Structure and Function of the Catalytic Domain of the Dihydrolipoyl Acetyltransferase Component in Escherichia coli Pyruvate Dehydrogenase Complex

The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s⁻¹, comparable with the rate of NADH production in the PDHc reaction. Hence, neither reductive acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4′-aminopyrimidine tautomer of bound thiamin diphosphate (AP).     

Influence of nucleotide effectors on the kinetics of the quaternary structure transition of allosteric aspartate transcarbamylase

We report the effects of allosteric effectors, ATP, CTP and UTP on the kinetics of the quaternary structure change of Escherichia coli ATCase during the enzyme reaction with physiological substrates. Time-resolved, small-angle, X-ray scattering of solutions allows direct observation of structural transitions over the entire time-course of the enzyme reaction initiated by fast mixing of the enzyme and substrates. In the absence of effectors, all scattering patterns recorded during the reaction are consistent with a two-state, concerted transition model, involving no detectable intermediate conformation that differs from the less active, unliganded T-state and the more active, substrate-bound R-state. The latter predominates during the steady-state phase of enzyme catalysis, while the initial T-state is recovered after substrate consumption. The concerted character of the structural transition is preserved in the presence of all effectors. CTP slightly shifts the dynamical equilibrium during a shortened steady state toward T while the additional presence of UTP makes the steady state vanishingly short. The return transition to the T conformation is slowed significantly in the presence of inhibitors, the effect being most severe in the presence of UTP. While ATP increases the apparent T to R rate, it also increases the duration of the steady-state phase, an apparently paradoxical observation. This observation can be accounted for by the greater increase in the association rate constant of aspartate, promoted by ATP, while the nucleotide produces a lesser degree of increase in the dissociation rate constant. Under our experimental conditions, using high concentrations of both enzyme and substrate, it appears that this very mechanism of activation turns the activator into an efficient inhibitor. The scattering patterns recorded in the presence of ATP support the view that ATP alters the quaternary structure of the substrate-bound enzyme, an effect reminiscent of the reported modification of PALA-bound R-state by Mg-ATP.     

Solution Structure and Characterisation of the Human Pyruvate Dehydrogenase Complex Core Assembly

Mammalian pyruvate dehydrogenase complex (PDC) is a key multi-enzyme assembly that is responsible for glucose homeostasis maintenance and conversion of pyruvate into acetyl-CoA. It comprises a central pentagonal dodecahedral core consisting of two subunit types (E2 and E3BP) to which peripheral enzymes (E1 and E3) bind tightly but non-covalently. Currently, there are two conflicting models of PDC (E2 + E3BP) core organisation: the ‘addition’ model (60 + 12) and the ‘substitution’ model (48 + 12). Here we present the first ever low-resolution structures of human recombinant full-length PDC core (rE2/E3BP), truncated PDC core (tE2/E3BP) and native bovine heart PDC core (bE2/E3BP) obtained by small-angle X-ray scattering and small-angle neutron scattering. These structures, corroborated by negative-stain and cryo electron microscopy data, clearly reveal open pentagonal core faces, favouring the ‘substitution’ model of core organisation. The native and recombinant core structures are all similar to the truncated bacterial E2 core crystal structure obtained previously. Cryo-electron microscopy reconstructions of rE2/E3BP and rE2/E3BP:E3 directly confirm that the core has open pentagonal faces, agree with scattering-derived models and show density extending outwards from their surfaces, which is much more structurally ordered in the presence of E3. Additionally, analytical ultracentrifugation characterisation of rE2/E3BP, rE2 (full-length recombinant E2-only) and tE2/E3BP supports the substitution model. Superimposition of the small-angle neutron scattering tE2/E3BP and truncated bacterial E2 crystal structures demonstrates conservation of the overall pentagonal dodecahedral morphology, despite evolutionary diversity. In addition, unfolding studies using circular dichroism and tryptophan fluorescence spectroscopy show that the rE2/E3BP is less stable than its rE2 counterpart, indicative of a role for E3BP in core destabilisation. The architectural complexity and lower stability of the E2/E3BP core may be of benefit to mammals, where sophisticated fine-tuning is required for cores with optimal catalytic and regulatory efficiencies.     

乳腺癌易感蛋白2的结构和功能

乳腺癌易感蛋白2是由乳腺癌易感基因2编码的一种在维持哺乳动物细胞染色体的稳定及DNA损伤生物应答中发挥重要作用的蛋白质。文章通过介绍近几年来对乳腺癌易感蛋白2的结构研究,阐述其在双链DNA损伤修复中的作用模型及其在肿瘤抑制中的功能。     

病毒融合蛋白的结构和功能

病毒融合蛋白可以分为三种类型,不同类型的病毒融合蛋白的结构差异很大,但是会采用相似的"发卡"构象实现融合.在一定条件下,病毒融合蛋白的疏水结构域,融合环或融合肽插入靶膜中,通过其自身折叠形成发卡使病毒和宿主的膜靠近.与此同时,融合蛋白构象变化会释放出足够的能量将双方膜打破并完成融合.本文中,我们总结了三种类型病毒融合蛋白的特征,并对其中央发卡三聚体结构域、跨膜结构域以及近膜结构域在融合过程中的作用进行了论述.     

植物COP1蛋白的结构与功能

组成型光形态建成1(constitutively photomorphogenic 1,COP1)蛋白是一个分子量为76 kD的核蛋白,它由3个特殊的结构域组成即环形锌指结合域、卷曲螺旋形结构域和WD_40 重复序列,并含有一个核定位信号和一个新型细胞质定位信号,它是一个光形态建成的抑制子,是一个光调控植物发育的分子开关。当植物在暗环境下生长时,COP1蛋白聚集在细胞核内,抑制光形态的建成,而在光环境下,COP1蛋白则分散到细胞质中,解除其抑制作用,恢复光形态建成。COP1蛋白在细胞内的核质分布受多个因素的影响,核内COP1通过与特异转录因子相互作用来调节光形态建成。继从植物中分离鉴定出COP1蛋白之后,动物体内也发现有COP1蛋白的存在,提示COP1蛋白可能在调节动物和植物的发育及信号转导等方面具有共同作用模式。     

植物COP1蛋白的结构与功能

组成型光形态建成 1 (constitutivelyphotomorphogenic 1 ,COP1 )蛋白是一个分子量为 76kD的核蛋白 ,它由 3个特殊的结构域组成即环形锌指结合域、卷曲螺旋形结构域和WD_40重复序列 ,并含有一个核定位信号和一个新型细胞质定位信号 ,它是一个光形态建成的抑制子 ,是一个光调控植物发育的分子开关。当植物在暗环境下生长时 ,COP1蛋白聚集在细胞核内 ,抑制光形态的建成 ,而在光环境下 ,COP1蛋白则分散到细胞质中 ,解除其抑制作用 ,恢复光形态建成。COP1蛋白在细胞内的核质分布受多个因素的影响 ,核内COP1通过与特异转录因子相互作用来调节光形态建成。继从植物中分离鉴定出COP1蛋白之后 ,动物体内也发现有COP1蛋白的存在 ,提示COP1蛋白可能在调节动物和植物的发育及信号转导等方面具有共同作用模式。     

细菌全局性调控因子CsrA的结构与功能

碳存储调控因子A (carbon storage regulator, CsrA) 是一种RNA结合蛋白,在细菌的碳代谢、生物被膜形成、运动性、病原菌毒力、群体感应、环二鸟苷酸信号合成、应激感应等多种生理过程中具有重要调节功能,是全局性调控蛋白.它通过与靶标mRNA的特异结合,抑制其翻译或增强其稳定性来调控下游基因的表达,属于转录后调控因子的范畴.CsrA蛋白的表达与活性受碳存储调控(Csr)系统本身多个自主调节回路的精密控制：　一些小的非编码RNA (snmRNAs,如CsrB/C）作为拮抗因子与CsrA二聚体结合并抑制其活性;而这些snmRNAs在体内又可在CsrD的辅助下被核糖核酸内切酶E和多核苷酸磷酸化酶降解,释放CsrA的活性.当前,对于Csr系统的调节作用、调控通路与机制的研究是细菌学研究的热点,本文综述了该蛋白及Csr系统的结构、功能和作用机制的最新研究进展.     

蛋白激酶Cα相互作用蛋白的结构与功能

蛋白激酶Cα相互作用蛋白（protein interacting with Cα kinase,PICK1）是蛋白激酶Cox（protein kinase Cα,PKCα）的靶蛋白之一,也是在PKCα和突触后膜受体蛋白间起重要作用的衔接蛋白。PICK1分别由PDZ结构域、BAR结构域以及卷曲螺旋区和酸性氨基酸区组成。PICK1中的PDZ结构域和受体蛋白、转运蛋白、衔接蛋白的相互作用报道较多,BAR结构域则与支架蛋白、质膜等相互作用。PICK1在突触可塑性、神经递质传递、外周神经感觉、细胞生长和黏连等方面发挥重要作用。本文对PICK1的结构和功能进行综述。     

植物中钙依赖蛋白激酶（CDPKs）的结构与功能

在植物细胞中,钙离子作为第二信使,通过钙依赖蛋白激酶（CDPKs)发挥功能是其传递信号的主要途径之一。CDPKs广泛存在于植物体中,是目前植物体内研究最深入的蛋白激酶,在简要阐述CDPKs于植物体内的分布定位的基础上,介绍了CDPKs的结构特点,生化性质及其在植物细胞生理功能中的作用,并就该领域的研究前景作了展望。     

蛋白质二硫键异构酶家族的结构与功能

蛋白质二硫键异构酶（protein disulfide isomerase,PDI）家族是一类在内质网中起作用的巯基-二硫键氧化还原酶.它们通常含有CXXC（Cys-Xaa-Xaa-Cys,CXXC）活性位点,活性位点的两个半胱氨酸残基可催化底物二硫键的形成、异构及还原.所有PDI家族成员包含至少一个约100个氨基酸残基的硫氧还蛋白同源结构域.PDI家族的主要职能是催化内质网中新生肽链的氧化折叠,另外在内质网相关的蛋白质降解途径（ERAD）、蛋白质转运、钙稳态、抗原提呈及病毒入侵等方面也起重要作用.     

植物中钙依赖蛋白激酶(CDPKs)的结构与功能

在植物细胞中,钙离子作为第二信使,通过钙依赖蛋白激酶（CDPKs）发挥功能是其传递信号的主要途径之一。CDPKs广泛存在于植物体内,是目前植物体内研究最深入的蛋白激酶。在简要阐述CDPKs于植物体内的分布定位的基础上,介绍了CDPKs的结构特点、生化性质及其在植物细胞生理功能中的作用,并就该领域的研究前景作了展望。     

Structure of the UHRF1 Tandem Tudor Domain Bound to a Methylated Non-histone Protein,LIG1, Reveals Rules for Binding and Regulation

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高等植物中蛋白磷酸酶2C的结构与功能

蛋白质磷酸化／去磷酸化是生物信号级联传递的重要方式之一,主要通过生化性质互为对立的蛋白激酶和蛋白磷酸酶实现。蛋白磷酸酶2C(PP2C)是蛋白磷酸酶的一个分支,其生化性质、蛋白质组成与结构都和其他磷酸酶显著不同,但都在生物信号传递中扮演重要角色。高等植物中PP2C广泛参与脱落酸(ABA)的各种信号途径,包括ABA诱导的种子萌发／休眠、保卫细胞及离子通道调控和气孔关闭、逆境胁迫等。PP2C也多样地参与植物创伤反应、生长发育以及抗病性等各个途径。作为大多数信号途径的负调控因子,PP2C能直接与激酶结合,与其他调控蛋白结合,以及直接与DNA结合调控相关基因的表达。     

接头蛋白Gab1的结构及功能研究进展

Gab1蛋白属于接头蛋白Gab家族,该家族蛋白因能与生长因子受体结合蛋白2（Grb2）相结合而得名。作为接头蛋白,Gab1蛋白能被多种受体酪氨酸激酶或非受体酪氨酸激酶激活,接受胞外多种生长因子、细胞因子和一些T/B细胞抗原受体的刺激,介导PI3K/Akt和Ras/MAPK等多条信号转导途径,具有促进细胞生长、迁移、调节免疫等多种生物学功能,与糖尿病、肿瘤、心血管疾病等的发生发展密切相关。     

叶脉结构与功能及其对叶片经济谱的影响

叶脉由贯穿于叶肉内部的维管组织及其外围机械组织构成,多样化的脉序及网络结构使叶脉系统发生变异和功能分化。该文综述了叶脉系统结构与功能的最新研究进展。通过聚焦叶脉分级系统的结构与功能及其在叶片经济谱(LES)中的重要性,解释叶脉性状与其它叶片功能性状之间的关系及机制。不同等级叶脉在机械支撑与水分运输方面存在功能分化,其中1–3级粗脉在维持叶片形状和叶表面积以及物理支撑方面发挥重要作用,有利于维持叶片最大受光面积;4级及以上细脉具有水分调节功能,它们与气孔相互协调,影响叶片水分运输、蒸腾散热和光合作用速率。叶片生长过程与叶脉发育的动态变化模式决定叶脉密度,并影响叶脉密度与叶片大小之间的关系:叶面积与粗脉密度呈显著负相关,与粗脉直径呈显著正相关,而与细脉密度无关。与叶脉性状相关的叶片经济谱框架模型预测,叶脉密度较高的叶片寿命短、比叶重较小,叶片最大碳同化速率、代谢速率以及资源获取策略潜力较高。     

Structure and Function of N-Terminal Zinc Finger Domain of SARS-CoV-2 NSP2

Virologica Sinica - SARS-CoV-2 has become a global pandemic threatening human health and safety. It is urgent to find effective therapeutic agents and targets with the continuous emergence of novel...