AAA Proteins, Their Functions and Related Diseases

AAA 蛋白、其功能和相关疾病

基本信息

批准号：
10926043
负责人：
di s xia
金额：
$ 86.03万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
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/10926043
关键词：
ATP Hydrolysis ATP phosphohydrolase ATPase Domain Address Adenylyl Imidodiphosphate Adopted Affect Affinity Amino Acid Substitution Amino Acids Back Binding Biochemical Biological Calorimetry Cancer cell line Cell Cycle Regulation Cell physiology Cells Clinical Clinical Research Communication Complex Contracts Coupling DNA biosynthesis Deubiquitination Devices Disease Electron Transport Complex III Endoplasmic Reticulum Event Extravasation Family Fingers Goals Hand Homo Human Inclusion Body Myopathy with Early-Onset Paget Disease Induction of Apoptosis Inner mitochondrial membrane Investigation Iron-Sulfur Proteins Kinetics Link Location Longevity Membrane Membrane Fusion Membrane Fusion Activity Membrane Proteins Merozoite Surface Protein 1 Mitochondria Mitochondrial Matrix Modeling Molecular Molecular Chaperones Molecular Conformation Molecular Machines Motion Movement Mus Mutagenesis Mutation Myopathy N-terminal Names Neurodegenerative Disorders Nucleic Acids Nucleotides Organelles Pathogenicity Pathway interactions Peptides Pharmaceutical Preparations Phase Physiological Play Positioning Attribute Process Protein Subunits Protein translocation Proteins Protons Quality Control Reaction Regulation Reporting Research Respiratory Process Role Series Side Site Structure Time Titrations Ubiquitination Visualization Work Yeasts cancer therapy cofactor density drug development experimental study extracellular fungus indexing inhibitor insight mitochondrial membrane mutant prevent protein complex protein degradation protein folding recruit respiratory seal segregation stoichiometry stress management three dimensional structure translocase unfoldase valosin-containing protein

项目摘要

Our recent work has been focusing on two mammalian AAA proteins: the human AAA protein p97 and the mouse mitochondrial AAA protein bcs1. The human p97 is a major cytosolic AAA chaperone. Although it has been known that D2 ring of p97 contributes most to the overall ATPase activity of p97, the function of the D1 ring is not clear. Our work has contributed significantly to our understanding the function of the D1 ring, which is the regulatory domain of p97. We focus our study on one type of p97 mutants that cause IBMPFD or MSP1. IBMPFD mutants have single amino acid substitutions at the interface between the N-terminal domain (N-domain) and the adjacent AAA domain (D1) and our work suggests that the mutations result in a reduced affinity for ADP. The structures of p97 N-D1 fragments bearing IBMPFD mutations adopt an Up N-domain conformation or Up-conformation in the presence of Mg2+-ATPgS, which is reversible by ADP (Down-conformation), demonstrating for the first time the nucleotide-dependent conformational change of the N-domain. We further found that wild type p97 also undergoes nucleotide-dependent Up- and Down-N-domain conformational change in solution. Using isothermal titration calorimetry (ITC), we determined a Kd value of 0.88 uM towards ADP for the wild type N-D1 with a stoichiometry of 0.35, suggesting only 2 out of 6 sites are available for binding, which is consistent with previously reports of the number of occluded ADP in wild-type p97. By contrast, mutant p97 N-D1 fragments displayed reduced binding affinities for ADP. For example, the R155H mutant showed a maximum reduction with a Kd of 4.25 uM. Notably, the number of occluded ADP in mutant p97 is dramatically reduced. Unexpectedly, the titration profiles with ATPgS for mutants were biphasic and can only be fitted to a two-site model. The Kd values for the high affinity site were well determined and close to 0.1 uM for all mutants, whereas those for the low affinity site were associated with significant errors. Again, mutant p97 displayed higher stoichiometry than wild type in the ATPgS titration experiments. A model with four nucleotide-binding states for the ATP cycle in the D1-domain was proposed. We also investigated how IBMPFD mutations affect the molecular mechanism that governs the function of p97. We showed that within the hexameric ring of a mutant p97, D1 domains fail to regulate their respective nucleotide-binding states, as evidenced by the lower amount of prebound ADP, weaker ADP binding affinity, full occupancy of ATP-gS binding, and elevated overall ATPase activity, indicating a loss of communication among subunits. Defective communication between subunits is further illustrated by altered conformation in the side chain of residue Phe-360 that probes into the nucleotide-binding pocket from a neighboring subunit. Consequently, conformations of N-domains in a hexameric ring of a mutant p97 become uncoordinated, thus impacting its ability to process substrate. Our investigation into the intra-molecular communication pathway also led to the discovery that the presence of a 22 amino acid peptide at the end of N-D1 truncate, named D1-D2 linker, of the human AAA+ protein p97 has been shown to activate ATP hydrolysis of the D1 domain, but the mechanism of activation remains unclear. We identified the N-terminal half of this D1-D2 linker, which is ubiquitously conserved from human to fungi, is essential for the activation of the ATPase. Based on the analysis of all available p97 structures, we observed that the presence of the D1-D2 linker affects the way subunits of p97 associate to form hexameric rings, which was manifested in the crystal symmetry. The presence of the linker leads to lower crystal symmetry, an observation that is reinforced by the two new crystal structures, a wild-type N-D1 truncate with the linker and a L198W mutant N-D1 truncate without the linker, determined in the present work. The lack of activity of the D1 ATPase domain in the absence of D1-D2 linker implies the functional importance of asymmetric subunit arrangement, which we suggest to be estimated quantitatively by the metrics Asymmetirc Index. Structure comparison correlates the conformation of the D1-D2 linker to conformation of the Arg-finger from a neighboring subunit, suggesting a regulatory role of the D1-domain in the conformation of D2-domain. More recently, we studied the association of cytosolic AAA protein p97 to membranes, which is essential for various cellular processes including the endoplasmic reticulum (ER)-associated degradation. The N-domain of p97 is known for undergoing large nucleotide-dependent conformational change but the physiological relevance this conformational change has not been established. We showed p97 is recruited to the ER membrane predominantly by interacting with VIMP, an ER resident protein. The recruitment can be regulated through a nucleotide-dependent conformation switch of the N-domain in wild-type p97 and this regulation is obliterated in pathogenic mutants. The molecular mechanism of the regulation is revealed by a series of structures of p97, VIMP and their complex, thus suggesting a physiological role of the nucleotide-dependent conformational change of the N-domain of p97. In addition, intermediate positions of the N-domain are seen when AMP-PNP occupies the D1-domain, allowing construction of a trajectory for the N-domain movement. Our findings suggest the nucleotide-dependent membrane interaction cycle may be applicable to other p97-dependent events. Another AAA protein that are being actively pursued in the lab is called bcs1 that, unlike the functions of most AAA proteins known to date, involves in folded protein translocation across the membrane. Having determined the structures of mouse Bcs1 (mBcs1) in different nucleotide states and conformations, we now have acquired a structural framework from which more detailed mechanistic insights into the transport mechanism of Bcs1 can be expected. Currently, we focus on studies that will likely reveal how Bcs1 recognizes and binds the folded ISP-ED, capture its action in translocating the substrate across the membrane, and visualize how it releases the substrate into the membrane. To achieve these goals, a combination of various research approaches will have to be employed. From a structural point of view, it is necessary to obtain the structure of Bcs1 in complex with the substrate ISP in order to address the questions on how substrate binding trigger changes in Bcs1 and whether binding of substrate is sufficient to induce nucleotide exchange. Structures are also needed to determine whether subunits of Bcs1 functions in a sequential fashion or in a concerted manner. The former is the hallmark of the hand-over-hand or split wash mechanism of translocation displayed by many hexameric AAA proteins. In the apo and ADP bound structures, the unknown density plugging the small pore in the center of the Bcs1-specific domains should also be investigated. Biochemically, kinetic study of the life span of different nucleotide states will provide clues on the rate limiting steps in the reaction landscape. Coupling these studies with mutagenesis will likely play a major role in verifying various mechanistic hypotheses. For example, to prevent proton leakage during translocation, an airlock-like mechanism was proposed. However, how the opening and closure of the seal pore is controlled requires further elucidation. Mutagenesis studies will allow functional and structural characterizations of many documented disease-related mutants. The structures should also facilitate development of drugs to modulate function of Bcs1.

我们最近的工作一直集中在两种哺乳动物AAA蛋白上：人AAA蛋白p97和小鼠线粒体AAA蛋白BCS1。人P97是主要的胞质AAA伴侣。尽管众所周知，p97的D2环对p97的整体ATPase活性做出了最大的贡献，但D1环的功能尚不清楚。我们的工作为我们理解D1环的功能做出了重大贡献，D1环是P97的调节域。我们将研究重点放在导致IBMPFD或MSP1的一种P97突变体上。 IBMPFD突变体在N末端结构域（N-域）和相邻的AAA结构域（D1）之间的界面上具有单氨基酸取代（D1），我们的工作表明该突变导致对ADP的亲和力降低。携带IBMPFD突变的p97 N-D1片段的结构在存在Mg2+-ATPG的情况下采用了N域构象或上符号，这是通过ADP（下键合）可逆的，这是第一次证明N-Domain的核苷酸依赖性构象变化。我们进一步发现，野生型p97还经历了溶液中核苷酸依赖性的上下构域构象变化。使用等温滴定量热法（ITC），我们确定了野生型N-D1的KD值为0.88 UM，其化学计量学为0.35，这表明6个站点中只有2个可用于绑定，这与以前曾与野生型P97中闭塞性ADP的数量保持一致。相比之下，突变体P97 N-D1片段显示出ADP的结合亲和力降低。例如，R155H突变体显示最大降低，KD为4.25 um。值得注意的是，突变体p97中的ADP的数量大大减少。出乎意料的是，用于突变体的ATPG的滴定曲线是双相的，只能适合两个站点模型。高亲和力位点的KD值均得到很好的确定，所有突变体的KD值接近0.1 UM，而低亲和力位点的KD值与重大误差有关。同样，在ATPGS滴定实验中，突变体P97比野生型显示出更高的化学计量法。提出了一个具有四个核苷酸结合状态的模型，用于D1域中的ATP循环。我们还研究了IBMPFD突变如何影响控制p97功能的分子机制。我们表明，在突变体p97的六聚体环内，D1结构域无法调节其各自的核苷酸结合态，这证明了较低量的预堆ADP，ADP结合亲和力较弱，ATP-GS的完全占用率，ATP-GS的全部占用率，表明总ATPase活性升高，表明子nits之间的通信丧失。亚基之间的有缺陷的通信通过在残基PHE-360的侧链中的改变构象进一步说明，该构型从邻近的亚基探测到核苷酸结合口袋中。因此，突变体P97的六聚体环中N域的构象变得不协调，从而影响其处理底物的能力。我们对分子内通信途径的研究还导致发现，人类AAA+蛋白p97的N-D1截断末端的22个氨基酸肽，称为D1-D2接头，称为D1-D2连接器，已证明可以激活D1域的ATP ATP ATP水解，但仍然不清楚激活机制。我们确定了该D1-D2连接器的N末端一半，该末端从人到真菌无处不在，对于ATPase的激活至关重要。基于对所有可用p97结构的分析，我们观察到D1-D2接头的存在会影响p97助理的亚基形成甲式化环的方式，这在晶体对称性中表现出来。接头的存在导致较低的晶体对称性，这一观测值是由两个新的晶体结构加强的，即接头的野生型N-D1截断，而L198W突变体N-D1截断，没有接头，在目前的工作中确定。在没有D1-D2链接器的情况下，D1 ATPase结构域缺乏活性，这意味着非对称亚基排列的功能重要性，我们建议这是由指标不对称指数定量估计的。结构比较将D1-D2连接器的构象与邻近亚基的Arg Finger的构象相关，这表明D1域在D2域构象中的调节作用。最近，我们研究了胞质AAA蛋白p97与膜的关联，这对于包括内质网（ER）相关的降解在内的各种细胞过程至关重要。 p97的N域以经历较大的核苷酸依赖性构象变化而闻名，但生理相关性尚未确定。我们表明，P97主要通过与ER常驻蛋白VIMP相互作用，主要通过与ER膜相互作用。可以通过野生型p97中N域的核苷酸依赖性构象转换来调节募集，并且该调节在致病性突变体中被消除。调节的分子机制通过p97，vimp及其复合物的一系列结构揭示，因此表明p97的N域的核苷酸依赖性构象变化具有生理作用。此外，当AMP-PNP占D1域时，可以看到N域的中间位置，从而为N域运动构建轨迹。我们的发现表明，核苷酸依赖性膜相互作用周期可能适用于其他依赖P97的事件。在实验室中积极追求的另一种AAA蛋白称为BCS1，与迄今已知的大多数AAA蛋白的功能不同，它涉及整个膜上的折叠蛋白易位。在确定了不同核苷酸状态和构象中小鼠BCS1（MBCS1）的结构之后，我们现在已经获得了一个结构框架，可以从中可以从中对BCS1的传输机制有了更详细的机理见解。当前，我们专注于研究可能会揭示BCS1如何识别和结合折叠的ISP-ED，捕获其在跨膜上易位的作用，并可视化底物如何将基板释放到膜中。为了实现这些目标，必须采用各种研究方法的结合。从结构的角度来看，有必要获得与底物ISP复杂成分的BCS1的结构，以解决有关BCS1中底物结合触发方式的问题，以及底物的结合是否足以诱导核苷酸交换。还需要结构来确定BCS1的亚基是以顺序的方式还是以协调的方式函数。前者是许多六聚体AAA蛋白显示的易位机理的标志或分裂洗涤机制。在APO和ADP结构的结构中，还应研究未知的密度塞在BCS1特异性域中心的小孔。从生化上，对不同核苷酸状态的寿命的动力学研究将为反应格局中的速率限制步骤提供线索。将这些研究与诱变耦合可能在验证各种机械假设方面起主要作用。例如，为了防止在易位过程中质子泄漏，提出了一种类似气闸的机制。但是，如何控制密封孔的开口和闭合需要进一步阐明。诱变研究将允许许多与疾病相关的突变体的功能和结构表征。结构还应促进药物的开发以调节BCS1的功能。

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）

Chaperone-tip adhesin complex is vital for synergistic activation of CFA/I fimbriae biogenesis.

分子伴侣-尖端粘附素复合物对于 CFA/I 菌毛生物发生的协同激活至关重要。

DOI：
10.1371/journal.ppat.1008848
发表时间：
2020-10
期刊：
PLoS pathogens
影响因子：
6.7
作者：
He LH;Wang H;Liu Y;Kang M;Li T;Li CC;Tong AP;Zhu YB;Song YJ;Savarino SJ;Prouty MG;Xia D;Bao R
通讯作者：
Bao R

A Mighty "Protein Extractor" of the Cell: Structure and Function of the p97/CDC48 ATPase.