Tambayoyi na intersubunit interface na tashar Hv1 proton mai buɗewa tare da bincike mai haɗaka.

Na gode da ziyartar Nature.com. Sigar burauzar da kuke amfani da ita tana da iyakataccen tallafi ga CSS. Don ƙwarewa mafi kyau, muna ba da shawarar ku yi amfani da sabunta burauza (ko kashe yanayin dacewa a cikin Internet Explorer). ci gaba da goyon baya, za mu nuna shafin ba tare da salo da JavaScript ba. Tashar tashar proton mai ƙarfin wutar lantarki ta Hv1 wani hadadden dimeric ne wanda ya ƙunshi yankuna biyu masu ɗaukar ƙarfin lantarki (VSDs), kowannensu yana ƙunshe da hanyar shiga cikin gated proton. yanayin budewa a cikin VSD guda biyu an san yana faruwa tare da haɗin gwiwa; duk da haka, an san kadan game da hanyoyin da ke da tushe.Intersubunit musaya suna taka muhimmiyar rawa a cikin matakai na allosteric; duk da haka, ba a gano irin waɗannan hanyoyin sadarwa a cikin tashoshin Hv1 masu buɗewa ba. Anan mun nuna cewa abubuwan da suka samo asali na 2-guanidinothiazole sun toshe Hv1 VSD guda biyu a cikin hanyar haɗin gwiwa kuma suna amfani da ɗaya daga cikin waɗannan mahadi a matsayin bincike don haɗin haɗin gwiwa tsakanin budewa. Mun gano cewa extracellular. Ƙarshen ɓangaren ɓarna na farko na VSD yana samar da hanyar sadarwa ta intersubunit wanda ke daidaita haɗin kai tsakanin shafukan dauri, yayin da yanki mai naɗaɗɗen ba ya shiga cikin wannan tsari kai tsaye. Mun kuma sami shaida mai ƙarfi cewa tashar ta proton-zaɓin tacewa yana sarrafa haɗin gwiwar toshe-dauri. . Tashoshin proton masu ƙarfin lantarki suna taka muhimmiyar rawa a cikin nau'ikan halittu daban-daban, daga phytoplankton zuwa ɗan adam1.A cikin mafi yawan sel, waɗannan tashoshi suna yin sulhu tsakanin proton efflux daga membrane na proton kuma suna daidaita ayyukan NADPH oxidase.Babban tashar tashar proton da aka sani kawai a cikin mutane. shine Hv1, wanda shine samfurin HVCN1 gene2,3.Hv1 (aka VSOP) an nuna yana taka rawa a cikin yaduwar kwayar halitta ta B4, samar da nau'in oxygen mai amsawa ta hanyar tsarin rigakafi na asali5,6,7,8, kwayar maniyyi. motility9 da tsarin pH na ruwan saman hanyar iska10. Wannan tashar tana da hannu a cikin da yawa Overexpressed a cikin nau'in ciwon daji irin su B-cell malignancies4,11 da nono da kuma ciwon daji na launi12,13. An sami aikin Hv1 mai yawa don ƙara yawan ƙwayar cutar ciwon daji na 11, 12. A cikin kwakwalwa, an bayyana Hv1. ta hanyar microglia, kuma an nuna aikin sa don ƙara lalacewar kwakwalwa a cikin nau'in bugun jini na ischemic. Protein Hv1 ya ƙunshi yanki mai ɗaukar wutar lantarki (VSD), wanda ya ƙunshi sassa huɗu na transmembrane da ake kira S1 zuwa S414. VSD yayi kama da wuraren da suka dace na tashoshin wutar lantarki-gated Na+, K+ da Ca2+ da phosphatases masu ƙarfin lantarki, kamar CiVSP daga Ciona gutis15.A cikin waɗannan sauran sunadaran, C-terminus na S4 yana haɗe zuwa wani samfurin tasiri, yankin pore ko enzyme. .Tashar tashar dimeric hadaddun da ke kunshe da VSD guda biyu, kowannensu yana dauke da gated proton permeation pathway16,17,18.Waɗannan subunits Hv1 guda biyu an samo su don buɗe haɗin gwiwa19,20,21,22, suna nuna cewa haɗin gwiwar allosteric da haɗin gwiwa tsakanin subunit suna wasa. muhimmiyar rawa a cikin tsarin gating.Maganar da ke tsakanin sassan da ke cikin yanki na coil coil yana da kyau a bayyana saboda tsarin crystal na yankuna biyu da aka keɓe suna samuwa22,23. A gefe guda, haɗin tsakanin VSDs a cikin membrane ba da kyau fahimta.The crystal tsarin na Hv1-CiVSP chimeric gina jiki ba ya samar da bayanai game da wannan dubawa, kamar yadda trimeric kungiyar na crystallized tashar hadaddun iya haifar da maye na 'yan qasar Hv1 CCD da yisti leucine zik din GCN424. Wani binciken da aka yi a kwanan nan na ƙungiyar subunit na tashar Hv1 ya kammala cewa S4 helices guda biyu sun shiga cikin CCD ba tare da tsangwama mai tsanani na tsarin sakandare ba, wanda ya haifar da dogon lokaci mai tsawo wanda ya fara daga membrane kuma ya shiga cikin cytoplasm.Based akan cysteine ​​​​crosslinking analysis, wannan binciken ya ba da shawarar cewa Hv1 VSDs suna tuntuɓar juna tare da sashin S4. Duk da haka, wasu nazarin sun ba da shawarar hanyoyin da za a iya amfani da su a tsakanin VSDs.Wadannan hanyoyin haɗin gwiwar sun haɗa da S1 sassan 17, 21, 26 da ƙananan ƙarshen S2 kashi 21. Wani dalili mai yiwuwa na rikici. Sakamakon waɗannan karatun shine cewa an bincika haɗin haɗin gwiwa tsakanin VSD dangane da tsarin gating, wanda ya dogara da rufaffiyar jihohi da buɗewa, kuma ma'amala tsakanin VSD na iya bambanta a cikin canje-canjen yanayi daban-daban a cikin daidaituwa. Anan, mun gano cewa 2-guanidinothiazole yana hana tashoshi na Hv1 ta hanyar haɗakarwa ta hanyar haɗin gwiwa zuwa VSD guda biyu masu buɗewa, kuma sun yi amfani da ɗayan mahaɗan, 2-guanidinobenzothiazole (GBTA), don bincika hulɗar tsakanin subunits a cikin bude jihar. interface.Mun gano cewa GBTA daurin kwana za a iya kwatanta shi da kyau ta hanyar ƙididdige ƙididdiga wanda ɗaurin mai hanawa zuwa wani yanki ɗaya yana haifar da karuwa a cikin haɗin haɗin haɗin da ke kusa. Mun kuma gano cewa ragowar D112, masu zaɓin zaɓi don tashar 27, 28 da wani ɓangare na gunkin dauri mai ɗauri na guanidine 29 sarrafa GBTA haɗin haɗin gwiwa. Muna nuna cewa ana kiyaye haɗin haɗin gwiwa a cikin Hv1 dimer, inda CCD ya rabu da sashin S4, yana nuna cewa haɗin haɗin gwiwa a cikin CCD ba ya kai tsaye. daidaita haɗin haɗin haɗin gwiwa tsakanin GBTA-dauri sites.Ya bambanta, mun gano cewa guntun S1 wani ɓangare ne na haɗin kai tsakanin sassan da kuma ba da shawara na tsarin VSD na kusa tare da ƙarshen S4 helix daga tsakiyar dimer don ba da damar. guntun S1 ya kasance a cikin yanayin buɗewa. Ƙananan masu hana ƙwayoyin ƙwayoyin cuta na Hv1 suna da amfani a matsayin magungunan anticancer da magungunan neuroprotective. Duk da haka, har zuwa yau, ƙananan mahadi sun iya hana tashar tashar 30,31,32,33. Daga cikinsu, 2-guanidinobenzimidazole (2GBI, fili [1] a cikin Fig. . 1a) da kuma abubuwan da suka samo asali don toshe VSD29,32 permeation na protons ta hanyar tashar. An yi tunanin daurin irin waɗannan mahadi na faruwa da kansa a cikin subunits guda biyu.2-guanidinobenzothiazole (GBTA, fili [2] a cikin Hoto 1a) ya kasance. wanda aka nuna a baya don hana Hv1 kusan kamar yadda ya kamata kamar 2GBI lokacin da aka gwada shi a matakin 200 μM (Figure 1b) .Mun bincika sauran abubuwan da suka samo asali na thiazole kuma mun gano cewa wasu daga cikinsu sun hana tashar tare da irin wannan iko ko mafi girma fiye da GBTA (Fig. 1 da Ƙarin Ƙari). rubutu).Mun ƙaddara matakan mayar da martani na abubuwan da suka samo asali na thiazole guda huɗu (GBTA da mahadi [3], [6] da [11], siffa 1c) kuma mun gano cewa sun fi na 2GBI. The Hill coefficients (h) na thiazole abubuwan da suka samo asali daga 1.109 ± 0.040 zuwa 1.306 ± 0.033 (Fig. 1c da Ƙarin Fig. 1).Ya bambanta, Ƙaƙwalwar Hill don 2GBI shine 0.975 ± 0.024 29, Fig. 2a da Ƙarin Hoto. Daure site,29,32 mun yi tunani cewa daurin wani thiazol zuwa wani subunit zai iya inganta daurin na biyu inhibitor kwayoyin zuwa kusa da subunit.GBTA shi ne gwajin fili tare da mafi girma Hill coefficient.Saboda haka, mun zabi wannan fili don ci gaba da nazarin tsarin dauri na haɗin gwiwa kuma an yi amfani da 2GBI azaman kulawa mara kyau. (a) Abubuwan gwaji: [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 igiyoyin da aka auna a ciki-fita plaques na Xenopus oocytes a mayar da martani ga depolarization daga iyawa rike na -80 mV zuwa +120 mV.Kowane inhibitor an kara zuwa wanka a wani taro na 200 μM.pHi = pHo = 6.0 .Bayani suna ma'anar ± SEM (n≥4) .(c) Ƙaddamar da haɗin kai na Hv1 na mutum ta hanyar mahadi [2], [3], [6] da [11] .Kowane ma'ana yana wakiltar ma'anar hanawa ± SD na 3. zuwa ma'auni 15. Layin shine Dutsen Dutsen da aka yi amfani da shi don samun alamun Kd da aka ruwaito a cikin Ƙarin Ƙarin 1.Hill coefficients an ƙaddara daga daidaitattun da aka ruwaito a Ƙarin Hoto. 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 (duba Hanyoyi). (a,b) Haɗaɗɗen 2GBI da GBTA sun hana dimeric da monomeric Hv1 a cikin hanyar da aka dogara da hankali.Kowane batu yana wakiltar ma'anar hanawa ± SD na 3 zuwa 8 ma'auni kuma kullun shine Hill fit. Ƙididdigar Hill (h) da aka nuna a ciki An ƙaddara lissafin bayanan shigar daga daidaitattun da aka ruwaito a Ƙarin Figs 3 da 4. Amsar maida hankali na GBTA da aka nuna a cikin (a) daidai yake da wanda ke cikin siffa. haɗin haɗin gwiwa na GBTA zuwa dimeric Hv1. Ƙarfin baƙar fata yana wakiltar dacewa ga bayanan gwaji ta hanyar ƙididdiga (6), wanda ya kwatanta samfurin ɗaurin da aka nuna a cikin (d) Layukan da aka yi wa lakabin Sub 1 da Sub 2 suna wakiltar ƙungiyar bimolecular. -masu daidaita ma'auni na farko da na biyu na abubuwan ɗaurewa, bi da bi (Sub 1: OO + B ⇄ BO*, Kd1 = 290 ± 70 μM; Sub 2: BO* + B ⇄ B*O*, Kd2 = 29.3 ± 2.5 μM .(d) Tsarin tsari na tsarin da aka tsara na toshe Hv1. A cikin GBTA, ɗaure zuwa ɗayan buɗaɗɗen buɗaɗɗen yana ƙara kusancin sashin buɗewa kusa (Kd2 Ana iya daidaita tashoshin Hv1 ta hanyar maye gurbin N- da C-terminal cytoplasmic domains tare da daidai rabo na Ciona Intestinalis ƙarfin lantarki-m phosphatase CiVSP (Hv1NCCiVSP) 18,34.Mun auna maida hankali dogara na hana monomeric Hv2GBI da GBTA da kuma idan aka kwatanta su da na dimeric tashar (nau'in daji) hanawa (Fig. 2a,b) . Mun gano cewa an kawar da bambanci a cikin haɗin gwiwar haɗin kai tsakanin mahadi guda biyu a cikin Hv1 na monomeric, yana goyan bayan bayanin cewa GBTA daure zuwa ɗayan ɗayan yana ƙara haɓaka. Mun yi tunanin cewa haɗin GBTA zuwa ɗayan Hv1 na iya haifar da sake tsara wurin daurin (samar da fit35), wanda zai haifar da sake tsara wurin daurin da ba kowa na kusa da shi, wanda zai haifar da ƙara ɗaurin. kusanci. Don ƙididdige adadin haɗin gwiwar haɗin gwiwa na GBTA zuwa tashar, mun yi amfani da samfurin wanda ko dai subunit zai iya ɗaure kwayoyin hanawa na farko da ke biyo bayan amsawar bimolecular tare da ƙaddamarwa akai-akai Kd1 (Fig. 2c, Sub 1, OO+ B ⇆ BO *). Daure yana haifar da tashar don ɗaukar yanayin da sauran ƙananan abubuwan da ba su da komai suna ɗaure ga mai hanawa bin wani yanayi na musamman na bimolecular tare da rarrabuwa akai-akai Kd2, inda Kd2 Da zarar an ɗaure ƙwayoyin inhibitor na biyu, duka sassan biyu suna da rarrabuwa akai-akai Kd2 (Fig. 2d) . An auna hanawar tashar a ƙarƙashin yanayin lalata (+ 120 mV) .Channel nau'in tare da ɗaya bude da ɗayan rufaffiyar (duba CC ⇄ OC ⇄) OO a cikin siffa 2d) an samo a baya suna ba da gudummawar rashin kulawa ga halin yanzu na Hv1 a ƙarƙashin waɗannan sharuɗɗan19,20 don haka ba a haɗa su cikin ƙirar ɗauri ba. Layin baƙar fata mai ƙarfi a cikin Hoto 2c shine dacewa da ƙirar ƙira zuwa madaidaicin maida hankali na gwaji, yana ba da Kd1 na ~ 290 μM da Kd2 na ~ 29 μM (Sashe na hanyoyin, daidaito (6)) .Haka kuma samfurin ya bayyana. dauri na 2GBI, inda Kd2 ≈ Kd1 = Kd (Fig. 2d) .A monomeric Hv1, akwai kawai ɗauri site da Kdm daya (O ° + B ⇆ B °) .A cikin yanayin GBTA, Kdm yana kusa da 54. μM, wanda ya fi kama da Kd1 fiye da Kd2 (Fig. 2d) . A takaice dai, wurin dauri na monomer yana cikin tsari (O °) ya fi kama da yanayin haɗin gwiwa (BO *) fiye da ƙananan- yanayin kusanci (OO) na dimer, mai yuwuwa saboda kawar da mu'amala tsakanin subunits. Kamar yadda aka nuna a baya don 2GBI32, mun gano cewa GBTA yana danne magudanar ruwa na Hv1 ta hanyar toshe tashar lokacin da yake buɗewa maimakon ta sanya shi wahalar buɗewa (Ƙarin Hoton 2a, b, d). a mayar da martani ga membrane repolarization, amma wannan sabon abu ba a lura a GBTA (Ƙarin Fig. 2c) .A gaban Hv1 inactivation a gaban 2GBI, ƙofofin a cikin kowane subunit ba zai iya rufe har sai blocker ya bar wurin dauri (da" ƙafa gate” inji), da 2GBI unbinds a hankali fiye da ƙofofin da ke kusa. Idan ɗayan Hv1 ba a toshe shi kuma an rufe shi, yayin da sashin da ke kusa da shi ya kasance a toshe, cire sauran ƙwayoyin 2GBI ya zama sannu-sannu (kamun block). Subunit guda daya kawai da aka toshe yana gudanar da protons na wucin gadi kafin rufewa da ba da gudummawa mai mahimmanci ga halin yanzun wutsiya mai rubewa a hankali. Binciken cewa lalatawar igiyoyin wutsiya na Hv1 ba a ragu sosai ba a gaban GBTA (Ƙarin Fig. 2c) ya dace da tsarin haɗin gwiwar haɗin gwiwa don wannan blocker (Fig. 2d) .Da zarar kwayar GBTA ta ɓace daga sashin Hv1. , Dangantakar mai toshewa zuwa sashin da ke kusa da shi ya ragu kusan sau 10, yana fifita tsarin da ba zai iya ba. Wannan yana nufin cewa kwayoyin na biyu na GBTA yana da damar da ya fi girma na kwancewa kafin ya kama shi a cikin tashar. samar da subunit guda ɗaya kawai ana tsammanin ya fi yawa a gaban GBTA fiye da kasancewar 2GBI, yana haifar da saurin ruɓewar wutsiya na yanzu. Domin GBTA ta ɗaure tare da haɗin gwiwa tare da sassan Hv1 guda biyu, dole ne a haɗa rukunin yanar gizon dauri. canzawa daga ƙananan alaƙa zuwa haɗin kai mai girma.Mun yi la'akari da cewa idan takamaiman sharuɗɗan da ke cikin rukunin yanar gizon sun ba da gudummawa ga tsarin haɗin gwiwar, maye gurbin su ya kamata ya canza madaidaicin Hill na madaidaicin amsawar maida hankali na hana GBTA na Hv1.Residues D112, F150 , S181 da R211 sun kasance a baya an nuna su zama wani ɓangare na yanayin 2GBI29 mai ɗauri kuma mun yi tsammanin cewa za su kasance da hannu a cikin haɗin GBTA (Fig. 3A) . Mun auna ma'auni na amsawar maida hankali na GBTA don tashoshin mutant D112E, F150A, S181A. , da R211S (Hoto 3) kuma idan aka kwatanta su Hill coefficients zuwa na Hv1 daji-type, ta yin amfani da 2GBI a matsayin tunani. Residue V109 yana a kan fuska guda na S1 kashi kamar D112 kuma yana da wani karin haske a cikin tantanin halitta. V109 bai shiga cikin ɗaurin 2GBI29 ba, mun yi amfani da mutant V109A azaman sarrafawa (Fig. 3 b). (a) Shawarwarin Hv1 da aka ba da shawarar da ke cikin haɗin haɗin guanidine. Ƙunƙarar shuɗi mai lankwasa suna kewaye da sarƙoƙi na gefe da aka tsara a baya waɗanda ke hulɗa da sassa daban-daban na fili 2GBI29. duhu ja) .Kowane batu yana wakiltar ma'anar hanawa na 3 zuwa 12 ma'auni ± SD V109 a matsayin iko mara kyau.Curves are Hill fits amfani da su don samun bayyanar Kd dabi'u (duba Ƙarin Table 1) .The Hill coefficients (h) da aka nuna a ciki An ƙididdige ƙididdiga na inset ɗin daga daidaitattun da aka ruwaito a Ƙarin Figs 3 da 4. Ana nuna ƙimar h don Hv1 WT azaman layukan da aka yanke. Alamu suna nuna bambance-bambance masu mahimmanci tsakanin mutant da sassan WT (p 0.05) a cikin Hill coefficient na GBTA daure zuwa GGG tashoshi idan aka kwatanta da nau'in daji (Fig. 5c), yana nuna cewa duk da rawar da yake takawa wajen kiyayewa. ɓangarorin biyu tare masu mahimmanci, amma CCD ba ta yin sulhu kai tsaye tsakanin haɗin gwiwar haɗin gwiwa tsakanin GBTA. (a) Tsarin tsari na Hv1 dimer tare da maye gurbi na triglycine a ƙarshen ciki na S4 wanda aka ƙera don tarwatsa haɗin haɗin gwiwa wanda ke daidaitawa ta hanyar yanki na coiled-coil na cytoplasmic (kiban shuɗi). maye gurbi da aka nuna, wanda aka tsara don gwada ɓangarorin S1 waɗanda ke cikin haɗawa tsakanin subunits (kibiyoyi masu shuɗi).(c-h) 2GBI (cyan) da GBTA (ja mai duhu) sun hana abubuwan da aka nuna a cikin hanyar dogaro da hankali.Kowane batu yana wakiltar ma'anar hanawa. ± SD na ma'auni 3 zuwa 10. Layin ya kasance Tudun da aka yi amfani da shi don samun ƙimar Kd na bayyane (duba Ƙarin Teburin 1) .Hill coefficients a cikin interpolated histograms an ƙaddara kamar yadda aka bayyana a cikin sashe hanyoyin (duba Ƙarin Figs 3 da 4) .Reference h dabi'u don Ana nuna Hv1 WT azaman layukan da aka yanke. Alamomi suna nuna mahimman bambance-bambance tsakanin mutant da nassin WT (p 0.05, Fig. 5d,i) ). A gefe guda, maye gurbin D123A ya rage mahimmancin ƙimar Hill na GBTA (p 0.05/14). Tun da neutralizing da cajin a matsayi na 123 a kan duka sassan biyu ya haifar da canji mai ƙarfi a cikin haɗin gwiwar haɗin gwiwar GBTA, yayin da mayar da cajin akan duka sassan biyu yana da ƙananan tasiri kawai, mun tsawaita bincike don haɗawa ɗaya kawai tare da tashar juyawa na caji. ya haifar da dimers masu haɗin Hv1 tare da maye gurbin D123R a cikin sashin C-terminal (Fig. 5b) kuma auna hanawar maida hankali ta GBTA da 2GBI. Mun gano cewa Hill coefficient na GBTA daure zuwa WT-D123R tashoshi ya fi girma fiye da haka. na nau'in daji na Hv1 (p