ライフサイエンスコーパス: exosite

1 ytic triad), and K(420) (part of a substrate exosite).

2 tes as well as with secondary binding sites (exosites).

3 mechanistic insight into the binding of the exosite.

4 surface loops of FD that form part of the FD exosite.

5 recognizes the C4 C345C domain through a CCP exosite.

6 ctive site, or the exosite, and to fX at the exosite.

7 tor VIIIa) via interaction with a factor IXa exosite.

8 which are critical for the formation of the exosite.

9 and activators occupying a ubiquitin-binding exosite.

10 e active site that may serve as a regulatory exosite.

11 the binding of ligands to its two principal exosites.

12 y around the catalytic domain to reach novel exosites.

13 llostery and the presence of putative FXIIIa exosites.

14 eases by generating new protease interaction exosites.

15 teraction of inhibitor with protease through exosites.

16 udied thrombin inhibition in the presence of exosite 1 and 2 ligands.

17 however, the thermodynamic coupling between exosite 1 and the active site has not been fully explore

18 Competitive studies using a hirudin peptide (exosite 1 ligand) and unfractionated heparin, heparin oc

19 Different exosite 1 ligands with widely varied thermodynamic signa

20 f the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both

21 Replacement of exosite 1 or 2 with analogous residues from cathepsin L

22 avy chain, the gamma-loop, and anion-binding exosite 1, the main allosteric binding site, retain mus-

23 ligand binding to either the active site or exosite 1.

24 atures for binding to the active site and to exosite 1.

25 ions at the catalytic site when complexed to exosite 1.

26 inity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between e

27 The binding of thrombomodulin (TM) to exosite-1 and the binding of Na(+) to 225-loop allosteri

28 ofactor binding-site, both Na(+)-binding and exosite-1 are energetically linked.

29 in mutants in which either the 70-80 loop of exosite-1 or the 225-loop of the Na(+)-binding site was

30 e kinetic studies in the presence of the two exosite-1-specific ligands Tyr(63)-sulfated hirudin(54-6

31 site-directed mutagenesis studies identified exosite 2 as the site of binding for the most potent sul

32 We reasoned that an exosite 2 directed allosteric thrombin inhibitor should

33 t that multiple avenues are available within exosite 2 for inducing thrombin inhibition.

34 t arises from the occlusion of anion-binding exosite 2 in the catalytic domain by the covalently reta

35 l insights into the prominent role played by exosite 2 in the rate-limiting step of factor V activati

36 tasaccharide, and gamma'-fibrinogen peptide (exosite 2 ligands) demonstrated exosite 2 recognition in

37 Exosite 2 of human thrombin contributes to two opposing

38 gen peptide (exosite 2 ligands) demonstrated exosite 2 recognition in a manner different from that of

39 anning mutagenesis of 12 Arg/Lys residues of exosite 2 revealed a defect in 9a potency for Arg233Ala

40 lacement of several arginines and lysines of exosite 2 with alanine did not affect thrombin inhibitio

41 Thermodynamic linkage between anion-binding exosite 2, the Na(+)-binding site, and the active site a

42 agment 1.2 (F12), a ligand for anion-binding exosite 2, to probe the zymogenicity of thrombin by isot

43 more than 60% of them being performed by the exosite 2-composing residues.

44 SbO4L binds to Arg233, Lys235, and Lys236 of exosite 2.

45 These results suggest that, while exosite-2 of thrombin is an independent cofactor binding

46 We recently designed a group of novel exosite-2-directed sulfated, small, allosteric inhibitor

47 is thought to involve thrombin anion binding exosite (ABE) I. alpha-Thrombin can undergo additional p

48 ease thrombin (IIa) employ two anion binding exosites (ABE-I and -II) to aid in binding.

49 xtended active site region and anion-binding exosites (ABEs) I and II.

50 exosites and to investigate the presence of exosite-active site and exosite-exosite interactions.

51 Identification of specific exosites also provides targets for selective inhibitors.

52 ort peptide substrates was affected by these exosite alterations, underscoring the importance of this

53 tide region that engages a substrate docking exosite and a C-terminal transition-state analog moiety

54 lectivity of one of the aptamers towards its exosite and a further negative allosteric effect upon sa

55 earrangement upon activation, and reveals an exosite and a sugar-rich channel, both of which possibly

56 bstrate interacts with the deacetylase at an exosite and contributes to the activity of the substrate

57 IDE reveals the binding of bradykinin to the exosite and not to the catalytic site.

58 N-terminal domain of caspase-7 form such an exosite and promote the rapid proteolysis of the poly(AD

59 can be engineered by incorporating factor Xa exosite and reactive site recognition determinants in a

60 xperiments demonstrate that both the docking exosite and the active site are engaged by the bipartite

61 ic activity but does not preclude thrombin's exosites and binding to fibrinogen.

62 to characterize ligand binding to individual exosites and to investigate the presence of exosite-acti

63 es circumscribe the catalytic cleft, form an exosite, and are distinctive features available for targ

64 5 can bind to fXa at the active site, or the exosite, and to fX at the exosite.

65 ing the enzymes' specificity pockets, nearby exosites, and downstream domains.

66 onstrate that RCL-primed residues, strand 3C exosites, and the furin(298-300) loop are critical deter

67 ts demonstrate that fXIa activates fIX by an exosite- and Ca(2+)-mediated release-rebind mechanism in

68 This exosite appears to be unique to caspase-6, as the residu

69 Exosites are emerging as one of the mechanisms by which

70 t the role of the factor IXa heparin-binding exosite as a critical regulator of coagulation and novel

71 nhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

72 and establish the factor IXa heparin-binding exosite as the relevant molecular target for inhibition

73 nfolds the VWF A2 domain and reveals cryptic exosites as well as the scissile bond.

74 Although enzyme-substrate interactions at exosites away from the active site are mapped in detail

75 If both exosites bind to GpIbalpha, thrombin could potentially a

76 between heavy and light chains for thrombin exosite binding and subsequent proteolysis with binding

77 y be used for identifying novel, potentially exosite binding compounds.

78 the P1-P1' substrate residues while defining exosite binding domains.

79 Exosite binding drives substrate affinity and is indepen

80 bstrate-like binding in the active site with exosite binding on the protease surface.

81 y the intrinsic Xase complex by showing that exosite binding plays a critical role in this process, w

82 In the present work a glycosylated, exosite-binding substrate of ADAM10 and ADAM17 was utili

83 genolytic activities and was achieved via an exosite-binding triple-helical peptide.

84 not caspase-3 or a caspase-7 with a mutated exosite, binds nucleic acids.

85 may serve to regulate the properties of the exosite-bound proteinase.

86 the ADAMTS13 cysteine-rich and spacer domain exosites bring enzyme and substrate into proximity.

87 In addition, the use of exosites by maspin and plasminogen activator inhibitor-1

88 man serpins, this minireview examines use of exosites by nine serpins in the initial complex-forming

89 Here we show that functional exosites can be engineered at homologous positions in a

90 Targeting secondary substrate binding sites (exosites) can potentially work as an alternative strateg

91 oth free enzyme and initial substrate-enzyme exosite complex but would be excluded by the final Micha

92 ate complex significantly favors the initial exosite complex.

93 Additionally, we identified an exosite containing an acidic patch in Asf1 and show that

94 Evaluation of these chimeras revealed two exosites contributing to the elastolytic activity of cat

95 A careful balance between active-site and exosite contributions is critically important for the sp

96 C1s is achieved through both active site and exosite contributions.

97 on how the binding of short peptides at the exosite could regulate substrate recognition.

98 results demonstrate that these loops contain exosites critical for interaction with and processing of

99 Exosite-dependent binding of prothrombin to prothrombina

100 It is not known if a similar exosite-dependent interaction contributes to the specifi

101 One region implicated in this exosite-dependent interaction is the factor VIII a2 segm

102 antithrombin (AT) by heparin facilitates the exosite-dependent interaction of the serpin with factors

103 ise from a loss in the membrane component of exosite-dependent tethering of substrate to prothrombina

104 Further characterization of exosite determinants that govern interactions of MMPs wi

105 hat mutation of specific residues within the exosite differentially affects MKK and NLRP1B cleavage i

106 and in vitro, and the properties of a unique exosite-directed prothrombinase inhibitor.

107 ition reveals that the inhibitor binds to an exosite, displays noncompetitive partial inhibition, and

108 the presence of one zinc/monomer bound at an exosite distal from the active site.

109 etween factor X and intrinsic Xase occurs at exosites distant from the active site, followed by activ

110 Through exosite engagement, GPIbalpha may influence FIIa-depende

111 Binding studies showed that the exosites enhance the Michaelis complex interaction of al

112 gate the presence of exosite-active site and exosite-exosite interactions.

113 tivation requiring binding of Fbg through an exosite expressed on the activated ProT*.VWbp complex.

114 domain plays a key role in the regulation of exosite expression and prothrombinase assembly.

115 complex and the expression of a weaker "pro"-exosite for binding of a second Pg in the substrate mode

116 ime, yielding valuable information about the exosite for C4 binding located at this position.

117 residues 236-246), which serves as a general exosite for caspase-6-specific substrate recruitment.

118 These results demonstrate the presence of an exosite for FIX binding on the FXIa-LC remote from its a

119 s required for the fVa-dependent recognition exosite for fXa in prothrombinase within the amino acid

120 antithrombins to S195A proteases showed that exosites generated by conformationally activating antith

121 Together these results show that exosites generated by heparin activation of antithrombin

122 different aptamers, binding to two different exosites, have been selected.

123 , interactions with secondary binding sites (exosites) helped direct the specificity of these enzymes

124 ften bind noncovalently and weakly to Ub at "exosites." However, identification of such sites is typi

125 To define the role of this exosite, human factor IXa with alanine substituted for c

126 cating the involvement of both anion-binding exosite I (ABE-I) and anion-binding exosite II (ABE-II).

127 the catalytic site and the precursor form of exosite I (proexosite I).

128 ir-(54-65)(SO(3)(-)), characterized thrombin exosite I and II interactions with HCII and heparin in t

129 cal turn that directs the backbone away from exosite I and over the autolysis loop.

130 ults in the formation of the active site and exosite I and the exposure of exosite II.

131 t thrombin activation of PAR4 may occur with exosite I available to bind cofactor molecules, like the

132 Occupancy of exosite I by PAR3 allosterically changes the conformatio

133 nd, and whether binding of thrombomodulin to exosite I can allosterically shift the E* form to the ac

134 ed a concerted action of the active site and exosite I during ternary complex formation.

135 tivity to the platelet surface while leaving exosite I free for PAR-1 recognition.

136 * form and explain why binding of ligands to exosite I has only a modest effect on the E*-E equilibri

137 The involvement of exosite I in alpha-thrombin (FIIa) binding to platelet g

138 .48), suggesting a role for alpha-thrombin's exosite I in PAR4 activation.

139 -fold, reflecting the contribution of direct exosite I interaction with HCII.

140 compared with heparin binding alone and that exosite I is still available for ligand or HCII binding

141 Moreover, three exosite I ligands--aptamer HD1, hirugen, and lepirudin--

142 ermine the pathway of expression of Na+-(pro)exosite I linkage during ProT activation, the effects of

143 HCII binding to exosite I of heparin-bound [4'F]FPR-T caused a saturable

144 ome amino acids located in the anion binding exosite I of the protein in aptamer-thrombin interaction

145 hydrophobic and electrostatic contacts with exosite I of thrombin through the fragment (47)FEEFPLSDI

146 final complex disorders the active site and exosite I of thrombin, but exosite II is thought to rema

147 en alter Trp(148) orientation in a loop near exosite I preventing contacts with the sulfate oxygen at

148 thrombins, [4'F]FPR-T and [6F]FFR-T, and the exosite I probe, Hir-(54-65)(SO(3)(-)), characterized th

149 g Tyr(278) or Tyr(279), which mostly contact exosite I residues, reduced FIIa complexing in solution

150 t structures of human and murine WE bound to exosite I with a fragment of the platelet receptor PAR1,

151 rthologue anophelin, cE5 binds both thrombin exosite I with segment Glu-35-Asp-47 and the catalytic s

152 t of the protease activated receptor PAR1 to exosite I, 30-A away from the active site region, causes

153 uses three principal sites, the active site, exosite I, and exosite II, for recognition of its many c

154 tream residues A35-P45 shield the regulatory exosite I, defining a unique reverse-binding mode of an

155 ex with serpins and find that in addition to exosite I, exosite II is also disordered, as reflected b

156 g the long cleft between the active site and exosite I.

157 raction mainly by establishing contacts with exosite I.

158 strates that bridge both the active site and exosite I.

159 ing of hirudin-(54-65)(SO(3)(-)) and HCII to exosite I.

160 teraction of its NH(2)-terminal segment with exosite I.

161 ve binding mode bridging the active site and exosite I.

162 the exodomain of PAR1 that binds thrombin's exosite I.

163 retains the capacity to bind to PAR1 through exosite-I and may modulate its function independent of r

164 interacts with the thrombin active site and exosite-I.

165 -binding exosite I (ABE-I) and anion-binding exosite II (ABE-II).

166 Heparin binding to exosite II and a second weaker site caused fluorescence

167 that bind primarily in the region defined by exosite II and allosterically induce thrombin inhibition

168 e other hand, binds specifically to thrombin exosite II and has no affinity to prothrombin at all.

169 were inactivated at comparable rates, and an exosite II aptamer had no effect on the inactivation, su

170 fect on the inactivation, suggesting limited exosite II involvement.

171 pins and find that in addition to exosite I, exosite II is also disordered, as reflected by a loss of

172 e active site and exosite I of thrombin, but exosite II is thought to remain functional.

173 ly as much as aptamer HD22 and heparin, both exosite II ligands.

174 This disordering of exosite II occurs for all tested natural thrombin-inhibi

175 referentially binds in or near anion-binding exosite II of thrombin.

176 complementarity between the gamma' chain and exosite II or if there are critical charged gamma' chain

177 Mutating Tyr(276), which mostly contacts exosite II residues, markedly reduced FIIa interaction w

178 cipal sites, the active site, exosite I, and exosite II, for recognition of its many cofactors and su

179 cleft to a region corresponding to thrombin exosite II, which is known to interact with allosteric e

180 t of a basic interface that is also known as exosite II.

181 ibrinogen gamma' chain through anion-binding exosite II.

182 rin template between the serpin and thrombin exosite II.

183 ctive site and exosite I and the exposure of exosite II.

184 llular domain bind exclusively to thrombin's exosite II.

185 site-directed mutagenesis to identify a new exosite in caspase-6 at the hinge between the disordered

186 The role of the factor IXa heparin-binding exosite in coagulation was assessed with mutations that

187 omplex is mediated through a heparin-binding exosite in the FX serine protease domain.

188 An exosite in the hemopexin domain, which binds the leucine

189 shed more light on the critical role of the exosite in the spacer domain in substrate recognition.

190 We hypothesize that a modification of an exosite in the spacer domain may generate ADAMTS13 varia

191 Mutagenesis of the exosite in the V-B loop at Thr-205 and His-206 that vary

192 and Arg(150) residues that interact with the exosite in the x-ray structure of the Michaelis complex

193 r, there are only a few reports of potential exosites in ADAM protease structures.

194 Potential exosites in ADAM structures have been reported, but no s

195 bin, abrogated the ability of the engineered exosites in alpha1PI to promote factor Xa inhibition.

196 determine the involvement of thrombin's two exosites in GpIbalpha binding, we employed the complemen

197 n utilized to dissect the roles of potential exosites in MMP-9 collagenolytic behavior.

198 B8-furin Michaelis complex identified serpin exosites in strand 3C close to the 298-300 loop whose su

199 ose areas are designated substrate-dependent exosites, in that they accommodate different peptide str

200 The combined effect of the two exosites increased the association rate constant for the

201 two ADAM17 analogs, while a non-zinc-binding exosite inhibitor of ADAM17 showed significantly lower p

202 nopyranose from beechwood, is a multifaceted exosite inhibitor of the aggrecanases and protects carti

203 ve site to secondary substrate binding site (exosite) inhibitor discovery in order to identify non-zi

204 rtunities for therapeutic intervention using exosite inhibitors.

205 We hypothesized that secondary binding site (exosite) inhibitors should provide a viable alternative

206 , an anticoagulant activity that requires an exosite interaction between its basic C terminus and an

207 s studies have suggested the existence of an exosite interaction between LF and MKKs that promotes cl

208 y TFPIalpha mediated through a high-affinity exosite interaction between the basic region of TFPIalph

209 aggrecan cleavage predominately reflects the exosite interaction.

210 Multiple ADAMTS13 exosite interactions are involved in recognition of the

211 describing substrate features necessary for exosite interactions exist.

212 h a balance between attractive and repulsive exosite interactions in the native state is shifted to f

213 Gln(1624) and Arg(1668), and together these exosite interactions increase the rate of substrate clea

214 ation of C1s by this enzyme, indicating that exosite interactions were also important.

215 without altering the MMP-1 structure or the exosite interactions, by axial rotation of the collagen

216 gest a general strategy for mapping protease exosite interactions.

217 721, Glu724, and Asp725 likely constitute an exosite-interactive region in factor VIII facilitating c

218 This "exosite" interface endows an additional function for the

219 Additionally, interaction with the caspase-7 exosite involves both the Zn3 and BRCT domains of PARP-1

220 These results demonstrate that the exosite is a key determinant of antithrombin reactivity

221 show that the overall positive charge of the exosite is the critical feature of this evolutionarily c

222 Our data suggest that binding of zinc at the exosite is the primary route of inhibition, potentially

223 ght interactions with substrates occur at an exosite located approximately 30 A away from the catalyt

224 In MASP-2, an exosite located within the CCP domains recognizes the C4

225 Identification of these exosites may contribute to the design of inhibitors that

226 between C1s and C4 involves active site and exosite-mediated events, but the molecular details are u

227 In closed system models of fibrin formation, exosite-mediated thrombin binding to fibrin contributes

228 r Xa modestly enhanced the reactivity of the exosite mutant inhibitor with factor Xa by approximately

229 by selected mutations in the heparin-binding exosite (N178A, K126A, R165A).

230 e, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures

231 omains in ADAMTS13 bind to the spacer domain exosite of a truncated ADAMTS13 variant, MDTCS (KD of 13

232 mutagenesis experiments show that the alpha-exosite of BoNT/C1 plays a less stringent role in substr

233 thermore, if FIX binding via the heavy chain exosite of FXIa determines the affinity of the enzyme-su

234 s to properly anchor their N-terminus to the exosite of IDE and undergo a conformational switch upon

235 reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for c

236 unique size, shape, charge distribution, and exosite of the IDE catalytic chamber contribute to its h

237 These findings suggest that targeting exosites of ADAM17 can be used to obtain highly desirabl

238 ibitors targeting the active site as well as exosites of glutamate carboxypeptidase II (GCPII), a pro

239 development, we have probed several distinct exosites of NS3/4A which are either outside of or partia

240 chanism in which binding occurs when the two exosites of one FIIa molecule independently interact wit

241 C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues.

242 hrombin binding site, and both anion binding exosites of thrombin have been implicated in GpIbalpha b

243 binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as

244 In summary, we have identified an exosite on caspase-6 that is critical for protein substr

245 Binding is at an exosite on maspin close to, but outside of, the reactive

246 tial binding of fIX and fIXalpha requires an exosite on the fXIa A3 domain, but not the A2 or catalyt

247 Binding of factor IX (FIX) to an exosite on the heavy chain of factor XIa (FXIa) is essen

248 oes allosteric modification on binding to an exosite on the heavy chain of FXIa (FXIa-HC) required fo

249 ne that four Fabs simultaneously occupy four exosites on the beta-tryptase tetramer, inducing alloste

250 To resolve the effects of these exosites on the initial Michaelis docking step and the s

251 bstrate access by binding to active sites or exosites or by allosteric modulation.

252 suggest that the factor IXa heparin-binding exosite participates in both cofactor binding and protea

253 inspects both normal and damaged bases in an exosite pocket that is distant from the active site.

254 Moreover, additional bridging exosites provided by a hexadecasaccharide heparin activa

255 anine mutations of six putative antithrombin exosite residues and three complementary protease exosit

256 g-150 in factor Xa, which interacts with the exosite residues in heparin-activated antithrombin, abro

257 te residues and three complementary protease exosite residues on antithrombin reactivity with these p

258 ations of antithrombin Tyr(253) and His(319) exosite residues produced massive 10-200-fold losses in

259 f Asn(233), Arg(235), Glu(237), and Glu(255) exosite residues showed that these residues made both re

260 istent with previous studies and a potential exosite resulting from putative receptor trimerization.

261 between Ixolaris and FX heparin-binding (pro)exosite, resulting in an allosteric switch in the cataly

262 regulation of inhibitory activity unless the exosite(s) are engaged.

263 Exosite(s) on fXIa are required for fIX binding, however

264 clude that substrate recognition by the FXIa exosite(s) requires disulfide-linked heavy and light cha

265 recognizes ligands at the active site or at exosites separate from the active site region, but remar

266 rt to occupy both the catalytic site and the exosite simultaneously.

267 d have identified the MT-LOOP as a potential exosite target region to develop selective MT1-MMP inhib

268 iscovery of highly selective nonzinc-binding exosite-targeting inhibitors of ADAM17 that exhibited no

269 Exosite tethering of the substrate in either the zymogen

270 ops of activated protein C (APC) comprise an exosite that contributes to the binding and subsequent p

271 tease domain that appear to form a catalytic exosite that is required for efficient cleavage of C4.

272 proximal side of the heme in hIDO1 and in an exosite that is ~40 angstrom away from the active site i

273 The remote alpha-exosite that was previously identified in the complex of

274 ADAMTS13 function is dependent upon multiple exosites that specifically bind the unraveled VWF A2 dom

275 When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while

276 site-directed mutagenesis, we have mapped an exosite to a non-catalytic region of LF.

277 l how IDE utilizes its catalytic chamber and exosite to engulf and bind up to two NPs leading to bias

278 dly, we also found that Serpinb6b employs an exosite to specifically inhibit dimeric but not monomeri

279 tion, the oxoG lesion has transited from the exosite to the active site pocket, but has not undergone

280 ding of the ADAMTS13 disintegrin-like domain exosite to VWF allosterically activates the adjacent met

281 However, many serpins exploit additional exosites to generate the exquisite specificity that make

282 u-255 in the serpin antithrombin function as exosites to promote the inhibition of factor Xa and fact

283 ubstrate-binding pocket, caspase-7 also uses exosites to select specific substrates.

284 wly identified ligands binding at unique IDE exosites together to construct a potent series of novel

285 e centre loop (RCL) of PAI-1 and at the same exosite used by both tissue and urokinase plasminogen ac

286 2 AIP reactive loop residues in the alpha1PI exosite variant with a preferred IEG substrate sequence

287 Results from SPR, using a panel of APC exosite variants where basic residues were mutated, in b

288 The zinc in the exosite was liganded by Lys-36, Glu-244, and His-287 wit

289 Thus, by recombining exosites, we engineered ADAMTS5 to cleave a new bond in

290 analyses and database searches for candidate exosites, we utilized site-directed mutagenesis to ident

291 The effects of the engineered exosites were specific, alpha1PI inhibitor reactions wit

292 that this APC2 surface is also a Ub-binding exosite with preference for K48-linked chains.

293 hows how IDE utilizes the interaction of its exosite with the N terminus of the insulin A chain as we

294 E mutants reveal that the interaction of the exosite with the N-terminus of Ub guides the unfolding o

295 ibution of basic residues comprising the APC exosite, with significant contributions from Lys39, Arg6

296 Exosites within the MMP-9 fibronectin II inserts were fo

297 Most studies have focused on the role of exosites within the VWF A2 domain, involved in interacti

298 the binding of RelA to a negatively charged "exosite" within the SET domain of Set9.

299 rences in substrate secondary binding sites (exosites) within the MMP family.

300 Replacement of both exosites yielded a non-elastase variant similar to that