key: cord-0933691-tuhld32x
authors: Liu, Peiyuan; Zhang, Yanfeng; Li, Zibin; Huang, Jianwen; Wang, Tao; Chen, Cheng
title: Structural insights revealed by crystal structure of B38-CAP, an isoenzyme of carboxypeptidase ACE2, the receptor of SARS-CoV-2
date: 2022-03-18
journal: Biochem Biophys Res Commun
DOI: 10.1016/j.bbrc.2022.03.077
sha: 5c3c1255f7270aa2598cb298de1102ae7d000eef
doc_id: 933691
cord_uid: tuhld32x

The worldwide pandemic of Coronavirus disease 2019 (COVID-19) is triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and further worsened by the emergence of a variety of SARS-CoV-2 variants. Angiotensin-converting enzyme 2 (ACE2), a carboxypeptidase of M32 family, serves as the receptor of SARS-CoV-2 and key regulator of host renin-angiotensin system (RAS), both of which are mainly mediated via the carboxypeptidase domain of ACE2 (sACE2) or its activity. sACE2 is thus promising in the treatment of COVID-19 but unfortunately weakened by its unstrigent substrate preference and complex interplay with host RAS. B38-CAP, an isoenzyme of ACE2, partically compensates these defects but still encounters the problem related to carboxypeptidase activity and specificity. In this study, we firstly determined the crystal structure of B38-CAP at a resolution of 2.44 Å which exists in dimeric form with the non-crystallographic two-fold axis being in coincidence with the crystallogrphic two-fold axis. Further structural analysis revealed the structural conservatism feature among M32 family, particularly the catalytic core and moreover lead us to hypothesize that conformational flexibility might play an pivotal role in the catalysis of B38-CAP and ACE2. The work provided here presents key features of the M32 family carboxypeptidase and provides structural basis for further development of B38-CAP-based anti-SARS-CoV-2 drugs.

SARS-CoV-2 drug development due to its multiple functions in SARS-CoV-2 1 pathogenesis [12, 13] . During the course of SARS-CoV-2 infection, ACE2 (or more 2 specifically, its membrane-bound form) firstly serves as the receptor of SARS-CoV-2.

To enter into host cells, the receptor binding domain (RBD) of SARS-CoV-2 Spike 4 protein needs to first bind to the carboxypeptidase domain of ACE2, immediately 5 followed by the cleavage of Spike protein by TMPRSS2 [12, 14] . In the meantime, ACE2, 6 as a key regulator of host renin-angiotensin system (RAS), also employs its 7 carboxypeptidase activity to reduce host inflammatory response and restrict the 8 deterioration of SARS-CoV-2 [13, 15] . This is also mediated by the carboxypeptidase 9 domain of ACE2 through the digestion of substrate Ang II to Ang (1-7), despite a dozens 10 of other substrates having been identified for ACE2 [16] . Thus, the soluble 11 carboxypeptidase domain of ACE2 (sACE2) which possesses both RBD-binding (in 12 other way, SARS-CoV-2 neutralizing) and RAS-modulating ability regardless of 13 whether and how SARS-CoV-2 mutates, is a natural drug candidate to treat COVID-19. 14 Currently, a series of sACE2-based anti-SARS-CoV-2 strategy are in investigation [17-15 19 ]. However, it's not until recently that researchers find that sACE2 surprisingly 16 mediate the cell entry of SARS-CoV-2 via interaction with proteins related to RAS [20] . 17 This finding, in addition to indicating the antagonistic role of ACE2 in SARS-CoV-2 18 pathogenesis, further promotes people to re-examine the clinical complexity of ACE2.

B38-CAP, another member of M32 carboxypeptidase family and in fact an isoenzyme 20 of ACE2's carboxypeptidase domain, serves as a better start for developing anti-SARS- 21 CoV-2 drug, due to its non interacting property with RAS proteins and moreover, an 22 J o u r n a l P r e -p r o o f extra ability of digesting Ang(1-9) into Ang(1-8) compared to ACE2 [21] . However, 1 B38-CAP is found to display weaker carboxypeptidase activity compared to ACE2 and 2 retain the shortcoming of unstrigent substrate property of ACE2 [21] . To aids our 3 understanding of the carboxypeptidase activity of B38-CAP, we firstly determined the 4 three-dimensional structure of B38-CAP in this study and then performed structural 5 comparison between B38-CAP and other M32-family carboxypeptidases, particularly 6 ACE2. It was shown that B38-CAP which exists in dimeric form shares structural rather 7 than sequence conservatism with ACE2 particularly in the catalytic core, while in the 8 meantime, displays little structural flexibility in dimeric form compared to ACE2, which 9 may have a potential effect and thus contribute to their difference in carboxypeptidase 10 activity. The coding sequence of B38-CAP was synthetized and cloned into the vector pGEX- containing the ACE2 were collected [14] . The purified B38-CAP protein was concentrated to 20 mg/ml in the above buffer 7 containing 10 mM Tris-HCl, pH 8.0, 100 mM NaCl. In the primary stage, commercial 8 kits including were used to screen for preliminary crystallization conditions for B38- 9 CAP. Crystallization trials were set in 48-well crystallization plates at 291 K using the 10 sitting-drop vapour diffusion method. Crystallization drops were carefully set by mixing Table 1 . 2 

The crystal structure of B38-CAP was solved by molecular replacement method in 4 Phaser using the structure of BsuCP (PDB entry 3HQ2) as the search model [22] . The 5 initial model was then subjected to manual rebuilt in COOT[23] and automatic 6 refinement in PHENIX [24] iteratively until the stereochemical parameters suit the 7 criteria of Molprobity [25] . The refinement strategy includes individual_sites refinement 8 in real-space and reciprocal-space, atomic displacement refinement and TLS refinement. 9 In the final stage of refinement, solvent molecules were added at a contour level of 3.0 noteworthy that an real-time mode is employed here rather than the average mode 10 reported in earlier literature [21] . It turns out that B38-CAP displays considerably weak 11 carboxypeptidase activity compared to ACE2 carboxypeptidase domain (Fig. 1B) , 12 rasing the concern that how this can be linked to their respective structural differences. packing reveals that the non-crystallographic two-fold axis between the two molecules 20 of B38-CAP dimer is in perfect coincidence to the crystallographic two-fold axis of the 21 C2 space group (Fig. 2A) . The interface area between the two molecules constituting 22 J o u r n a l P r e -p r o o f B38-CAP dimer is as high as 984.5 Å 2 . An extensive hydrogen bond, salt bridge and 1 hydrophobic interaction network exists between these two B38-CAP molecules, 2 contributing to the stabilization of B38-CAP dimer (Fig. 2B) . Inside of the structure, 3 there is a zinc ion, which, by coordination with residues H269, H273, E273 and water 4 molecules, forms the catalytic core ( Fig. 2A) . To assess the conservative property of 5 B38-CAP, we then performed DALI structural similarity search and identified eight M32 6 carboxypeptidase in PDB database, all bacteria-derived, with r.m.s. deviation between 7 them ranging from 1.1 to 2.9 Å (Table S1 ). Stutural alignment clearly shows that B38-8 CAP adopts a typical M32 carboxypeptidase domain fold (Fig. 2C) . This is in good 9 accordance with the sequence comparison result, which means B38-CAP and the eight 10 carboxypeptidases share both structural and sequence conservation (Table S1). 11 3.3. The structural conservation and diversity between B38-CAP and ACE2

Sequence comparison displays little, if there is, identity between B38-CAP and 13 ACE2 carboxypeptidase domain (Fig. 3A) , raising the question that whether ACE2 has 14 evolved a brand-new model for carboxypeptidase catalysis. Considering that structural 15 conservatity is more reliable, a DALI analysis was performed using the structure of B38- 16 CAP monomer and ACE2 carboxypeptidase domain, which however gives an r.m.s. 17 deviation of 5 Å with 437 Cα atoms aligned. It's surprising to find that further inspection 18 of their fine structure reveals that the catalytic core of B38-CAP and ACE2 are 19 completely conservative between each other and so is the spatial configuration of the 20 structural elements adjacent to catalytic core (Fig. 3B) . This strongly indicates that the 

In this study, we characterized the dimeric state of B38-CAP which is in accordance 15 with its solved crystal structure. We also characterized the real-time carboxypeptidase 16 activity of B38-CAP which is weaker compared to ACE2. Structural comparison 17 between B38-CAP and other M32-family carboxypeptidase showed highly sequential 18 and structural similarity while only structural conservatism was retained between B38-19 CAP and ACE2 carboxypeptidase domain, particularly in their catalytic core. Moreover, 20 the dimeric architecture was shown to be strictly conservative among bacteria-derived 21 carboxypeptidases while ACE2 did not obey this rule. This marked difference is [5] First oral antiviral for COVID-19, Lagevrio (molnupiravir), approved by MHRA. 7 https://www.gov.uk/government/news/first-oral-antiviral-for-covid-19-lagevrio-8 molnupiravir-approved-by-mhra, 2021 (accessed 5 November 2021). 9

[6] Pfizer's novel COVID-19 oral antiviral treatment candidate reduced risk of hospitalization 10 of death by 89% in interim analysis of phase 2/3 EPIC-HR study. 11 https://www.pfizer.com/news/press-release/press-release-detail/pfizers-novel-covid-19-12 oral-antiviral-treatment-candidate, 2021 (accessed 5 November 2021). 13 Table 1 20 Data collection and refinement statistics. 

A Novel Coronavirus 17 from Patients with Pneumonia in China

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 12 receptor

Classic and Nonclassic Renin-Angiotensin Systems in the 14 Critically Ill

Hydrolysis of biological 18 peptides by human angiotensin-converting enzyme-related carboxypeptidase

B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses 14 hypertension and cardiac dysfunction

Insight into the substrate length restriction of M32 carboxypeptidases: 18 Characterization of two distinct subfamilies

Coot: Model-building tools for molecular graphics

PHENIX: A 26 comprehensive Python-based system for macromolecular structure solution

MolProbity: More and better 3 reference data for improved all-atom structure validation

Dali server update

W355

The PyMOL Molecular Graphics System

Nicotianamine is a novel 10 angiotensin-converting enzyme 2 inhibitor in soy-bean

ACE2 X-Ray Structures Reveal a 14

Large Hinge-bending Motion Important for Inhibitor Binding and Catalysis

ACE2-like carboxypeptidase B38-CAP protects from SARS-CoV-2-induced lung 21 injury