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Determinants of the Inhibition of a Taiwan Habu Venom

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Determinants of the Inhibition of a Taiwan Habu Venom Eur. J. Biochem. 269, 3047–3056 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02982.x Determinants of the inhibition of a Taiwan habu venom metalloproteinase by its endogenous inhibitors revealed by X-ray crystallography and synthetic inhibitor analogues Kai-Fa Huang1, Shyh-Horng Chiou1,2, Tzu-Ping Ko1 and Andrew H.-J. Wang1,2 1Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; 2Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan Venoms from crotalid and viperid snakes contain several of the proteinase.Results from the study of synthetic peptide inhibitors which regulate the proteolytic activities of inhibitor analogues showed the primary specificity of Trp their snake-venom metalloproteinases (SVMPs) in a residue of the inhibitors at the P)1 site, corroborating the reversible manner under physiological conditions.In this stacking effect observed in our structures.Furthermore, we report, we describe the high-resolution crystal structures of a have made a detailed comparison of our structures with the SVMP, TM-3, from Taiwan habu (Trimeresurus mucro- binding modes of other inhibitors including batimastat, a squamatus) cocrystallized with the endogenous inhibitors hydroxamate inhibitor, and a barbiturate derivative.It pyroGlu-Asn-Trp (pENW), pyroGlu-Gln-Trp (pEQW) or suggests a close correlation between the inhibitory activity of pyroGlu-Lys-Trp (pEKW).The binding of inhibitors causes an inhibitor and its ability to fill the S)1 pocket of the pro- some of the residues around the inhibitor-binding environ- teinase.Our work may provide insights into the rational ment of TM-3 to slightly move away from the active-site design of small molecules that bind to this class of zinc- center, and displaces two metal-coordinated water molecules metalloproteinases. by the C-terminal carboxylic group of the inhibitors.This Keywords: snake-venom metalloproteinase; Trimeresurus binding adopts a retro-manner principally stabilized by four mucrosquamatus; endogenous tripeptide inhibitor; TNFa possible hydrogen bonds.The Trp indole ring of the inhib- ) converting enzyme; retro-binding mode. itors is stacked against the imidazole of His143 in the S 1 site Venoms secreted from the glands of crotalid and viperid the following.(a) SVMPs in crude venoms might exist snakes are able to elicit shock, intravascular clotting, originally as a large multidomain precursor, in which the systemic and local hemorrhage, edema and necrosis upon central zinc-metalloproteinase domain is flanked by an victimized preys following snakebites [1].The major com- N-terminal propeptide and a C-terminal disintegrin-like plication arising from snake envenomation is hemorrhagic domain [4].A cysteine residue in a conserved PKMCGV effects, which are generally thought to result from the region of the propeptide is believed to bind to the catalytic structural destruction of capillary basement membranes via zinc ion in the inactive proenzyme, prior to activation by a proteolytic degradation by snake-venom metalloproteinases cysteine-switch mechanism [5].(b) Venom secretions con- (SVMPs) [2,3].In order to avoid auto-digestion of the tain several endogenous small peptides, e.g. pyroGlu-Asn- venom gland itself from its secreted metalloproteinases after Trp and pyroGlu-Gln-Trp [6].They could selectively bind in vivo generation of venom proteases, several strategies are to SVMPs, thereby partially inhibiting their proteolytic presumably employed by snakes to regulate the proteolytic activities [7,8].(c) A variety of crude snake venoms have activities of SVMPs in their venom secretions.These include been reported to have citrate at high concentration, in the range 30–150 mM, which is thought to play a role of chelating the active-site zinc ion of SVMPs, thus keeping Correspondence to S.-H. Chiou, Institute of Biological Chemistry, their activities low [9]. Academia Sinica, Nankang, Taipei, Taiwan. Interestingly, many proteinase inhibitors (commonly Fax: + 886 226530014, called hemorrhagin neutralizing factors) were purified from E-mail: [email protected] the blood sera of some mammals and snakes, e.g. oprin and A.H.-J.Wang, Institute of Biological Chemistry, Academia from Didelphis virginiana [10], DM43 from Didelphis Sinica, Nankang, Taipei, Taiwan. marsupialis [11], HSF from Trimeresurus flavoviridis [12], Fax: +886 227882043, E-mail: [email protected] BJ46a from Bothrops jararaca [13], and TMI from Trim- Abbreviations: SVMP, snake-venom metalloproteinase; MMP, eresurus mucrosquamatus [14].These plasma inhibitors matrix metalloproteinase; ADAM, a disintegrin-like and could act by noncovalently binding to SVMPs, and thus, metalloproteinase protein; TNFa, tumor necrosis factor-a;TACE, neutralizing their hemorrhagic activities, and endowing TNFa converting enzyme; HNC, human neutrophil collagenase; these animals with resistance to envenomation by crotalid pENW, pyroGlu-Asn-Trp; pEQW, pyroGlu-Gln-Trp; pEKW, and viperid snakebites. pyroGlu-Lys-Trp. Together with the matrixins (vertebrate collagenases, or (Received 5 February 2002, revised 24 April 2002, denoted as matrix metalloproteinases, MMPs), serralysins accepted 7 May 2002) (large bacterial zinc-endopeptidases) and astacins, SVMPs 3048 K.-F. Huang et al. (Eur. J. Biochem. 269) Ó FEBS 2002 are grouped in a superfamily of metzincin which exhibits FITC (fluorescein isothiocyanate)-labeled casein (FITC- some typical structural features, such as the Met-turn and casein, 38 lg FITC per mg protein) was procured from active-site consensus HExxHxxGxxH sequence [15–17]. Sigma (St Louis, MO, USA).The membranes (Centricon, Some organisms and mammalian tissues recently have been YM-10) for ultrafiltration and concentration was obtained reported to contain a number of multidomain proteins, from Millipore (Amicon bioseparation, Bedford, MA, which are related to the fertilization, neurogenesis and USA). inflammation processes [18–20].They are generally called ADAMs (a disintegrin-like and metalloproteinase domain) Preparation of inhibitor analogues and proteinase with the same central catalytic domain as SVMPs and inhibition assays MMPs, especially at the active-site structure [21,22].A well known example is the TACE, also known as ADAM 17, Inhibitor analogues were synthesized using 4-(2¢,4¢-dimeth- responsible for the release of a major proinflammatory oxyphenyl-Fmoc-amino methyl)phenoxyl-resins and Fmoc- cytokine, tumor necrosis factor-a (TNFa), from its mem- amino acid derivatives by an automatic peptide synthesizer brane-anchored precursor into extracellular space [23,24]. (Applied Biosystems, Foster City, CA, USA).At the end of The crystal structure of the catalytic domain in TACE was synthesis cycles, peptides on the resin were cleaved off by a reported and revealed a characteristic polypeptide fold solvent mixture of trifluoroacetic acid and ethanedithiol, containing a catalytic zinc environment resembling that of and solvent was evaporated to dryness.The resins were then the SVMP family [22].Moreover, two SVMPs isolated from washed with cold ether and the peptides were extracted with the venoms of Bothrops jararaca and Echis carinatus laekeyi, 5% acetic acid.Combined solutions were lyophilized to respectively,wereshowntobeabletoreleasetheactive yield crude peptides which were used for further purification TNFa at the envenomation site [25], corroborating the on HPLC.Inhibition activity of each peptide was assayed structural similarities between SVMPs and TACE as using purified TM-3 and a fluorescence substrate FITC- mentioned above.Before the TACE structure was solved, casein as described previously [35].The inhibition constants, adamalysin II had been considered to be a good starting Ki values, were calculated according to the Dixon plot [36]. model in SVMP family for the rational design of drugs against TACE-involved inflammatory diseases.Based on Crystallization of TM-3 the crystal structure of adamalysin II and modeled on an endogenous venom tripeptide, several peptidic inhibitors TM-3 was isolated from the venom of Taiwan habu were synthesized, such as Furoyl-Leu-Trp (pol647) and its (Trimeresurus mucrosquamatus) and purified to high homo- cyclic and phosphonate derivatives [26–28]. geneity as described previously [37].Crystals were obtained In our laboratory, the crystal structure of a snake-venom using the crystallization screening kits of Hampton metalloproteinase TM-3 from Trimeresurus mucrosquamatus Research (Laguna Niguel, CA, USA).Finally, 4 lL mother ˚ was solved and refined to 1.35 A resolution [29].It is more liquid [0.1 M CdCl2,0.1M sodium acetate and 30% (v/v) similar to TACE than adamalysin II in terms of the poly(ethylene glycol) 400 at pH 4.6] was mixed with 3.5 lL )1 )1 disulfide configurations and the S -pocket dimension. TM-3 (10.5 mgÆmL in 0.2 M ammonium acetate buffer, Currently, some macrocyclic and succinate-based hydroxa- pH 6.0) and 0.5 lL of the synthetic inhibitor, followed by mic acids have been reported to directly block the release of cocrystallization at 4 °C using hanging-drop vapor diffusion TNFa in vitro and in vivo by inhibiting the activity of TACE method.Crystals started to appear with their dimensions [30,31].However, most designs for inhibitors were of the reaching 0.6 · 0.8 · 1.6 mm within 1 week. The concen- type that mimicks the structural features of substrate tration of inhibitors used are: pENW, 114.3 mM; pEQW, binding described for MMPs, or through the screening of 107.1 mM; pEKW, 101.6 mM. libraries of MMP inhibitors in-house [32–34].Investigations
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