Certain amino acids changes in the human Na^(+)/K^(+)-ATPase pump,ATPase Na^(+)/K^(+)transporting subunit alpha 1(ATP1A1),cause Charcot-Marie-Tooth disease type 2(CMT2)disease and refractory seizures.To develop in viv...Certain amino acids changes in the human Na^(+)/K^(+)-ATPase pump,ATPase Na^(+)/K^(+)transporting subunit alpha 1(ATP1A1),cause Charcot-Marie-Tooth disease type 2(CMT2)disease and refractory seizures.To develop in vivo models to study the role of Na^(+)/K^(+)-ATPase in these diseases,we modified the Drosophila gene homolog,Atpα,to mimic the human ATP1A1 gene mutations that cause CMT2.Mutations located within the helical linker region of human ATP1A1(I592T,A597T,P600T,and D601F)were simultaneously introduced into endogenous Drosophila Atpαby CRISPR/Cas9-mediated genome editing,generating the Atpα^(TTTF)model.In addition,the same strategy was used to generate the corresponding single point mutations in flies(Atpα^(I571T),Atpα^(A576T),Atpα^(P579T),and Atpα^(D580F)).Moreover,a deletion mutation(Atpα^(mut))that causes premature termination of translation was generated as a positive control.Of these alleles,we found two that could be maintained as homozygotes(Atpα^(I571T)and Atpα^(P579T)).Three alleles(Atpα^(A576T),Atpα^(P579)and Atpα^(D580F))can form heterozygotes with the Atpαmut allele.We found that the Atpαallele carrying these CMT2-associated mutations showed differential phenotypes in Drosophila.Flies heterozygous for Atpα^(TTTF)mutations have motor performance defects,a reduced lifespan,seizures,and an abnormal neuronal morphology.These Drosophila models will provide a new platform for studying the function and regulation of the sodium-potassium pump.展开更多
MgATP is a stable complex formed by the chelation of Mg^(2+)with deprotonated adenosine-5'-triphosphate(ATP).In the cellular environment,MgATP plays a critical role in ATP hydrolysis,releasing substantial energy t...MgATP is a stable complex formed by the chelation of Mg^(2+)with deprotonated adenosine-5'-triphosphate(ATP).In the cellular environment,MgATP plays a critical role in ATP hydrolysis,releasing substantial energy to support essential biological functions.To understand the structure and stabilization mechanism of MgATP,we conducted a joint negative ion photoelectron spectroscopic and computational study of the[ATP^(4-)·Mg^(2+)]^(2-)complex dianion,using[ATP^(4-)·2H^(+)]^(2-)as a reference.The experimentally determined adiabatic and vertical detachment energies(ADE and VDE)of[ATP^(4-)·Mg^(2+)]^(2-)at 20 K are 3.51±0.05 eV and 3.82±0.05 eV,respectively.The major spectral features of[ATP^(4-)·Mg^(2+)]^(2-)are attributed to two theoretically identified isomers with unfolded geometries,which are stabilized primarily by electrostatic interactions between Mg^(2+)and the triphosphate and ribose groups,with four deprotonated oxygens forming a pseudo-tetrahedral coordination.In contrast,[ATP^(4-)·2H^(+)]^(2-)exhibits a fundamentally different stabilization mechanism.Although most of the fifteen identified[ATP^(4-)·2H^(+)]^(2-)isomers also adopt unfolded geometries,they are primarily stabilized by intramolecular hydrogen bonds within the triphosphate group and between triphosphate and ribose groups.The interaction between ATP^(4-)and two protons is found to be much weaker than that with Mg^(2+),and[ATP^(4-)·2H^(+)]^(2-)exhibits substantial structural flexibility compared to[ATP^(4-)·Mg^(2+)]^(2-)due to the conformational constraint of the triphosphate chain by Mg^(2+).Thirteen[ATP^(4-)·2H^(+)]^(2-)isomers with unfolded geometries likely account for the major high-EBE(electron-binding-energy)spectral features.Notably,for the first time,a low EBE and temperature-dependent spectral feature is observed and attributed to two folded isomers of[ATP^(4-)·2H^(+)]^(2-),which exist at 20 K but disappear at room temperature.This study provides valuable molecular-level insights into cellular MgATP that resides within the hydrophobic pockets of proteins.展开更多
植物细胞质雄性不育(cytoplasmic male sterility,CMS)与线粒体ATP合成酶亚基基因功能异常有密切关系。为了揭示烟草atp2基因在其生长发育过程中的功能,本研究利用在线软件分析了烟草ATP2蛋白的组成结构和理化性质,绘制了atp2基因和ATP...植物细胞质雄性不育(cytoplasmic male sterility,CMS)与线粒体ATP合成酶亚基基因功能异常有密切关系。为了揭示烟草atp2基因在其生长发育过程中的功能,本研究利用在线软件分析了烟草ATP2蛋白的组成结构和理化性质,绘制了atp2基因和ATP2蛋白的分子进化树,通过在烟草中瞬时表达atp2-gfp基因明确其蛋白的亚细胞定位。结果表明,ATP2蛋白全长包含560个氨基酸,等电点6.05,相对分子质量59.7 kD,氨基酸组成中甘氨酸(Gly)、丙氨酸(Ala)、亮氨酸(Leu)含量最高;其二级结构中,螺旋、折叠片、环状结构分别占比为37.95%、14.03%、48.02%;三级结构预测表明第1~51位氨基酸是线粒体信号肽序列;不同高等植物atp2基因序列一致性达到85.7%,其ATP2蛋白序列C端高度保守,但是N端信号肽序列差异显著。分子进化分析表明,atp2基因同义(dS)替换速率显著高于非同义(dN)替换速率,且不同植物中的非同义替换速率差异显著。亚细胞定位结果表明,烟草ATP2蛋白定位于线粒体内,叶绿体内未发现其定位信号。本研究结果为后续深入探讨烟草雄性生殖发育过程中atp2的基因功能提供了理论依据。展开更多
基金supported by the Natural Science Foundation of Fujian Province,No.2020J02027the National Natural Science Foundation of China,No.31970461the Foundation of NHC Key Laboratory of Technical Evaluation of Fertility Regulation for Non-human Primate,Fujian Maternity and Child Health Hospital,No.2022-NHP-05(all to WC).
文摘Certain amino acids changes in the human Na^(+)/K^(+)-ATPase pump,ATPase Na^(+)/K^(+)transporting subunit alpha 1(ATP1A1),cause Charcot-Marie-Tooth disease type 2(CMT2)disease and refractory seizures.To develop in vivo models to study the role of Na^(+)/K^(+)-ATPase in these diseases,we modified the Drosophila gene homolog,Atpα,to mimic the human ATP1A1 gene mutations that cause CMT2.Mutations located within the helical linker region of human ATP1A1(I592T,A597T,P600T,and D601F)were simultaneously introduced into endogenous Drosophila Atpαby CRISPR/Cas9-mediated genome editing,generating the Atpα^(TTTF)model.In addition,the same strategy was used to generate the corresponding single point mutations in flies(Atpα^(I571T),Atpα^(A576T),Atpα^(P579T),and Atpα^(D580F)).Moreover,a deletion mutation(Atpα^(mut))that causes premature termination of translation was generated as a positive control.Of these alleles,we found two that could be maintained as homozygotes(Atpα^(I571T)and Atpα^(P579T)).Three alleles(Atpα^(A576T),Atpα^(P579)and Atpα^(D580F))can form heterozygotes with the Atpαmut allele.We found that the Atpαallele carrying these CMT2-associated mutations showed differential phenotypes in Drosophila.Flies heterozygous for Atpα^(TTTF)mutations have motor performance defects,a reduced lifespan,seizures,and an abnormal neuronal morphology.These Drosophila models will provide a new platform for studying the function and regulation of the sodium-potassium pump.
基金was supported by the U.S.Department of Energy(DOE),Office of Science,Office of Basic Energy Sciences,Division of Chemical Sciences,Geosciences,and Biosciences,Condensed Phase and Interfacial Molecular Science program,FWP 16248.
文摘MgATP is a stable complex formed by the chelation of Mg^(2+)with deprotonated adenosine-5'-triphosphate(ATP).In the cellular environment,MgATP plays a critical role in ATP hydrolysis,releasing substantial energy to support essential biological functions.To understand the structure and stabilization mechanism of MgATP,we conducted a joint negative ion photoelectron spectroscopic and computational study of the[ATP^(4-)·Mg^(2+)]^(2-)complex dianion,using[ATP^(4-)·2H^(+)]^(2-)as a reference.The experimentally determined adiabatic and vertical detachment energies(ADE and VDE)of[ATP^(4-)·Mg^(2+)]^(2-)at 20 K are 3.51±0.05 eV and 3.82±0.05 eV,respectively.The major spectral features of[ATP^(4-)·Mg^(2+)]^(2-)are attributed to two theoretically identified isomers with unfolded geometries,which are stabilized primarily by electrostatic interactions between Mg^(2+)and the triphosphate and ribose groups,with four deprotonated oxygens forming a pseudo-tetrahedral coordination.In contrast,[ATP^(4-)·2H^(+)]^(2-)exhibits a fundamentally different stabilization mechanism.Although most of the fifteen identified[ATP^(4-)·2H^(+)]^(2-)isomers also adopt unfolded geometries,they are primarily stabilized by intramolecular hydrogen bonds within the triphosphate group and between triphosphate and ribose groups.The interaction between ATP^(4-)and two protons is found to be much weaker than that with Mg^(2+),and[ATP^(4-)·2H^(+)]^(2-)exhibits substantial structural flexibility compared to[ATP^(4-)·Mg^(2+)]^(2-)due to the conformational constraint of the triphosphate chain by Mg^(2+).Thirteen[ATP^(4-)·2H^(+)]^(2-)isomers with unfolded geometries likely account for the major high-EBE(electron-binding-energy)spectral features.Notably,for the first time,a low EBE and temperature-dependent spectral feature is observed and attributed to two folded isomers of[ATP^(4-)·2H^(+)]^(2-),which exist at 20 K but disappear at room temperature.This study provides valuable molecular-level insights into cellular MgATP that resides within the hydrophobic pockets of proteins.
文摘植物细胞质雄性不育(cytoplasmic male sterility,CMS)与线粒体ATP合成酶亚基基因功能异常有密切关系。为了揭示烟草atp2基因在其生长发育过程中的功能,本研究利用在线软件分析了烟草ATP2蛋白的组成结构和理化性质,绘制了atp2基因和ATP2蛋白的分子进化树,通过在烟草中瞬时表达atp2-gfp基因明确其蛋白的亚细胞定位。结果表明,ATP2蛋白全长包含560个氨基酸,等电点6.05,相对分子质量59.7 kD,氨基酸组成中甘氨酸(Gly)、丙氨酸(Ala)、亮氨酸(Leu)含量最高;其二级结构中,螺旋、折叠片、环状结构分别占比为37.95%、14.03%、48.02%;三级结构预测表明第1~51位氨基酸是线粒体信号肽序列;不同高等植物atp2基因序列一致性达到85.7%,其ATP2蛋白序列C端高度保守,但是N端信号肽序列差异显著。分子进化分析表明,atp2基因同义(dS)替换速率显著高于非同义(dN)替换速率,且不同植物中的非同义替换速率差异显著。亚细胞定位结果表明,烟草ATP2蛋白定位于线粒体内,叶绿体内未发现其定位信号。本研究结果为后续深入探讨烟草雄性生殖发育过程中atp2的基因功能提供了理论依据。
