AAA Proteins, Their Functions and Related Diseases

AAA 蛋白、其功能和相关疾病

• 批准号: 10262117
• 负责人: di s xia
• 金额: $ 74.1万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/10262117
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATPase Domain; Adaptor Signaling Protein; Adenosine; Adenylyl Imidodiphosphate; Adopted; Affect; Affinity; Amino Acid Substitution; Amino Acids; Apoptosis; Behavior; Binding; Binding Sites; Biochemical; Biological; Biophysics; Calorimetry; Cancer cell line; Cell Cycle Regulation; Cell physiology; Cells; Clinical; Communication; Complex; Crystallization; DNA biosynthesis; Defect; Deubiquitination; Devices; Dimensions; Disease; Endoplasmic Reticulum; Event; Family; Fingers; Goals; Human; Inclusion Body Myopathy with Early-Onset Paget Disease; Investigation; Link; Location; Measures; Membrane; Membrane Fusion; Membrane Fusion Activity; Merozoite Surface Protein 1; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Machines; Movement; Mutation; Myopathy; N-terminal; Names; Neurodegenerative Disorders; Nucleotides; Organelles; Pathogenicity; Pathway interactions; Patients; Peptides; Phenotype; Physiological; Play; Positioning Attribute; Process; Protein Subunits; Proteins; Quality Control; Regulation; Reporting; Research; Resolution; Roentgen Rays; Role; Series; Side; Site; Structure; Structure-Activity Relationship; Techniques; Time; Titrations; Ubiquitination; Work; base; cancer therapy; cofactor; experimental study; fungus; indexing; inhibitor/antagonist; mitochondrial membrane; molecular phenotype; mutant; protein complex; protein degradation; protein function; recruit; stoichiometry; stress management; unfoldase; valosin-containing protein

Our recent work has been focusing on the human AAA protein p97, the 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 function of the D1 ring, which is the regulatory domain of the p97. This result came from our study 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. The transition from the ADP- to the ATPgS-bound state is accompanied by a loop-to-helix conversion in the N-D1 linker and by an apparent re-ordering in the N-terminal region of p97. X-ray scattering experiments suggest that wild type p97 subunits undergo a similar nucleotide-dependent N-domain conformational change. We propose that IBMPFD mutations, by destabilizing the ADP bound form, alter the timing of the transition between nucleotide states and consequently interfere with the interactions between the N-domains and their substrates. Wild type and mutant N-D1 fragments were also studied in the presence of ATPgS or ADP in solution by SAXS, suggesting that nucleotide-dependent Up- and Down-N-domain conformational change also takes place 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 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 amount 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. First, there is an ATP state, with ATP bound and the N-domain in the Up-conformation. In a wild type p97 hexamer, due to non-exchangeable, pre-bound ADP, not all subunits will have their N-domains in the Up-conformation even with an excess amount of ATP in solution. We therefore hypothesize that there is an ADP-locked state, with non-exchangeable, pre-bound ADP at the D1 site and the N-domain in the Down-conformation. This state appears to be important for wild type p97 function and the pre-bound ADP is particularly difficult to exchange. The structure of the N-D1 fragment of wild type p97 may represent this conformation. In a third state, termed ADP-open, ADP is bound but exchangeable. This state was observed for mutant p97 by its biphasic ITC titration profile and is presumably in equilibration with the ADP-locked state. The structure of R155H with bound ADP represents this conformation. The fourth state is the Empty state, with nucleotide-binding sites unoccupied and the N-domain in an unknown position. The difference between the wild type and mutants, however, lies in the transition between the ADP-locked state and the ADP-open state. We propose that in the wild type protein this transition is tightly controlled and characterized by the asymmetry in nucleotide binding states in D1-domains of different subunits, resulting in a low concentration of the ADP-open state, whereas in IBMPFD mutants, this control mechanism is altered, leading to a high concentration of subunits in the ADP-open state. 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 adenosine-5_-O-(3-thiotriphosphate) 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.

我们最近的工作重点是人类 AAA 蛋白 p97，它是主要的胞质 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 D1 环的功能做出了重大贡献，D1 环是 p97 的调控域。这一结果来自我们对导致 IBMPFD 或 MSP1 的 p97 突变体的研究。 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。从 ADP 到 ATPgS 结合状态的转变伴随着 N-D1 连接子中的环到螺旋的转换以及 p97 N 末端区域的明显重新排序。 X 射线散射实验表明野生型 p97 亚基经历类似的核苷酸依赖性 N 结构域构象变化。我们提出 IBMPFD 突变通过破坏 ADP 结合形式的稳定性，改变核苷酸状态之间转换的时间，从而干扰 N 结构域与其底物之间的相互作用。还通过 SAXS 在溶液中存在 ATPgS 或 ADP 的情况下研究了野生型和突变型 N-D1 片段，表明核苷酸依赖性上行和下行 N 结构域构象变化也发生在溶液中。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。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 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。首先，存在 ATP 状态，其中 ATP 结合且 N 结构域处于向上构象。在野生型 p97 六聚体中，由于不可交换的预结合 ADP，即使溶液中存在过量 ATP，并非所有亚基的 N 结构域都处于向上构象。因此，我们假设存在 ADP 锁定状态，在 D1 位点和 Down 构象中的 N 结构域具有不可交换的预结合 ADP。这种状态似乎对于野生型 p97 功能很重要，并且预结合的 ADP 特别难以交换。野生型p97的N-D1片段的结构可能代表这种构象。在第三种状态（称为 ADP-open）中，ADP 是绑定的但可交换。通过其双相 ITC 滴定曲线观察到突变体 p97 的这种状态，并且可能与 ADP 锁定状态平衡。结合有 ADP 的 R155H 的结构代表了这种构象。第四种状态是空状态，核苷酸结合位点未被占据，N 结构域处于未知位置。然而，野生型和突变体之间的差异在于ADP锁定状态和ADP开放状态之间的转变。我们认为，在野生型蛋白质中，这种转变受到严格控制，其特征在于不同亚基的 D1 结构域中核苷酸结合状态的不对称性，导致 ADP 打开状态浓度较低，而在 IBMPFD 突变体中，这种控制机制被改变，导致亚基处于 ADP 打开状态的高浓度。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、完全占据腺苷-5_-O-(3 -硫代三磷酸）结合，并且总体 ATP 酶活性升高，表明亚基之间失去了通讯。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。