High affinity binding of SARS-CoV-2 spike protein enhances ACE2 carboxypeptidase activity

A novel coronavirus (SARS-CoV-2) has emerged to a global pandemic and caused significant damages to public health. Human angiotensin-converting enzyme 2(ACE2) was identified as the entry receptor for SARS-CoV-2. As a carboxypeptidase, ACE2 cleaves many biological substrates besides Ang II to control vasodilatation and permeability. Given the nanomolar high affinity between ACE2 and SARS-CoV-2 spike protein, we wonder how this interaction would affect the enzymatic activity of ACE2. Surprisingly, SARS-CoV-2 trimeric spike protein increased ACE2 proteolytic activity ~3–10 fold when fluorogenic caspase-1 substrate and Bradykinin-analog peptides were used to characterize ACE2 activity. In addition, the enhancement was mediated by ACE2 binding of RBD domain of SARS-CoV-2 spike. These results highlighted the altered activity of ACE2 during SARS-CoV-2 infection and would shed new lights on the pathogenesis of COVID-19 and its complications for better treatments.


Introduction
permeability and vasodilatation during SARS-CoV-2 infection. However, there was no direct assessment of ACE2 enzymatic activity during coronavirus infection. Here we examined how the binding of SARS-CoV-2 spike protein would affect the intrinsic enzymatic activity of ACE2 using one well characterized fluorogenic ACE2 substrate, the caspase-1 substrate (Mca-YVADAPK-Dnp) 13 . In comparison, ACE substrate, a bradykinin analog (Mca-RPPGFSAFK-Dnp) was also included in the enzymatic activity assessment 16 . To our surprise, SARS-CoV-2 spike enhanced ACE2 proteolytic activity to cleave Mca-YVADAPK-Dnp, which was mediated by ACE2 binding of RBD. Furthermore, SARS-CoV-2 RBD enhanced ACE2 activity to hydrolyze the bradykinin-analog better than SARS-CoV RBD. Measurements of kinetic constants also showed that SARS-CoV-2 spike protein altered the binding affinity (K m ) of ACE2 to the caspase-1 substrate or Bradykinin-analog. We propose that this new line of evidence that SARS-CoV-2 spike protein significantly alters ACE2 activity and specificity could be clinically relevant to the understanding of the pathogenesis of COVID-19. and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted July 1, 2020. . https://doi.org/10.1101/2020.07.01.182659 doi: bioRxiv preprint

SARS-CoV-2 spike protein enhances ACE2 activity
Genetic analysis revealed that SARS-CoV-2 was highly homologous to severe acute respiratory syndrome coronavirus (SARS-CoV) that infected human cells with angiotensin-converting enzyme 2(ACE2) receptor 2,8 . After the outbreak of COVID-19, ACE2 was quickly identified as the entry receptor for SAR-Cov-2 as well. Further structural studies demonstrated that both SARS-CoV-2 and SARS-CoV utilized their RBD domain to interact with ACE2 at a very similar binding mode. However, SARS-CoV-2 spike protein displays a much higher binding affinity to ACE2 compared with SARS-CoV. Given the nanomolar affinity between ACE2 and SARS-CoV-2 S spike protein, we wonder how the binding of SARS-CoV-2 spike protein to ACE2 would affect its enzymatic activity since ACE2 is a critical component in both the Rennin-angiotensin and Kinin-kallikrein system as a carboxypeptidase 14,17 .
As previously reported, ACE2 efficiently hydrolyzes biological peptides with a consensus sequence of Pro-X(1-3 residues)-Pro-hydrophobic at P5-P1' positions 13 , with cleavage between proline and the hydrophobic residue as exemplified by Ang II (DRVYIHP↓F) and des-Arg 9 -BK (RPPGFSP↓F). ACE2 also cleaves peptides with a basic residue at P1' position such as Dynorphin A (YGGFLRRIRPKL↓K) and Neurotensin 1-8(pE-LYENKP↓R). To rapidly and continuously assess enzyme activity, fluorogenic peptide substrates were normally used. Therefore, we monitored ACE2 proteolytic activity by measuring the degradation of fluorogenic capase-1 substrate Mca-YVADAPK(Dnp) 13 . In addition, a bradykinin produced ~70,000 RFU in the presence of SARS-CoV-2 spike protein ( Figure 1A). Similarly, SARS-CoV-2 spike protein enhanced ACE2 cleavage of bradykinin analog Mca-RPPGFSAFK(Dnp)-OH to produce ~3 times more RFU within 2 hours ( Figure 1B). Previous enzymatic assays showed that ACE2 activity increased with the concentrations of NaCl between 0.15M to 1M. We then carried out the same enzymatic assays at increased concentration of NaCl. Consistently, SARS-CoV-2 spike protein enhanced ACE2 activity at 0.3M and 1M NaCl and enabled ACE2 to cleave bradykinin-analog as well and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted July 1, 2020. . https://doi.org/10.1101/2020.07.01.182659 doi: bioRxiv preprint (supplementary Figure 1), indicating the enhancement was resulted by the interaction between SARS-CoV-2 spike protein and ACE2.
Furthermore, the enhancement of ACE2 activity by SARS-CoV-2 spike protein was at a concentration dependent manner. In the enzymatic assays, we added various concentrations of SARS-CoV-2 spike protein ranging from 70ug/ml to 0.4ug/ml. The dilution of SARS-CoV-2 spike protein in the assays gradually mitigated the enhancement of ACE2 activity with a clear transition between 3.5ug/ml and 7ug/ml of SARS-CoV-2 spike protein (Figure 1 C and D). The dose dependent enhancement of ACE2 activity by SARS-CoV-2 spike protein was in line with the binding affinity between ACE2 and SARS-CoV-2 spike protein, which was reported to be 14.7nM, equivalent to ~7ug/ml. Previous studies showed that SARS-CoV infection may lead to the internalization of ACE2 receptor and thus decrease the cell surface level of ACE2 6 . However, examining the lung tissue 3 days post infection of SARS-CoV-2 virus in ACE2 transgenic mice showed colocalization of SARS-Cov-2 spike protein and ACE2 receptor on the cell surface 7, 18 , further supporting SARS-CoV-2 spike formed a stable interaction with ACE2 for the cell entry. In addition, the lasting interaction between SARS-CoV-2 spike and ACE2 on the cell surface suggests that the observed enhancement of ACE2 activity in solution might happen in vivo as well.

SARS-CoV-2 RBD enhanced ACE2 activity better than SARS-CoV RBD
SARS-CoV-2 and SARS-CoV spike protein were highly homologous in their prefusion trimeric architectures with one receptor binding domain (RBD) for ACE2 binding. However, SARS-CoV-2 spike protein binds to ACE2 at ~5-10 fold higher affinity than SARS-CoV spike 9,11 . We then determined whether RBD domain itself would be sufficient to boost ACE2 activity. The results showed that both SARS-CoV-2 RBD and SARS-CoV RBD enhanced ACE2 cleavage of caspase-1 substrate (Figure 2 A and B), demonstrating that SARS-CoV-2 and SARS-CoV RBD alone is sufficient to enhance ACE2 activityACE2. The enhancement of ACE2 activity by SARS-CoV-2 RBD protein showed a concentration dependent saturation with half maximal enhancement at ~70nM, whereas SARS-CoV RBD protein enhances ACE2 activity almost linearly between 63nM and 1000nM with half maximal enhancement at ~170nM ( Figure 2E). Nevertheless, only SARS-CoV-2 RBD but not SARS-CoV RBD could increase the cleavage of Mca-RPPGFSAFK(Dnp)-OH by ACE2 (Figure 2 C, D and F). As recently reported, SARS-CoV-2 RBD bound to ACE2 at nanomolar affinities(~30-50nM), while SARS-CoV RBD bound to ACE2 at sub-micromolar affinities(~180-400nM). The different capabilities of SARS-CoV-2 and SARS-CoV RBD proteins to enhance ACE2 enzymatic activity suggested that a more stable interaction between RBD and ACE2 was necessary for its cleavage of non-optimal substrates such as bradykinin analog.

Determination of kinetic constants of ACE2 in the presence of SARS-CoV-2 spike
and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted July 1, 2020. . https://doi.org/10.1101/2020.07.01.182659 doi: bioRxiv preprint To further characterize how efficiently ACE2 cleaves fluorogenic caspase-1 substrate and bradykinin analog in the presence of SARS-CoV-2 spike protein, we determined the kinetic constants for ACE2, particularly under the physiological condition at pH7.5 with 150mM NaCl. The measurements were carried out with 20ng ACE2 in the presence or absence of 14ug/ml SARS-CoV-2 spike protein and the hydrolysis was limited to 15% product formed as initial velocity conditions. Michaelis-Menten plots showed that SARS-CoV-2 spike protein resulted in a ~4 fold reduction of K m from 10. showed higher activities at 0.3M NaCl as previously reported. It is worth mention that k cat /K m value for ACE2 hydrolysis of bradykinin analog Mca-RPPGFSAFK(Dnp)-OH in the presence of SARS-CoV-2 spike protein was 7.55 x 10 3 M -1 s -1 , which is slightly higher than that of ACE2 hydrolysis of Angiotensin I decapeptide (DRVYIHPFH↓L) that was a biological substrate for ACE2 13 . Taken together, SARS-CoV-2 increased the substrate binding affinities and catalytic rate of ACE2.

Competitive ACE2 cleavage in the presence of BK, desBK, and Ang II
From the measurements of kinetic constants of ACE2, we revealed that the binding of SARS-CoV-2 spike protein not only altered the substrate affinity (K m ) to ACE2 but also changed its enzymatic efficiency. In the presence of SARS-CoV-2 spike protein, ACE2 cleaved bradykinin-analog with a K m of ~2uM. As previously reported, ACE2 could also cleave des-Arg 9 -bradykinin but with a much higher K m (290uM). Therefore, we wonder how the binding of SARS-CoV-2 spike protein would alter the cleavage dynamics among different substrates. We then designed substrate competition assays to assess the selectivity and activity of ACE2 upon binding of SARS-CoV-2 RBD. In the enzymatic reactions, we utilized non-fluorogenic BK, desBK or Ang II peptide to compete for ACE2 cleavage of fluorogenic substrates (Figure 4). In the absence of SARS-CoV-2 RBD binding, only Ang II but not BK or desBK was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted July 1, 2020. . https://doi.org/10.1101/2020.07.01.182659 doi: bioRxiv preprint enhanced ACE2 binding to BK and desBK. As for ACE2 cleavage of the BK-analog, Ang II competed with a Ki constant of 57 µM and 4.5 µM in the absence and presence of the SARS-CoV-2 RBD, respectively (supplement figure 3), similar to that from the caspase-1 substrate cleavage. However, BK and desBK failed to inhibit ACE2 cleavage of BK-analog. As shown before, BK-analog bound to endothelin converting enzyme-1 (ECE-1) much better than BK due to its C-terminal modification 16 . It is likely that BK-analog also bound better to ACE2 than BK.

Discussion
The new coronavirus SARS-CoV-2 infects human cells via the binding of its spike (S) glycoprotein to the human angiotensin-converting enzyme-2 (ACE2). Here we examined the influence of the high affinity binding of SARS-CoV-2 spike protein on the enzymatic activity of ACE2. Surprisingly, our enzymatic assay showed that the SARS-CoV-2 spike protein significantly enhanced the enzymatic activity of ACE2 to cleave YVADAPK peptide, suggesting ACE2 upon SARS-CoV-2 binding would likely cleave more efficiently other substrates with proline at P1 position such as Ang II (DRVYIHP↓F), Apelin-13(QRPRLSHKGPMP↓F). Similarly, binding of SARS-CoV-2 spike enhanced ACE2 to cleave bradykinin analog RPPGFSAFK. In addition, bradykinin (RPPGFSPFR) competed with YVADAPK peptide for ACE2 cleavage to a similar extent as des-Arg 9 -bradykinin. Our results showed that SARS-CoV-2 spike protein not only bound to ACE2 as the entry receptor but also hijacked its enzymatic activities.
We then compared various structures of ACE2 to reveal what structural changes on ACE2 proteolytic domain might happen upon SARS-CoV-2 spike binding to affect the enzymatic activity of ACE2. The native structure of ACE2 extracellular proteolytic domain comprised two subdomains, the N terminal and C terminal subdomain with a wide cleft in between for substrate binding and catalysis 19  was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted July 1, 2020. . https://doi.org/10.1101/2020.07.01.182659 doi: bioRxiv preprint ACE2 structures onto native ACE2 structure. Structural alignment revealed that both SARS-CoV-2 and SARS-CoV RBD binding did not result in significant conformational changes on the N-domain of ACE2 with r.m.s.d less than 0.5Å ( Figure 5 D and E). However, the SARS-CoV-2 RBD but not SARS-CoV RBD binding caused the hinge movement between the N-and C-terminal domains reminiscent of a closing clam. We then assessed such relative movement by measuring the angle formed by Asn136 on the rim of native ACE2 C-terminal domain, zinc atom at the catalytic center, and Asn136 of superimposed ACE2 bound by RBD of SARS-CoV-2 or SARS-CoV. Compared to SARS-CoV RBD binding (0.3°), this angle upon SARS-CoV-2 RBD binding was increased to 5°. Particularly, the binding of a chimeric SARS-CoV-2 RBD to ACE2 11 , could cause significant movement up to ~12°of C-terminal subdomain toward the N-terminal domain of ACE2 ( Figure 5F). Furthermore, this angle caused by inhibitor MLN-4760 binding was 29° to form a closed conformation of ACE2, suggesting that SARS-CoV-2 spike binding initiated a progressive conformational change to energetically facilitate ACE2 catalysis. The comparison of substrate binding pocket on ACE2 showed that residues F274, H345, F504, H505, Y510 and Y515 in SARS-CoV-2-ACE2 complexes moved closer to aligned Ang II or MLN-4760 inhibitor, suggesting that SARS-CoV-2 RBD binding to ACE2 could increase the substrate binding ( Figure 5G). This is consistent with our kinetic constant measurement, which revealed that the K m values of ACE2 hydrolysis were decrease upon SARS-CoV-2 spike binding. In addition, in SARS-CoV-2 RBD bound ACE2, H345 residue that was proposed to stabilize the hybridized nitrogen upon catalysis adopted the same conformation as that bound by inhibitor. This active conformation of H345 was not observed in native or SARS-CoV RBD bound ACE2 structure, suggesting that the tighter binding of SARS-CoV-2 RBD could also induce a conformation change to position ACE2 substrate pocket residues for efficient catalysis.
With the increasing number of confirmed cases worldwide, the major manifestation of SARS-CoV-2 mediated COVID-19 disease and its complications generated critical concerns due to the lack of highly effective antiviral drugs or vaccines. Our current analysis of ACE2 enzymatic activity upon SARS-CoV-2 spike protein binding would be clinically relevant. However, a limitation of our study is that we could not systemically measure the levels of various ACE2 substrates and their cleaved products in COVID-19 patients currently, but we would propose that the ratios between vasodilators and vasoconstrictors would change during SARS-CoV-2 infection due to the high affinity interaction between ACE2 and SARS-CoV-2 spike protein and subsequently altered ACE2 enzymatic activity and substrate selectivity.
Compared to previous outbreaks of SARS-CoV and MERS, SARS-CoV-2 is more contagious and clinical symptoms of COVID-19 are more variable 4 . In addition to the accumulation of fluid in the lung, a considerable fraction of critically ill COVID-19 patients developed deep venous thrombosis or pulmonary and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted July 1, 2020. . https://doi.org/10.1101/2020.07.01.182659 doi: bioRxiv preprint embolism 5 . These clinical observations share a common prognostic feature that pointed to the cardiovascular system. Vasodilation, vasoconstriction, and permeability are the essential components of the cardiovascular system, which are regulated by the crosstalk of many hormones and biological active peptides. Among them, renin-angiotensin system and kinin-kellikrein system have major effects upon the cardiovascular systems 14,17 . Importantly, ACE2 catalyzes the conversion of these regulatory peptides in these systems to maintain the homeostatic state. Among the biological active substrates, ACE2 cleaves Ang II (DRVYIHP↓F), Apelin-13(QRPRLSHKGPMP↓F), and dynorphin A 1-13(YGGFLRRIRPKL↓K) with high proteolytic efficiency (K m <10uM and k cat /K m >1×10 6 M -1 s -1 ). ACE2 also has substantial activity on Ang I (DRVYIHPFH↓L) des-Arg 9  Among the peptide components of the rennin-angiotensin system, ACE2 could efficiently hydrolyze Ang II (DRVYIHP↓F), and partially cleave Ang I(DRVYIHPFH↓L). During SARS-CoV-2 infection, the enhancement of ACE2 activity could produce more of their cleaved products, Ang 1-9 and Ang 1-7.
Beside the opposing effects of Ang II and Ang 1-7 on vasoconstriction and vasodilatation, they regulated the expression of ACE2. Several in vivo and in vitro studies documented that Ang II decreased the expression of ACE2 through activation of MAPK pathways, whereas Ang 1-7 counteracted the inhibitory effect of Ang II to boost the expression of ACE2 through MAPK phosphatase activation 23,24 . Previous studies showed that SARS-CoV infection caused the internalization of ACE2 6 . However, analyzing the lung tissue in ACE2 mice 3 days post SARS-CoV-2 infection showed substantial expression of ACE2 7 .
More importantly, ACE2 expression was increased in the lungs of severe COVID-19 patients with comorbidities, compared to control individuals 18,25 . The enhanced ACE2 activity to cleave Ang II upon SARS-CoV-2 spike binding might be one of the mechanisms whereby SARS-CoV-2 utilized to boost the expression of ACE2.
Apelin 1-13(QRPRLSHKGPMP↓F) is the endogenous ligand for the angiotensin II protein J receptor (APJR), which is a homolog of the angiotensin receptor AT1 26 . Apelin 1-13 has nanomolar affinity to APJR and the major effect of Apelin in vivo was nitric oxide dependent arterial dilatation to counterbalance Ang II effects. The cleavage of its carboxyl-terminal phenylalanine of Apelin 1-13 cause the loss of critical interactions between Phe13 and a large hydrophobic cavity on APJR 27 , resulting in 2-5 and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted July 1, 2020. . https://doi.org/10.1101/2020.07.01.182659 doi: bioRxiv preprint fold decrease of its potency 28 . The enhancement of ACE2 activity would accelerate the metabolism of Apelin 1-13, further dampening its beneficial effects on overall cardiac functions. The proteolysis of bradykinin may be particularly relevant to COVID-19. Normally ACE is responsible for the hydrolysis of bradykinin, whereas ACE2 hydrolyzes des-Arg 9 -bradykinin 14 . Bradykinin selectively binds to B2 receptors and des-Arg 9 -bradykinin selectively binds to B1 receptor. Compared to the constitutive expression of B2 receptor, expression of B1 receptor is upregulated during inflammation. In addition, previous studies showed that all of the reactions for bradykinin degradation occurred normally but the production rate of des-Arg 9 -bradykinin was accelerated fivefold when blood was clotted 29 . Our results showed that SARS-CoV-2 spike protein enhanced bradykinin and des-Arg 9 -bradykinin binding to ACE2. Furthermore, neuropeptides Dynorphin A 1-13(YGGFLRRIRPKL↓K) and -Casomorphin(YPFVEP↓I) are good ACE2 substrate, which are endogenous ligands for opioid receptors and have antinociceptive effects 30 . Interestingly, Dynorphin A 1-13 was also implicated as an appetite stimulant 31 . The removal of the C-terminal lysine residue of Dynorphin A 1-13 resulted in 90% loss of its potency 32 . Since symptoms of COVID-19 also include body aches and loss of taste, it would be intriguing to speculate that altered ACE2 hydrolysis of these neuropeptides would affect the homeostatic functions of these opioid receptors as well.
Collectively, the homeostatic regulation of cardiovascular system controlled by ACE2 would be out of and development of better treatment of this disease and its complications. and is also made available for use under a CC0 license.
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SARS-CoV-2 trimeric spike protein expression and purification
The vector to express the prefusion S ectodomain, a gene encoding residues 1-1208 of SARS-CoV-2(GenBank: MN908947) was kindly provided by Jason S. McLellan (the University of Texas at Austin) and the protein was expressed as described previously 9 . In brief, FreeStyle293 F cells (Thermo Fisher) were transiently transfected with this expression vector using polyethylenimine (PEI). Protein was purified from filtered cell supernatants using StrepTactin XT resin (IBA, Inc) and subsequently subjected to further purification by size exclusion chromatography using a Superose 6 10/300 column (GE Healthcare) with the buffer of 75mM Tris (pH7.5), 0.15M NaCl.

Enzymatic assays of ACE2 hydrolysis of biological peptides
Reactions were performed in black microtiter plates at ambient temperature (26°C) according to manufacturer's instruction. To each well, 50ul of 0.2 or 0.4ug/ml ACE2 in assay buffer containing 75mM Tris(pH7.5) plus NaCl at 0.15M, 0.3M or 1.0M were added respectively. Then 10ul dialysis buffer or SARS-CoV-2 S spike/RBD domain proteins at various final concentrations ranging from 70ug/ml to 0.4ug/ml were added to wells and incubated for 20min after mixing by shaking. The reactions were initiated by adding 50ul of fluorogenic peptides at 40uM or with 2-fold serial dilutions ranging from 160uM to 0.6uM to determine the kinetic constants for ACE2 hydrolysis. The relative fluorescence units and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted July 1, 2020. . https://doi.org/10.1101/2020.07.01.182659 doi: bioRxiv preprint (RFU) were read at excitation and emission wavelengths of 320nm and 405nm (top read), respectively in kinetic mode at 1-minute intervals for 8 hours.
To calculate specific activity of ACE2, the substrate blank adjusted relative fluorescent units were converted to molarities according to the conversion factor on Synergy H1 plate reader that was calibrated by standard Mca-pro-Leu-OH (supplement figure 2). To obtain the kinetic constants, the hydrolysis was limited to ≤15% product formed as the initial velocity conditions. Initial velocities were plotted versus

Substrate competition assays using non-fluorogenic BK, desBK and Ang II peptides
To compare the relative proteolytic activity of ACE2 to different substrates, various concentrations of non-fluorogenic BK, desBK or Ang II peptides were added to the reaction mixture with fluorogenic peptides as competitive substrates. As mentioned above, 50ul of ACE2 at 0.4ug/ml was preincubated with buffers or SARS-CoV-2 RBD protein at a final concentration of 125nM for 20min. Subsequently, 50ul of fluorogenic peptides at 20uM supplemented with a serial dilution of non-fluorogenic BK, desBK or Ang II peptides ranging 640uM to 80uM were added to initiate enzymatic cleavage. Fluorescence changes were measured at 1min intervals for 1 hour at 26 degree as the initial velocity conditions.

Statistical analysis
Student's t-Test was used to determine the statistical significance in each pair wise comparison. Each experiment was repeated at least with duplicates. P<0.05 was considered statistically significant(* P<0.05; ** P<0.01; ***P<0.001; **** P<0.0001). and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted July 1, 2020. . https://doi.org/10.1101/2020.07.01.182659 doi: bioRxiv preprint

Disclosure
The authors declare that no competing interests exist.

Author contributions
J. Lu conceived this project and carried out all the experiments. J. Lu and P. Sun wrote the manuscript.

Acknowledgement
The funding of this work is provided by the Intramural Research Program (IRP) of National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health. and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted July 1, 2020. . https://doi.org/10.1101/2020.07.01.182659 doi: bioRxiv preprint and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted July 1, 2020. . https://doi.org/10.1101/2020.07.01.182659 doi: bioRxiv preprint and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted July 1, 2020. . https://doi.org/10.1101/2020.07.01.182659 doi: bioRxiv preprint and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted July 1, 2020. . https://doi.org/10.1101/2020.07.01.182659 doi: bioRxiv preprint and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted July 1, 2020. . https://doi.org/10.1101/2020.07.01.182659 doi: bioRxiv preprint  (14 ug/ml) and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted July 1, 2020. . https://doi.org/10.1101/2020.07.01.182659 doi: bioRxiv preprint and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted July 1, 2020. . https://doi.org/10.1101/2020.07.01.182659 doi: bioRxiv preprint Figure 5 Conformational changes of ACE2 upon SARS-CoV-2 binding. A) apo structure of ACE2 (PDB ID: 1R42).The N-terminal subdomain was colored in cyan with the secondary structures of SARS-CoV-2 spike binding sites highlighted in blue. The C-terminal domain of apo ACE2 was colored in wheat. All subsequent structural superpositions were based the alignment of ACE2 residues 20-84 that formed the first 2 helix for RBD domain interaction. The ACE2 inhibitor MLN-4760 (purple) and Angiotensin II (yellow) were positioned in the substrate pocket based on the structural alignment. B) ACE2 structure with inhibitor MLN-4760 binding (PDB ID: 1R4L). The C-terminal domain was highlighted in orange. C) hACE in complex with Ang II(PDB ID: 4APH). The C-terminal domain was highlighted in orange. D)ACE2 structure in complex with SARS-CoV RBD(PDB ID: 2AJF). The C-terminal domain was highlighted in yellow. E) ACE2 structure in complex with SARS-CoV-2 RBD (PDB ID: 6M0J). The Cterminal domain was highlighted in lime green. F) ACE2 structure in complex with a chimeric SARS-CoV-2 RBD domain (PDB ID: 6VW1). The C-terminal domain was highlighted in green. G) Enlarged view of ACE2 substrate binding pocket. One additional ACE2-SARS-CoV-2 RBD complex(PDB ID: 6LZG) was included. The residue color scheme was listed in the bottom panel. and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted July 1, 2020. .