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.

我们最近的工作一直集中在人类AAA蛋白P97上，这是主要的胞质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。我们的工作对D1环的功能做出了重大贡献，D1环是P97的调节域。该结果来自我们对引起IBMPFD或MSP1的P97突变体的研究。 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。从ADP-到ATPGS结合状态的过渡伴随着N-D1链接器中的循环至螺旋转换，并在p97的N末端区域中明显重新排序。 X射线散射实验表明，野生型P97亚基经历了类似的核苷酸依赖性N域构象变化。我们建议通过破坏ADP结合形式的IBMPFD突变改变核苷酸状态之间的过渡时间，并因此干扰N-域与其底物之间的相互作用。还研究了SAXS中ATPGS或ADP的存在，野生型和突变的N-D1片段也被研究，这表明在溶液中也发生了依赖核苷酸依赖性的上下N核苷构象变化。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，而野生型N-D1的化学计量计为0.35，这表明6个位点中只有2个可用于绑定，这与野生型P97中咬伤的ADP的先前报道一致。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中的闭塞ADP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。首先，有一个ATP状态，具有ATP绑定和上符号中的N-域。在野生型P97六聚体中，由于不可交换，预遇到的ADP，并非所有亚基都在上符号中具有N域，即使溶液中的ATP过量过量。因此，我们假设存在ADP锁定状态，在D1位点具有不可交换的预击ADP，而在下符号中则具有N域。该状态对于野生型p97功能似乎很重要，并且预陷的ADP尤其难以交换。野生型p97的N-D1片段的结构可能代表这种构象。在第三个状态下，称为ADP-OPEN，ADP是约束但可以交换的。突变体P97通过其双相ITC滴定曲线观察到了这种状态，并且大概与ADP锁定状态平衡。具有绑定ADP的R155H的结构代表了这种构象。第四个状态是空状态，核苷酸结合位点未占用，而N域位于未知位置。然而，野生型和突变体之间的差异在于ADP锁定状态与ADP开放状态之间的过渡。我们建议，在野生型蛋白质中，这种过渡是由不同亚基D1域中的核苷酸结合状态中的不对称状态进行紧密控制和特征的，导致ADP开放状态较低，而在IBMPFD突变体中，这种控制机制被改变，导致该控制机制的浓度很高，导致adp-op-pop-pop-pop-popopen。我们还研究了IBMPFD突变如何影响控制p97功能的分子机制。 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.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。