Structures of Adamalysin I1 with Peptidic Inhibitors. Implications for the Design of Tumor Necrosis Factor CY Convertase Inhibitors
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Protein Science (1998), 7283-292. Cambridge University Press. Printed in the USA Copyright 0 1998 The Protein Society Structures of adamalysin I1 with peptidic inhibitors. Implications for the design of tumor necrosis factor CY convertase inhibitors E XAVIER GOMIS-RUTH,’ EDGAR E MEYER,2 LAWRENCE E KRESS,3 AND VmCENZO POLIT14 ‘Department de Biologia Molecular i Cel.lular, Centre d’Investigaci6 i Desenvolupament C.S.I.C., Jordi Girona, 18-26, 08034 Barcelona, Spain 'Biographies Laboratory, Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843 ’Molecular and Cellular Biology Department, Roswell Park Cancer Institute, Buffalo, New York 14263 4Polifa~a.Via Tor Sapienza, 138, 00155 Roma, Italy (RECEIVEDJuly 23, 1997; ACCEPTEDOctober 14, 1997) Abstract Croralus adamanteus snake venom adamalysin I1 is the structural prototype of the adamalysin or ADAM family comprising proteolytic domains of snake venom metatloproteinases, multimodular mammalian reproductive tract pro- teins, and tumor necrosis factor a convertase, TACE, involved in the release of the inflammatory cytokine, TNFa. The structure of adamalysin I1 in noncovalent complex with two small-molecule right-hand side peptidomimetic inhibitors (Pol 647 and Pol 656) has been solved using X-ray diffraction data up to 2.6 and 2.8 8, resolution. The inhibitors bind to the S’-side of the proteinase, inserting between two protein segments, establishing a mixed parallel-antiparallel three-stranded P-shest and coordinate the central zinc ion in a bidentate manner via their two C-terminal oxygen atoms. The proteinase-inhibitor complexes are described in detail and are compared with other known structures. An adamalysin- based model of the active site of TACE reveals that these small molecules would probably fit into the active site cleft of this latter metalloproteinase, providing a starting model for the rational design of TACE inhibitors. Keywords: ADAM; adamalysin; snake venom prokinase; X-ray structure; zine endopeptidase Adamalysin I1 (E.C. 3.4.24.46) is a 203-amino acid metalloendo- Thisenzyme is the structuralprototype of the adamalysin- peptidase (MEP) isolated from the venom of the Eastern diamond- family of zinc-dependent endopeptidases (Stocker et al., 1995), back rattlesnake Crotalus adamanteus (Kurecki et al., 1978; Kress which have also been called reprolysins or ADAMS (Bjamason & & Catanese, 1981). It has been shown to exhibit no significant Fox, 1994; Wolfsberg et al., 1995a, 1995b). Together with the serra- hemorrhagic activity at the envenomation site upon snake bite, but lysins, astacins, vertebrate collagenases/matrix metalloproteinases/ to readily inactivate serpins by limited proteolysis of their reactive matrixins (MMPs), leishmanolysin (Schlagenhauf et al., 1995), and site loops (Kurecki et al., 1978; Kress, 1986). Adamalysin I1 is a a zinc endoprotease from Streptomyces caespitosus (Kurisu et al., single-chain 24-kDa protein requiring zinc and calcium for its 1997), the adamalysins are grouped together in the metzincin clan activity. It displays a close sequential similarity to the low molec- of AEPs (Bode et al., 1993; Stocker et al., 1995).Like the other metz- ular weight Croralus atrox MEPs Ht-c and Ht-d, which are capable incins, the adamalysins comprise an elongated zinc binding con- of degrading type IV collagen and various gelatins, and are con- sensus sequence HEXXHXXGXXH and a so-called “Met-turn” sidered as locally (at the envenomation site) or systemically hem- forming the hydrophobic base of the catalytic zinc-binding site. The orrhagic (affecting heart, lungs, kidneys, and brain; Shannon et al., three histidine residues of the consensus sequence are involved in 1989; Hite et al., 1994). Adamalysin I1 displays proteolytic activity zinc ligation, and the glutamic acid residue presumably is the gen- only toward longer (penta-, hexa-) peptides, i.e., requires an ex- eral base in catalysis (Matthews, 1988; Bode et al., 1993; Stocker tended substrate binding site interaction (Kurecki et al., 1978; et al., 1995). Kress & Catanese, 1981), and prefers medium-sized hydrophobic Adamalysins include hemorrhagic and nonhemorrhagic protein- residues at Pi and Pi (Schechter & Berger, 1967). ases from snake venoms (Hite et al., 1992, 1994; Kini & Evans, 1992),mammalian reproductive tract and cell-fusion proteins Reprint requests to: EX.Gomis-Ruth, Department de Biologia Molec- (Wolfsberg et al., 1995b; Huovila et al., 1996), proteins involved ular i Cel.lular, Centre d’hvestigacib i Desenvolupament C.S.I.C., Jordi in embryonic development (Alfandari et al., 1997), and a tumor Girona, 18-26, 08034 Barcelona, Spain; e-mail: [email protected]. suppressor gene product (Emi et al., 1993). Recently, three new 283 284 EX. Gomis-Ruth et al. proteinases isolated from bovine and porcine spleen and human MEP family, C. atrox atrolysin C (Zhang et al., 1994) and Hz- monocytes (Black et al., 1997; Lunn et al., 1997;Moss et al., proteinase from Trimeresurus flavoviridis, have been published 1997) with the ability to release soluble tumor necrosis factor-a (Kumasaka et al., 1996). In order to study the functional determi- (TNFa), an important inflammatory cytokine, from its membrane- nants of inhibition of peptidomimetic inhibitors in complex with bound precursor by limited proteolysis of bond Ala 76-Val 77 have adamalysins, we analyzed the interactions of atrolysin C (Ht-d) been described at the primary structure level. These TNFa-con- with its natural inhibitor, PyroGlu-Asn-Trp, and with the synthetic verting enzymes, TACE, have been putatively proposed to be mem- compound, SCH47890 (Zhang et al., 1994). With this publication, bers of the adamalysin family. This task has been shown to be we intend to continue these investigations in a systematic manner performed also by members of the MMP family, whereas MMP and extend it to adamalysin 11, which exhibits a higher affinity to inhibitors block the release of active TNFa in vitro and the devel- most of these inhibitors and represents a working model for TACE, opment of acute lipopolysaccharide (endotoxin) shock in vivo (Gear- in absence of a crystallographic structure of this enzyme. We show ing et al., 1994; McGeehan et al., 1994; Mohler et al., 1994). and analyze the crystal structures of adamalysin I1 in complex with Recently, two hemorrhagic snake-venom adamalysins, isolated from two inhibitors, compare their interactions with the substrate-like Bothrops juraracu and Echis curinatus luekeyi venoms, have been and propeptide-like binding in MMPs, and discuss the interaction shown to be able to release active TNFa at the envenomation site, of S’-right-hand side inhibitors with the various subsites of the establishing an in vivo correlation between cytokine release and enzyme moiety. Furthermore, we discuss a TACE active site model local inflammation and venom-induced necrosis upon snake bite, based on the adamalysin I1 structure in light of the presently de- because the envenomation events are partially reduced by the ad- scribed enzyme-inhibitor complexes. These crystal structures are ministration of ovine TNFa Fab antibodies (Moura da Silva et al., appropriate starting models for the development of new types of 1996). inhibitors by rational drug design. Adamalysins are mostly multidomainal proteins with an ap- proximately 24-kDa central catalytic zinc-containing proteinase Results and discussion domain flanked by an activation peptide, and followed by a dis- integrin-like domain and a cystein-rich domain (Neeper & Jacob- Figure 1 displays the chemical formulae of the two inhibitors son, 1990; Hite et al., 1992, 1994; Kini & Evans, 1992). Additional investigated in complex with adamalysin I1 along with the residue modules, such as EGF-like, crambin-like, transmembrane, and cy- notation used. Their ICso values for adamalysin I1 have been de- toplasmic domains, have been described (Wolfsberg et al., 1995b; termined to be l. (Pol647) and l + M (Pol 656). Both Black et al., 1997; Moss et al., 1997). In these larger multidomain inhibitors are closely related to and reminiscent of the naturally enzymes, activation is believed to occur as in MMPs, i.e., follow- found endogenous venom tripeptide inhibitor PyroGlu-Asn-Trp ing a cysteine-switch mechanism (Springman et al., 1990; Hite present in venoms at millimolar concentrations (Robeva et al., et al., 1992; Grams et al., 1993). A cysteine residue in a strictly 1991). In Pol 647, the first residue (PyroGlu) is replaced by a conserved PKMCGV region, approximately 20 residues N-terminal furoyl group and, in Pol 656, additionally, the indole moiety of the from the start of the mature protein sequence, is believed to block tryptophan residue is crosslinked with the amide nitrogen through the catalytic zinc in the proenzyme, preventing substrate accessi- a methylene group, rendering a more rigid three-membered ring bility of the active site cleft. Upon proteolytic activation cleavage, system. This modification results in a decrease of the ICso value by the peptide and, thus, the sulfur-zinc ligand, are removed, the one order of magnitude, i.e., a tighter binding to the active site of latter being replaced in the active enzyme by a solvent molecule. the enzyme moiety. Protein inhibitors directed against adamalysins have been found in the blood sera of some mammals and snakes, the most note- worthy being the American marsupial opossum (Didelphis virgini- I una) and the Habu snake (Catanese & Kress, 1992; Yamakawa & Residue FleIl I Residue TrplCtrl2 Omori-Satoh,1992). Some syntheticpeptides, with cysteine- I switch consensus sequences, of mammalian vertebrate collagenses and snake venoms were shown to inhibit snake venom a-proteinase (Nomura & Suzuki, 1993). Pyroglutamate (PyroG1u)-containing tripeptides, such as isolated from C. utrox venom, have been shown to inhibit MEPs, with K, values in the lower micromolar range: two tripeptides (PyroGlu-Asn-Trp and PyroGlu-Gln-Trp) are usually found in very high concentrations M and higher) in venom samples also containing MEPs, suggesting that this is the peculiar manner of inhibition selected by snakes in order to avoid autolysis before envenomation or TNFarelease (Robeva etal., 1991).