The P2/P2′ sites affect the substrate cleavage of TNF-α converting enzyme (TACE)

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<ul><li><p>Te</p><p>Sa</p><p>b</p><p>c</p><p>a</p><p>ARRA</p><p>KTAPS</p><p>1</p><p>sMue2mat12</p><p>eHbSC</p><p>v</p><p>h0</p><p>Molecular Immunology 62 (2014) 122128</p><p>Contents lists available at ScienceDirect</p><p>Molecular Immunology</p><p>j ourna l ho me pa g e : www.elsev ier .com/ locate /mol imm</p><p>he P2/P2 sites affect the substrate cleavage of TNF- convertingnzyme (TACE)</p><p>en Liua,b,, Song Liua,b, Yanlin Wanga,b, Zhaojiang Liaoc</p><p>Institute of Molecular Biology, China Three Gorges University, Yichang 443002, PR ChinaCollege of Medical Science, China Three Gorges University, Yichang 443002, PR ChinaCollege of Biological and Pharmaceutical Science, China Three Gorges University, Yichang 443002, PR China</p><p> r t i c l e i n f o</p><p>rticle history:eceived 14 April 2014eceived in revised form 28 May 2014ccepted 29 May 2014</p><p>eywords:NF- converting enzyme</p><p>a b s t r a c t</p><p>Tumor necrosis factor-alpha converting enzyme (TACE) is a proteinase that releases over eighty solubleproteins from their membrane-bound forms, and it has long been an intriguing therapeutic target inauto-immune diseases, and recently, in cancers. However, a haunting question is how TACE recognizesits substrates. In this work, we applied computational and experimental methods to study the role of theP2 site and the P2 site of the substrate peptide in the substrate cleavage of TACE. In the computationalcomplex model, the sidechains of these residues do not form key interactions with TACE, but experimen-DAM17eptideprotein dockingubstrate recognition</p><p>tally, the mutations at these two positions largely affect the peptide cleavage efficiency in the enzymaticassay. We then showed that the P2/P2 sites could affect the efficiency of the conformation search for thecorrect peptide orientation, which in turn affects the substrate cleavage efficiency. Our result providesnew information to the better understanding of the enzymatic mechanism of TACE, and could be usefulin the design of novel TACE inhibitors.</p><p> 2014 Elsevier Ltd. All rights reserved.. Introduction</p><p>TACE (TNF- converting enzyme) is an enzyme that releasesoluble TNF- from its membrane-bound form (Black et al., 1997;oss et al., 1997), and has been found to be able to release the sol-ble form of about another 80 proteins (Black et al., 1997; Schellert al., 2011; Moss et al., 1997; Gooz, 2010; Stephenson and Avis,012). Therefore, TACE has been a highly attractive target in inflam-atory diseases (Lichtenthaler, 2012; Scheller et al., 2011; Saftignd Reiss, 2011; Bahia and Silakari, 2010) and, recently, in cancerherapy (Black et al., 1997; Guinea-Viniegra et al., 2012; Moss et al.,997; Richards et al., 2012; Scheller et al., 2011; Saftig and Reiss,011; Baumgart et al., 2010).TACE, also called ADAM17, is one of the most well-studied</p><p>nzymes in the ADAM (a disintegrin and metalloproteinase) family.owever, the mechanism of substrate recognition and processing</p><p>y TACE remains elusive (Scheller et al., 2011; Gooz, 2010;tephenson and Avis, 2012). Caescu et al. (Lichtenthaler, 2012;aescu et al., 2009; Scheller et al., 2011; Saftig and Reiss, 2011;</p><p> Corresponding author at: College of Medical Science, China Three Gorges Uni-ersity, Yichang 443002, PR China. Tel.: +86 717 6397179; fax: +86 717 6397179.</p><p>E-mail address: senliu.ctgu@gmail.com (S. Liu).</p><p>ttp://dx.doi.org/10.1016/j.molimm.2014.05.017161-5890/ 2014 Elsevier Ltd. All rights reserved.Bahia and Silakari, 2010) and Lambert et al. (2005) found that theresidue identities of the substrate peptide can affect the cleavageefficiency of TACE, but Wang et al. (2002) and Hinkle et al. (2004)suggested that the position of the cleavage site relative to the trans-membrane region and the first globular part of the protein is moredeterminant than the sequence of the cleavage site. It was even sug-gested that the substrate recognition and cleavage is governed byTACEs interactions with some unfound adaptor proteins (Mohanet al., 2002). Therefore, a structural view of the complex betweenthe substrate peptide and TACE would be very helpful for a betterunderstanding of the mechanism (Hartmann et al., 2013), whereasan experimental complex structure is still not available.</p><p>Recent advances in computational modeling has provided agood alternative to understand the molecular mechanisms ofproteinpeptide specificity (Smith and Kortemme, 2011; Baboret al., 2011; Kaufmann et al., 2011; Smith and Kortemme, 2010;Goldschmidt et al., 2010; Walshe et al., 2009; Grigoryan et al.,2009; Mandell and Kortemme, 2009; Kota et al., 2009; Humphrisand Kortemme, 2008; Fu et al., 2007) and enzymepeptide activity(Chaudhury and Gray, 2009; London et al., 2011). Computationally,Manzetti et al. (2003) modeled substrate-enzyme complexes forADAM-9 and ADAM-10 to conclude that the S1 pocket and the</p><p>S2/S3 region of the enzymes dominate the substrate specificity.Therefore, when an experimental complex structure is currentlyabsent, computational modeling could be a good way to provide</p><p>dx.doi.org/10.1016/j.molimm.2014.05.017http://www.sciencedirect.com/science/journal/01615890http://www.elsevier.com/locate/molimmhttp://crossmark.crossref.org/dialog/?doi=10.1016/j.molimm.2014.05.017&amp;domain=pdfmailto:senliu.ctgu@gmail.comdx.doi.org/10.1016/j.molimm.2014.05.017</p></li><li><p>muno</p><p>sr</p><p>capoetfo</p><p>2</p><p>2</p><p>TT(PeI(cRcawgrFaa</p><p>2</p><p>u1tInCpcmazaswf(w</p><p>2</p><p>sSaifAB</p><p>S. Liu et al. / Molecular Im</p><p>ome useful clues to the molecular mechanism of the substrateecognition of TACE.</p><p>In this study, we computationally modeled and optimized theomplex structure of TACEcat (the catalytic domain of TACE) and</p><p> substrate peptide, based on a preliminary docking analysis by usreviously (Liu, 2012). We noticed that the P2 and the P2 sitesf the substrate peptide distinctly affect the docking results, andxperimentally verified that the P2/P2 sites have big impacts onhe substrate cleavage by TACE. Finally, we showed that the con-ormation search of the substrate peptide for the correct bindingrientation could be the reason.</p><p>. Methods</p><p>.1. TACEcat-peptide docking</p><p>The substrate peptide was docked to the catalytic domain ofACE (TACEcat) in the protein design software Rosetta (version 3.4).he peptide docking process was similar to previously describedLiu, 2012). Briefly, a 9-residue peptide (corresponding to the5P4 sites of the recognizing peptide) was prepared as a lin-ar chain and then docked to the structure of TACEcat (from PDBD: 1BKC) using the FlexPepDocking-AbInitio protocol in RosettaRaveh et al., 2011). During the docking steps, the catalytic zinc wasoordinated by three His residues (the residues 187, 191, and 197 inosetta numbering). To keep the geometry of the catalytic center,onstraints were applied on the distances between the zinc atomnd the NE2 atoms of the three coordinating His residues (2.4 Aith a variation of 0.2 A respectively), as well as the hydroxyl oxy-en atom of the catalytic-water-coordinating glutamate acid (theesidue 188 in Rosetta numbering; 4.6 A with a variation of 0.3 A).inally, 54,000 docking models were generated for each peptide,nd the 500 lowest-score models were clustered for model evalu-tion.</p><p>.2. Molecular dynamics optimization of the complex model</p><p>The lowest-score model in the largest cluster from Rosetta wassed for molecular dynamics simulation in NAMD (GPU version.9) (Phillips et al., 2005). Periodic water (TIP3P) box was addedo wrap the complex structure with 10 A of boundary distances.ons (Na+ and Cl) were added to 0.15 mol/L and counteracted theet charger of the water box including the protein complex. Theharmm parameters from c35b2 c36a2 were used, and the smootharticle-mesh Ewald (PME) method was enabled. To minimize theomplex model, a 3-step procedure was applied. First, 2000 steps ofinimization were applied with constraints applied on the heavytoms of the proteins, followed by another 2000 steps of minimi-ation with constraints only applied on the C-alpha atoms. Finally,nother 2000 steps of minimization were run without any con-traints on the system. After the minimization step, the systemas equilibrated and a 10-ns molecular dynamics run was per-</p><p>ormed, and the atom coordinates were recorded per picosecondps). The analysis of the molecular dynamics trajectory and modelsere done in VMD (version 1.9.1) (Humphrey et al., 1996).</p><p>.3. In vitro real-time binding assay</p><p>Peptides were synthesized and covalently linked to bovineerum albumin (BSA) at the C-termini (ChinaPeptides Co., Ltd,hanghai, China) with a Cystein residue. The real-time bindingssay was performed on the BLItz system (ForteBio, PALL), which</p><p>s powered by BLI (bio-layer interferometry), a powerful label-ree assay technology. The catalytic domain of TACE (residuesrg215-Asn671; R&amp;D Systems) was biotinylated using NHS-LC-iotin (succinimidyl-6-(biotinamido)hexanoate; Sangon Biotechlogy 62 (2014) 122128 123</p><p>(Shanghai) Co., Ltd.) at a 1:2 molar ratio for 2 h at 4 C in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,2 mM KH2PO4, pH 7.4). A streptavidin-coated (SA) biosensor (Forte-Bio, PALL) was mounted to the BLItz system and used to immobilizebiotinylated TACEcat after being hydrated for 10 min in PBS. Thebinding buffer (10 mM Hepes, 100 mM NaCl, pH 7.4, 0.05% (v/v)Tween-20) was used to dissolve the peptide samples as well as forthe real-time binding assay. The binding assay was performed at25 C with 600 s of association and 420 s of disassociation. The con-centration of the peptides was 10 M, and BSA was used as theblank control, and the response signal was subtracted in the finalanalysis.</p><p>2.4. Enzymatic cleavage of the peptides</p><p>The peptides were synthesized and assayed for cleavage by TACEwith LCMS. Synthetic peptide stock solutions were prepared inAssay buffer (25 mM TrisHCl, 2.5 M ZnCl2, 0.005% Brij-35; pH9.0) at a concentration of 1 mM respectively. The catalytic domain ofTACE (residues Arg215-Asn671) was purchased from R&amp;D Systems,Inc., Minneapolis, USA. Both protein and peptide substrates wereincubated at 500 M final concentrations with TACE at 2 ng/L inAssay buffer at 37 C. Aliquots from each cleavage reaction weretaken at 3 h after incubation, and stopped by ice incubation. Thealiquots (20 L/test) were analyzed by RP-HPLC on a SinochromODS-BP 4.6 mm 150 mm column (Dalian Elite Analytical Instru-ments Co., Ltd.) at 25 C, using a 15 min linear gradient from 0.1%TFA in water (Buffer A) to 0.1% TFA in acetonitrile (Buffer B) for eachpeptide.</p><p>3. Results</p><p>3.1. The peptideprotein docking analysis</p><p>As described in Section 2, a nine-residue peptide from proTNF-(referred as TNFtide hereafter), PLAQA|VRSS, was docked to TACE-cat in Rosetta. Bertini et al. (2006) had previously done a beautifulwork in which they captured several transition states of a matrixmetalloproteinase, MMP-12, with its peptide product. Matrix met-alloproteinases (MMPs) are a family of proteins involved in cellsignaling and tissue remodeling, and the zinc-dependent activecenter of MMPs shows remarkable homology with that of TACE,including the conserved amino acid sequence of -HExGHxxGxxH-(Murphy and Lee, 2005). Therefore, the enzymatic mechanism ofTACE could be similar to that of MMPs, and indeed, some substratescan be cleaved by both TACE and MMPs, and some MMP inhibitorscan also inhibit the activity of TACE (de Meijer et al., 2010; DasGuptaet al., 2009; Pirard and Matter, 2006). So we reasoned that the pre-sented models by Bertini et al. should be a good reference for TACEtoo. In their model of the enzyme/peptide complex, the carbonicoxygen of the P1 residue of the peptide substrate was coordinatedby the zinc atom with a distance of around 2.4 A between them,which is also true in the available complex structures of TACE withother molecules in PDB and other similar proteases (Lingott et al.,2009). So, in addition to the Rosetta scores of the models (Ravehet al., 2011), the following extra two rules were applied to pick outthe reasonable complex model: first, the distance between the zincatom and the carbonic oxygen of the P1 residue of the peptide isless than 2.8 A; second, the P1 residue of the peptide locates in theS1 pocket of TACEcat, which is known well as the requirement of</p><p>the activity (Black et al., 2003; Caescu et al., 2009; Lambert et al.,2005; Yang et al., 2010; DasGupta et al., 2009). The docked complexmodel was further optimized with molecular dynamics modeling,which produced restrain-free complex models (Fig. 1).</p></li><li><p>124 S. Liu et al. / Molecular Immunology 62 (2014) 122128</p><p>Fig. 1. (A) The 100 complex models from the molecular dynamics simulation are shown in gray. The starting docked model is shown in magenta. The TACEcat chains ares in sphr rom ler</p><p>csi(ome2cmaetLtt</p><p>3</p><p>stttancTarctrrcw</p><p>cleavage by TACE.</p><p>0 200 400 600 800 10000</p><p>0.1</p><p>0.2</p><p>0.3</p><p>0.4</p><p>0.5</p><p>Res</p><p>po</p><p>nse</p><p> Un</p><p>it (</p><p>RU</p><p>)</p><p>TNFtide (PLAQAVRSS)</p><p>TENtide (PRYEAYKMG)</p><p>Pep1 (PLAAAVRSS)</p><p>Pep2 (PLAQAVFSS)hown in lines, and the TNFtide chains shown in sticks. The zinc atoms are shownepresentative model from the molecular dynamics simulation. The TNFtide runs feferences to color in text, the reader is referred to the web version of this article.)</p><p>Structurally, the peptide fits in the crevice running across theatalytic site of TACEcat. The peptide adopts an anti-parallel -heet conformation against a nearby -sheet of TACEcat, whichs quite similar to the docked models in ADAM-9 and ADAM-10Manzetti et al., 2003). As expected, the sidechain of the P1 residuef the peptide goes into the S1 pocket deeply, which mainly deter-ines the specificity of the substrate processing by TACE (Blackt al., 2003; Caescu et al., 2009; Lambert et al., 2005; Yang et al.,010; DasGupta et al., 2009). What interested us was the sidechainonformation of the P2 residue and the P2 residue. In the complexodel, the sidechains of these two residues are largely exposed,nd do not form highly favorable interactions with TACEcat. How-ver, it has been shown that distinct residue preferences exist athese two sites from peptide library screening (Caescu et al., 2009;ambert et al., 2005). Therefore, we were interested to know if thesewo sites really affect the substrate processing by TACE, and why ifhey do.</p><p>.2. The P2 and P2 sites affect the substrate processing by TACE</p><p>To experimentally test the effect of the P2/P2 sites on the sub-trate processing of TACE, we firstly tried introducing mutations athese sites and evaluating the binding affinities of different peptideso TACEcat. In one peptide, a Gln Ala mutation was introduced athe P2 site (PLAAAVRSS; named as TNFtide4A); in another peptide,n Arg Phe mutation was made at the P2 position (PLAQAVFSS;amed as TNFtide7F). The TNFtide peptide was used as the positiveontrol, and a reported peptide that cannot be cleaved by TACE,ENtide (PRYEAYKMG) (Caescu et al., 2009), was used as the neg-tive control. The peptides were synthesized and subjected to theeal-time binding assay (Section 2). As shown in Fig. 2, at the sameoncentration, these peptides have different bindi...</p></li></ul>

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