Design And Verification Of Recombinant ACE2 Proteins With Enhanced Binding Capacity To SARS-CoV-2 Spike And Its Molecular Basis

BackgroundSevere acute respiratory syndrome coronavirus 2(SARS-CoV-2),whose genomic sequence shows high similarity to that of severe acute respiratory syndrome coronavirus(SARS-CoV),is an important member of the Betacoronavirus genus.It is one of the three coronaviruses that can cause severe human infections.SARS-CoV-2,which is more contagious than SARS-CoV and Middle East respiratory syndrome coronavirus(MERS-CoV),has caused more than 110 million infections and over 2.6 million deaths worldwide,posing a severe threat to the global public health.Similar to SARS-CoV,SARS-CoV-2 also recognizes angiotensin converting enzyme 2(ACE2)via its spike(S)protein to mediate cell entry and thereby initiate the virus infection.Thus far,several cryo-EM structures of the full-length S protein bound to ACE2 and the crystal structures of the S protein receptor binding domain(S-RBD)in complex with the ACE2 extracellular domain have already been reported.As an important antiviral drug target,S protein is a key in the development of antibody therapies and vaccines.So far,several antibody drugs have already entered clinical trials,and their efficacies have been widely acknowledged.In addition,some vaccines have been approved for emergency use around the world,and hundreds of candidate vaccines are currently in clinical assays and/or preclinical studies.These advances in vaccine and drug development have dawned on the future elimination of SARS-CoV-2.Nevertheless,while the pandemic is continuing,the virus is constantly mutating.Among the virus encoded proteins,S is one in which mutations could be easily accumulated.As a result,a variety of SARS-CoV-2 variants(eg.the Brazilian strain,South African strain,India strain and etc.)have quickly emerged.These mutant strains are resistant to various neutralizing antibodies,thus significantly compromising or even completely depriving the efficacy of antivirals such as convalescent plasma,polyclonal serum,monoclonal antibodies and vaccines.It has been reported that recombinant human ACE2 protein can be used as a"decoy" to compete with the ACE2 protein on the cell surface for SARSCoV-2 binding,thereby showing antiviral effects without immune escape.In addition,recombinant human ACE2 protein,which has already passed clinical safety evaluations in many countries,is safe in vivo.Both in-vitro and in-vivo data have shown that recombinant human ACE2 has a good antiviral efficacy.As a potential antiviral drug,it would be of great significance to modify ACE2 to enhance its binding affinity to the S protein of SARS-CoV-2 and thereby to improve its antiviral effect.ObjectivesThis dissertation focuses on the recombinant human ACE2 protein and aims to modify ACE2 to enhance its S-RBD binding capacity towards SARS-CoV-2 via a combination of the computational biology,molecular biology,cellular biology and protein engineering methods.Our ultimate goal is to identify ACE2 mutants with both increased SRBD binding affinity and improved entry-inhibition activity against SARS-CoV-2.In addition,we will also try to delineate the molecular mechanism and the structural basis for the enhanced binding via structural biology.MethodsVia literature review and PDB search for available crystal structures of the SARS-CoV-2 S-RBD/ACE2 complex,key amino acids mediating the S-RBD/ACE2 engagement were firstly identified.The MOE software was then used for calculation and prediction to screen out potential mutations in ACE2 that might enhance its binding to SARSCoV-2 S-RBD.SARS-CoV-2 S-RBD,wild-type(WT)ACE2 and ACE2 mutant proteins were subsequently expressed in insect cells via the Bacto-Bac baculovirus expression system and purified by affinity chromatography,gel filtration chromatography and/or ion exchange chromatography.The protein binding affinities were then determined by surface plasmon resonance(SPR).Those mutations in ACE2 that could enhance S-RBD binding were subsequently identified and combined to further increase its binding affinity.We then used the protein crystallographic method to solve the structures of the affinity-enhanced ACE2 mutants bound to SARS-CoV-2 S-RBD,via which the basis for the enhanced binding could be investigated at the atomic level.In the next step,the pseudovirus of SARS-CoV-2 were packaged,and the inhibitory efficacy of the affinity-enhanced ACE2 mutants against SARS-CoV-2 infection was determined.Finally,these identified ACE2 mutants were further investigated for their binding affinities to SARSCoV S-RBD and their antiviral efficacy against SARS-CoV infection.Results(1)Via MOE analyses and prediction,a total of seven mutation options,including S19W,T27W,H34E,G326W,G326K,N330Y and K353R,were identified.The proteins of SARS-CoV-2 S-RBD and wildtype(ACE2/WT)and mutant ACE2(ACE2/S19W,ACE2/T27W,ACE2/H34E,ACE2/G326W,ACE2/N330Y,ACE2/K353R),as well as the peptidase active-site mutated ACE2 protein(ACE2/active-sitemutant with H374A,H378A and E402A mutations),were subsequently produced in insect cells and purified to high homogeneity through a series of chromatographic purification methods.The binding affinities between SARS-CoV-2 S-RBD and the indicated ACE2 proteins were then determined via surface plasma resonance(SPR)in three independent repeated experiments,which showed that ACE2/S19W,ACE2/T27W,ACE2/N330Y and ACE2/active-site-mutant exhibited higher S-RBD binding affinities than ACE2/WT.Guided by the results of our initial SPR screening,the ACE2 mutations of S19W,T27W,and N330Y were further combined in pairs or simultaneously in the context of ACE2/active-site-mutant.The resultant mutant proteins(designated as ACE2[W19/W27],[W19/Y330],[W27/Y330]and[W19/W27/Y330]hereafter)were then prepared via the same expression and purification strategy as before and subjected to SPR binding assay in parallel with the wild-type protein.The determined affinities were 10.1 ± 1.2 nM,13.6 ± 1.8 nM,76.7 ±10.3 nM,and 19.3 ± 0.9 nM for ACE2[W19/Y330],ACE2[W27/Y330],ACE2[W19/W27],and ACE2[W19/W27/Y330],respectively.In comparison to ACE2/WT whose affinity is calculated to be 81.8±4.7 nM,ACE2[W19/Y330],ACE2[W27/Y330]and ACE2[W19/W27/Y330]all exhibited significantly enhanced binding towards SARS-CoV-2 S-RBD(by about 4-8 fold).However,no affinity improvement was observed for ACE2[W19/W27].The results,therefore,clearly showed a synergistic effect in binding enhancement when N330Y was combined with either S19W or T27W but the mutual incompatibility between S19W and T27W.(2)By using the commercial crystallization screening kits,we successfully obtained high-quality and diffractable crystals of SARSCoV-2 S-RBD in complex with both ACE2[W19/Y330]and ACE2[W27/Y330].The X-ray diffraction data were subsequently collected,and the two structures were solved,via molecular displacement,to 2.7-A and 2.5-A,respectively.Each structure contained a single S-RBD/ACE2 complex bound in a 1:1 binding mode.ACE2 itself is composed of two subdomains(subdomains I and II).In each structure,the viral ligand directly engaged subdomain I of the receptor.In comparison to the previously reported complex structure of SARS-CoV-2 S-RBD bound to wild-type ACE2(pdb code:6LZG),the receptor subdomain Ⅱ in ACE2[W19/Y330]and ACE2[W27/Y330]was observed to move for a long distance towards subdomain Ⅰ,causing the ACE2 receptor to shift from a commonly-seen "open" state to a completely "close" state.Such closed conformation is highly similar to that when the MLN-4760 inhibitor bounds.These were,to our knowledge,the first structures of ACE2 in a closed state in complex with any viral ligands reported thus far.(3)In order to learn the basis for the binding enhancement associated with the S19W,T27W and N330Y substitutions and to gain insight into altered affinity for selectively combined mutations(S19W and T27W were mutual incompatible but both showed a synergistic effect with N330Y),the detailed amino acid interactions between SARS-CoV-2 S-RBD and the ACE2 mutants were comprehensively characterized and compared.We found that the aromatic-residue substitutions at these three positions did not induce obvious conformational changes to the neighboring interface residues in ACE2.The enhanced bindings were,therefore,almost completely contributed by the van der Waals(vdw)interactions mediated by the aromatic sidechains.In addition,Y330 also provided an extra hydrogen-bond binding.Sterically,Y330 and W19/W27 were distantly located from each other and devoid of any steric interference,correlating well with their synergistic characteristics.W19 and W27,however,were observed to orient their side-chains towards each other,leading to strong steric conflicts.Such conflicts in the side-chain orientations between W19 and W27 well explains the mutual incompatibility between the two mutations towards further enhancement in S-RBD binding.(4)Since ACE2[W19/Y330],ACE2[W27/Y330],and ACE2[W19/W27/Y330]were shown to exhibit the best enhancement in S-RBD binding,we further tested the entry-inhibition efficacy of these ACE2 mutants using the SARS-CoV-2 pseudoviruses.We initially used the WT pseudovirus.The IC50 of the wild-type ACE2/WT protein was determined to be 15.44 μg/mL.As expected,all the three mutants showed much improved entry inhibition,with IC50s of 1.21 μg/ml,1.52μg/ml and 2.04 μg/ml for ACE2[W19/Y330],ACE2[W27/Y330]and ACE2[W19/W27/Y330],respectively.We also prepared the divalent protein forms for these ACE2 mutants via fusion with the Fc tag.Several recent studies have shown that ACE2 bivalency can increase both the protein stability and its entry-inhibition capacity against SARS-CoV-2.Expectedly,fusion with Fc indeed further improved the inhibition efficacy.The three Fc-tagged ACE2 mutants showed an IC50 of 0.09μg/ml for ACE2[W19/Y330]-Fc,0.25 μg/ml for ACE2[W27/Y330]-Fc,and 0.53 μg/ml for ACE2[W19/W27/Y330]-Fc,respectively.These values represented approximately 29-173 fold improvement in comparison to the ACE2/WT protein.In the next step,we further investigated the entry-inhibition capacity of our ACE2 mutants towards a series of known antibodyresistant viruses.Because S-RBD is one of the major targets for neutralizing antibodies,the mutations in S-RBD,including N439K,G446V,N450K,L452R,A475V,S477N,V483A,E484K,F490L and Y508H,were finally selected.The resultant S-RBD mutant proteins were subsequently prepared and further analyzed using SPR.For each S-RBD mutants tested,ACE2[W19/Y330],ACE2[W27/Y330]and ACE2[W19/W27/Y330]exhibited approximately 4-20 fold higher affinities than ACE2/WT.We also prepared the pseudoviruses containing these antibody-resistant mutations and investigated the inhibition activity of ACE2[W19/Y330],ACE2[W27/Y330]and ACE2[W19/W27/Y330]against the mutated viruses.The data showed that all the three ACE2 mutants exhibited evidently increased entry inhibition in comparison to the ACE2/WT protein.These results demonstrated that the enhanced S-RBD-binding associated with ACE2 S19W,T27W and N330Y substitutions would not be affected by those S-RBD mutations attenuating antibody neutralization.Moreover,we also investigated the decoy effect of our modified ACE2s towards SARS-CoV-2 South Africa(B.1.351)and India(B.1.617)variant strains.Towards the S-RBDs of B.1.351 and B.1.617,ACE2[W19/Y330],ACE2[W27/Y330],and ACE2[W19/W27/Y330]all exhibited higher affinities than ACE2/WT.Using the pseudo viruses of the B.1.351 and B.1.617 variants,the three ACE2 mutants expectedly showed increased entry-inhibition activities in comparison to the ACE2/WT protein.These results indicated that,towards these circulating SARS-CoV-2 variants,our ACE2 mutants also act as a better decoy than ACE2 WT.(5)In light of the spike-sequence similarity and the resembled SRBD binding mode by ACE2 between SARS-CoV and SARS-CoV-2,we then set out to investigate if these aromatic-residue substitutions could similarly increase the ACE2 binding to SARS-CoV S-RBD.Using SPR,the binding affinity between ACE2/WT and SARS-CoV SRBD was determined to be 523.0 ± 41.0 nM.In the parallel experiments,ACE2[W19/Y330],ACE2[W27/Y330]and ACE2[W19/W27/Y330]all showed greatly elevated binding(by about 6-16 fold),whose affinities were determined to be 33.5 ± 0.5 nM,88.9 ± 0.6 nM,and 69.8± 3.8 nM,respectively.The efficacy of the three ACE2 mutants to inhibit SARSCoV entry was also investigated using the SARS-CoV pseudovirus.While ACE2/WT inhibited the virus entry with an IC50 of 54.88 μg/ml,ACE2[W19/Y330],ACE2[W27/Y330]and ACE2[W19/W27/Y330]showed an IC50 of 2.10 μg/ml,5.11 μg/ml and 3.82 μg/ml,respectively.The inhibition efficacy could be further improved by fusion ACE2 with Fc,whose IC50s were determined to be 0.34 μg/ml for ACE2[W19/Y330]-Fc,1.04 μg/ml for ACE2[W27/Y330]-Fc and 0.49μg/ml for ACE2[W19/W27/Y330]-Fc,respectively.These values represented approximately 56-172 fold improvement in comparison to the ACE2/WT protein.(6)Finally,we also investigated if the open/close conformation of ACE2 might affect its binding to SARS-CoV-2 and SARS-CoV SRBDs.The well-known ACE2 inhibitor of MLN-4760 was incubated with ACE2/WT to trap the protein in the closed conformation.The resultant ACE2/MLN-4760 complex was then analyzed in parallel with ACE2/WT for their interactions with the two viral S-RBDs via SPR.For SARS-CoV-2 S-RBD,ACE2 in the closed conformation was observed to exhibit an affinity that was about 1.2-fold higher than that of ACE2 in the open conformation.For SARS-CoV S-RBD,however,ACE2 in the closed conformation exhibited an affinity that was about 2-fold lower than that of ACE2 in the open conformation.ConclusionsThe current study focuses on the recombinant human ACE2 protein variants with enhanced binding capacity towards viral ligands.Three residue substitutions(S19W,T27W and N330Y)in the receptor are identified to be able to increase its SARS-CoV-2 S-RBD binding affinity.In addition,N330Y could be synergistically combined with either S19W or T27W to further enhance the S-RBD binding,whereas S19W and T27W are mutually unbeneficial.The complex structures of SARS-CoV S-RBD bound to both ACE2[W19/Y330]and ACE2[W27/Y330]are successfully solved,via which the basis underlying the enhanced binding and the mechanism for the observed residue-combination features are delineated at the atomic level.Using the SARS-CoV-2 pseudoviruses,we further show that the affinity-enhanced ACE2 proteins exhibit significantly-increased inhibitory activity in blocking virus entry towards both the wild-type and the antibody-resistant viruses.Additionally,we demonstrate that the identified residue-substitutions could also dramatically enhance the ACE2 binding to SARS-CoV SRBD and improve the antiviral efficacy of the resultant ACE2 proteins against SARS-CoV infection.Finally,we also provide,to our knowledge,the first evidence that the open/close conformation of ACE2 could affect S-RBD binding for both SARS-CoV-2 and SARS-CoV.