key: cord-297747-kifqgskc authors: Lupala, Cecylia S.; Li, Xuanxuan; Lei, Jian; Chen, Hong; Qi, Jianxun; Liu, Haiguang; Su, Xiao-dong title: Computational simulations reveal the binding dynamics between human ACE2 and the receptor binding domain of SARS-CoV-2 spike protein date: 2020-03-27 journal: bioRxiv DOI: 10.1101/2020.03.24.005561 sha: doc_id: 297747 cord_uid: kifqgskc A novel coronavirus (the SARS-CoV-2) has been identified in January 2020 as the causal pathogen for COVID-19 pneumonia, an outbreak started near the end of 2019 in Wuhan, China. The SARS-CoV-2 was found to be closely related to the SARS-CoV, based on the genomic analysis. The Angiotensin converting enzyme 2 protein (ACE2) utilized by the SARS-CoV as a receptor was found to facilitate the infection of SARS-CoV-2 as well, initiated by the binding of the spike protein to the human ACE2. Using homology modeling and molecular dynamics (MD) simulation methods, we report here the detailed structure of the ACE2 in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The predicted model is highly consistent with the experimentally determined complex structures. Plausible binding modes between human ACE2 and the RBD were revealed from all-atom MD simulations. The simulation data further revealed critical residues at the complex interface and provided more details about the interactions between the SARS-CoV-2 RBD and human ACE2. Two mutants mimicking rat ACE2 were modeled to study the mutation effects on RBD binding to ACE2. The simulations showed that the N-terminal helix and the K353 of the human ACE2 alter the binding modes of the CoV2-RBD to the ACE2. The outbreak of a new type of severe pneumonia COVID-19 started in December 2019 1 has been going on world-wide, and caused over 15,000 fatalities, infected more than 350,000 individuals globally. Although the earlier infected cases were mainly found in China before March, 2020, particularly in Hubei Province, the confirmed COVID-19 cases have been reported in more than 160 countries or territories by the end of March, 2020, and still increasing rapidly. One urgent desire in coping with this global crisis is to develop or discover drugs that can treat the diseases caused by the novel coronavirus, the SARS-CoV-2 (also known as 2019-nCoV)2. According to the genome comparative studies, the SARS-CoV-2 belongs to the genus beta-coronavirus, with nucleotide sequence identity of about 96% compared to the closest bat coronavirus RaTG13, about 89% compared to two other bat SARS-like viruses (Bat-SL-CoVZC45 & Bat-SL-CoVZXC21), and 79% compared to the SARS-CoV 3,4. Furthermore, the SARS-CoV-2 spike protein has a protein sequence identity of 73% for the receptor binding domain (RBD) with the SARS-CoV RBD (denoted as SARS-RBD in the following). The SARS-CoV and SARS-CoV-2 both utilize the human Angiotensin converting enzyme 2 protein (ACE2) to initiate the spike protein binding and facilitate the fusion to host cells 5-9. The 193-residue RBD of the SARS-CoV spike protein has been found to be sufficient to bind the human ACE2 6. Based on this fact, the RBD of SARS-CoV-2 becomes a critical protein target for drug development to treat the COVID-19. When this study was started, neither the crystal structure of the SARS-CoV-2 spike protein nor the RBD segment were determined, so the homology modeling approach was applied to construct the model of the SARS-CoV-2 spike RBD in complex with the human ACE2 binding domain (denoted as CoV2-RBD/ACE2 in the following). Similar approach has been applied to predict the complex structure and estimate the binding energies 3. Because of the high sequence similarity between CoV2-RBD and SARS-RBD, the predicted structure was found to be highly consistent with the resolved crystal structures 10 (see another crystal structure at http://nmdc.cn/nCoV entry:NMDCS0000001, PDBID: 6LZG). These structures laid the foundation for the dynamics investigation of the CoV2-RBD/ACE2 complex using computational simulation method. The predicted CoV2-RBD/ACE2 model was subjected to all-atom molecular dynamics (MD) simulations to study the binding interactions. Although the crystal structure and the predicted model of the CoV2-RBD/ACE2 complex provide important information about the binding interactions at the molecular interfaces, MD simulations can extend the knowledge to a dynamics regime in a fully solvated environment. The importance of the ACE2 residues was investigated by simulating the complexes with ACE2 mutants, in which partial dissociation from the ACE2 was observed within 500 ns simulations. The control simulations of the SARS-RBD/ACE2 complexes allowed the detailed comparison in receptor binding for the two different types of viruses. The results showed that the wild type CoV2-RBD/ACE2 complex is stable in 500 ns simulations, especially in the well-defined binding interface. On the other hand, the mutations on the helix-1 or K353 of the ACE2 can alter the binding, revealing new binding poses with reduced contacts compared to those in the crystal structures. The analysis of the interaction energy showed that the binding is enhanced by adjusting conformations to form more favorable interactions as the simulation progressed, consistent with the increased hydrogen bonding patterns. Furthermore, the analysis also showed that SARS-RBD and CoV2-RBD have comparable binding affinities to the ACE2, with the former slightly stronger than the latter. The dynamic information obtained by this study shall be useful in understanding SARS-CoV-2 host interaction and for designing inhibitors to block CoV2-RBD binding. The computer model of the SARS-CoV-2 spike RBD in complex with human ACE2 The spike RBD of SARS-CoV-2 (GenBank: MN908947 2) comprises Cys336-Gly526 residues according to the sequence homology analysis with SARS-CoV spike RBD. The predicted three-dimensional structure model of these residues was obtained with the SWISS-MODEL server 11. This predicted SARS-CoV-2 RBD model was subsequently superimposed into the X-ray structure of SARS-CoV RBD in complex with human ACE2 (PDB code:2AJF, Chain D 7). Finally, the computer model of SARS-CoV-2 RBD with human ACE2 (CoV2-RBD/ACE2) was obtained for further simulations and analysis. Based on the analysis of the predicted model, sequence alignment, and literature survey, two other systems containing mutations in the human ACE2 were prepared and subject to MD simulation studies. The mutant construct is based on the fact that Rat ACE2 markedly diminishes interactions with SARS spike protein 12, and it was proposed that the rat ACE2 likely has reduced binding affinity to the CoV2-RBD 13. To investigate the roles of critical residues on the ACE2, we created two mutants of the human ACE2 (see Table 1 ): (1) mutant mut_h1, with the ACE2 N-terminal (residue 19-40) mutated to the residues of rat ACE2; and (2) mutant K353H, in which the highly conserved K353 was mutated to histidine (the corresponding amino acid in rat and mouse ACE2 proteins). To focus on the impact of these two binding sites, the rest of the ACE2 were kept to be the same as human ACE2. The predicted model of CoV2-RBD/ACE2 complex was used as the starting models for MD simulations. The spike protein RBD domain is composed of 180 residues (323-502), while the ACE2 protein contains 597 residues from the N-terminal domain. The simulation parameterization and equilibration were prepared for complex structures including the mutant systems, using the CHARMM-GUI webserver 14. Each system was solvated in TIP3P water and sodium chloride ions to neutralize the systems to a salt concentration of 150 mM. Approximately, each system was composed of about 220,000 atoms that were parametrized with the CHARMM36 force field 15. After energy minimization using the steepest descent algorithm, each system was equilibrated at human body temperature 310.15 K, which was maintained by Nose-Hoover scheme with Three independent trajectories starting from random velocities based on Maxwell distributions were simulated for both CoV2-RBD/ACE2 and SARS-RBD/ACE2 complex systems in their wild types. In all simulations, a time step of 2.0 fs was used and the PME (particle mesh Ewald) 20 was applied for long-range electrostatic interactions. The van der Waals interactions were evaluated within the distance cutoff of 12.0 Å. Hydrogen atoms were constrained using the LINCS algorithm 21. The human ACE2 mutants in complex with CoV2-RBD were constructed as described previously. Each mutant complex model was simulated in two independent trajectories. Furthermore, as the crystal structure of the CoV2-RBD/ACE2 complex became available, two additional simulations were carried out using the crystal structure as the starting model to cross-validate the simulation results based on the homology model. Each trajectory was propagated to 500 ns by following the same protocol as the wild type CoV2-RBD/ACE2 complex simulations. Analyses were carried out with tools in GROMACS (such as rmsd, rmsf, energy, and pairdist) to examine the system properties, such as the overall stability, local residue and general structure fluctuations through the simulations. The g_mmpbsa program 22 was applied to extract the molecular mechanics energy EMM (Lennard Jones and electrostatic interactions) between ACE2 and the RBD of spike proteins. VMD and Chimera were applied to analyze the hydrogen bonds, molecular binding interface, water distributions, visualization and rending model images 23, 24 . The homology structure of the CoV2-RBD/ACE2 was compared to the SARS-RBD/ACE2 crystal structure and the newly resolved crystal structure of the CoV2-RBD/ACE2. The results indicated that the homology model is accurate, especially at the binding interface. The MD simulations further refined the side chain orientations to improve the model quality. The simulation data revealed the stable binding between the CoV2-RBD and the ACE2, in spite of the conformational changes of the ACE2. The relative movement between the CoV2-RBD and the ACE2 mainly exhibited as a swinging motion pivoted at the binding interface. Simulations also revealed the roles of water molecules in the binding of the RBD to the ACE2 receptor. The MD simulation of complex with ACE2 mutants suggested that mutation to the ACE2 helix-1 and the K353 can alter the binding modes and binding affinity. The predicted CoV2-RBD/ACE2 complex structure is highly similar to the SARS-RBD/ACE2, as shown in Figure 1 . The RBD domain has an RMSD of 0.99 Å for the aligned residues (1.53 Å for all 174 residue pairs), indicating that the homology model of the CoV2-RBD is in good agreement with the SARS-RBD. For ACE2 residues near the binding interface (within 4.0 Å of the RBD), the RMSD is smaller than 0.43 Å compared to the SARS-RBD/ACE2 complex. The superposed structures revealed that the RBD/ACE2 interfaces are almost identical in two complexes (Figure 1c ). In a retrospective comparison, the predicted complex structure was superposed to the newly resolved crystal models (see Figure 2d for a detailed comparison at the interface). The results indicated that the homology model is very accurate, especially for the binding interface. The residues near the CoV2-RBD/ACE2 interface (defined as the combined set of ACE2 residues within 4.0 Å of RBD and the RBD residues within 4.0 Å of the ACE2) exhibited a difference of 0.43 Å RMSD, which is comparable to the difference between the two independently reported crystal models (an RMSD of 0.25 Å for the same comparison). The RMSD is about 0.77 Å for residues in an extended region within 10.0 Å of the binding interface. The RBD domain of the spike protein showed an overall RMSD values less than 1.5 Å, and the ACE2 domain with an RMSD about 2.0 Å between the predicted model and the crystal structures. In three simulations of the CoV2-RBD/ACE2 systems, the binding interface was highly stable, exhibiting very small conformational changes, especially for the interfacing residues of the ACE2 protein. The RMSD for the residues at the RBD binding interface is 0.85Å (+/-0.13Å) on average. Side chain atom positions were refined to form more favorable interactions (Figure 2d) . One outstanding example is the K31 side-chain, which pointed in the wrong orientation in the predicted structure, was quickly refined to the correct orientation, consistent with the crystal structure (right panel of Figure 2d ). In terms of collective conformational changes, the CoV2-RBD/ACE2 complex showed two interesting movements: (1) the loop (L67) between β6 and β7 (residues between S477 and G485 in particular) of CoV2-RBD was found to expand its interactions with the Nterminal helix (the helix-1) of the ACE2 (Figure 2a) , while it pointed away from the helix-1 in the predicted and the crystal structures (Figure 2d, left) ; (2) a tilting movement of the RBD relative to the ACE2 was observed, which can be depicted as a swinging motion with the binding interface as the pivot (see Figure 4 for an illustration). In both predicted and the crystal structures of the CoV2-RBD/ACE2 complex, the L67 does not form close contacts with the ACE2. The analysis of the crystal packing revealed that this loop participated in the interaction with another asymmetric unit (see Supplemental Information). Interestingly, the simulation data suggested that the L67 could move towards the ACE2 and form contacts with the helix-1. This can potentially enhance the binding, as reflected in the change of interaction energies. In the crystal structure, the C480 and C488 of the RBD are cross-linked via a disulfide bond that reduces the flexibility of the L67 region, limiting its access to the ACE2. On the other hand, it has been reported that the binding of SARS-RBD to ACE2 is insensitive to the redox states of the cysteines to a high extend 26. Based on the simulation results, we hypothesize that the reduced form of C480 and C488 can also exist during the virus invasion to host cells, and the reduced cysteines can potentially enhance the binding to ACE2. In the other two simulation trajectories, we found that the L67 remained in conformations similar to that in the crystal structure and the cysteines (C480 and C488) were close enough for disulfide bond formation. By examining the binding interface of CoV2-RBD and the ACE2, we found the polar and charged residues account for a large fraction, therefore the electrostatic interactions play critical roles for the complex formation. Based on the distances between the two proteins, the key residues at the binding interface were identified and summarized in Table 2 for the three representative models (see Figure 2 ). Majority of these residues are conserved for these three models, except that the model#1 (Figure 2a) has additional contacts to the ACE2 from residues in the L67 region (highlighted with green color in Table 2 ). As shown in Figure 2 , the L67 remained in the starting position for the other two representative models (Figure 2b,c) . The same simulations were carried out for the SARS-RBD/ACE2 complex, serving as a comparative system. Interestingly, the SARS-RBD counterpart of the L67 in CoV2-RBD did not form close contacts with the ACE2 in three simulations. It is worthwhile to mention that the sequence identity between CoV2-RBD and SARS-RBD is low in this loop region, suggesting the loop region might be partially responsible for the difference in the receptor binding. The hydrogen bonds between the CoV2-RBD and ACE2 were extracted using VMD Based on the statistics of three simulation trajectories, the CoV2-RBD/ACE2 complex has 2.7 hydrogen bonds between the subunits on average with stringent criteria. In comparison, the SARS-RBD/ACE2 has 3.2 hydrogen bonds on average (see Supplementary Information ). The statistics of hydrogen bonds suggest a slightly weaker binding between the CoV2-RBD and the ACE2, relative to the SARS-RBD/ACE2 complex. It is also noteworthy to point out the important roles of water molecules at the complex interface for CoV2-RBD/ACE2 complex. At any instant time, there are approximately 15 water molecules at the binding interface, simultaneously located within 2.5 Å of both the CoV2-RBD and the ACE2 (Figure 6 ). These water molecules can function as bridges by forming hydrogen bonds with the residues from the RBD or the ACE2. The dwelling time of water molecules at the interface can be a few nanoseconds, as revealed by the MD simulations. This results is also consistent with the crystal structure, which has 12 water molecules at the interface (Figure 6c ). These discoveries emphasize the role of the water molecules, which desires detailed quantification to understand the interactions between the RBD and the ACE2. It has been demonstrated that the ACE2 from several mammalian species possess high sequence similarities, yet their binding to the SARS-RBD differs significantly. In particular, the binding of SARS-RBD to the rat ACE2 is much weaker as discovered in experiments 12. Inspired by these information, two mutants of the CoV2-RBD/ACE2 were constructed: (1) ACE2-mut-h1 by mutating N-terminal helix-1 to that of the rat ACE2; (2) ACE2-K353H by mutating K353 to Histidine (the amino acid in wild type Rat ACE2). Two 500ns MD simulations were carried out for each mutant system. The simulation showed that the mutations in ACE2-mut-h1 reduced the interaction between the CoV2-RBD and the helix-1, and the ACE2-K353H showed weaker binding between the CoV2-RBD and the β-hairpin centered at the H353. Using the clustering analysis, the representative structures were identified from each simulation trajectory (Figure 7) . Although the overall topology is very similar to the wild type complex structure, there are pronounced differences. For the ACE2-mut-h1 system, the CoV2-RBD tilted further away from the ACE12 helix-1 in one simulation (Figure 7a) ; and the CoV2-RBD lost its contact with helix-13 (G326 to N330) in another simulation for the ACE2-K353H (Figure 7c ). In the wild type ACE2, the K353 is a hydrogen donor, and its mutant H353 cannot form the hydrogen bond with the CoV2-RBD as in the wild type CoV2-RBD/ACE2 complex. The number of contacting residue pairs was significantly reduced in the ACE2-K353H mutant system. This is in line with the report that K353 is more critical than the other residues, as its hydrophobic neighborhood enables this positively charged residue high selectivity to the RBD 27,28. The physical interactions between the RBD and the ACE2 were quantified for the simulated structures. We considered the molecular mechanics energy EMM , which is composed of the van der Waals and the electrostatic interactions. Furthermore, the number of residue contacts (NC) between (RBD and ACE2) was extracted from simulated structures. Both the EMM and NC indicate that the RBD interactions with the ACE2 are comparable for CoV2 and SARS spike proteins (Figure 8) . From the simulations, the We would like to point out that the energy EMM is the physical interaction between the RBD and the ACE2, rather than the binding energy, which requires accurately incorporating solvation energy and entropy. Furthermore, the standard deviations of EMM are 70.2 kJ/mol and 65.5 kJ/mol for the two complexes. Therefore, we infer that the binding affinities are comparable for CoV2-RBD/ACE2 and SARS-RBD/ACE2. The simulations started from the predicted and crystal models yielded very similar results (purple triangles). This is in line with a recent study, in which the authors showed similar binding affinity to human ACE2 for both SARS-CoV-2 and SARS-CoV spike proteins 29. They found the association rate constants kon to be the same at 1.4x105 M-1s-1, while the SARS-CoV spike protein showed a faster dissociation, with the rate constant koff to be 7.1x10-4 s-1, about 4.4 times larger than the SARS-CoV-2 spike protein koff =1.6x10-4 s-1. similar kon values and the equilibrium dissociation constants KD in nanomolar range were reported in other studies for SARS-CoV-2 spike protein (or RBD) binding to human ACE210,30. More interestingly, the mutation impacts were reflected in the EMM and NC analysis: the ACE2-mut_h1 is likely to reduce the binding to the ACE2 due to the tilting movement of CoV2-RBD, making it further from the ACE2 helix-1 (the blue triangle symbol at lower right, see Figure 7a for the representative structure). In the other simulation trajectory for the CoV2-RBD/ACE2-mut_h1 complex (blue triangle at the left upper corner), the largest NC was observed among all simulations. For simulations of the complex with ACE2-K353H mutants (green diamonds), the number of contacts were both reduced compared to the wild type system. In one simulation, the contacts between the CoV2-RBD and the Helix-13 of the ACE2 were completely lost (see Figure 7c) , consistent with the less favorable interactions reflected on an increase of EMM. For the SARS-RBD interaction with the ACE2-mut_h1, both simulations revealed fewer contacts compared to the wild type SARS-RBD/ACE2 complex (purple stars in Figure 8 ). The homology modeling of the CoV2-RBD/ACE2 complex yielded highly consistent models compared to the crystal structures. All-atom molecular dynamics simulations were carried out to study the dynamic interactions of CoV2-RBD with human ACE2, the results were compared to the SARS-RBD/ACE2 system. The human ACE2 mutants were also constructed to mimic the rat ACE2 to investigate the roles of critical residues, and possible binding modes in other mammals. It is observed that MD simulations improved the structure at the binding interface and strengthened the interactions between the subunits. The structure of the complex interface is highly stable for all simulations of CoV2-RBD/ACE2 complex in the wild type. The loop region between β6 and β7 can potentially form more contacts with the ACE2 as observed in one simulation trajectory. The simulations results also reveal that the interactions between CoV2-RBD and the ACE2 are mediated by water molecules at the interfaces, stressing the necessity of accounting for the explicit water molecules when quantifying the binding affinity. The interactions between the RBD and the ACE2 were quantified by physical interaction energies (molecular mechanics energy) and the number of contacting residues. The detailed comparison results suggest that the CoV2-RBD and the SARS-RBD bind to human ACE2 with comparable affinity. The comparison between the SARS-RBD/ACE2 and the CoV2-RBD/ACE2 complexes, with the former forms fewer contacts than the latter (Figure 8 ), yet exhibiting stronger interactions. The decomposition of the EMM to the van der Waals and the electrostatic interactions revealed that the major difference is attributed to the electrostatic interactions. Furthermore, we compared the major contacting residues and found that the SARS-RBD has two charged residues (R426 and D463) and the CoV2-RBD has only one charged residue (K417) at the complex interface. The polar and hydrophobic residues are comparable in the two RBDs. This is consistent with the statistics of hydrogen bonds at the complex interfaces. This study was started with a structure predicted using homology modeling method, which later found to be highly consistent with the crystal structure, demonstrating the potentiality of structure prediction and dynamics simulation in revealing molecular details before the availability of high resolution experimental information. Furthermore, the interactions between CoV2-RBD and the ACE2 mutants mimicking rat ACE2 protein were investigated. The results provide valuable information at the atomic level for the reduced binding affinity. The recent report on the SARS-CoV-2 infection to a dog 31 Remark mut_h1 T20L, Q24K, K26E, T27S, D30N, H34Q, F40S L20, N30, Q34, and S40 are conserved between rat and mouse. K353H K353H H353 is conserved between rat and mouse Table 2 . Contact residues between the CoV2-RBD and the ACE2. Green color denotes new interaction not observed in crystal structure. Model#2 T27 F28 D30 K31 H34 E35 E37 D38 Y41 Q42 M82 Y83 N330 K353 G354 D355 R357 K417 Y453 L455 F456 Q474 A475 G476 S477 T478 G485 F486 N487 Y489 Q493 Y495 G496 Q498 T500 N501 G502 Y505 S19 Q24 T27 F28 D30 K31 H34 E35 D38 Y41 Q42 L45 M82 Y83 N330 K353 G354 D355 R357 K417 G446 Y449 Y453 L455 F456 A475 F486 N487 Y489 Q493 Y495 G496 Q498 T500 N501 G502 Y505 Q24 T27 F28 D30 K31 H34 E35 E37 D38 Y41 Q42 M82 Y83 N330 K353 G354 D355 R357 K417 G446 Y449 Y453 L455 F456 A475 F486 N487 Y489 Q493 Y495 G496 Q498 T500 N501 G502 Y505 Table S1. Contact residues at the SARS-COV-RBD/ACE2 interface Traj 1 Traj2 Traj 3 ACE2 CoV ACE2 CoV ACE2 CoV S19 Q24 T27 K31 H34 E37 D38 Y41 Q42 L45L L79L M82 Y83 Q325 E329 N330 K353 G354 D355 R357 R426 Y436 Y440 Y442 L443 D463 L472 N473 Y475 N479 G482 Y484 T486 T487 G488 I489 Y491 Q24 T27 D30 K31 H34 E37 D38 Y41 Q42 L45L L79L M82 Y83 Q325 E329 N330 K353 G354 D355 R357 R426 Y436 Y440 Y442 L443 P462 D463 G464 L472 N473 Y475 N479 G482 Y484 T486 T487 G488 I489 Y491 Q24 T27 K31 H34 E37 D38 Y41 Q42 L45L L79L M82 Y83 Q325 E329 N330 K353 G354 D355 R357 R426 Y440 Y442 L443 P462 D463 G464 P470 L472 N473 Y475 L478 N479 G482 Y484 T486 T487 G488 I489 Y491 M82 T27 K31 K353 Y41 E329 M82 T27 K31 K353 Y41 E329 Fig.1 CoV2-RBD/ACE2 SARS-RBD/ACE2 Superposed a. b. c. Fig.2 a. b. c. 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Science (80-. ) Coronavirus: Hong Kong confirms a second dog is infected The authors declare no competing interests.