High affinity binding of SARS-CoV-2 spike protein enhances ACE2 carboxypeptidase activity
2020; Elsevier BV; Volume: 295; Issue: 52 Linguagem: Inglês
10.1074/jbc.ra120.015303
ISSN1083-351X
Autores Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoThe novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) has emerged to a pandemic and caused global public health crisis. 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 angiotensin II to control vasodilatation and vascular permeability. Given the nanomolar high affinity between ACE2 and SARS-CoV-2 spike protein, we investigated how this interaction would affect the enzymatic activity of ACE2. Surprisingly, SARS-CoV-2 trimeric spike protein increased ACE2 proteolytic activity ∼3-10 fold against model peptide substrates, such as caspase-1 substrate and Bradykinin-analog. The enhancement in ACE2 enzymatic function was mediated by the binding of SARS-CoV-2 spike RBD domain. These results highlighted the potential for SARS-CoV-2 infection to enhance ACE2 activity, which may be relevant to the cardiovascular symptoms associated with COVID-19. The novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) has emerged to a pandemic and caused global public health crisis. 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 angiotensin II to control vasodilatation and vascular permeability. Given the nanomolar high affinity between ACE2 and SARS-CoV-2 spike protein, we investigated how this interaction would affect the enzymatic activity of ACE2. Surprisingly, SARS-CoV-2 trimeric spike protein increased ACE2 proteolytic activity ∼3-10 fold against model peptide substrates, such as caspase-1 substrate and Bradykinin-analog. The enhancement in ACE2 enzymatic function was mediated by the binding of SARS-CoV-2 spike RBD domain. These results highlighted the potential for SARS-CoV-2 infection to enhance ACE2 activity, which may be relevant to the cardiovascular symptoms associated with COVID-19. The novel coronavirus, SARS-CoV-2 (1Wu F. Zhao S. Yu B. Chen Y.-M. Wang W. Song Z.-G. Hu Y. Tao Z.-W. Tian J.-H. Pei Y.-Y. Yuan M.-L. Zhang Y.-L. Dai F.-H. Liu Y. Wang Q.-M. et al.A new coronavirus associated with human respiratory disease in China.Nature. 2020; 579 (32015508): 265-26910.1038/s41586-020-2008-3Crossref PubMed Scopus (6424) Google Scholar, 2Zhou P. Yang X.-L. Wang X.-G. Hu B. Zhang L. Zhang W. Si H.-R. Zhu Y. Li B. Huang C.-L. Chen H.-D. Chen J. Luo Y. Guo H. Jiang R.-D. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; 579 (32015507): 270-27310.1038/s41586-020-2012-7Crossref PubMed Scopus (12486) Google Scholar, 3Zhu N. Zhang D. Wang W. Li X. Yang B. Song J. Zhao X. Huang B. Shi W. Lu R. Niu P. Zhan F. Ma X. Wang D. Xu W. China Novel Coronavirus Investigating and Research Team et al.A novel coronavirus from patients with pneumonia in China, 2019.N. Engl. J. 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Xia P. et al.Pathological findings of COVID-19 associated with acute respiratory distress syndrome.Lancet Respir. Med. 2020; 8: 420-42210.1016/S2213-2600(20)30076-XAbstract Full Text Full Text PDF PubMed Scopus (5614) Google Scholar). In addition, abnormal blood clots were observed in many hospitalized patients (6Wichmann D. et al.Autopsy findings and venous thromboembolism in patients with COVID-19.Ann. Intern. Med. 2020; 173: 268-277Crossref PubMed Scopus (1490) Google Scholar). However, the mechanistic understanding of the pathogenicity of SARS-CoV-2 and its complications is still lacking. ACE2 was identified as the entry receptor for both SARS-CoV-2 2, and SARS-CoV (7Kuba K. Imai Y. Rao S. Gao H. Guo F. Guan B. Huan Y. Yang P. Zhang Y. Deng W. Bao L. Zhang B. Liu G. Wang Z. Chappell M. et al.A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury.Nat. Med. 2005; 11 (16007097): 875-87910.1038/nm1267Crossref PubMed Scopus (2395) Google Scholar, 8Bao L. et al.The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice.Natjure. 2020; Google Scholar, 9Li W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzuriaga K. Greenough T.C. Choe H. Farzan M. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus.Nature. 2003; 426 (14647384): 450-45410.1038/nature02145Crossref PubMed Scopus (4100) Google Scholar). Structural studies revealed that both SARS-CoV-2 and SARS-CoV spike (S) glycoproteins bind ACE2 with higher affinity (10Wrapp D. Wang N. Corbett K.S. Goldsmith J.A. Hsieh C.-L. Abiona O. Graham B.S. McLellan J.S. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.Science. 2020; 367 (32075877): 1260-126310.1126/science.abb2507Crossref PubMed Scopus (74) Google Scholar, 11Lan J. Ge J. Yu J. Shan S. Zhou H. Fan S. Zhang Q. Shi X. Wang Q. Zhang L. Wang X. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor.Nature. 2020; 581 (32225176): 215-22010.1038/s41586-020-2180-5Crossref PubMed Scopus (3256) Google Scholar, 12Shang J. Ye G. Shi K. Wan Y. Luo C. Aihara H. Geng Q. Auerbach A. Li F. Structural basis of receptor recognition by SARS-CoV-2.Nature. 2020; 581 (32225175): 221-22410.1038/s41586-020-2179-yCrossref PubMed Scopus (2176) Google Scholar, 13Wang Q. Zhang Y. Wu L. Niu S. Song C. Zhang Z. Lu G. Qiao C. Hu Y. Yuen K.-Y. Wang Q. Zhou H. Yan J. Qi J. Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2.Cell. 2020; 181 (e899) (32275855): 894-90410.1016/j.cell.2020.03.045Abstract Full Text Full Text PDF PubMed Scopus (1713) Google Scholar). The overall structure of SARS-CoV-2 S resembles that of SARS-CoV S with the spike RBD domain contacting the extracellular region of ACE2. Physiologically, ACE2 is a zinc metalloprotease (carboxypeptidase), a homolog to dipeptidase angiotensin-converting enzyme (ACE) but with different substrate specificity (14Vickers C. Hales P. Kaushik V. Dick L. Gavin J. Tang J. Godbout K. Parsons T. Baronas E. Hsieh F. Acton S. Patane M. Nichols A. Tummino P. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase.J. Biol. Chem. 2002; 277 (11815627): 14838-1484310.1074/jbc.M200581200Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar). ACE cleaves the C-terminal of angiotensin I (Ang I) to produce the potent vasopressor octapeptide angiotensin II (Ang II), which is further cleaved at its C terminus by ACE2 to deactivate Ang II and produce Ang 1-7. Together, ACE and ACE2 regulate vasoconstriction and vasodilatation in the rennin-angiotensin system (RAS). In addition, ACE and ACE2 regulate kinin-kallikrein system to control vascular permeability and vasodilatation (15Kaplan A.P. Joseph K. Pathogenic mechanisms of bradykinin mediated diseases: dysregulation of an innate inflammatory pathway.Adv. Immunol. 2014; 121 (24388213): 41-8910.1016/B978-0-12-800100-4.00002-7Crossref PubMed Scopus (89) Google Scholar). ACE deactivates Bradykinin (BK) nonapeptide, the ligand for constitutively expressed bradykinin receptor B2. Bradykinin can be further processed by carboxypeptidase N or M to form des-Arg9-bradykinin (desBK), a potent ligand for bradykinin receptor B1(16van de Veerdonk F.L. Netea M.G. van Deuren M. van der Meer J.W. de Mast Q. Brüggemann R.J. van der Hoeven H. Kallikrein-kinin blockade in patients with COVID-19 to prevent acute respiratory distress syndrome.Elife. 2020; 910.7554/eLife.57555Crossref PubMed Google Scholar). Beyond renin-angiotensin and kinin-kallikrein systems, ACE2 also cleaves other biological peptides such as Apelin-13 that activates apelin receptor to cause vasodilatation (17Yang P. et al.Apelin-13(1-12) Is a Biologically Active ACE2 Metabolite of the Endogenous Cardiovascular Peptide [Pyr(1)].Apelin-13. Front. Neurosci. 2017; 11 ([Pyr(1)]): 92PubMed Google Scholar). Despite the importance of ACE2 in RAS, there is limited understanding to the impact of coronavirus infection to the physiological function of ACE2. ACE2 is predominantly expressed on type II pneumocytes in lung (18Hou Y.J. Okuda K. Edwards C.E. Martinez D.R. Asakura T. Dinnon K.H. Kato T. Lee R.E. Yount B.L. Mascenik T.M. Chen G. Olivier K.N. Ghio A. Tse L.V. Leist S.R. et al.SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract.Cell. 2020; 182: 429-446.e1410.1016/j.cell.2020.05.042Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar). Clinical observations showed that COVID-19 patients often had dyspnea and accumulation of fluid in lung resembling local angioedema (7Kuba K. Imai Y. Rao S. Gao H. Guo F. Guan B. Huan Y. Yang P. Zhang Y. Deng W. Bao L. Zhang B. Liu G. Wang Z. Chappell M. et al.A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury.Nat. Med. 2005; 11 (16007097): 875-87910.1038/nm1267Crossref PubMed Scopus (2395) Google Scholar, 16van de Veerdonk F.L. Netea M.G. van Deuren M. van der Meer J.W. de Mast Q. Brüggemann R.J. van der Hoeven H. Kallikrein-kinin blockade in patients with COVID-19 to prevent acute respiratory distress syndrome.Elife. 2020; 910.7554/eLife.57555Crossref PubMed Google Scholar, 19Imai Y. Kuba K. Rao S. Huan Y. Guo F. Guan B. Yang P. Sarao R. Wada T. Leong-Poi H. Crackower M.A. Fukamizu A. Hui C.-C. Hein L. Uhlig S. et al.Angiotensin-converting enzyme 2 protects from severe acute lung failure.Nature. 2005; 436 (16001071): 112-11610.1038/nature03712Crossref PubMed Scopus (1834) Google Scholar), suggesting a pathology driven by changes in vascular permeability and vasodilatation during SARS-CoV-2 infection. However, there was no direct assessment of ACE2 enzymatic activity during the coronavirus infection. Here we examined the effect of the binding of SARS-CoV-2 spike protein to the intrinsic enzymatic activity of ACE2 using two fluorogenic substrates, the caspase-1 substrate (Mca-YVADAPK-Dnp) (14Vickers C. Hales P. Kaushik V. Dick L. Gavin J. Tang J. Godbout K. Parsons T. Baronas E. Hsieh F. Acton S. Patane M. Nichols A. Tummino P. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase.J. Biol. Chem. 2002; 277 (11815627): 14838-1484310.1074/jbc.M200581200Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar), and a bradykinin analog (Mca-RPPGFSAFK-Dnp) (20Johnson G.D. Ahn K. Development of an internally quenched fluorescent substrate selective for endothelin-converting enzyme-1.Anal. Biochem. 2000; 286 (11038281): 112-11810.1006/abio.2000.4772Crossref PubMed Scopus (48) Google Scholar). To our surprise, SARS-CoV-2 spike enhanced ACE2 proteolytic activity on both caspase-1 substrate and bradykinin-analog, and the enzymatic enhancement was mediated by the spike RBD domain binding. The ability of SARS-CoV-2 spike protein to alter ACE2 enzymatic activity may result in dysregulation of RAS and contribute to the pathogenesis of COVID-19. SARS-CoV-2 is highly homologous to SARS-CoV and both use ACE2 as their entry receptor (2Zhou P. Yang X.-L. Wang X.-G. Hu B. Zhang L. Zhang W. Si H.-R. Zhu Y. Li B. Huang C.-L. Chen H.-D. Chen J. Luo Y. Guo H. Jiang R.-D. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; 579 (32015507): 270-27310.1038/s41586-020-2012-7Crossref PubMed Scopus (12486) Google Scholar, 9Li W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzuriaga K. Greenough T.C. Choe H. Farzan M. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus.Nature. 2003; 426 (14647384): 450-45410.1038/nature02145Crossref PubMed Scopus (4100) Google Scholar). Further structural studies demonstrated that both SARS-CoV-2 and SARS-CoV used their spike protein RBD domain to interact with ACE2 in similar binding modes (11Lan J. Ge J. Yu J. Shan S. Zhou H. Fan S. Zhang Q. Shi X. Wang Q. Zhang L. Wang X. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor.Nature. 2020; 581 (32225176): 215-22010.1038/s41586-020-2180-5Crossref PubMed Scopus (3256) Google Scholar). However, SARS-CoV-2 spike protein exhibited higher binding affinity to ACE2 than that of SARS-CoV. Given the nanomolar affinity between ACE2 and SARS-CoV-2 S spike protein, we wonder if the binding of SARS-CoV-2 spike protein to ACE2 affected its function as a carboxypeptidase regulating both the rennin-angiotensin and kinin-kallikrein systems (15Kaplan A.P. Joseph K. Pathogenic mechanisms of bradykinin mediated diseases: dysregulation of an innate inflammatory pathway.Adv. Immunol. 2014; 121 (24388213): 41-8910.1016/B978-0-12-800100-4.00002-7Crossref PubMed Scopus (89) Google Scholar, 21Burrell L.M. Johnston C.I. Tikellis C. Cooper M.E. ACE2, a new regulator of the renin-angiotensin system.Trends Endocrinol. Metab. 2004; 15 (15109615): 166-16910.1016/j.tem.2004.03.001Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). As previously reported, ACE2 efficiently hydrolyzes peptides with Pro-X(1-3 residues)-Pro-hydrophobic sequences between proline and the hydrophobic residue as exemplified by Ang II (DRVYIHP↓F) and des-Arg9-BK (RPPGFSP↓F) (14Vickers C. Hales P. Kaushik V. Dick L. Gavin J. Tang J. Godbout K. Parsons T. Baronas E. Hsieh F. Acton S. Patane M. Nichols A. Tummino P. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase.J. Biol. Chem. 2002; 277 (11815627): 14838-1484310.1074/jbc.M200581200Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar). ACE2 also cleaves peptides with a basic residue at P1′ position such as Dynorphin A (YGGFLRRIRPKL↓K) and Neurotensin 1-8(pE-LYENKP↓R). We measured ACE2 proteolytic activity using fluorogenic capase-1 substrate Mca-YVADAPK(Dnp) (14Vickers C. Hales P. Kaushik V. Dick L. Gavin J. Tang J. Godbout K. Parsons T. Baronas E. Hsieh F. Acton S. Patane M. Nichols A. Tummino P. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase.J. Biol. Chem. 2002; 277 (11815627): 14838-1484310.1074/jbc.M200581200Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar), and bradykinin analog Mca-RPPGFSAFK(Dnp)-OH(20Johnson G.D. Ahn K. Development of an internally quenched fluorescent substrate selective for endothelin-converting enzyme-1.Anal. Biochem. 2000; 286 (11038281): 112-11810.1006/abio.2000.4772Crossref PubMed Scopus (48) Google Scholar). Indeed, ACE2 efficiently hydrolyzed Mca-YVADAPK(Dnp) but cleaved bradykinin analog Mca-RPPGFSAFK(Dnp)-OH less (Fig. 1A and B). Within 2 h, ACE2 cleaved ∼0.5 μm of Mca-YVADAPK (Dnp), and ∼ 0.05 μm of Mca-RPPGFSAFK(Dnp)-OH. Surprisingly, addition of SARS-CoV-2 spike protein at 14 µg/ml (∼30nM) concentration to the enzymatic assay resulted in ∼3 fold increase in ACE2 cleavage of both the caspase-1 substrate and the BK-analog (Fig. 1A and B). Consistent with reported NaCl concentration-dependent ACE2 activity (14Vickers C. Hales P. Kaushik V. Dick L. Gavin J. Tang J. Godbout K. Parsons T. Baronas E. Hsieh F. Acton S. Patane M. Nichols A. Tummino P. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase.J. Biol. Chem. 2002; 277 (11815627): 14838-1484310.1074/jbc.M200581200Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar), SARS-CoV-2 spike protein further enhanced ACE2 cleavage of both peptides at 0.3M and 1M NaCl (Fig. S1), indicating the enhancement was resulted by the interaction between SARS-CoV-2 spike protein and ACE2. In addition, the spike protein-mediated enhancement of ACE2 activity depended on the concentration of the spike. Dilution of SARS-CoV-2 spike protein from 60 nm to 0.9 nm gradually mitigated the enhancement to ACE2 activity with a transition between 7.5 nm and 15 nm of SARS-CoV-2 spike protein (Fig. 1C and D). This dose dependent enhancement of ACE2 activity was consistent with a 24 nm binding affinity between ACE2 and SARS-CoV-2 spike protein (Fig. S2B). Previous studies showed that SARS-CoV infection may lead to internalization of ACE2 receptor and thus decrease cell surface level of ACE2 (7Kuba K. Imai Y. Rao S. Gao H. Guo F. Guan B. Huan Y. Yang P. Zhang Y. Deng W. Bao L. Zhang B. Liu G. Wang Z. Chappell M. et al.A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury.Nat. Med. 2005; 11 (16007097): 875-87910.1038/nm1267Crossref PubMed Scopus (2395) Google Scholar). However, immunostaining of SARS-CoV-2 infected ACE2 transgenic lung showed cell surface colocalization of the viral spike and ACE2 receptor 3 days post infection (8Bao L. et al.The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice.Natjure. 2020; Google Scholar, 18Hou Y.J. Okuda K. Edwards C.E. Martinez D.R. Asakura T. Dinnon K.H. Kato T. Lee R.E. Yount B.L. Mascenik T.M. Chen G. Olivier K.N. Ghio A. Tse L.V. Leist S.R. et al.SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract.Cell. 2020; 182: 429-446.e1410.1016/j.cell.2020.05.042Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar), supporting the presence of a stable spike-ACE2 complex on the surface of infected cells. SARS-CoV-2 and SARS-CoV spike protein are highly homologous in their prefusion trimeric architectures and both bind ACE2 with one receptor binding domain (RBD). We then determined whether RBD domain was sufficient to boost ACE2 activity. Indeed, both SARS-CoV-2 RBD and SARS-CoV RBD enhanced ACE2 cleavage of caspase-1 substrate (Fig. 2A and B), demonstrating that RBD alone was sufficient to enhance ACE2 activity. However, SARS-CoV-2 spike protein exhibited ∼5-10-fold higher ACE2 binding affinity than SARS-CoV (10Wrapp D. Wang N. Corbett K.S. Goldsmith J.A. Hsieh C.-L. Abiona O. Graham B.S. McLellan J.S. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.Science. 2020; 367 (32075877): 1260-126310.1126/science.abb2507Crossref PubMed Scopus (74) Google Scholar, 12Shang J. Ye G. Shi K. Wan Y. Luo C. Aihara H. Geng Q. Auerbach A. Li F. Structural basis of receptor recognition by SARS-CoV-2.Nature. 2020; 581 (32225175): 221-22410.1038/s41586-020-2179-yCrossref PubMed Scopus (2176) Google Scholar). The enhancement of ACE2 activity by SARS-CoV-2 RBD showed a concentration dependent saturation with half maximal en-hancement at ∼70nM, whereas the enhancement by SARS-CoV RBD was almost linearly correlated with the RBD concentration with half maximal enhancement at ∼170nM (Fig. 2E). Interestingly, only SARS-CoV-2 RBD but not SARS-CoV RBD enhanced ACE2 cleavage of the BK-analog (Fig. 2C, D, and F). SARS-CoV-2 RBD bound to ACE2 at nanomolar affinities (∼30–50 nM), ∼5–10 fold better than that of SARS-CoV RBD (∼180–400 nM) (12Shang J. Ye G. Shi K. Wan Y. Luo C. Aihara H. Geng Q. Auerbach A. Li F. Structural basis of receptor recognition by SARS-CoV-2.Nature. 2020; 581 (32225175): 221-22410.1038/s41586-020-2179-yCrossref PubMed Scopus (2176) Google Scholar). The different capabilities of SARS-CoV-2 and SARS-CoV RBD proteins to enhance ACE2 enzymatic activity suggested that the stronger interaction between RBD and ACE2 is necessary for its cleavage of nonoptimal substrates such as bradykinin analog. Further, the viral spike protein-mediated cleavage enhancement is specific to carboxypeptidase activity of ACE2 rather than potential contaminating protease present in the spike preparation as the enzymatic enhancement was abolished in the presence of a potent ACE2 specific inhibitor MLN-4760 (10 μm) (Fig. 3A and B). In contrast, none of the category protease inhibitors specific for serine protease (AEBSF), asparagine protease (pepstatin A) and cysteine protease (leupeptin) inhibited ACE2 cleavage at 40 μm concentrations nor its enhancement by the spike RBD (Fig. 3C–E). Thus, the spike RBD specifically enhanced ACE2 carboxypeptidase activity.Figure 3A and B, ACE2 specific inhibitor MLN-4760 (10 μm) completely blocked ACE2 activity and abolished the enhancement mediated by SARS-CoV-2 spike and RBD protein binding. C–E, various protease inhibitors at 40 μm (AEBSF, pepstatin A, leupeptin) had no effects on ACE2 activity.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 rate constants of ACE2 cleavage under physiological pH7.5 with 150 mm NaCl. The measurements were carried out with 20 ng ACE2 in the presence or absence of 14 µg/ml (∼30nM) 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 in binding constant Km from 10.2 μm to 2.4 μm for ACE2 hydrolysis of the BK-analog (Fig. 4 and Table 1). Likewise, SARS-CoV-2 spike protein reduced the Km for ACE2 hydrolysis of Mca-YVADAPK(Dnp) from 46.6 μm to 28.2 μm. This results in a ∼3- and 10-fold increase to kcat/Km for ACE2 hydrolysis of the caspase-1 substrate and BK-analog, respectively. It is worth mention that kcat/Km value for ACE2 hydrolysis of bradykinin analog Mca-RPPGFSAFK(Dnp)-OH in the presence of SARS-CoV-2 spike protein was 7.55 × 103 M−1s−1, similar to that of ACE2 hydrolysis of Angiotensin I decapeptide (DRVYIHPFH↓L), a biological substrate for ACE214. Taken together, SARS-CoV-2 increased the substrate binding and catalytic rate of ACE2.Table 1Kinetics constants for hydrolysis of fluorogenic peptides Mca-YVADAPK-Dnp and Mca-RPPGFSAFK-Dnp by ACE2 in the presence of SARS-CoV-2 spike protein (30 nM)SubstrateSARS-CoV-2 Spike (30nm)NaCl (mm)Km (µM)Kcat (s−1)Kcat/Km (M−1s−1)IC50 (µM) for Competitive substratesBKdesBKAng IIYVADAPK-15046.6 ± 10.30.33 ± 0.057.08 × 103n.dn.d47 ± 9+15028.2 ± 0.60.58 ± 0.072.06 × 104680 ± 871428 ± 6556.4 ± 0.9-30024.8 ± 9.90.058 ± 0.011.66 × 104+30021.1 ± 6.80.11 ± 0.0143.71 × 104RPPGFSAFK-15010.2 ± 2.70.007 ± 0.0027.06 × 102n.dn.d129 ± 52+1502.41 ± 0.850.018 ± 0.0027.55 × 103n.dn.d23 ± 5.0-30010.0 ± 3.00.01 ± 0.0051.02 × 103+3001.3 ± 0.550.015 ± 0.0041.14 × 104Notes: blank cell (not measured); n.d (not detectable). Open table in a new tab Notes: blank cell (not measured); n.d (not detectable). Although SARS-CoV-2 spike protein enhanced ACE2 cleavage of caspase-1 substrate and BK-analog, it was not clear if the spike binding enhanced ACE2 cleavage of its physiological substrates desBK and Ang II. Because of the difficulty to conjugate FRET fluorophores to the physiological ligands without affecting their enzymatic cleavage, we used an unlabeled substrate competition assay to assess the effect of the spike RBD binding on ACE2 cleavage of caspase-1 substrate in the presence of nonfluorogenic BK, desBK or Ang II peptides. In the absence of SARS-CoV-2 RBD binding, Ang II but not desBK inhibited the cleavage of caspase-1 substrate (Mca-YVADAPK(Dnp)-OH) with an inhibition constant IC50 of 47 μm, consistent with observed better ACE2 binding to Ang II (Km ∼2.0 μm) than to desBK (Km ∼290 μm) (Fig. 5) (14Vickers C. Hales P. Kaushik V. Dick L. Gavin J. Tang J. Godbout K. Parsons T. Baronas E. Hsieh F. Acton S. Patane M. Nichols A. Tummino P. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase.J. Biol. Chem. 2002; 277 (11815627): 14838-1484310.1074/jbc.M200581200Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar). In the presence of SARS-CoV-2 RBD, however, desBK inhibited the ACE2 cleavage with a IC50 of 1428 μm (Fig. 5A), suggesting that SARS-CoV-2 enhanced ACE2 binding to desBK. Surprisingly, despite no detectable ACE2 cleavage of BK under physiological condition (14Vickers C. Hales P. Kaushik V. Dick L. Gavin J. Tang J. Godbout K. Parsons T. Baronas E. Hsieh F. Acton S. Patane M. Nichols A. Tummino P. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase.J. Biol. Chem. 2002; 277 (11815627): 14838-1484310.1074/jbc.M200581200Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar), BK inhibited ACE2 cleavage of caspase-1 substrate in the presence of the spike RBD with a IC50 of 680 μm (Fig. 5). As for ACE2 cleavage of the BK-analog, Ang II competed with a IC50 constant of 129 μm and 23 μm in the absence and presence of the SARS-CoV-2 RBD, respectively (Fig. S3), similar to that of caspase-1 substrate cleavage. However, both 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 because of its C-terminal modification (20Johnson G.D. Ahn K. Development of an internally quenched fluorescent substrate selective for endothelin-converting enzyme-1.Anal. Biochem. 2000; 286 (11038281): 112-11810.1006/abio.2000.4772Crossref PubMed Scopus (48) Google Scholar). It is likely that BK-analog also bound better to ACE2 than BK. 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, SARS-CoV-2 spike protein significantly enhanced the enzymatic activity of ACE2 to cleave caspase-1 (YVADAPK) peptide in a concentration and RBD dependent manner. It is likely that the spike protein binding to ACE2 would lead to more efficient cleavage of similar substrates such as Ang II (DRVYIHP↓F) and Apelin-13(QRPRLSHKGPMP↓F). Similarly, the binding of SARS-CoV-2 spike enhanced ACE2 cleavage of bradykinin analog, and bradykinin (RPPGFSPFR) and des-Arg9-bradykinin competed similarly for ACE2 cleavage of the caspase-1 substrate. To further gain insight into how the binding of the viral spike protein affected the catalytic activity of ACE2, we compared structures of ACE2 in the presence and absence of the viral spike protein. 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 (22Towler P. et al.ACE2 X-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis.J. Biol. Chem. 2004; 279 (14754895): 17996-1800710.1074/jbc.M311191200Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar) (Fig. 6A). The structure of ACE2 with a potent inhibitor MLN-4760 bound at the active site showed a clear ligand-induced hinge bending movement between the N- and C-terminal domains (Fig. 6B). This closed conformation of ACE2 proteolytic domain on inhibitor binding was consistent with that of ACE bound to its hydrolyzed product Ang II (23Masuyer G. Schwager S.L. Sturrock E.D. Isaac R.E. Acharya K.R. Molecular recognition and regulation of human angiotensin-I converting enzyme (ACE) activity by natural inhibitory peptides.Sci. 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The structural superposition between various RBD bound ACE2 and nativ
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