Structure and Function of Membrane Proteins

膜蛋白的结构和功能

基本信息

批准号：
10915963
负责人：
MIGUEL HOLMGREN
金额：
$ 161.68万
依托单位：
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10915963
关键词：
2019-nCoV ATP1A1 gene ATP1A3 gene Action Potentials Affinity Amino Acids Animals Arrhythmia Behavioral Binding Binding Sites Biology Biophysics Birth COVID-19 pandemic Calcium Calcium ion Cardiac Catalytic Domain Cations Cell Death Cell membrane Cell physiology Cells Cerebellar Ataxia Childhood Coronavirus Cryoelectron Microscopy Disease Dystonia Dystonia 12 E protein Electronics Electrophysiology (science)Encephalopathies Environment Enzymes Epilepsy Eye Movements Fever Genes Genome Goals Health Heart Homologous Gene Human Hydrolysis Impaired cognition Impairment Integral Membrane Protein Intellectual functioning disability Ion Channel Ion Transport Ions K ATPase Knock-in Mouse Mammalian Cell Membrane Membrane Potentials Membrane Proteins Membrane Structure and Function Mitochondria Molecular Molecular Conformation Monitor Movement Movement Disorders Muscle hypotonia Mutation Neurologic Neurological outcome Neurotransmitters Nonstructural Protein Nucleocapsid Nutrient Open Reading Frames Optic Atrophy Organelles Paralysed Pathogenesis Pathogenicity Pathway interactions Patients Phenotype Phosphorylation Potassium Probability Property Protein Conformation Protein Dephosphorylation Protein Dynamics Protein Isoforms Proteins Proteomics Pump Recurrence Research Resistance SARS coronavirus SARS-CoV-2 genome Sensorineural Hearing Loss Series Severe Acute Respiratory Syndrome Side Signal Transduction Site Site-Directed Mutagenesis Sodium Structural Protein Structure Symptoms Syndrome Talipes cavus Techniques Tissues Transmembrane Domain Transport Process Variant Viral Viral Proteins Virus Virus Replication Work alternating hemiplegia bat-borne experimental study extracellular falls large-conductance calcium-activated potassium channels nervous system disorder neural particle recruit sensor spasticity transcription factor voltage

项目摘要

Calcium ions increase the open probability of the large conductance potassium (BK) channels by binding to high affinity sites within the large cytosolic Ca2+ sensing structure called the gating ring. This Ca2+ sensing structure is formed by eight Regulator of K+ Conductance (RCK) domains, two from each subunit of the channel, termed them RCK1 and RCK2. Other divalent species, like Ba2+ ions, are also able to activate BK channels by interacting with the Ca2+ sensor. Unlike Ca2+ that activate BK channel through all RCK domains, Ba2+ does it selectively through the RCK2 domain. In addition to activation, Ba2+ can also block K+ permeation by binding to the selectivity filter of BK channels. We have solved Cryo-EM structures of BK channels with various concentrations of Ba2+. Structurally, blockade arises from one Ba2+ occupying the traditional K+ site S3 in the selectivity filter of the channel. In addition, electronic densities attributed to K+ ions are detected at K+ sites S2 and S4. In the gating ring, eight Ba2+ ions are bound to the corresponding high affinity Ca2+ binding sites. Thereby, the lack of functional significance of the RCK1 on the activation by Ba2+ indicates that the RCK1 is not in the activated configuration. Finally, these Ba2+ structures reveal an intricate series of concerted changes of the RCK 1 and RCK2 from the same subunit, suggesting coordinated intra-subunit dynamics. The Na+/K+-ATPase is a membrane embedded enzyme responsible for the exchange of 3 intracellular Na+ for 2 extracellular K+, fueled by the hydrolysis of 1 molecule of ATP. By performing this transport process, the Na+/K+-ATPase maintains the electrochemical gradients of these ions between the internal and external environments of animal cells, which are used for action potential signaling and for cotransport of substrates such as calcium, nutrients, and neurotransmitters. Reduction of Na+/K+-ATPase activity thus causes disease symptoms attributable to impairment of action potentials, disruption of other cellular functions, and cell death. Pathogenic variants in the ATP1A3 Na+/K+-ATPase gene, which encodes an isoform of the catalytic subunit that is highly expressed in neural and heart tissues, cause disabling neurological diseases with varying cardiac involvement. When ATP1A3 residues that participate in ion binding are involved, the resulting phenotypes often fall within the spectrum of rapid-onset dystonia-parkinsonism (RDP) to alternating hemiplegia of childhood (AHC). AHC and RDP are broadly characterized by asymmetric movement disorders plus other features such as plegia, hypotonia, intellectual disability, epilepsy, and cardiac arrhythmia in patients toward the more severe AHC end of the spectrum. The core enzyme is an obligatory heterodimer of an and subunit, in which the subunit contains the ATP binding domain as well as all three ion binding sites. Normal export of Na+ through the Albers-Post enzymatic cycle starts with the binding of 3 intracellular Na+ to the ATP-bound E1 form of the enzyme, which has high Na+ affinity (Na3-E1-ATP). These Na+ occupy 3 sites, numbered I-III in the crystal structure. Binding of all 3 Na+ triggers occlusion, enzyme phosphorylation with release of ADP, and change to a conformation that has an extracellular ion pathway and high affinity for K+ (P-E2-Na3). Extracellular Na+ release occurs sequentially with deocclusion followed by stepwise unbinding of each of the 3 Na+ at different rates. After releasing Na+, the phosphorylated E2 enzyme binds 2 K+ at sites I and II and imports them in a series of steps involving dephosphorylation and ATP binding. We studied mutations of three ATP1A3 residues thought to exclusively coordinate Na+ but not K+: Y768, T771, and D923. Missense variants of the first two (Y768C, Y768H, T771I, T771N) cause the severe phenotype, AHC, but their functional effects have not previously been characterized in ATP1A3. Missense variants of D923 (D923N, D923Y) cause phenotypes ranging across the AHC/RDP spectrum and reduce ion transport, with a reduction in the overall apparent affinity for Na+ observed for D923N. From Na+-bound crystal structures, the side chains of D923, Y768 and T771 specifically participate in the coordination of one Na+ at site III. Unlike sites I and II which are shared by K+ during the transport cycle, site III is uniquely used by one Na+. We also studied a new mutation, P775L that causes a cation leak through the ATP1A3 leading to spasticity and intellectual disability, symptoms that do not fulfil criteria for ATP1A3 related syndromes. The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) constituted a serious threat to global human health and underscored the need of detailed research into the SARS-CoV-2 virus. This virus which had high sequence similarity to SARS-CoV and bat-borne coronaviruses encodes four structural proteins, sixteen non-structural proteins and nine accessory proteins for viral replication and release. Structural proteins, including spike (S) protein, membrane (M) protein, envelope (E) protein, and nucleocapsid (N) protein, are incorporated into the structurally complete viral particles. Non-structural proteins act as various enzymes and transcription factors for viral replication and pathogenicity. Accessory proteins perform a diverse range of functions, involved in viral release, stability, and pathogenesis. Among these accessory proteins in SARS-CoV-2, ORF3a which is located between S and E proteins on the 3 terminal genome is the largest. It is also a membrane protein, present exclusively in SARS-like coronavirus but no other viruses. SARS-CoV-2 ORF3a had a high sequence identity (73%) with its SARS-CoV homolog, particularly in functional domains, such as the 3 transmembrane (TM) segments, suggesting functional similarity among the homologous ORF3a proteins. We are focusing our study to four aspects of the biology of ORF3a: 1) its oligoramization state in mammalian cells, 2) its functional properties as an ion channel, 3) its localization within the infected cells, and 4) its proteomic vicinity.

钙离子通过与大型胞质Ca2+传感结构内的高亲和力位点结合，增加了大电导钾（BK）通道的开放概率。该Ca2+传感结构由八个K+电导（RCK）域的调节剂形成，两个来自通道的每个亚基的两个调节器称为RCK1和RCK2。其他二价物种（如BA2+离子）也能够通过与CA2+传感器相互作用来激活BK通道。与通过所有RCK域激活BK通道的Ca2+不同，BA2+通过RCK2域选择性地进行。除了激活外，BA2+还可以通过与BK通道的选择性过滤器结合来阻止K+渗透。我们已经解决了具有不同浓度BA2+的BK通道的冷冻EM结构。从结构上讲，封锁是由一个BA2+在通道的选择性滤波器中占据传统的K+位点S3引起的。另外，在K+位点S2和S4检测到归因于K+离子的电子密度。在门控环中，八个BA2+离子与相应的高亲和力Ca2+结合位点结合。因此，RCK1在BA2+激活上缺乏功能意义，表明RCK1不在激活的配置中。最后，这些BA2+结构揭示了来自同一亚基的RCK 1和RCK2的一系列复杂的一系列协同变化，这表明了协调的亚基内部动力学。 Na+/K+-ATPase是一种膜嵌入的酶，负责将3个细胞内Na+交换为2个细胞外K+，并由ATP的1个分子的水解加油。通过执行此运输过程，Na+/K+-ATPase在动物细胞的内部和外部环境之间保持了这些离子的电化学梯度，这些梯度用于动作电位信号传导以及钙，营养素和神经递质等基板的共转移。因此，Na+/K+-ATPase活性的减少会导致疾病症状归因于动作电位受损，其他细胞功能的破坏和细胞死亡。 ATP1A3 Na+/K+-ATPase基因中的致病变异，该基因编码在神经和心脏组织中高度表达的催化亚基的同工型，引起与心脏受累不同的神经系统疾病。当涉及参与离子结合的ATP1A3残基时，所产生的表型通常属于快速发作的肌张力障碍 - 帕金森氏症（RDP）与儿童交替偏瘫（AHC）的交流。 AHC和RDP的特征是不对称运动障碍以及其他特征，例如Plegia，低调，智力障碍，癫痫和心律不齐的患者，均在更严重的AHC端端。核心酶是AN和亚基的强制性异二聚体，其中亚基包含ATP结合域以及所有三个离子结合位点。通过ALBER-POST酶循环的Na+正常出口始于3个细胞内Na+与具有高Na+亲和力（Na3-E1-ATP）的酶的ATP结合的E1形式的结合。这些Na+占据了3个位点，在晶体结构中编号I-III。所有3个Na+触发器闭塞，酶磷酸化的结合随释放的ADP结合，并变为具有细胞外离子途径和对K+的高亲和力的构象（P-E2-NA3）。细胞外Na+释放依次发生，并以除外咬合，然后以不同的速率逐步解开3 Na+的逐步释放。释放Na+后，磷酸化的E2酶在位点I和II上结合2 K+，并在涉及去磷酸化和ATP结合的一系列步骤中进口它们。我们研究了三个ATP1A3残基的突变，该突变被认为仅坐标为Na+，而不是K+：Y768，T771和D923。前两个（Y768C，Y768H，T771I，T771N）的错义变体会导致严重的表型，但是它们的功能效应以前尚未在ATP1A3中表征。 D923（D923N，D923Y）的错义变体导致跨AHC/RDP频谱范围内的表型并减少离子转运，并且观察到D923N的Na+的总体亲和力降低了D923N。从Na+实现的晶体结构中，D923，Y768和T771的侧链专门参与了Site III的一个Na+的协调。与位点I和II不同，在运输周期中由K+共享，位点III由一个Na+唯一使用。我们还研究了一种新的突变P775L，该突变通过ATP1A3导致阳离子泄漏，导致痉挛和智力障碍，无法满足ATP1A3相关综合征的标准的症状。由严重的急性呼吸综合症2（SARS-COV-2）引起的19009年大流行构成了对全球人类健康的严重威胁，并强调了对SARS-COV-2病毒的详细研究。该病毒与SARS-COV和BAT-BORONAVIRASE具有很高的序列相似性，编码四种结构蛋白，16种非结构性蛋白质和九种辅助蛋白，用于病毒复制和释放。结构蛋白，包括尖峰蛋白，膜（M）蛋白，包膜（E）蛋白和核素蛋白（N）蛋白，都掺入结构完整的病毒颗粒中。非结构蛋白充当病毒复制和致病性的各种酶和转录因子。辅助蛋白执行各种功能，涉及病毒释放，稳定性和发病机理。在SARS-COV-2中的这些辅助蛋白中，位于3个末端基因组上的S和E蛋白之间的ORF3A是最大的。它也是一种膜蛋白，仅存在于SARS样冠状病毒中，但没有其他病毒。 SARS-COV-2 ORF3A具有与其SARS-COV同源物，特别是在功能结构域（例如3个跨膜（TM）段）中具有高序列身份（73％），这表明同源物ORF3A蛋白之间的功能相似性。我们将研究重点放在ORF3A生物学的四个方面：1）其在哺乳动物细胞中的寡瘤状态，2）其功能性能作为离子通道，3）其在受感染细胞中的定位，4）其蛋白质组学附近。

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Analysis of SARS-CoV-2 ORF3a structure reveals chloride binding sites.

DOI：
10.1101/2020.10.22.349522
发表时间：
2020-10-22
期刊：
bioRxiv : the preprint server for biology
影响因子：
0
作者：
Marquez-Miranda, Valeria;Rojas, Maximiliano;Gonzalez-Nilo, Fernando D
通讯作者：
Gonzalez-Nilo, Fernando D

Restoration of proper trafficking to the cell surface for membrane proteins harboring cysteine mutations.

恢复含有半胱氨酸突变的膜蛋白向细胞表面的正确运输。

DOI：
10.1371/journal.pone.0047693
发表时间：
2012
期刊：
PloS one
影响因子：
3.7
作者：
Lopez-Rodriguez,Angelica;Holmgren,Miguel
通讯作者：
Holmgren,Miguel

Quasi-specific access of the potassium channel inactivation gate.

DOI：
10.1038/ncomms5050
发表时间：
2014-06-09
期刊：
NATURE COMMUNICATIONS
影响因子：
16.6
作者：
Venkataraman, Gaurav;Srikumar, Deepa;Holmgren, Miguel
通讯作者：
Holmgren, Miguel

A Structural Model of the Inactivation Gate of Voltage-Activated Potassium Channels

电压激活钾通道失活门的结构模型

DOI：
10.1016/j.bpj.2019.06.008
发表时间：
2019
期刊：
Biophysical Journal
影响因子：
3.4
作者：
Vergara-Jaque, Ariela;Palma-Cerda, Francisco;Lowet, Adam S.;de la Cruz Landrau, Angel;Poblete, Horacio;Sukharev, Alexander;Comer, Jeffrey;Holmgren, Miguel
通讯作者：
Holmgren, Miguel

Disease mutations of human α3 Na+/K+-ATPase define extracellular Na+ binding/occlusion kinetics at ion binding site III.

人类 α3 Na /K -ATP 酶的疾病突变定义了离子结合位点 III 的细胞外 Na 结合/闭塞动力学。