key: cord-0820721-xsm95l6l
authors: Healy, Eamonn F.; Lilic, Marko
title: A model for COVID-19-induced dysregulation of ACE2 shedding by ADAM17
date: 2021-08-15
journal: Biochem Biophys Res Commun
DOI: 10.1016/j.bbrc.2021.08.040
sha: 79200e4fb123065e59a1ec80ab4861cfa3068841
doc_id: 820721
cord_uid: xsm95l6l

The angiotensin Converting Enzyme 2 (ACE2) receptor is a key component of the renin-angiotensin-aldesterone system (RAAS) that mediates numerous effects in the cardiovascular system. It is also the cellular point of contact for the coronavirus spike protein. Cleavage of the receptor is both important to its physiological function as well as being necessary for cell entry by the virus. Shedding of ACE2 by the metalloprotease ADAM17 releases a catalytically active soluble form of ACE2, but cleavage by the serine protease TMPRSS2 is necessary for virion internalization. Complicating the issue is the observation that circulating ACE2 can also bind to the virus effectively blocking attachment to the membrane-bound receptor. This work investigates the possibility that the inflammatory response to coronavirus infection can abrogate shedding by ADAM17, thereby favoring cleavage by TMPRSS2 and thus cell entry by the virion.

Similar to the earlier SARS-CoV (Severe Acute Respiratory Syndrome CoronaVirus) SARS-CoV-2, the causative agent of COVID-19, invades the cell through attachment of the spike (S) protein to the ACE2 receptor, leading to internalization of the virion [1] . ACE2 is a type I transmembrane protein that can exist in either cellular (membrane-bound) or circulating (soluble) forms [2] . Circulating ACE2 is cleaved, or shed, from full length ACE2 on the cell membrane by ADAM17 (A Disintegrin And Metalloprotease), and it has been demonstrated that recombinant soluble ACE2 effectively blocks the association of the SARS-CoV spike protein with ACE2 [3], suggesting that high levels of shed ACE2 could inhibit COVID-19 infectivity.

By contrast the serine protease TMPRSS2 (TransMembranePRotease2) augments the viral infectivity of SARS-CoV by both priming the S protein for membrane fusion as well as cleaving the ACE2 receptor [4] . Cleavage of ACE2 by ADAM17 was found to be dispensible for activation of SARS-CoV cell entry, but it remains unclear how TMPRSS2 transcends ADAM17 for cleavage of ACE2 following infection by the virus.

ADAM17 is expressed as a zymogen, where autoinhibition by a large prodomain maintains the enzyme in a closed, or inactive, state [5] . Conformational in the cell membrane and its translocation to the outer leaflet of the lipid bilayer is implicated in ADAM sheddase activity [6] . The PS-binding motif is a triplet cluster of cationic residues, J o u r n a l P r e -p r o o f -3 -R_KK in ADAM10 [7] and RK_K in ADAM17 [8] , that mediates the interaction of the protease with the membrane. In ADAM17 this motif is located in the MPD. Given that most ADAM17

substrates are cleaved at positions very close to the membrane of the same cell [9] , and that increased shedding in vivo does not follow from overexpression of the protease [10] , this leads to a scenario for PS-activation whereby interaction of the cationic motif in the MPD with the PSexposed bilayer reorients the catalytic domain proximate to both its substrate and the cell membrane, Fig. S1B [11] . In the quiescent state the protease is assumed to orient freely at the cell surface and coordination of these spatio-temporal events is critical for PS-activation.

Dysregulation of the sheddase activity of ADAM17 through disruption of this repositioning of the catalytic domain would be expected to influence the severity of COVID-19, since ADAM17 is responsible for the regulated shedding of ACE2.

Fever is a complex cytokine-mediated physiological response to infection, and the initial presentation of fever in COVID-19 is likely a manifestation of the body's immune response to viral replication. However a persistent high fever (> 39 o C) is considered to be an indicator of severe infection [12] . The heat shock response (HSR) is a highly conserved evolutionary response not just to thermal stress but is also critical to the resolution of the inflammatory response to infection. Whereas the major risk factors for fatal outcomes in COVID-19 patients share the characteristic of being inflammatory diseases linked to a defective HSR, the small heat shock proteins (sHsp) Hsp20 (or HspB6) and Hsp27 (or HspB1) exhibit widely recognized antiinflammatory capabilities linked to cardiprotection [13] , protection against free radicals and environmental toxins [14] and against the consequences of viral infection [15] . The sHsp subunit has a mass of 14-40 kDa and is characterized by a highly conserved -crystallin domain (ACD) flanked by an N-terminal domain (NTD) and a C-terminal extension (CTE). sHsps form large polydisperse assemblies of varying subunit size through the extension of the CTE from one J o u r n a l P r e -p r o o f -4 -homodimer into the -crystallin domain of the neighboring one, a motif called "patching" [16] .

A temperature-regulated subunit exchange releases the chaperone-active dimer from the oligomeric assembly, exposing substrate binding sites that allow dynamic and reversible interaction with a wide array of targets (over 450 in the case of Hsp27), providing stress tolerance through the maintenance of cellular proteostasis [17] . In the case of tubulin and Factin this interaction serves to stabilize the cytoskeleton by affecting the spatio-temporal organization of the components and preventing reorganization [18] . We have previously demonstrated how the M. tuberculosis Hsp Acr can exploit the host innate response by dysregulating shedding of the inflammatory cytokine CXCL16 by ADAM10 [19] . Among the over 80 cell-bound substrates of ADAM17 at least 9 are cytokines or cytokine receptors that have been reported as triggering inflammation when dysregulated [20] [21] [22] .

This work investigates whether the HSR in COVID-19 can also serve to dysregulate the cleavage of ACE2 by ADAM17. By mapping substrate binding sites in the ACD of human Hsp20 to non-canonical patching sequences in ADAM17 we postulate that temporal accessibility of these sites allows for dynamic formation of an ADAM17-Hsp20 complex. Modelling of the full ectodomain of ADAM17 explains how this association can disrupt the repositioning of the ADAM17 catalytic domain, thus preventing ACE2 cleavage from the membrane, Fig S1C. peptide docking using the DINC 2.0 (Docking INCrementally) protocol was used to characterize the strength of the protein-protein interactions (PPI) responsible for the putative complexation of sHsps with ADAM17. DINC is an incremental, meta-docking method optimized for the docking of longer peptides [26] . The approach was validated by confirming that for the experimental assemblies the lowest energy pose obtained by docking differed from the crystal structure by a heavy-atom root mean square difference (RMSD) of less than 1.0 Å, Fig 1. Homology modeling was used to generate a complete structure of the ADAM17 ectodomain. Two models were generated, one using a recently characterized x-ray structure of the ADAM10 ectodomain [27] , available as Protein Data Bank (pdb) code entry 6BE6, and the other using vascular apoptosis-inducing protein-1 (VAP1) [28] , a snake venom homolog of mammalian ADAM (pdb id 2ERO) as templates. Whereas the VAP1 template, Fig 2A, displays the unique C-shaped structure implicated in ADAM17 regulation [7] , the ADAM10 ectodomain, Fig 2B, exhibits a more compact arrangement where the part of the MPD occludes key elements of the catalytic domain. After insertion and refinement of missing residues using the MODELLER loop refinement algorithm [29] the query sequence of hADAM17 (Uniprot ID P78536) was aligned to the template, and the alignment used to create a set of 20 homology models. The best model is selected as the structure with the smallest value of the normalized discrete optimized molecule energy (DOPE) [30] . Finally the catalytic domain was replaced by grafting the x-ray crystal structure of the domain (1BKC) [31] in place of the homology model, hydrogens were added and the structures subjected to energy minimization using the CHARMm force field.

The ClusPro docking server [32] was employed to generate ACE2/ADAM17 complex, using the homology models for the ADAM17 ectodomain and the complete ACE2 receptor, available as pdb id 6M1D [33] . This direct, rigid-body protocol involves Fast Fourier Transform J o u r n a l P r e -p r o o f -6 -(FFT)-based global sampling of the rotational/translational space, followed by clustering of the one thousand lowest-energy structures, finishing with a CHARMM minimization to remove steric clashes. Ranking is based on cluster population, using the cluster center as the model complex. The structure generated in Fig. 3 represents the center of the most populated cluster when scored using either electrostatic-favored, hydrophobic-favored, Van der Waalselectrostatic-favored, or balanced weight coefficients. The ACE2 attractor set included residues Arg652, Lys657, Arg659, Arg708, Ser709 and Arg710 . The attractor set for the ligand was chosen as the His405, Glu406, His409, Gly412 and His415 residues that incorporate the zinc-binding consensus motif for ADAM17.

The orientation of the complex relative to the cell membrane was determined by embedding the ACE2 transmembrane (TM) region of the complex in an explicit membrane configuration containing a 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer. The optimal orientation was defined as the tilt angle of the TM relative to the membrane normal that corresponds to the minimum solvation energy [34] . After embedding and orienting the ADAM17 TM, the C-terminus of ADAM17 was extended to add the -helical CANDIS domain [35] .

Finally the CANDIS and TM domains of ADAM17 were connected using the MODELLER loop refinement algorithm.

The quaternary structure of sHsps derives from the extension of the CTE of one homodimer into the ACD of the neighboring subunit, patching a hydrophobic groove in the ACD with a conserved IXI/V motif. This hydrophobic groove is a cleft defined by the 4 and 8

sheets running anti-parallel. Because of two short 310 helices the CTE must run at an angle to the 4 / 8 strands covering one pocket, P1 , defined in Hsp20 by the side chains of V4/V4/V8/S8 , J o u r n a l P r e -p r o o f -7 -and a second pocket, P2 defined by I4/V4/S8/L8. In addition to this canonical patch mediated by the CTE crystal structures have revealed ACD tetrameric assemblies for a variety of human sHsps that arise from extensive inter-dimer patching of the hydrophobic groove by tripeptide motifs found in the NTD directly adjacent to the ACD, that resemble but are distinct from the canonical IXI sequence. Table 1 . Because of the angle of the patch to the 4/8 fold some of these sequences only cover a single pocket. Similar sequences (shown in bold) are to be found in both ADAM10 ( 651DGPLAR_KK659) and ADAM17 (621NLFLRK_K628) proximate to the cationic sequence identified as responsible for PS-activation. By facilitating a protein-protein interaction (PPI) between the ADAM10 and the pathogenic sHsp Mtb Acr we have previously shown how these residues provide a mechanism for the heat shock activation of ADAM10, providing an immunomodulatory role for the mycobacterium in the formation of the tubercular granuloma [19] . The Gbinding , i.e. free energy of binding for the comparable ADAM17 sequence relative to that calculated for the experimental patch, Table 1 , confirms the feasibility of forming such a dynamic sHsp/ADAM17 complex. However even with leucine residues isomeric to the canonical patching isoleucine the flexibility of the Phe632 methylene group allows the aryl ring to occupy one of the pockets in preference to the leucine, Figs. 1A-1C. This same flexibility also allows for a unique double patching scheme where the leucine side chains cover the P1 pockets of two different ACD subunits, Fig 1D, a motif not mirrored in the crystal structures. Homology models of the ectodomain provide insight as to how these potential interactions might affect the proteolytic activity of ADAM17.

The VAP1 and ADAM10 ectodomain structures provide markedly different templates for homology modeling of ADAM17. Comparing the MPDs of both templates with the ADAM17 MPD structure previously characterized using heteronuclear NMR spectroscopy [36] it can be seen that there is dramatically better alignment for the more compact ADAM10 structure, Interestingly protein disulfide isomerase (PDI), a stress protein essential in maintaining homeostasis [37] , induces a conformational change in the MPD from the active to inactive states, but not the reverse. While the MPD of ADAM10 has disulfide linkages (C594-C639 and C632-C645) aligned to those found in the inactive state of ADAM17, two of the disulfide linkages in the crystal structure of VAP1, C556-C598 and C592-C603 show no such alignment, suggesting a closed state in the former and an open, or active, conformation for the latter. Congruent with this analysis is the result that whereas no functional complex was generated for the docking of the ADAM10-generated homology model to ACE2, the model generated using the snake venom homologVAP1 successfully docked to the ACE2 neck region that encompasses residues of the cleavage motif, Fig. 3 .

The interface contacts for the ACE2/ADAM17 complex are summarized in Table S1 , and can be characterized as representing three distinct regions centering on the ACE2 residues Lys657, Arg652 and Arg710 respectively, Fig.S2 . While sharing many of the same contacts found in an earlier model of the complex, composed of the ACE2 receptor and just the catalytic domain of ADAM17 [38] , the model in Fig. 3 , previously identified as essential for binding recognition, neither predicts any participation for residues Arg708 and Ser709, the proposed site for cleavage of ACE2 by ADAM17 [39] The orientation of the 622LFL624 sequence capable of patching ADAM17 to a sHsp is shown in Fig. 2 inset for both the closed state of the MPD , as characterized by NMR, and in the MPD of the homology-modeled ADAM17 ectodoamin capable of docking to ACE2. To better understand the effect of sHsp interaction on ADAM17 proteolysis the orientation of the complex relative to the cell membrane was modeled by extending the C-terminus of ADAM17 to add the -helical CANDIS domain and connected to the ADAM17 TM embedded an in a POPC bilayer.

As well as being involved in substrate recognition the CANDIS segment also plays a role in regulating sheddase activity, with the hydrophobic side of the CANDIS helix being essential for ADAM17 activity [40] . This effect is primarily hydrophobic, with the 652FWDF655 segment having a significant impact on membrane binding and ADAM17 activity. In our membranebound model of the ACE2/ADAM17 complex these hydrophobic residues are more or less parallel to the plane of the membrane, as are the RK_K side chains critical for PS-activation, (1) hADAM17 peptide NLFLR+4JUS (2) Human Hsp20 ACD complex a ( Details for the interface contacts for the ACE2/ADAM17 complex are summarized in Table S1 , and the three distinct regions centering on the ACE2 residues Lys657, Arg652 and Arg710 respectively are shown in Fig.S2 . Schematics relating to PS-activation and sHSP dysregulation of ADAM17 sheddase activation are shown in Fig. S1 . 

Cell entry mechanisms of SARS-CoV-2

Chaperone-like properties of the prodomain of TNFalpha-converting enzyme (TACE) and the functional role of its cysteine switch

Phosphatidylserine exposure is required for ADAM17 sheddase function

& Bhakdi, S. ADAM10 sheddase activation is controlled by cell membrane asymmetry

How membrane asymmetry regulates ADAM17 sheddase function

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Systemic overexpression of TNFalpha-converting enzyme does not lead to enhanced shedding activity in vivo

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A Mechanism of Action for Small Heat Shock Proteins

Crystallin proteins and amyloid fibrils

Dynamic localization of αB-crystallin at the microtubule cytoskeleton network in beating heart cells

An immunomodulatory role for the Mycobacterium tuberculosis Acr protein in the formation of the tuberculous granuloma

The shedding protease ADAM17: physiology and pathophysiology

The role of ADAMmediated shedding in vascular biology

ADAM17: a molecular switch to control inflammation and tissue regeneration

Structural basis for the interaction of a human small heat shock protein with the 14-3-3 universal signaling regulator

Molecular structure and dynamics of the dimeric human small heat shock protein HSPB6

CHARMM: A program for macromolecular energy, minimization, and dynamics calculations

DINC 2.0: a new protein-peptide docking webserver using an incremental approach

Structural basis for regulated proteolysis by the α-secretase ADAM10

Crystal structures of VAP1 reveal ADAMs' MDC domain architecture and its unique C-shaped scaffold

Modeling of loops in protein structures

Statistical potential for assessment and prediction of protein structures

The ClusPro web server for protein-protein docking

Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2

Introducing an implicit membrane in generalized Born/solvent accessibility continuum solvent models

A disintegrin and metalloprotease 17 dynamic interaction sequence, the sweet tooth for the human interleukin 6 receptor

Membrane-proximal domain of a disintegrin and metalloprotease-17 represents the putative molecular switch of its shedding activity operated by proteindisulfide isomerase

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TMPRSS2 and ADAM17 interactions with ACE2 complexed with SARS-CoV-2 and B0AT1 putatively in intestine, cardiomyocytes, and kidney

Angiotensin-converting enzyme 2 ectodomain shedding cleavage-site identification: determinants and constraints

Extracellular juxtamembrane segment of ADAM17 interacts with membranes and is essential for its shedding activity

The authors wish to acknowledge the Welch Foundation (Grant# BH-0018) for its continuing support of the Chemistry Department at St. Edward's University.