Ajuju nke intersubunit interface nke ọwa proton Hv1 mepere emepe nwere nyocha jikọtara ọnụ.

Daalụ maka ileta Nature.com. Ụdị ihe nchọgharị ị na-eji nwere nkwado dị nta maka CSS. Maka ahụmịhe kachasị mma, anyị na-akwado ka ị jiri ihe nchọgharị emelitere (ma ọ bụ gbanyụọ ọnọdụ ndakọrịta na Internet Explorer) ka ọ dị ugbu a, iji hụ na nkwado na-aga n'ihu, anyị ga-egosipụta saịtị na-enweghị ụdị na Javascript. Hv1 voltaji-gated proton channel bụ dimeric mgbagwoju nke nwere ngalaba abụọ voltaji-sensing (VSDs), nke ọ bụla n'ime ha nwere a gated proton permeation ụzọ.Dimerization na-achịkwa cytoplasmic coiled-coil domain.Ntughari si na mechiri emechi na steeti. steeti mepere emepe na VSD abụọ a mara na ọ na-emekọ ọnụ; Otú ọ dị, a maghị nke ọma banyere usoro ndị dị n'okpuru. Intersubunit interfaces na-ekere òkè dị mkpa na usoro allosteric; Otú ọ dị, a chọpụtabeghị oghere ndị dị otú ahụ na ọwa Hv1 na-emeghe. N'ebe a, anyị na-egosi na 2-guanidinothiazole ndị na-emepụta ihe na-egbochi Hv1 VSD abụọ n'otu n'otu ma jiri otu n'ime ogige ndị a dị ka nyocha maka njikọta allosteric n'etiti oghere ndị mepere emepe. Anyị chọpụtara na extracellular extracellular. njedebe nke mpempe transmembrane nke mbụ nke VSD na-etolite interface intersubunit nke na-emekọrịta njikọ n'etiti saịtị njikọ, ebe ngalaba mkpuchi mkpuchi anaghị etinye aka na usoro a. Anyị chọpụtakwara ihe akaebe siri ike na proton-selective filter ọwa na-achịkwa ihe mgbochi-njikọ nkwado. . Ọwa proton voltaji na-arụ ọrụ dị mkpa n'ụdị dị iche iche, site na phytoplankton ruo mmadụ1. N'ọtụtụ mkpụrụ ndụ, ọwa ndị a na-emezi proton efflux site na akpụkpọ ahụ proton ma na-achịkwa ọrụ NADPH oxidase. Naanị ihe a ma ama na ọwa proton voltaji gated n'ime mmadụ. bụ Hv1, nke bụ ngwaahịa nke HVCN1 gene2,3.Hv1 (aka VSOP) ka egosiri na ọ na-ekere òkè na mgbasa ozi B cell4, mmepụta nke ụdị oxygen na-emeghachi omume site na usoro ahụ ji alụso ọrịa ọgụ5,6,7,8, sperm cell. motility9 na ụkpụrụ pH nke mmiri elu elu ikuku10. Ọwa a na-etinye aka na ọtụtụ Overexpressed na ụdị ọrịa cancer dị ka B-cell malignancies4,11 na ara na ọrịa cancer colorectal12,13. Achọpụtara ọrụ Hv1 dị ukwuu iji mee ka ikike metastatic nke mkpụrụ ndụ cancer 11, 12 dịkwuo elu. N'ime ụbụrụ, a na-egosipụta Hv1. site na microglia, na ọrụ ya egosipụtara na-eme ka mmebi ụbụrụ dịkwuo njọ n'ụdị ọrịa strok ischemic. The Hv1 protein nwere a voltaji-sensing ngalaba (VSD), nke mejupụtara akụkụ anọ transmembrane a na-akpọ S1 ka S414. VSD yiri ngalaba kwekọrọ ekwekọ nke voltaji-gated Na +, K + na Ca2 + ọwa na voltaji-sensitive phosphatases, dị ka CiVSP si Ciona gutis15. N'ime protein ndị ọzọ, C-terminus nke S4 na-ejikọta ya na modul mmetụta, ngalaba pore ma ọ bụ enzyme. Na Hv1, S4 jikọtara ya na ngalaba coiled-coil (CCD) nke dị n'akụkụ cytoplasmic nke akpụkpọ ahụ. .Ọwa ahụ bụ mgbagwoju anya dimeric nke nwere VSD abụọ, nke ọ bụla nwere ụzọ gated proton permeation pathway16,17,18. Achọpụtara ihe abụọ Hv1 abụọ a ka ha na-emepe nkwado19,20,21,22, na-atụ aro na njikọ njikọ allosteric na mmekọrịta dị n'etiti-subunit na-egwu egwu. Ọrụ dị mkpa na usoro gating. A na-akọwapụta ihe dị n'etiti subnits n'ime ngalaba mkpuchi mkpuchi nke ọma n'ihi na ihe owuwu kristal nke ngalaba abụọ dịpụrụ adịpụ dị22,23. N'aka nke ọzọ, interface dị n'etiti VSD n'ime akpụkpọ ahụ adịghị. Aghọtara nke ọma. Ọdịdị kristal nke protein Hv1-CiVSP chimeric adịghị enye ozi gbasara interface a, dịka nhazi trimeric nke mgbagwoju anya ọwa crystal nwere ike ime site na ngbanwe nke Hv1 CCD nke ala site na yist leucine zipa GCN424. A na-adịbeghị anya ọmụmụ nke subunit nzukọ nke Hv1 ọwa kwubiri na abụọ S4 helices mgbanwe n'ime CCD na-enweghị oké ọgba aghara nke abụọ Ọdịdị, na-akpata ogologo helices na-amalite site akpụkpọ ahụ na oru ngo n'ime cytoplasm.Dabere na cysteine ​​​​crosslinking analysis. Ọmụmụ ihe a na-atụ aro na Hv1 VSD na-akpọtụrụ ibe ya n'akụkụ akụkụ S4. Otú ọ dị, ọmụmụ ihe ndị ọzọ akwadoro ụzọ ndị ọzọ dị n'etiti VSDs. Ndị a interfaces gụnyere S1 akụkụ 17, 21, 26 na mpụta nsọtụ nke S2 ​​akụkụ 21.A nwere ike ime ihe na-emegiderịta onwe ya. Nsonaazụ nke ọmụmụ ndị a bụ na a na-enyocha njikọ nke allosteric n'etiti VSD n'ihe gbasara usoro gating, nke dabere na steeti mechiri emechi na nke mepere emepe, na interface dị n'etiti VSD nwere ike ịdịgasị iche na mgbanwe steeti dị iche iche na nhazi. N'ebe a, anyị chọpụtara na 2-guanidinothiazole na-egbochi ọwa Hv1 site na synergistically na-ejikọta na VSD abụọ mepere emepe, ma jiri otu n'ime ogige ahụ, 2-guanidinobenzothiazole (GBTA), nyochaa mmekọrịta dị n'etiti subunits na steeti mepere emepe. interface.We chọpụtara na GBTA adịgide adịgide curve nwere ike kọwara nke ọma site a quantitative nlereanya nke njide nke inhibitor na otu subunit pụta na-abawanye na njikọ njikọ nke n'akụkụ subunit.We chọpụtakwara na residues D112, selectivity nzacha maka. ọwa 27, 28 na akụkụ nke guanidine derivative adịgide adịgide saịtị 29 akara GBTA adịgide adịgide cooperativity. Anyị na-egosi na nkwado njikọ na-nọgidere na Hv1 dimer, ebe CCD kewapụrụ na S4 akụkụ, na-atụ aro na intersubunit interface na CCD adịghị ozugbo. mediate allosteric njikọ n'etiti GBTA-njikọ saịtị. N'ụzọ dị iche, anyị na-ahụ na S1 iberibe bụ akụkụ nke interface n'etiti subunits na-atụ aro nhazi nke n'akụkụ VSDs na extracellular njedebe nke S4 helix pụọ n'etiti dimer na-ekwe ka. iberibe S1 ga-adị na steeti mepere emepe. Obere ihe mgbochi molecule nke Hv1 bara uru dị ka ọgwụ anticancer na ndị na-ahụ maka neuroprotective. Otú ọ dị, ka ọ dị ugbu a, ogige ole na ole nwere ike igbochi ọwa30,31,32,33. N'ime ha, 2-guanidinobenzimidazole (2GBI, compound [1] na Fig. . 1a) na ihe ndị na-emepụta ya ka achọpụtara na-egbochi VSD29,32 permeation nke protons site na ọwa. A na-eche na a na-ejikọta ogige ndị dị otú ahụ n'onwe ya na mpaghara abụọ mepere emepe.2-guanidinobenzothiazole (GBTA, compound [2] na Figure 1a). E gosipụtara na mbụ iji gbochie Hv1 fọrọ nke nta ka ọ dị irè dị ka 2GBI mgbe a nwalere ya na ntinye nke 200 μM (Njirimara 1b) .Anyị nyochara ihe ndị ọzọ na-emepụta thiazole ma chọpụta na ụfọdụ n'ime ha na-egbochi ọwa ahụ nwere ikike yiri nke ahụ ma ọ bụ karịa karịa GBTA (Fig 1 na mgbakwunye). ederede) .Anyị kpebisiri ike na ntụgharị nzaghachi uche nke thiazole anọ (GBTA na ogige [3], [6] na [11], Fig. 1c) wee chọpụta na ha dị elu karịa nke 2GBI. The Hill coefficients (h) nke thiazol sitere na 1.109 ± 0.040 ruo 1.306 ± 0.033 (Fig. 1c na Mgbakwunye Fig. 1).N'ụzọ dị iche, ọnụ ọgụgụ ugwu maka 2GBI bụ 0.975 ± 0.024 29, Fig. 2a na Fig. ebe a na-ekekọta,29,32 anyị chere na ijikọ nke thiazole sitere na otu subunit nwere ike ịkwalite njikọ nke molecule inhibitor nke abụọ n'akụkụ subunit dị n'akụkụ.GBTA bụ ihe nyocha nke nwere ọnụ ọgụgụ ugwu kachasị elu. Ya mere, anyị họọrọ ogige a ka gaa n'ihu mụọ usoro nke njikọta mmekọrịta ma jiri 2GBI mee ihe dịka akara nchịkwa na-adịghị mma. (a) Ngwakọta ule: [1] Reference Hv1 inhibitor 2-guanidino-benzimidazole (2GBI).[2] 2-guanidino-benzothiazole (GBTA), [3] (5-trifluoromethyl-1,3-benzothiazol-2-yl) guanidine, [4] naphtho[1,2-d] [1, 3] Thiazol-2-yl guanidine, [5] (4-methyl-1,3-thiazol-2-yl) guanidine, [6] (5-bromo-4-methyl-1,3-thiazol- 2-yl) guanidine, [7] famotidine, [8] 2-guanidino-5-methyl-1,3-thiazole-4-carboxylic acid ethyl ester, [9] 2-guanidino-4-methyl Ethyl-1,3-thiazole-5-carboxylate, [10] ](2-guanidino-4-methyl-1,3-thiazol-5-yl) ethyl acetate, [11] 1-[4- (4 -Chlorophenyl) -1,3-thiazol-2-yl] guanidine, 12] 1- [4- (3,4-dimethoxyphenyl) -1,3-thiazol-2-yl] guanidine. .Hv1 proton currents na-atụle n'ime plaques nke Xenopus oocytes na-azaghachi na nkwụsịtụ site na ikike ijide -80 mV ruo +120 mV. A na-agbakwunye onye na-emechi ihe ọ bụla na bat na ntinye nke 200 μM.pHi = pHo = 6.0 .Data bụ pụtara ± SEM (n≥4) .(c) Mgbochi na-adabere na ntinye uche nke mmadụ Hv1 site na ogige [2], [3], [6] na [11] . Ihe ọ bụla na-anọchite anya nkwụsịtụ ± SD nke 3. ruo 15 nha. Ahịrị bụ ugwu dabara adaba iji nweta ụkpụrụ Kd pụtara ìhè akọwara na tebụl mgbakwunye 1.Hill coefficients e kpebisiri ike site na dabara nke akọwara na Fig. 1: h (1) = 0.975 ± 0.024 h (2) = 1.306 ± 0.033, h (3) = 1.25 ± 0.07, h (6) = 1.109 ± 0.040, h (11) = 1.179 ± 0.036 (lee Ụzọ). (a,b) Ngwakọta 2GBI na GBTA na-egbochi dimeric na monomeric Hv1 n'ụzọ na-adabere na ntinye uche. Isi ihe ọ bụla na-anọchite anya nkwụsị nke ± SD nke 3 ruo 8 nha na usoro ahụ bụ ugwu fit. The Hill coefficients (h) gosiri na. Achọpụtara histogram inset site na dabara nke akọwara na fig 3 na 4 nke mgbakwunye. Nzaghachi nke GBTA nke egosiri na (a) bụ otu ihe ahụ dị na Fig. njikọ nkwado nke GBTA na dimeric Hv1. Akara ojii siri ike na-anọchi anya ihe dabara adaba na data nnwale site na nhata (6), nke na-akọwa ihe njiri mara nke egosipụtara na (d) .Akara ndị a gbajiri agbaji akpọrọ Sub 1 na Sub 2 na-anọchite anya mkpakọrịta bimolecular. -dissociation equilibrium curves of the first and second links, n'otu n'otu (Sub 1: OO + B ⇄ BO*, Kd1 = 290 ± 70 μM; Sub 2: BO* + B ⇄ B*O*, Kd2 = 29.3 ± 2.5 μM .(d) Eserese eserese nke usoro a tụrụ aro nke ngọngọ Hv1. N'ihe gbasara GBTA, ijikọ na otu ngalaba mepere emepe na-eme ka njikọ nke ngalaba mepere emepe dị nso (Kd2 Hv1 ọwa nwere ike monomerized site na-edochi ha N- na C-ọnụ cytoplasmic ngalaba na kwekọrọ ekwekọ akụkụ nke Ciona Intestinalis voltaji phosphatase CiVSP (Hv1NCCiVSP) 18,34.We tụrụ na ịta ndabere nke inhibition nke monomeric Hv2GBI na GBTA. tụnyere ha na nke dimeric ọwa (ụdị anụ ọhịa) mgbochi (Fig. 2a,b) .Anyị chọpụtara na ọdịiche dị na njikọ njikọta n'etiti ogige abụọ ahụ na-ekpochapụ na monomeric Hv1, na-akwado nkọwa na GBTA na-ejikọta na otu akụkụ na-abawanye. Affinity nke ngalaba nke ọzọ maka inhibitor. Anyị chere na ijikọ GBTA na otu akụkụ Hv1 nwere ike iduga nhazigharị saịtị nke njikọ (na-ebute fit35), nke ga-eme ka nhazigharị nke oghere oghere nke mpaghara dị n'akụkụ, na-eduga n'ịbawanye njikọ. mmekọ. Iji kọwaa n'ụzọ zuru ezu njikọ njikọta nke GBTA na ọwa ahụ, anyị na-eji ihe atụ nke nke ọ bụla n'ime ha nwere ike jikọta molekul inhibitor nke mbụ na-esote mmeghachi omume bimolecular na nkesa Kd1 mgbe niile (Fig. 2c, Sub 1, OO+ B ⇆ BO *). Njide na-eme ka ọwa ahụ nweta ọnọdụ nke akụkụ ndị fọdụrụ na-adịghị efu na-ejikọta na inhibitor na-eso mmeghachi omume bimolecular pụrụ iche na nkesa mgbe niile Kd2, ebe Kd2 Ozugbo molecule inhibitor nke abụọ na-ejikọta, ma subunits nwere dissociation mgbe niile Kd2 (Fig. 2d) .Channel mgbochi a tụrụ n'okpuru ọnọdụ depolarizing (+ 120 mV) .Channel ụdị nwere otu oghere na otu nke mechiri emechi (lee mgbanwe CC ⇄ OC ⇄). OO dị na Fig. 2d) ka achọpụtara na ọ na-enye aka n'ụzọ na-ezighi ezi na Hv1 dị ugbu a n'okpuru ọnọdụ ndị a19,20 ma ya mere etinyeghị ha n'ụdị njide. Ahịrị ojii siri ike dị na Figure 2c bụ ihe dabara adaba nke nha nhata na ntụgharị nzaghachi nzaghachi nnwale, na-enye Kd1 nke ~ 290 μM na Kd2 nke ~ 29 μM (Ngalaba Usoro, nha nha (6)) . njide nke 2GBI, ebe Kd2 ≈ Kd1 = Kd (Fig. 2d) .Na monomeric Hv1, e nwere nanị otu saịtị njikọ na otu Kdm (O ° + B ⇆ B °) .N'ihe gbasara GBTA, Kdm dị gburugburu 54. μM, nke yiri Kd1 karịa Kd2 (Fig. 2d) .N'ikwu ya n'ụzọ ọzọ, ebe a na-ejikọta monomer dị na nhazi (O °) dị ka ọnọdụ njikọ dị elu (BO *) karịa ala- Affinity state (OO) nke dimer, o yikarịrị ka n'ihi mkpochapụ nke interface n'etiti subunits. Dị ka e gosiri na mbụ maka 2GBI32, anyị chọpụtara na GBTA kwụsịrị Hv1 iyi site na igbochi ọwa mgbe ọ na-emeghe kama ime ka o sie ike imeghe (Mgbakwunye 2a, b, d) .2GBI na-marakwa na ọ na-eme ka ọdụ ụgbọ mmiri na-agbaji nwayọọ nwayọọ. na nzaghachi nke akpụkpọ ahụ repolarization, ma nke a na-ahụghị na GBTA (Mgbakwunye Fig. 2c) .N'ihu Hv1 inactivation na ọnụnọ nke 2GBI, ọnụ ụzọ ámá na nke ọ bụla n'okpuru nke ọ bụla enweghị ike imechi ruo mgbe onye na-egbochi na-ahapụ ebe a na-ekekọta (na "" ụkwụ ọnụ ụzọ ámá" usoro), na 2GBI unbinds nwayọọ karịa ọnụ ụzọ ámá nso. Ọ bụrụ na otu Hv1 subunit na-unlocked na mechie, mgbe n'akụkụ subunit na-anọgide na-egbochi, unbinding nke fọdụrụ 2GBI molekul na-aghọ nwayọọ nwayọọ (blocker njide). naanị otu subunit egbochiri omume protons nwa oge tupu emechie ma nye aka dị ukwuu na ọdụ ọdụ na-eji nwayọ na-emebi. Nchọpụta na nrụrụ nke ọdụ ụgbọ mmiri Hv1 adịghị ebelata nke ukwuu n'ihu GBTA (Mgbakwunye 2c) na-adabere na usoro njikọ njikọ maka ihe mgbochi a (Fig 2d) . Ozugbo GBTA molecule na-esi na Hv1 subnit. , Njikọ nke onye na-egbochi onye na-egbochi ya na mpaghara dị n'akụkụ na-adaba ihe dị ka okpukpu 10, na-akwado usoro nkwụsị ahụ. Nke a pụtara na molekul nke abụọ nke GBTA nwere ohere dị elu karị nke ịkọwapụta tupu ya ejide ya na ọwa. mepụta naanị otu subunit mgbochi ka a na-atụ anya na ọ ga-aba ụba karịa na ọnụnọ GBTA karịa ọnụnọ 2GBI, na-ebute ire ere ọdụ ngwa ngwa ugbu a. Ka GBTA wee jikọta ọnụ na mpaghara Hv1 abụọ ahụ, saịtị njikọ ahụ ga-ejikọrịrị nke ọma. Ebe njide ọ bụla ga-enwerịrị ike: 1) ịkpalite usoro ihe omume na-ekwurịta okwu na mpaghara ndị dị n'akụkụ jikọtara ya na inhibitor, yana 2) na-emekọrịta ihe. mgbanwe site na njikọ dị ala na njikọ njikọ dị elu. Anyị chere na ọ bụrụ na ihe ndị fọdụrụ n'ime ebe a na-ejikọta ya na-enye aka na usoro nkwado ahụ, ngbanwe ha kwesịrị ịgbanwe ngbanwe nke ugwu nke ntụgharị nzaghachi nzaghachi nke GBTA mgbochi nke Hv1.Residues D112, F150 , S181 na R211 e gosiri na mbụ ịbụ akụkụ nke 2GBI29 njide gburugburu ebe obibi na anyị chere na ha ga-n'otu aka ahụ na-etinye aka na GBTA njikọ (Fig. 3A) .We tụrụ inhibitory ịta-aza curves nke GBTA maka mutant ọwa D112E, F150A, S181A. , na R211S (Nyocha 3) ma jiri ha tụnyere Hill coefficients na nke ụdị anụ ọhịa Hv1, na-eji 2GBI dị ka ntụaka. Residue V109 dị n'otu ihu nke akụkụ S1 dị ka D112 ma nwee otu ntụgharị helical ọzọ n'ime cell. V109 etinyeghị aka na njikọ nke 2GBI29, anyị jiri V109A mutant dịka njikwa (Fig. 3b). (a) Atụ aro Hv1 residues na-etinye aka na guanidine derivative binding. Akpụkpọ anụ na-acha anụnụ anụnụ gbara gburugburu n'agbụ akụkụ ndị a tụrụ anya na mbụ nke na-emekọrịta na akụkụ dị iche iche nke ogige 2GBI29. ọchịchịrị uhie) .Nke ọ bụla na-anọchi anya ihe pụtara mgbochi nke 3 ka 12 nha ± SD V109 dị ka a na-adịghị mma control.Curves bụ Hill dabara iji nweta pụtara Kd ụkpụrụ (lee Mgbakwunye Tebụl 1) .The Hill coefficients (h) gosiri na The inset histograms e kpebisiri ike site na dabara nke kọrọ na Mmeju 3 na 4. Reference h ụkpụrụ maka Hv1 WT ka egosiri dị ka akara akara. Asterisks na-egosi statistically dị iche n'etiti mutant na WT amaokwu (p 0.05) mbelata na ugwu ọnụọgụ nke GBTA ejikọta na GGG ọwa ma e jiri ya tụnyere ụdị anụ ọhịa (Fig 5c), na-atụ aro na n'agbanyeghị ọrụ ya na-edebe akụkụ abụọ jikọtara ọnụ dị mkpa, mana CCD anaghị agbazi njikọ njikọta nke njikọ njikọ n'etiti saịtị GBTA. (a) Eserese eserese nke Hv1 dimer nwere ngbanwe triglycine na nsọtụ ime nke S4 emebere iji mebie njikọ njikọta nke ngalaba cytoplasmic coiled-coil (akụ na-acha anụnụ anụnụ) na-emekọrịta ihe. Mgbanwe egosipụtara, nke e mere iji nwalee iberibe S1 na-etinye aka na njikọta n'etiti subunits (akụ na-acha anụnụ anụnụ).(c-h) 2GBI (cyan) na GBTA (acha ọbara ọbara gbara ọchịchịrị) na-egbochi ihe ndị a na-egosi na ntinye uche. ± SD nke nha 3 ruo 10. Usoro ahụ bụ ugwu dabara adaba iji nweta ụkpụrụ Kd pụtara ìhè (lee tebụl mgbakwunye 1) .Ekpebiri ọnụọgụ ugwu dị na histogram interpolated dị ka akọwara na ngalaba ụzọ (lee fig 3 na 4 mgbakwunye) .Reference h ụkpụrụ maka A na-egosi Hv1 WT dị ka ahịrị ndị a kpụrụ akpụ. Asterisks na-egosi ọdịiche pụtara ìhè n'etiti mkpụrụedemede mutant na WT (p 0.05, Fig. 5d, i) ). N'aka nke ọzọ, mutation D123A budata ebelata ugwu ọnụọgụ nke GBTA (p 0.05/14). Ebe ọ bụ na neutralizing ụgwọ na ọnọdụ 123 na abụọ subunits mere ka a siri ike mgbanwe na GBTA adịgide adịgide cooperativity, mgbe reversing ụgwọ na abụọ subunits nwere nanị obere mmetụta, anyị na-agbatịkwu analysis na-agụnye nanị otu subunit na inversion ọwa nke charge. emepụtara Hv1-njikọ dimers na D123R nnọchi na ngalaba C-terminal (Fig 5b) wee tụọ mgbochi nzaghachi nzaghachi site na GBTA na 2GBI. Anyị chọpụtara na ọnụọgụ nke Hill nke GBTA na-ejikọ na ọwa WT-D123R dị elu karịa nke ahụ. nke ụdị anụ ọhịa Hv1 (p 0.05). Anyị chọpụtakwara na na enweghị βME, ọnụọgụ nke Hill nke GBTA na-ejikọta na D112E I127C Hv1 dimer dị elu karịa nke a tụrụ na ọnụnọ nke mbenata ndị ọrụ (Fig. 6e na Mgbakwunye Fig. 4), na-egosi na Mgbanwe D112E. emeghị ka mmụba nke GBTA na-ejikọta cooperativity sitere na cross-linking of cysteine ​​​​127. The Hill coefficient of 2GBI binding to D112E, I127C Hv1 dimers e nwekwara mmetụta dị ukwuu site na D112E mutation (Figure 1) .6d na Mmeju. Foto 3). Ejikọtara ọnụ, nchoputa ndị a na-egosi na njikọ GBTA na-abawanye site na mmekọrịta dị n'etiti nsọtụ mpụta nke iberibe S1 dị n'akụkụ Hv1 dị n'akụkụ site na mmekọrịta electrostatic na-adọrọ adọrọ ma ọ bụ site na nguzobe nke njikọ covalent n'etiti cysteine ​​​​alosteric njikọ n'etiti saịtị na-abawanye ma na-ebute nkwado nkwado. Ọ bụ ezie na mmetụta nke mmekọrịta electrostatic na-adọrọ adọrọ na imekọ ihe ọnụ nwere ike ịkwụsị site na mutation D112E, mmetụta nke agbụ covalent enweghị ike. Na nyocha anyị nke oghere kemịkalụ dị maka ijikọta ihe ndị na-emepụta guanidine na ọwa Hv1, anyị chọpụtara na 2-guanidinothiazoles dị ka GBTA nwere ntụkwasị obi dị elu karịa 2-guanidinobenzimidazoles (Fig 1c) .The Hill coefficient analysis of GBTA link to both the dimeric na ọwa monomeric (Fig. 2a,b) na ọwa dimeric nke otu subunit na-ejikọta ya na onye na-emechi ya (Fig 4) mere ka anyị kwubie na mgbochi nke Hv1 site na GBTA Ihe omume ahụ bụ usoro synergistic, na A na-ejikọta saịtị njikọ nke ogige ndị dị na mpaghara abụọ ahụ allosterically. Nchọpụta na GBTA na-ejikọta na ọwa oghere, dị ka e gosiri na mbụ maka njikọ 2GBI32 metụtara ya, na-egosi na enwere ike nyochaa njikọ njikọ allosteric kpọmkwem na steeti mepere emepe.Onyinye nkwado anyị. nwere ike ịkọwa ihe mgbochi nke HV1 site na GTTA (Fig 2) ma kọwaa nsonaazụ dị iche na GATRA na nbibi, nke na-akwado nkọwa anyị. Ikike kachasị elu nke enwere ike ibute na protein abụọ nwere saịtị abụọ (dịka HV1) nwere ọnụ ọgụgụ dị mma nke 1.81 na HV1 I127c.the Igwe na-enweghị isi, ọdịiche dị n'etiti ndị na-enweghị ike na saịtị ndị dị ala na nke kachasi ike (HCLIL / BLAMARY / Nile na HV1 I127c.the nke oxygenlobin bụ ihe atụ kachasị ewu ewu na nke ọma nke usoro mmekọrịta mmadụ na ibe ya) KCAL / MOL, dabere na ọnọdụ nnwale38.Tus, n'ihe gbasara ụwa, ndị na-emekọ ihe ọnụ na-enweghị isi na HV1 Na Syney Modey, njikọta nke GTTA Molecules na otu usoro na-arụpụta ihe na-arụ ọrụ na-arụ ọrụ na-eme ka a na-ekekọta ihe dị nso. Mgbanwe na mmekorita n'etiti nduru.in na mgbanwe ndị a, na-agbanwe saịtị ha, na-ebute usoro a, na-arụ ọrụ na-arụ ọrụ na-ahụ maka njikọta nke njikọta E gosipụtara na ọ bụ akụkụ nke HV1 Proton Ormodzọ na-eme ka ọ bụrụ ihe na-eme ka ọ bụrụ nhọrọ prove27,28. Nsonaazụ anyị na-egosi na a na-egosi ihe nzacha nke HV1 abụọ Na klọọkụ kristal nke HV1-Civersp Chimera.sugcals na-ahụ maka njikọta ojii na-egosi na ọ bụ akụ ojii. (a) atụmatụ nke HV1 vsd.Basal na kristal usoro nke HV1-Civensp Chimera, a na-egosi usoro enyemaka na-enweghị isi. A na-egosi ọkwa nke GTTA GTTA dị ka isi awọ .black akụ na-egosi na ị na-ejikọ ọnụ na mpaghara abụọ dị iche iche. na nhazi dị iche iche dị iche iche dị ka hụrụ site na akụkụ ahụ nke memplane. nkewa nke ozé nke oz o, àkù nke di egwu) Na nhazi ihe owuwu ahụ dị na P140 kristal 6.80 na Civsp dabara na ọnọdụ D123 na HV1. Nsonaazụ anyị na-egosi na mmekọrịta na-akpali akpali n'etiti ngbanwe 123 (123d / 123R / 123r) na-eme ka ọ bụrụ na a na-emeghe ihe na-agagharị (123d / 123R) , na-atụ anya nkwado (Fig 5g) Nkọwa bụ na ịkapụta ihe na-ebute ihe na-etinye hydrophilic nwere ike ịkachasị mmachi ihe dị n'etiti D123 na-eme ka ọ dị na Ngalaba Nchọpụta Ọnọdụ D171 nke ciona Guis CI-HV1 (nke kwekọrọ na DV1) na-arụ ọrụ, na-akwado echiche ahụ na mpaghara D123 dị na gburugburu hydropic na steeti hydrophilic na steeti hydropic. A maara ọwa nke HV1 na-eme site na ọtụtụ ntụgharị19,20,3,26,30,39,40,40,41.Quu na-eme ka mgbanwe nke ci-HV1 na-eme ka ọwa nwee ike , sochiri mgbanwe dị iche iche nke na-eme ka protons mepere emepe na ụzọ abụọ. Ihe ngosi nke mgbama ahụ na-agbanwe agbanwe na-agbanwe agbanwe na ọnụnọ nke njikọta nhọrọ nke usoro mgbanwe nke usoro mmekọrịta na-achọpụta na D123 residuect na steeti ma megharia mmekorita nke abuo n'etiti saịtị GTTA. Fujiwara Et25 tụpụtara na interface nke Cytoplasmic nke Cytoplasmic Coded-Counts Nchịkọta VSD na nyocha nke mpaghara na-ejikọ s4 Helix na CCDELS na-achọ ihe na-aga n'ihu na-emeghe, HV1 Corlink na-eme n'okpuru ọnọdụ ebe ọwa ahụ na-emechi. Ya mere, nyocha S4-S4 nwere ike igosipụta usoro nhazi obodo nke na-aga n'ihu na-eme ka ọ bụrụ na ọwa ahụ nwere ike ịnwe nhazi dị iche iche na emeghe Ọ na-ekwuchitere, echiche kwekọrọ na nchọta nke monat et al. S1 na-akpali 39 n'oge egwu. N'ebe a, anyị na-egosi nke a, na njedebe nke oghere, nsọtụ nke S1 Heallix dị nso iji kwado mmekọrịta elektrọnik.in onye na-emekọrịta ihe na-agbatị ụkwụ Ewezuga ibe ya na ibe ya. Nwanyi elekere 20 Celsius elekere nke abụọ Vsd Site na nchọpụta anyị (Fig 7b, aka nri). Ọ bụ ezie na a na-eche echiche a na-eche maka monomer, a na-ejikwa ya na-eme ihe ọ bụla. maka HV1 (Fig). 7C) Na Conspsp Dives na-atụ aro na vsds nke protein ndị a nwere ụdị ihe a na-eme na-etolite interface ebe ngwụcha nke S1 mụ na ya na-emekọrịta ihe. Ọrụ dị mkpa nke HV1 na Sperm Cell na-eme ka ọwa mara mma a na-eme ka ọ bụrụ nke na-eme ihe na-enweghị atụ. Enwere ike iji ndị ọrịa 12 ma ọ bụ ọrịa cancecan 13 ma echekwara ka ndị na-ahụ maka ihe na-arụ ọrụ na-aghọ ihe dị ka ndị na-ahụ maka ihe omume. Mepee usoro nhazi HV1, na-eduga iji nweta mmekọ nwoke na nwanyị, nwere ike ibute mmepe nke ọgwụ ndị ọzọ siri ike na-ezuru ọwa HV1. Emebere megharia saịtị HV1 na-eji usoro ọkọlọtọ nke HV1NCCCIVESP rụọ ọrụ, dochie 1-113 na 240-57 nke Civ1 - Njikọ HV1, A na-ejikọ C-Tecktus na-ejikọ na ntụtụ nke usoro nke abụọ site na ggsgsgsgsgsgsgsgrgsgsgsgsgsgs.The pgemigs na-ejikọ aka na-eme ya A na-etinye oge p7 missis nke MMACHIE (Abionbọchị). site na nduru bioscion.follolongung.follo ala na NDNA, 2 MM KACL, 1 mm HCL, 1 mm / mm / mm / mm / mm 7.2 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 Celsius C . 2-Guanidino-BenZidazole [1], 2-guanidino-benzotqole [2] -Tyazol-2-yl) guanidine) [6] 5-carnetylate [9] na (granidino-4-methy-1,3-yl) sitere na sigry.fomotidine [7] bụ [4) - (4-chlorophenylyl -1,3-tiazol-2-thididine 2- (3- (3-4-tiazol-2-yl] guaridine [12] si Matrix sayensi. opekata mpe 99% ọcha. Ka nkwusioru nke Amino-4- (trifluooomethyl) Benzenethylide (1.5 mm hydrochloric acid (380 mg, 4.5 mmlol), na heteogeneous ngwakọta A na-echegharị nke na-akpali agụụ mmekọahụ maka 4 awa. 65 Celsius C) ruo ọtụtụ awa, ma tinye ya na Ethl acetate / petrolem eart iji nye ihe siri ike (500 mg, 48%); MP 221-22 ° C (Light 225-226 Celsius C) 45; 1h nmr (500 mhz, DMSO-D6): 7.25 (nke ukwuu s, 1 h, 1 h, 11 h), 1 h), 7.92 (d) , 1 H, J = 8.1 nom nmr (200 mhz, DMSO-D6), 117.5 HZ), 121.6, J = 276.1 (2, 126.1 h = 272 hz), 134.5, 152.5.5.5.5.5.0.0416, achọtara : 261.0419. Naftho [1,2-d] Tiazol-2-AMG (300 mg, na-ekwupụta ihe a na-akọwara na mbụ, ọ na-ekwupụta ya na mbụ .300 mg) Cynamide na 1.0 ml conce.Tto a na-ekpo ọkụ na-ekpo ọkụ na hydrochloric acid ngwa ngwa na a na-edebe ngwakọta na mmiri ịsa ahụ. gbajiri n'ime obere iberibe ma jiri mmiri saa mmiri na-acha odo odo. (38 mg, 10%) MP 246-250 Celsius C; 1h nmr (500 mhz, dmso-d6, d2o): 7.59 (t) h, 1 h, 1 h, 1 h), 7.77), 7.77 (d, 1 h, 1 h , J = 8.6 HZ), 7.89 (d, 1 h, 1 h, 1 h, 1 h, 1 h, 1 h, 1 h, 1 h, 1 h) .13c nmr (15) MHz, DMSO-d6): δ = 119.9, 122.7, 123.4, 123.6, 126.5, 127.1, 128.7, 132.1, 140.7, 169.1.HRMS (ESI): m/z calculated value.For C12H11N4S (M + H)+: 243.0699 , achọtara: 243.0704. A tụlere ihe ndị na-eme ihe nkiri na mpụga nke oocytes na-ekwupụta ihe dị iche iche na-egosipụta usoro axon (ngwaọrụ molectifier na-achịkwa ya: 100 mm 2- (n-mo-morphonino ) Ethanesulfonic acid (mes), 30 mm Tetramyylatey (tii) Mescol-Bis (AMMENENENENENENT-BIS), n tatraagetic acid (egta), na-agbanwe PH 6.0 na tii up hydroxide.al na-arụ ọrụ na 22 ± 2 yhz, sampled na 5 khz na ngụkọta clampfit10.2 (ngwaọrụ molecular) ) na mbido.1 (Tymab). Ngwọta nke nwere ihe dị iche iche nke HV1 na ọnọdụ ụfọdụ na oge ụfọdụ 10 mm βMe ka a na-asa ahụ na VC-6 Persion site na Plampy Software TTL (Translistor- A na-arụ ọrụ plọgịs.rapid na-eme ihe ngosi site na iji scti-tu kwade.) +120 MV na-eme ihe nhata na-arụ ọrụ dị ka ndị akọwapụtara na mbụ na voltaji dị iche iche site na -20 mv ma ọ bụrụ na ọ ga-ekwu okwu ule. A na-ejizi ya maka ire ere dị ugbu a. Site na ugwu ugwu: ebe [m] bụ ịta ahụ na-enweghị uche nke m na H bụ Hill Cofieflable.To na-agbakọ usoro dị ka: