Cytoplasmic short linear motifs in ACE2 and integrin β3 link SARS-CoV-2 host cell receptors to endocytosis and autophagy

AbstractThe spike protein of the SARS-CoV-2 interacts with angiotensin converting enzyme 2 (ACE2) and enters the host cell by receptor-mediated endocytosis. Concomitantly, evidence is pointing to the involvement of additional host cell receptors, such as integrins. The cytoplasmic tails of ACE2 and integrin β3 contain a plethora of predicted binding motifs. Here, we confirm the functionality of some of these motifs through affinity measurements. The class I PDZ binding motif in the ACE2 cytoplasmic tail binds the first PDZ domain of the scaffold protein NHERF3. The clathrin-adaptor subunit AP2 μ2 interacts with an endocytic motif in the ACE2 with low affinity and the interaction is abolished by phosphorylation of Tyr781. Furthermore, the C-terminal region of integrin b3 contains a LC3-interacting region, and its interaction with ATG8 domains is enhanced by phosphorylation. Together, our data provides possible molecular links between host cell receptors and endocytosis and autophagy.One sentence summaryAffinity measurements confirmed binding of short linear motifs in the cytoplasmic tails of ACE2 and integrin β3, thereby linking the receptors to endocytosis and autophagy.

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Cytoplasmic short linear motifs in ACE2 and integrin β3 link SARS-CoV-2 host cell receptors to mediators of endocytosis and autophagy

Science Signaling ◽

10.1126/scisignal.abf1117 ◽

2021 ◽

Vol 14 (665) ◽

pp. eabf1117 ◽

Cited By ~ 2

Author(s):

Johanna Kliche ◽

Hanna Kuss ◽

Muhammad Ali ◽

Ylva Ivarsson

Keyword(s):

Binding Site ◽

Host Cell ◽

Viral Entry ◽

Tyrosine Kinases ◽

Host Cells ◽

Binding Motif ◽

Cell Receptors ◽

Short Linear Motifs ◽

Cytoplasmic Tails ◽

Linear Motifs

The spike protein of SARS-CoV-2 binds the angiotensin-converting enzyme 2 (ACE2) on the host cell surface and subsequently enters host cells through receptor-mediated endocytosis. Additional cell receptors may be directly or indirectly involved, including integrins. The cytoplasmic tails of ACE2 and integrins contain several predicted short linear motifs (SLiMs) that may facilitate internalization of the virus as well as its subsequent propagation through processes such as autophagy. Here, we measured the binding affinity of predicted interactions between SLiMs in the cytoplasmic tails of ACE2 and integrin β3 with proteins that mediate endocytic trafficking and autophagy. We validated that a class I PDZ-binding motif mediated binding of ACE2 to the scaffolding proteins SNX27, NHERF3, and SHANK, and that a binding site for the clathrin adaptor AP2 μ2 in ACE2 overlaps with a phospho-dependent binding site for the SH2 domains of Src family tyrosine kinases. Furthermore, we validated that an LC3-interacting region (LIR) in integrin β3 bound to the ATG8 domains of the autophagy receptors MAP1LC3 and GABARAP in a manner enhanced by LIR-adjacent phosphorylation. Our results provide molecular links between cell receptors and mediators of endocytosis and autophagy that may facilitate viral entry and propagation.

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SNX27 suppresses SARS-CoV-2 infection by inhibiting viral lysosome/late endosome entry

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2117576119 ◽

2022 ◽

Vol 119 (4) ◽

pp. e2117576119

Author(s):

Bo Yang ◽

Yuanyuan Jia ◽

Yumin Meng ◽

Ying Xue ◽

Kefang Liu ◽

...

Keyword(s):

Cell Surface ◽

Host Cell ◽

Viral Entry ◽

Complex Structure ◽

Pdz Domain ◽

Late Endosome ◽

Endocytic Pathway ◽

Cell Surface Receptor ◽

Binding Motif ◽

Type I

After binding to its cell surface receptor angiotensin converting enzyme 2 (ACE2), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell through directly fusing with plasma membrane (cell surface pathway) or undergoing endocytosis traveling to lysosome/late endosome for membrane fusion (endocytic pathway). However, the endocytic entry regulation by host cell remains elusive. Recent studies show ACE2 possesses a type I PDZ binding motif (PBM) through which it could interact with a PDZ domain-containing protein such as sorting nexin 27 (SNX27). In this study, we determined the ACE2-PBM/SNX27-PDZ complex structure, and, through a series of functional analyses, we found SNX27 plays an important role in regulating the homeostasis of ACE2 receptor. More importantly, we demonstrated SNX27, together with retromer complex (the core component of the endosomal protein sorting machinery), prevents ACE2/virus complex from entering lysosome/late endosome, resulting in decreased viral entry in cells where the endocytic pathway dominates. The ACE2/virus retrieval mediated by SNX27–retromer could be considered as a countermeasure against invasion of ACE2 receptor-using SARS coronaviruses.

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Network Proteins of Angiotensin-converting Enzyme 2 But Not Angiotensin-converting Enzyme 2 itself are Host Cell Receptors for SARS-Coronavirus-2 Attachment

Biology Engineering Medicine and Science Reports ◽

10.5530/bems.6.1.1 ◽

2020 ◽

Vol 6 (1) ◽

pp. 1-5

Author(s):

Sai Sailesh Kumar Goothy ◽

Arun HS Kumar

Keyword(s):

Angiotensin Converting Enzyme ◽

Host Cell ◽

Converting Enzyme ◽

Sars Coronavirus ◽

Angiotensin Converting Enzyme 2 ◽

Cell Receptors

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New insights into nCOVID-19 binding domain and its cellular receptors

10.1101/2020.09.06.285023 ◽

2020 ◽

Author(s):

Ankush Garg ◽

Gaurav Kumar ◽

Sharmistha Sinha

Keyword(s):

Receptor Binding ◽

Viral Entry ◽

Binding Domain ◽

Spike Protein ◽

Binding Motif ◽

Receptor Binding Domain ◽

Angiotensin Converting Enzyme 2 ◽

Cell Receptors ◽

High Propensity ◽

Multiple Receptor

AbstractnCOVID-19 virus makes cellular entry using its spike protein protruding out on its surface. Angiotensin converting enzyme 2 receptor has been identified as a receptor that mediates the viral entry by binding with the receptor binding motif of spike protein. In the present study, we elucidate the significance of N-terminal domain of spike protein in spike-receptor interactions. Recent clinical reports indicate a link between nCOVID-19 infections with patient comorbidities. The underlying reason behind this relationship is not clear. Using molecular docking, we study the affinity of the nCOVID-19 spike protein with cell receptors overexpressed under disease conditions. Our results suggest that certain cell receptors such as DC/L-SIGN, DPP4, IL22R and ephrin receptors could act as potential receptors for the spike protein. The receptor binding domain of nCOVID-19 is more flexible than that of SARS-COV and has a high propensity to undergo phase separation. Higher flexibility of nCOVID-19 receptor binding domain might enable it to bind multiple receptor partners. Further experimental work on the association of these receptors with spike protein may help us to explain the severity of nCOVID-19 infection in patients with comorbidities.

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Host-cell recognition through GRP78 is enhanced in the new variants of SARS-CoV-2; in silico perspective

10.21203/rs.3.rs-189975/v1 ◽

2021 ◽

Author(s):

Abdo Elfiky ◽

Ibrahim M Ibrahim ◽

Alaa M Elgohary

Keyword(s):

Cell Surface ◽

Host Cell ◽

South African ◽

Cell Receptor ◽

Binding Motif ◽

Angiotensin Converting Enzyme 2 ◽

Host Cell Receptor ◽

Binding Surface ◽

New Variant ◽

The Uk

Abstract New SARS-CoV-2 variants started in the UK and South Africa in December 2020 and currently spreading worldwide during the last few days. Additionally, another more recent variant sparked in Brazil (B.1.1.248 lineage) this month. The new variant 501.V2 (South African) bears three mutations in the receptor-binding domain (RBD) of the spike glycoprotein, K417N, E484K, and N501Y, while the Brazilian B.1.1.248 lineage have 12 mutations. The N501Y mutation is found in South African and Brazilian variants and is also shared with the UK variant VOC-202012/01 (1). This mutation may affect the host-cell receptor ACE2 (angiotensin-converting enzyme 2) recognition (2). Despite its presence in the ACE2 binding surface, we showed that the N501Y mutant shows a remarkable increase in binding of the ACE2-RBD complex to the host-cell surface Glucose Regulated Protein 78 (CS-GRP78) (3). On the other hand, the E484K is found in the spike RBD's binding motif that we reported earlier to be recognized by cell-surface GRP78 (C480-C488 region of the spike) (4). In this study, we simulate the complex ACE2-SARS-CoV-2 spike RBD system in which the RBD is in the wildtype and mutated (K417N, E484K, and N501Y) isoforms. Additionally, the CS-GRP78 association with the ACE2-SARS-CoV-2 spike RBD complex (ACE2-RBD) is modeled at the presence of these mutant variants of the viral spike.

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Network proteins of angiotensin-converting enzyme 2 but not angiotensin-converting enzyme 2 itself are host cell receptors for SARS-Coronavirus-2 attachment

10.21203/rs.3.rs-27149/v1 ◽

2020 ◽

Author(s):

Arun Kumar

Keyword(s):

Molecular Docking ◽

Angiotensin Converting Enzyme ◽

Host Cell ◽

Binding Affinity ◽

Surface Proteins ◽

Host Cells ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

Cell Receptors ◽

Dpp4 Inhibitors

Abstract Background: Coronaviruses causing severe acute respiratory syndrome (SARS-CoV) are known to enter the host cells by attaching to the membrane bound angiotensin-converting enzyme 2 (ACE2). Using molecular docking the efficiency of interaction between SARS-CoV-2 surface proteins and ACE2 network proteins was assessed. Materials and Methods: The ACE2 protein network was identified using the STRING database. The reported SARS-CoV-2 target proteins were searched in the protein data bank and uniport database. The protein-protein interactions were assessed by molecular docking using the Chimera software. The PubChem database was searched for known inhibitors of host cell receptors interacting with SARS-CoV-2 surface proteins. Molecular docking was performed to evaluate the binding efficacy of these compounds against the SARS-CoV-2 targets using AutoDock Vina and the docked protein-ligand complex were visualised using the Chimera and PyMOL software. Results: A low binding affinity was observed between SARS-CoV-2 spike proteins (protein S, M and 6YLA) and ACE2. Coronaviruses are also reported to bind to dipeptidyl peptidase 4 (DPP4), which is a network protein of ACE2. Network analysis showed five membrane proteins associated with ACE2. The ACE2 network proteins were assessed for their binding affinity with all known SARS-CoV-2 surface proteins. The SARS-CoV-2 surface proteins showed preferential binding to network proteins such as DPP4 and Meprin A alpha but not ACE2. The binding efficacy (affinity (-5.86 to -7.10 Kcal/mol), Ki (6.32 – 22.04 mM) and IC50 (12.63 – 113.71 mM) values) of DPP4 inhibitors (saxagliptin and sitagliptin) against SARS-CoV-2 surface proteins, was observed to be at a therapeutically feasible concentration to prevent SARS-CoV-2 attachment and entry into host cells. Conclusion: SARS-CoV-2 surface proteins has better interactions with DPP4 and Meprin A alpha host cells receptors rather than ACE2. DPP4 inhibitors (saxagliptin and sitagliptin) by binding with SARS-CoV-2 surface proteins may be helpful in preventing the virus entry into the host cells.

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Investigation of Critical Binding Pattern in SARS-CoV-2 Spike Glycoprotein with Angiotensin-Converting Enzyme 2: An in Silico Analysis

10.21203/rs.3.rs-113828/v1 ◽

2020 ◽

Author(s):

Farzaneh Jafary ◽

Sepideh Jafari ◽

Mohamad Reza Ganjalikhany

Keyword(s):

Angiotensin Converting Enzyme ◽

Host Cell ◽

Receptor Binding ◽

In Silico ◽

Lung Epithelial Cells ◽

Binding Motif ◽

Converting Enzyme ◽

Spike Glycoprotein ◽

Angiotensin Converting Enzyme 2 ◽

Free Energy Decomposition

Abstract Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a newly-discovered coronavirus and responsible for the spread of coronavirus disease 2019 (COVID-19). SARS-CoV-2 infected millions of people in the world and immediately became a pandemic in March 2020. SARS-CoV-2 belongs to the beta-coronavirus genus of the large family of Coronaviridae. It is now known that its surface spike glycoprotein binds to the angiotensin-converting enzyme-2 (ACE2), which is expressed on the lung epithelial cells, mediates the fusion of the cellular and viral membranes, and facilitates the entry of viral genome to the host cell. Therefore, blocking the virus-cell interaction could be a potential target for the prevention of viral infection. The binding of SARS-CoV-2 to ACE2 is a protein-protein interaction, and so, analyzing the structure of the spike glycoprotein of SARS-CoV-2 and its underlying mechanism to bind the host cell receptor would be useful for the management and treatment of COVID-19. In this study, we performed comparative in silico studies to deeply understand the structural and functional details of the interaction between the spike glycoprotein of SARS-CoV-2 and its cognate cellular receptor ACE2. According to our results, the affinity of the ACE2 receptor for SARS-CoV-2 was higher than SARS-CoV. According to the free energy decomposition of the spike glycoprotein-ACE2 complex, we found critical points in three areas which are responsible for the increased binding affinity of SARS-CoV-2 compared with SARS-CoV. These mutations occurred at the receptor-binding domain of the spike glycoprotein that play an essential role in the increasing the affinity of coronavirus to ACE2. For instance, mutations Pro462Ala and Leu472Phe resulted in the altered binding energy from -2 kcal·mol−1 in SARS-COV to -6 kcal·mol−1 in SARS-COV-2. The results demonstrated that some mutations in the receptor-binding motif could be considered as a hot-point for designing potential drugs to inhibit the interaction between the spike glycoprotein and ACE2.

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Short Linear Motifs Characterizing Snake Venom and Mammalian Phospholipases A2

Toxins ◽

10.3390/toxins13040290 ◽

2021 ◽

Vol 13 (4) ◽

pp. 290

Author(s):

Caterina Peggion ◽

Fiorella Tonello

Keyword(s):

Snake Venom ◽

Protein Interactions ◽

Biological Properties ◽

Protein Protein Interactions ◽

Sequence Alignments ◽

Toxic Activity ◽

Short Linear Motifs ◽

Phospholipases A2 ◽

Group I ◽

Linear Motifs

Snake venom phospholipases A2 (PLA2s) have sequences and structures very similar to those of mammalian group I and II secretory PLA2s, but they possess many toxic properties, ranging from the inhibition of coagulation to the blockage of nerve transmission, and the induction of muscle necrosis. The biological properties of these proteins are not only due to their enzymatic activity, but also to protein–protein interactions which are still unidentified. Here, we compare sequence alignments of snake venom and mammalian PLA2s, grouped according to their structure and biological activity, looking for differences that can justify their different behavior. This bioinformatics analysis has evidenced three distinct regions, two central and one C-terminal, having amino acid compositions that distinguish the different categories of PLA2s. In these regions, we identified short linear motifs (SLiMs), peptide modules involved in protein–protein interactions, conserved in mammalian and not in snake venom PLA2s, or vice versa. The different content in the SLiMs of snake venom with respect to mammalian PLA2s may result in the formation of protein membrane complexes having a toxic activity, or in the formation of complexes whose activity cannot be blocked due to the lack of switches in the toxic PLA2s, as the motif recognized by the prolyl isomerase Pin1.

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