Cholesterol inhibits assembly and activation of the EphA2 receptor.

The receptor tyrosine kinase EphA2 drives cancer malignancy by facilitating metastasis. EphA2 can be found in different self-assembly states: as a monomer, dimer, and oligomer. However, our understanding remains limited regarding which EphA2 state is responsible for driving pro-metastatic signaling. To address this limitation, we have developed SiMPull-POP, a single-molecule method for accurate quantification of membrane protein self-assembly. Our experiments revealed that a reduction of plasma membrane cholesterol strongly promoted EphA2 self-assembly. Indeed, low cholesterol caused a similar effect to the EphA2 ligand ephrinA1-Fc. These results indicate that cholesterol inhibits EphA2 assembly. Phosphorylation studies in different cell lines revealed that low cholesterol increased phospho-serine levels, the signature of oncogenic signaling. Investigation of the mechanism that cholesterol uses to inhibit the assembly and activity of EphA2 indicate an in-trans effect, where EphA2 is phosphorylated by protein kinase A downstream of beta-adrenergic receptor activity, which cholesterol also inhibits. Our study not only provides new mechanistic insights on EphA2 oncogenic function, but also suggests that cholesterol acts as a molecular safeguard mechanism that prevents uncontrolled self-assembly and activation of EphA2.

Local Structures in Proteins from Microsecond Molecular Dynamics Simulations: A Symmetry-Based Perspective.

We report on a new method for the characterization of local structures in proteins based on extensive molecular dynamics (MD) simulations, here, 1 µs in length. The N-H bond of the Rho GTPase binding domain of plexin-B1 (RBD) serves as a probe and the potential, u(MD), which restricts its internal motion, as a qualifier of the local dynamic structure. u(MD) is derived from the MD trajectory as a function of the polar angles, (θ, φ), which specify the N-H orientation in the protein. u(MD) is statistical in character yielding empirical descriptions. To establish more insightful methodical descriptions, we develop a comprehensive method which approximates u(MD) by combinations of analytical Wigner functions that belong to the D2h point group. These combinations, called u(simulated), make it possible to gain a new perspective of local dynamic structures in proteins based on explicit potentials/free energy surfaces and associated probability densities, entropy, and ordering. A simpler method was developed previously using 100 ns MD simulations. In that case, the traditional "perpendicular N-H ordering" setting centered at Cα-Cα with (θ, φ) = (90, 90) and generally, featuring positive φ, prevailed. u(MD) derived from 1 µs MD simulations is considerably more complex requiring substantial model enhancement. The enhanced method applies to the well-structured sections of the RBD. It only applies partly to its loops where u(MD) extends into the negative-φ region where we detect nonperpendicular N-H ordering. This arrangement requires devising new reference structures and making substantial algorithmic changes, to be performed in future work. Here, we focus on developing the comprehensive method and using it to investigate perpendicular ordering settings. We find that secondary structures (loops) exhibit varying (virtually invariant) potentials with Ag, B2u, and B1u (Ag and B2u) D2h symmetry. Application to RBD dimerization and RBD binding to the GTPase Rac1 is described in the subsequent article. Applications to other probes, proteins, and biological functions, based on explicit local potentials, probability densities, entropy, and ordering, are possible.

Local Structures in Proteins from Microsecond Molecular Dynamics Simulations: 2. The Role of Symmetry in GTPase Binding and Dimer Formation.

The Rho GTPase binding domain of Plexin-B1 (RBD) prevails in solution as dimer. Under appropriate circumstances, it binds the small GTPase Rac1 to yield the complex RBD-Rac1. Here, we study RBD dimerization and complex formation from a symmetry-based perspective using data derived from 1 µs long MD simulations. The quantities investigated are the local potentials, u(MD), prevailing at the N-H sites of the protein. These potentials are statistical in character providing an empirical description of the local structure. To establish more methodical description, a method for approximating them by explicit functions, u(simulated), was developed in the preceding article in this journal issue. These functions are combinations of analytical Wigner functions, DL,K, belonging to the D2h point group. The D2h subgroups Ag and B2u are found to dominate u(simulated); the B1u subgroup contributes in some cases. The Ag (B2u) functions have axial or rhombic symmetry. For the first time, local potentials in proteins can be quantitatively characterized in terms of their strength (rhombicity) evaluated by axial Ag (rhombic Ag and B2u) contributions. Until now, the chain-segment [ß3-L3-ß4] and to some extent the α2-helix have been associated with GTPase binding. Here, we find that this process causes an increase (decrease) in the potential strength of ß3 and ß4 (the preceding L2 loop and the remote chain-segment [(α2-helix)-(α2/ß5-turn)-(ß5-strand)]), suggesting effects of counterbalancing and allostery. There is evidence for the L2 loop being associated with RBD-GTPase binding. Until now only the L4 loop has been associated with RBD dimerization. The latter process is found to cause an increase (decrease) in the potential strength and rhombicity of the L4 loop (the adjacent chain-segment [(α2-helix)-(α2/ß5-turn)-(ß5-strand)]), suggesting counterbalancing activity. On average, the RBD dimer features stronger local potentials than RBD-Rac1. The novel information inherent in these findings is mesoscopic in character. Prospects of interest include exploring relation to atomistic force-field parameters.

Time-resolved live-cell spectroscopy reveals EphA2 multimeric assembly.

Ephrin type-A receptor 2 (EphA2) is a receptor tyrosine kinase that initiates both ligand-dependent tumor-suppressive and ligand-independent oncogenic signaling. We used time-resolved, live-cell fluorescence spectroscopy to show that the ligand-free EphA2 assembles into multimers driven by two types of intermolecular interactions in the ectodomain. The first type entails extended symmetric interactions required for ligand-induced receptor clustering and tumor-suppressive signaling that inhibits activity of the oncogenic extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) protein kinases and suppresses cell migration. The second type is an asymmetric interaction between the amino terminus and the membrane proximal domain of the neighboring receptors, which supports oncogenic signaling and promotes migration in vitro and tumor invasiveness in vivo. Our results identify the molecular interactions that drive the formation of the EphA2 multimeric signaling clusters and reveal the pivotal role of EphA2 assembly in dictating its opposing functions in oncogenesis.

Development of novel cytoprotective small compounds inhibiting mitochondria-dependent cell death.

We identified cytoprotective small molecules (CSMs) by a cell-based high-throughput screening of Bax inhibitors. Through a medicinal chemistry program, M109S was developed, which is orally bioactive and penetrates the blood-brain/retina barriers. M109S protected retinal cells in ocular disease mouse models. M109S directly interacted with Bax and inhibited the conformational change and mitochondrial translocation of Bax. M109S inhibited ABT-737-induced apoptosis both in Bax-only and Bak-only mouse embryonic fibroblasts. M109S also inhibited apoptosis induced by staurosporine, etoposide, and obatoclax. M109S decreased maximal mitochondrial oxygen consumption rate and reactive oxygen species production, whereas it increased glycolysis. These effects on cellular metabolism may contribute to the cytoprotective activity of M109S. M109S is a novel small molecule protecting cells from mitochondria-dependent apoptosis both in vitro and in vivo. M109S has the potential to become a research tool for studying cell death mechanisms and to develop therapeutics targeting mitochondria-dependent cell death pathway.

Allosteric inhibition of the epidermal growth factor receptor through disruption of transmembrane interactions.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) commonly targeted for inhibition by anticancer therapeutics. Current therapeutics target EGFR's kinase domain or extracellular region. However, these types of inhibitors are not specific for tumors over healthy tissue and therefore cause undesirable side effects. Our lab has recently developed a new strategy to regulate RTK activity by designing a peptide that specifically binds to the transmembrane (TM) region of the RTK to allosterically modify kinase activity. These peptides are acidity-responsive, allowing them to preferentially target acidic environments like tumors. We have applied this strategy to EGFR and created the PET1 peptide. We observed that PET1 behaves as a pH-responsive peptide that modulates the configuration of the EGFR TM through a direct interaction. Our data indicated that PET1 inhibits EGFR-mediated cell migration. Finally, we investigated the mechanism of inhibition through molecular dynamics simulations, which showed that PET1 sits between the two EGFR TM helices; this molecular mechanism was additionally supported by AlphaFold-Multimer predictions. We propose that the PET1-induced disruption of native TM interactions disturbs the conformation of the kinase domain in such a way that it inhibits EGFR's ability to send migratory cell signals. This study is a proof-of-concept that acidity-responsive membrane peptide ligands can be generally applied to RTKs. In addition, PET1 constitutes a viable approach to therapeutically target the TM of EGFR.

Transmembrane dimers of type 1 receptors sample alternate configurations: MD simulations using coarse grain Martini 3 versus AlphaFold2 Multimer.

Structures and dynamics of transmembrane (TM) receptor regions are key to understanding their signaling mechanism across membranes. Here we examine configurations of TM region dimers, assembled using the recent Martini 3 force field for coarse-grain (CG) molecular dynamics simulations. At first glance, our results show only a reasonable agreement with ab initio predictions using PREDDIMER and AlphaFold2 Multimer and with nuclear magnetic resonance (NMR)-derived structures. 5 of 11 CG TM structures are similar to the NMR structures (within <3.5 Å root-mean-square deviation [RMSD]) compared with 10 and 9 using PREDDIMER and AlphaFold2, respectively (with 8 structures of the later within 1.5 Å). Surprisingly, AlphaFold2 predictions are closer to NMR structures when the 2001 instead of 2020 database is used for training. The CG simulations reveal that alternative configurations of TM dimers readily interconvert with a predominant population. The implications for transmembrane signaling are discussed, including for the development of peptide-based pharmaceuticals.

A proteome-scale analysis of vertebrate protein amino acid occurrence: Thermoadaptation shows a correlation with protein solvation but less so with dynamics.

Despite differences in behaviors and living conditions, vertebrate organisms share the great majority of proteins, often with subtle differences in amino acid sequence. Here, we present a simple way to analyze the difference in amino acid occurrence by comparing highly homologous proteins on a subproteome level between several vertebrate model organisms. Specifically, we use this method to identify a pattern of amino acid conservation as well as a shift in amino acid occurrence between homeotherms (warm-blooded species) and poikilotherms (cold-blooded species). Importantly, this general analysis and a specific example further establish a broad correlation, if not likely connection between the thermal adaptation of protein sequences and two of their physical features: on average a change in their protein dynamics and, even more strongly, in their solvation. For poikilotherms, such as frog and fish, the lower body temperature is expected to increase the protein-protein interaction due to a decrease in protein internal dynamics. In order to counteract the tendency for enhanced binding caused by low temperatures, poikilotherms enhance the solvation of their proteins by favoring polar amino acids. This feature appears to dominate over possible changes in dynamics for some proteins. The results suggest that a general trend for amino acid choice is part of the mechanism for thermoadaptation of vertebrate organisms at the molecular level.

Computational analysis of interior mutations of SARS-CoV-2 Spike protein suggest a balance of protein stability and S2: S1 separation propensity.

SARS-CoV-2 variants often include surface mutations in the Spike protein that are important for viruses to recognize host receptors and evade antibody neutralization. The Spike protein also has mutations in the interior of the protein likely to affect the Spike protein S1 - S2 subunit's separation propensity, the most important of which is the D614G mutation. Remarkably, the Omicron variant contains a large number of internal mutations at the S2: S1 interface, which have not been investigated yet. In this study, we examined the effects of such interfacial mutations on the S2: S1 and subunit domain interactions and on the subunit's dissociation process. We found that the interaction with S2 is mainly contributed by the three encapsulation domains, named INT, ED1 and ED2 of S1, which are sandwiched between the S1 RBD and N-terminal NTD domain. We found that D614 is the strongest contributor for the S2: S1 interaction which is greatly weakened by the D614G mutation. Surprisingly, we found that, mutations T547K, H655Y, N764K, N856K, N969K, L981F in the Omicron variant largely enhance the S2: ED1 interaction, partially compensating the loss of S2: ED2 interaction due to the D614G mutation. Lastly, these results, together with biological considerations, allow us to suggest that in addition to the binding strength of between the RBD and ACE2, the stability of the Spike protein and the propensity of Spike protein S2: S1 separation are critical factors which likely exist in a balance for a particular infectivity and pathogenicity of the virus.

Microsecond MD Simulations of the Plexin-B1 RBD: 2. N-H Probability Densities and Conformational Entropy in Ligand-Free, Rac1-Bound, and Dimer RBD.

Orientational probability densities, Peq = exp(-u) (u, local potential), of bond-vectors in proteins provide information on structural flexibility. The related conformational entropy, Sk = -∫Peq(ln Peq)dΩ - ln ∫dΩ, provides the entropic contribution to the free energy of the physical/biological process studied. We have developed a new method for deriving Peq and Sk from MD simulations, using the N-H bond as probe. Recently we used it to study the dimerization of the Rho GTPase binding domain of Plexin-B1 (RBD). Here we use it to study RBD binding to the small GTPase Rac1. In both cases 1 µs MD simulations have been employed. The RBD has the ubiquitin fold with four mostly long loops. L3 is associated with GTPase binding, L4 with RBD dimerization, L2 participates in interdomain interactions, and L1 has not been associated with function. We find that RBD-Rac1 binding renders L1, L3, and L4 more rigid and the turns ß2/α1 and α2/ß5 more flexible. By comparison, RBD dimerization renders L4 more rigid, and the α-helices, the ß-strands, and L2 more flexible. The rigidity of L1 in RBDRAC is consistent with L1-L3 contacts seen in previous MD simulations. The analysis of the L3-loop reveals two states of distinct flexibility which we associate with involvement in slow conformational exchange processes differing in their rates. Overall, the N-H bonds make an unfavorable entropic contribution of (5.9 ± 0.9) kJ/mol to the free energy of RBD-Rac1 binding; they were found to make a favorably contribution of (-7.0 ± 0.7) kJ/mol to the free energy of RBD dimerization. In summary, the present study provides a new perspective on the impact of Rac1 binding and dimerization on the flexibility characteristics of the RBD. Further studies are stimulated by the results of this work.

Microsecond MD Simulations of the Plexin-B1 RBD: N-H Probability Density as Descriptor of Structural Dynamics, Dimerization-Related Conformational Entropy, and Transient Dimer Asymmetry.

Amide-bond equilibrium probability density, Peq = exp(-u) (u, local potential), and associated conformational entropy, Sk = -∫Peq (ln Peq) dΩ â”€ln ∫dΩ, are derived for the Rho GTPase binding domain of Plexin-B1 (RBD) as monomer and dimer from 1 µs MD simulations. The objective is to elucidate the effect of dimerization on the dynamic structure of the RBD. Dispersed (peaked) Peq functions indicate "flexibility" ("rigidity"; the respective concepts are used below in this context). The L1 and L3 loops are throughout highly flexible, the L2 loop and the secondary structure elements are generally rigid, and the L4 loop is flexible in the monomer and rigid in the dimer. Overall, many residues are more flexible in the dimer. These features, and their implications, are discussed. Unexpectedly, we find that monomer unit 1 of the dimer (in short, d1) is unusually flexible, whereas monomer unit 2 (in short, d2) is as rigid as the RBD monomer. This is revealed due to their engagement in slow-to-intermediate conformational exchange detected previously by 15N relaxation experiments. Such motions occur with rates on the order of 103-104 s-1; hence, they cannot be completely sampled over the course of 1 µs simulation. However, the extent to which rigid d2 is affected is small enough to enable physically relevant analysis. The entropy difference between d2 and the monomer yields an entropic contribution of -7 ± 0.7 kJ/mol to the free energy of RBD dimerization. In previous work aimed at similar objectives we used 50-100 ns MD simulations. Those results and the present result differ considerably. In summary, bond-vector Peq functions derived directly from long MD simulations are useful descriptors of protein structural dynamics and provide accurate conformational entropy. Within the scope of slow conformational exchange, they can be useful, even in the presence of incomplete sampling.

Trimeric receptor-binding domain of SARS-CoV-2 acts as a potent inhibitor of ACE2 receptor-mediated viral entry.

The COVID-19 pandemic has caused over four million deaths and effective methods to control CoV-2 infection, in addition to vaccines, are needed. The CoV-2 binds to the ACE2 on human cells through the receptor-binding domain (RBD) of the trimeric spike protein. Our modeling studies show that a modified trimeric RBD (tRBD) can interact with three ACE2 receptors, unlike the native spike protein, which binds to only one ACE2. We found that tRBD binds to the ACE2 with 58-fold higher affinity than monomeric RBD (mRBD) and blocks spike-dependent pseudoviral infection over 4-fold more effectively compared to the mRBD. Although mRBD failed to block CoV-2 USA-WA1/2020 infection, tRBD efficiently blocked the true virus infection in plaque assays. We show that tRBD is a potent inhibitor of CoV-2 through both competitive binding to the ACE2 and steric hindrance, and has the potential to emerge as a first-line therapeutic method to control COVID-19.

Computational studies of the principle of dynamic-change-driven protein interactions.

Dynamic allostery emphasizes a role of entropy change manifested as a sole change in protein fluctuations without structural changes. This kind of entropy-driven effect remains largely understudied. The most significant examples involve protein-ligand interactions, leaving protein-protein interactions, which are critical in signaling and other cellular events, largely unexplored. Here we study an example of how protein-protein interaction (binding of Ras to the Ras binding domain [RBD] of the effector protein Raf) affects a subsequent protein association process (Ras dimerization) by quenching Ras internal motions through dynamic allostery. We also investigate the influence of point mutations or ambient temperature, respectively, on the protein dynamics and interaction of two other systems: in adenylate kinase (ADK) and in the EphA2 SAM:Ship2 SAM complex. Based on these examples, we postulate that there are different ways in which dynamic-change-driven protein interactions are manifested and that it is likely a general biological phenomenon.

HBD-2 binds SARS-CoV-2 RBD and blocks viral entry: Strategy to combat COVID-19.

New approaches to complement vaccination are needed to combat the spread of SARS-CoV-2 and stop COVID-19-related deaths and medical complications. Human beta defensin 2 (hBD-2) is a naturally occurring epithelial cell-derived host defense peptide that has anti-viral properties. Our comprehensive in-silico studies demonstrate that hBD-2 binds the site on the CoV-2-RBD that docks with the ACE2 receptor. Biophysical measurements confirm that hBD-2 indeed binds to the CoV-2-receptor-binding domain (RBD) (KD ∼ 2µM by surface plasmon resonance), preventing it from binding to ACE2-expressing cells. Importantly, hBD-2 shows specificity by blocking CoV-2/spike pseudoviral infection, but not VSVG-mediated infection, of ACE2-expressing human cells with an IC50 of 2.8 ± 0.4 µM. These promising findings offer opportunities to develop hBD-2 and/or its derivatives and mimetics to safely and effectively use as agents to prevent SARS-CoV-2 infection.

Structural and Functional Insights into the Transmembrane Domain Association of Eph Receptors.

Eph receptors are the largest family of receptor tyrosine kinases and by interactions with ephrin ligands mediate a myriad of processes from embryonic development to adult tissue homeostasis. The interaction of Eph receptors, especially at their transmembrane (TM) domains is key to understanding their mechanism of signal transduction across cellular membranes. We review the structural and functional aspects of EphA1/A2 association and the techniques used to investigate their TM domains: NMR, molecular modelling/dynamics simulations and fluorescence. We also introduce transmembrane peptides, which can be used to alter Eph receptor signaling and we provide a perspective for future studies.

Conformational Clamping by a Membrane Ligand Activates the EphA2 Receptor.

The EphA2 receptor is a promising drug target for cancer treatment, since EphA2 activation can inhibit metastasis and tumor progression. It has been recently described that the TYPE7 peptide activates EphA2 using a novel mechanism that involves binding to the single transmembrane domain of the receptor. TYPE7 is a conditional transmembrane (TM) ligand, which only inserts into membranes at neutral pH in the presence of the TM region of EphA2. However, how membrane interactions can activate EphA2 is not known. We systematically altered the sequence of TYPE7 to identify the binding motif used to activate EphA2. With the resulting six peptides, we performed biophysical and cell migration assays that identified a new potent peptide variant. We also performed a mutational screen that determined the helical interface that mediates dimerization of the TM domain of EphA2 in cells. These results, together with molecular dynamic simulations, allowed to elucidate the molecular mechanism that TYPE7 uses to activate EphA2, where the membrane peptide acts as a molecular clamp that wraps around the TM dimer of the receptor. We propose that this binding mode stabilizes the active conformation of EphA2. Our data, additionally, provide clues into the properties that TM ligands need to have in order to achieve activation of membrane receptors.

NMR identification of a conserved Drp1 cardiolipin-binding motif essential for stress-induced mitochondrial fission.

Mitochondria form tubular networks that undergo coordinated cycles of fission and fusion. Emerging evidence suggests that a direct yet unresolved interaction of the mechanoenzymatic GTPase dynamin-related protein 1 (Drp1) with mitochondrial outer membrane-localized cardiolipin (CL), externalized under stress conditions including mitophagy, catalyzes essential mitochondrial hyperfragmentation. Here, using a comprehensive set of structural, biophysical, and cell biological tools, we have uncovered a CL-binding motif (CBM) conserved between the Drp1 variable domain (VD) and the unrelated ADP/ATP carrier (AAC/ANT) that intercalates into the membrane core to effect specific CL interactions. CBM mutations that weaken VD-CL interactions manifestly impair Drp1-dependent fission under stress conditions and induce "donut" mitochondria formation. Importantly, VD membrane insertion and GTP-dependent conformational rearrangements mediate only transient CL nonbilayer topological forays and high local membrane constriction, indicating that Drp1-CL interactions alone are insufficient for fission. Our studies establish the structural and mechanistic bases of Drp1-CL interactions in stress-induced mitochondrial fission.

Interactions between semaphorins and plexin-neuropilin receptor complexes in the membranes of live cells.

Signaling of semaphorin ligands via their plexin-neuropilin receptors is involved in tissue patterning in the developing embryo. These proteins play roles in cell migration and adhesion but are also important in disease etiology, including in cancer angiogenesis and metastasis. While some structures of the soluble domains of these receptors have been determined, the conformations of the full-length receptor complexes are just beginning to be elucidated, especially within the context of the plasma membrane. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy allows direct insight into the formation of protein-protein interactions in the membranes of live cells. Here, we investigated the homodimerization of neuropilin-1 (Nrp1), plexin A2, plexin A4, and plexin D1 using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy. Consistent with previous studies, we found that Nrp1, plexin A2, and plexin A4 are present as dimers in the absence of exogenous ligand. Plexin D1, on the other hand, was monomeric under similar conditions, which had not been previously reported. We also found that plexin A2 and A4 assemble into a heteromeric complex. Stimulation with semaphorin 3A or semaphorin 3C neither disrupts nor enhances the dimerization of the receptors when expressed alone, suggesting that activation involves a conformational change rather than a shift in the monomer-dimer equilibrium. However, upon stimulation with semaphorin 3C, plexin D1 and Nrp1 form a heteromeric complex. This analysis of interactions provides a complementary approach to the existing structural and biochemical data that will aid in the development of new therapeutic strategies to target these receptors in cancer.

Letting go: Deep computational modeling insights into pH-dependent calcium affinity.

Calcium and other cofactors can feature as key additions to a molecular interface, to the extent that the cofactor is completely buried in the bound state. How can such an interaction be regulated then? The answer: By facilitating a switch through an allosteric network. Although a number of unbinding mechanisms are being characterized, an extensive computational study by Joswig et al. reveals a detailed model for the pattern recognition receptor langerin.

Neuropilin-1 assists SARS-CoV-2 infection by stimulating the separation of Spike protein S1 and S2.

The cell surface receptor Neuropilin-1 (Nrp1) was recently identified as a host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry. The Spike protein of SARS-CoV-2 is cleaved into two segments, the S1 (residues (res.) 1-685) and the S2 (res. 686-1273) domains by furin protease. Nrp1 predominantly binds to the C-terminal RRAR amino acid motif (res. 682-685) of the S1 domain. In this study, we firstly modeled the association of an Nrp1 protein (consisting of domains a2-b1-b2) with the Spike protein. Next, we studied the separation of S2 from the S1 domain, with and without Nrp1 bound, by utilizing molecular dynamics pulling simulations. During the separation, Nrp1 stabilizes the S1 C-terminal region (res. 640-685) and thereby assists the detachment of S2 N-terminal region (res. 686-700). Without Nrp1 bound, S1 tends to become stretched, whereas the bound Nrp1 stimulates an earlier separation of S2 from the S1 domain. The liberated S2 domain is known to mediate the fusion of virus and host membranes; thus, Nrp1 likely increases virus infectivity by facilitating the S1 and S2 separation. We further analyzed the possible topological structure of the SARS-CoV-2 Spike protein when bound with Nrp1 and angiotensin-converting enzyme 2 (ACE2). Understanding of such an Nrp1-assisted viral infection opens the gate for the generation of protein-protein inhibitors, such as antibodies, which could attenuate the infection mechanism and protect certain cells in a future Nrp1-ACE2 targeted combination therapy.