Karthik S.; Rosita, Sherlin A. title: pH and Receptor Induced Conformational Changes-Implications Towards S1 Dissociation of SARS-CoV2 Spike Glycoprotein date: 2020-12-21 journal: bioRxiv DOI: 10.1101/2020.12.21.410357 sha: 004f4e81b5e8d92961e8e91980c4ad6a6f4b0dbe doc_id: 818475 cord_uid: ju95s37p Viruses, being obligate intracellular parasites, must first attach themselves and gain entry into host cells. Viral fusion machinery is the central player in the viral attachment process in almost every viral disease. Viruses have incorporated an array of efficient fusion proteins on their surfaces to bind efficiently to host cell receptors. They make use of the host proteolytic enzymes to rearrange their surface protein(s) into the form which facilitates their binding to host-cell membrane proteins and subsequently, fusion. This stage of viral entry is very critical and has many therapeutic implications. The current global pandemic of COVID-19 has sparked severe health crisis and economic shutdowns. SARS-CoV2, the etiological agent of the disease has led to millions of deaths and brought the scientific community together in an attempt to understand the mechanisms of SARS-CoV2 pathogenesis and mortality. Like other viral fusion machinery, CoV2 spike (S) glycoprotein- ‘The Demogorgon’ poses the same questions about viral-host cell fusion. The intermediate stages of S protein-mediated viral fusion are unclear owing to the lack of structural insights and concrete biochemical evidence. The mechanism of conformational transition is still unclear. S protein binding and fusion with host cell receptors, Eg., angiotensin-converting enzyme-2 (ACE2) is accompanied by cleavage of S1/S2 subunits. To track the key events of viral-host cell fusion, we have identified (in silico) that low pH-induced conformational change and ACE-2 binding events promote S1 dissociation. Deciphering key mechanistic insights of SARS-CoV2 fusion will further our understanding of other class-I fusion proteins Viruses constitute a vast majority of space under the infectious disease category. In the history of pandemics, viruses along with bacteria have conferred a major threat to world health as well as the economy. Unlike bacteria, they have higher mutation rates which make them easy to evolve into a better construct. HIV-I genome showed the highest mutation rate of (4.1 ± 1. 7 The spike glycoprotein of SARS-CoV2 is the large trimeric structure crucial for viral-cell fusion with the molecular weight of about 424 kDa. The structure has the three major topological domains which include Head, Stalk and Cytoplasmic Tail (CT). Each monomeric unit of the protein has two subunits namely S1 and S2 [16] . S1 has the regions responsible for This new region increases the rate of S1/S2 cleavage and thereby, elevates efficiency of viral entry into the host cells. Only for the Head domain, the experimental structure is available and the rest of the topological domains have been successfully modelled in accordance with many experimental data [19] . The stalk domain is responsible for global protein flexibility and allows the glycoprotein to bend in different angles [20] . It is the best example of a mechanistic protein i.e., it has exhibited many states which are shown in the Figure1.2 B. Followed by Angiotensin Collecting Enzyme-2 (ACE-2) Receptor binding in open form/ RBD 'Up' form (active form), the fusion events are initiated. The cleavage is likely to be mediated by host furin or TRPMSS2 at two distinct sites-S1/S2 and S2/S2' respectively [21] . As stated earlier, the polybasic cleavage site at the S1/S2 interface brings about effective proteolytic cleavage, thereby promoting the mechanistic tendency of the spike glycoprotein to mature into the post-fusion state. The steps of such transitions are unclear because very few biochemical evidence has been found for the intermediates. But it is well known that S1/S2 cleavage (Figure 1 i) Low pH creates unfavourable charge distribution in S1/S2 Interface and thereby promotes S1 Dissociation: S1 dissociation is a spontaneous step which occurs after S1/S2 Cleavage and the ACE-2 Binding event. S1 and S2 have two putative contact interfaces C1 and C2 (Supplementary On analysing the titratable residues in the S1/S2 Interface (Figure 1 ii) S1/S2 Cleavage Elevates Local Disorderliness in S1/S2 Interface Environment: We postcleaved as well as precleaved states, we came to find that postcleaved S1/S2 state showed increased RMSF (especially in S1/S2 interface) than that of precleaved S1/S2 state ( Figure 1.7) . It provided the preliminary insight that host-mediated proteolytic cleavage elevates relative disorderliness in S1/S2 interface and further promotes S1 dissociation. It is noteworthy that neither experimental structures nor models are capable of illustrating S2/S2' cleavage, which can be studied only through rigorous MD approaches. We also generated distant maps for the experimental structures in 'Closed' and 'ACE-2 Bound' states ( Figure 1.8) . It became clear that the contact distance in S1/S2 interface of the 'ACE-2 Bound' state appeared to have diminished. We could observe that the NTD and CTD-2 regions are fluctuating in nature whereas HR of S2 remained stable (Figure 1.9 ). This confirms that Host-Receptor binding also influences S1 dissociation. To observe pH-induced changes, we used as open-source. The pKa values of the titratable interface residues were then taken for further consideration, as our interest fits into the S1/S2 interface where possible initial events of S1 dissociation occur. Then the charge of the titratable residues upon decreasing pH i.e., 7 to 3 was calculated for the experimental and modelled structures to find the effect of pH in order to prove our hypothesis about unfavourable charge distribution. The charge values were obtained with the pH plot profiling module available at VMD 1.9.3. The titratable residues of S1/S2 interface were subjected for the charge profiling with the decreasing pH in the units of 0.01. The data points were then retrieved for comprehensive plotting with GNUPLOT 5.2. For better understanding the roles of S1/S2 cleavage, we need to compare the S1/S2 interface environment in both the states. We chose the models which clearly depict the precleaved The square fluctuations for both states were then calculated and resulting differential fluctuation values were compared. All these steps were performed with the Python-based tool-ProDy 1.10.11 [28] . For deriving a conclusive proof for the receptor-induced conformational changes in the S1/S2 interface, we built S1/S2 distance maps for the experimental structures in closed as well as in SARS-CoV2 spike glycoprotein forms a trimeric assembly in which each monomeric unit has two subunits S1 and S2. The main topological domains include head, stalk and cytoplasmic tail (CT). Spike has several key regions which are responsible for various functions that promote the viral entry into the host cell. Each region is coloured with respective to the one-dimensional legend. 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