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.

我们最近的工作重点是两种哺乳动物 AAA 蛋白：人类 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。尽管已知p97的D2环对p97的总ATP酶活性贡献最大，但D1环的功能尚不清楚。我们的工作对我们理解 D1 环的功能做出了重大贡献，D1 环是 p97 的调控域。我们的研究重点是一种导致 IBMPFD 或 MSP1 的 p97 突变体。 IBMPFD 突变体在 N 端结构域 (N-domain) 和相邻 AAA 结构域 (D1) 之间的界面处有单个氨基酸取代，我们的工作表明这些突变导致对 ADP 的亲和力降低。带有 IBMPFD 突变的 p97 N-D1 片段的结构在 Mg2+-ATPgS 存在下采用向上 N 结构域构象或向上构象，可通过 ADP（向下构象）可逆，首次证明了核苷酸依赖性N-结构域的构象变化。我们进一步发现野生型 p97 在溶液中也会经历核苷酸依赖性上行和下行 N 结构域构象变化。使用等温滴定量热法 (ITC)，我们确定野生型 N-D1 对 ADP 的 Kd 值为 0.88 uM，化学计量为 0.35，表明 6 个位点中只有 2 个可用于结合，这与之前的报道一致野生型 p97 中封闭的 ADP 数量。相比之下，突变的 p97 N-D1 片段显示出与 ADP 的结合亲和力降低。例如，R155H 突变体表现出最大的降低，Kd 为 4.25 uM。值得注意的是，突变体 p97 中封闭的 ADP 数量显着减少。出乎意料的是，突变体的 ATPgS 滴定曲线是双相的，只能拟合双位点模型。高亲和力位点的 Kd 值已确定，所有突变体均接近 0.1 uM，而低亲和力位点的 Kd 值则与显着误差相关。在 ATPgS 滴定实验中，突变体 p97 再次表现出比野生型更高的化学计量。提出了 D1 结构域中 ATP 循环的四种核苷酸结合状态的模型。我们还研究了 IBMPFD 突变如何影响控制 p97 功能的分子机制。我们发现，在突变体 p97 的六聚环内，D1 结构域无法调节其各自的核苷酸结合状态，如预结合 ADP 量较低、ADP 结合亲和力较弱、ATP-gS 结合完全占据以及整体升高所证明的那样。 ATP 酶活性，表明亚基之间通讯丧失。残基 Phe-360 侧链构象的改变进一步说明了亚基之间的缺陷通讯，该残基从相邻亚基探测到核苷酸结合口袋。因此，突变体 p97 六聚环中 N 结构域的构象变得不协调，从而影响其处理底物的能力。我们对分子内通讯途径的研究还发现，人类 AAA+ 蛋白 p97 的 N-D1 截短末端存在一个 22 个氨基酸的肽，称为 D1-D2 连接子，已被证明可以激活 ATP D1结构域的水解，但激活机制仍不清楚。我们确定了这个 D1-D2 连接子的 N 端一半，从人类到真菌都普遍保守，对于 ATP 酶的激活至关重要。基于对所有可用 p97 结构的分析，我们观察到 D1-D2 连接子的存在影响 p97 亚基结合形成六聚环的方式，这在晶体对称性中得到了体现。连接子的存在导致较低的晶体对称性，两种新的晶体结构强化了这一观察结果，即带有连接子的野生型 N-D1 截短物和没有连接子的 L198W 突变体 N-D1 截短子，目前已确定工作。在缺乏 D1-D2 连接体的情况下，D1 ATP 酶结构域缺乏活性意味着不对称亚基排列的功能重要性，我们建议通过不对称指数指标进行定量估计。结构比较将 D1-D2 连接子的构象与相邻亚基的 Arg 指构象相关联，表明 D1 结构域在 D2 结构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) 相关降解在内的各种细胞过程至关重要。众所周知，p97 的 N 结构域会发生大的核苷酸依赖性构象变化，但这种构象变化的生理相关性尚未确定。我们发现 p97 主要通过与内质网驻留蛋白 VIMP 相互作用而被招募到内质网膜上。在野生型 p97 中，可以通过 N 结构域的核苷酸依赖性构象开关来调节募集，并且这种调节在致病突变体中被消除。 p97、VIMP及其复合物的一系列结构揭示了该调节的分子机制，从而表明p97 N结构域的核苷酸依赖性构象变化的生理作用。此外，当 AMP-PNP 占据 D1 域时，可以看到 N 域的中间位置，从而可以构建 N 域运动的轨迹。我们的研究结果表明核苷酸依赖性膜相互作用循环可能适用于其他 p97 依赖性事件。实验室正在积极研究的另一种 AAA 蛋白称为 bcs1，与迄今为止已知的大多数 AAA 蛋白的功能不同，它涉及折叠蛋白跨膜易位。在确定了小鼠 Bcs1 (mBcs1) 在不同核苷酸状态和构象下的结构后，我们现在获得了一个结构框架，从中可以期望对 Bcs1 的转运机制有更详细的了解。目前，我们重点关注的研究可能会揭示 Bcs1 如何识别和结合折叠的 ISP-ED，捕获其在跨膜转运底物中的作用，并可视化它如何将底物释放到膜中。为了实现这些目标，必须结合使用各种研究方法。从结构角度来看，有必要获得Bcs1与底物ISP复合物的结构，以解决底物结合如何触发Bcs1变化以及底物结合是否足以诱导核苷酸交换的问题。还需要结构来确定 Bcs1 的亚基是否以顺序方式或以协调方式发挥作用。前者是许多六聚体 AAA 蛋白所表现出的交手或分流清洗易位机制的标志。在 apo 和 ADP 结合结构中，还应该研究堵塞 Bcs1 特异性结构域中心小孔的未知密度。从生物化学角度来看，不同核苷酸状态寿命的动力学研究将为反应环境中的限速步骤提供线索。将这些研究与诱变相结合可能会在验证各种机制假设方面发挥重要作用。例如，为了防止易位期间的质子泄漏，提出了一种类似气闸的机制。然而，如何控制密封孔的打开和关闭还需要进一步阐明。诱变研究将允许对许多已记录的疾病相关突变体进行功能和结构表征。这些结构还应有助于开发调节 Bcs1 功能的药物。

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.