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

4 recognizes the C4 C345C domain through a CCP exosite.

5 and activators occupying a ubiquitin-binding exosite.

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

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

8 rombin, a protease that does not utilize the exosite.

9 dulated by occupation of the heparin-binding exosite.

10 which are critical for the formation of the exosite.

11 mechanistic insight into the binding of the exosite.

12 the binding of ligands to its two principal exosites.

13 y around the catalytic domain to reach novel exosites.

14 llostery and the presence of putative FXIIIa exosites.

15 eases by generating new protease interaction exosites.

16 teraction of inhibitor with protease through exosites.

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

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

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

20 Different exosite 1 ligands with widely varied thermodynamic signa

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

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

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

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

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

26 he large loop corresponding to anion binding exosite 1.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

56 BoNT/A3 and BoNT/A4 will likely effect alpha-exosite and S1' subsite recognition, respectively.

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

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

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

60 5 can bind to fXa at the active site, or the exosite, and to fX at the exosite.

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

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

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

64 bin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion bindi

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

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

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

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

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

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

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

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

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

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

75 Exosite binding drives substrate affinity and is indepen

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

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

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

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

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

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

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

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

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

85 ate complex significantly favors the initial exosite complex.

86 ed AT and S195A fXa, revealing the extensive exosite contacts that confer specificity.

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

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

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

90 Importantly, the exosite-defective antithrombins bound heparin with nearl

91 or IXa with mutations in the heparin-binding exosite, demonstrated that relative affinity (K(i)) for

92 Exosite-dependent binding of prothrombin to prothrombina

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

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

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

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

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

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

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

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

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

102 Through exosite engagement, GPIbalpha may influence FIIa-depende

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

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

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

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

107 hich (Q315F(*149)) has the oxoG lesion in an exosite flanking the active site and the other of which

108 eavage where substrate initially binds at an exosite, followed by binding of the appropriate peptide

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

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

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

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

113 domain, which exposes the scissile bond and exosite for interaction with complementary sites on ADAM

114 osed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation.

115 255, or a residue proposed to constitute the exosite from modeling studies, Glu237, all produced mini

116 spacer domain from ADAMTS13 or deleting the exosite from the VWF substrate reduced the rate of cleav

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

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

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

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

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

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

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

124 her, the Na+ enhancement in MzT activity and exosite I affinity may function in directing the sequent

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

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

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

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

129 Occupancy of exosite I by PAR3 allosterically changes the conformatio

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

150 ctropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) t

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

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

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

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

155 raction mainly by establishing contacts with exosite I.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

176 n binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate tethe

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

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

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

180 rin template between the serpin and thrombin exosite II.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

195 rpin, antithrombin, by heparin generates new exosites in strand 3 of beta-sheet C, which promote the

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

197 Further exploration of MMP active sites and exosites, in combination with substrate conformation, ma

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

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

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

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

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

203 rtunities for therapeutic intervention using exosite inhibitors.

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

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

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

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

208 aggrecan cleavage predominately reflects the exosite interaction.

209 e native substrate, aggrecan, occurs through exosite interactions and peptide sequence recognition.

210 ization of LDA through the disruption of the exosite interactions between PAI-1 and tPA induced an in

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 ontributions to these interactions; and that exosite interactions reduced k(off) for the Michaelis co

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

216 owed that Tyr-253 was a critical mediator of exosite interactions with S195A factor Xa; that Glu-255,

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

218 gest a general strategy for mapping protease exosite interactions.

219 721, Glu724, and Asp725 likely constitute an exosite-interactive region in factor VIII facilitating c

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

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

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

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

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

225 of inhibition differs dramatically from the exosite mechanism of inhibition seen with the DNase coli

226 Rapid kinetic studies showed that these exosite-mediated enhancements in Michaelis complex affin

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

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

229 Competitive binding and kinetic studies with exosite mutant antithrombins showed that Tyr-253 was a c

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

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

232 that the ADAMTS13 spacer domain binds to an exosite near the C terminus of the VWF A2 domain.

233 act with protease secondary substrate sites (exosites), nonactive site-binding inhibitors can be iden

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

235 of NAP5 surprisingly interacts with the fXa exosite of a symmetry-equivalent molecule forming a shor

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

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

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

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

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

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

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

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

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

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

246 Requirement for both anion binding exosites of the enzyme has been suggested for the activa

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

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

249 ons of the Pro-1645-Lys-1668 region with the exosite on ADAMTS-13 play a significant role in mediatin

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

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

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

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

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

255 nclude that binding to two substrate-binding exosites one on the heavy chain and the other on the lig

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

272 Exosite tethering of the substrate in either the zymogen

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

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

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

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

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

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

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

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

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

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

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

284 Thrombin utilizes two anion binding exosites to supplement binding of fibrinogen to this ser

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

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 A cleavage product containing the exosite was a hyperbolic mixed-type inhibitor of ADAMTS1

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

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

291 To determine which residues comprise the exosites, we mutated strand 3C residues that are conserv

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

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