Study of AAA proteins by X-ray protein crystallography

X射线蛋白质晶体学研究AAA蛋白质

• 批准号: 8763147
• 负责人: di s xia
• 金额: $ 15.39万
• 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/8763147
• 关键词: ATP phosphohydrolase; ATPase Domain; Adaptor Signaling Protein; Adopted; Affinity; Agreement; Amino Acid Substitution; Bacterial Proteins; Behavior; Binding; Binding Sites; Biochemical; Calorimetry; Cell Cycle Regulation; Cell physiology; Cells; Crystallography; DNA biosynthesis; Defect; Devices; Family; Frontotemporal Dementia; Goals; Human; Inclusion Bodies; Length; Location; Measures; Membrane Fusion; Membrane Fusion Activity; Membrane Proteins; Modeling; Molecular Chaperones; Molecular Conformation; Molecular Machines; Mutation; Myopathy; N Domain; N-terminal; Neurodegenerative Disorders; Nucleotides; Osteitis Deformans; Patients; Play; Positioning Attribute; Process; Proteins; Radial; Reporting; Research; Roentgen Rays; Role; Site; Solutions; Structure; Structure-Activity Relationship; Techniques; Time; Titrations; Ubiquitination; Work; base; cofactor; mitochondrial membrane; mutant; protein complex; protein degradation; research study; stoichiometry; stress management; unfoldase; vector

Our recent work has been focusing on the human AAA protein p97. Mutations in p97, the major cytosolic AAA chaperone, cause inclusion body myopathy associated with Pagets disease of the bone and frontotemporal dementia (IBMPFD). IBMPFD mutants have single amino acid substitutions at the interface between the N-terminal domain (N-domain) and the adjacent AAA domain (D1), resulting in a reduced affinity for ADP. The structures of p97 N-D1 fragments bearing IBMPFD mutations adopt an atypical N-domain conformation in the presence of Mg2+-ATPgS, which is reversible by ADP, 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 by SAXS. The radii of gyration (Rg) are consistently 3-5 A smaller for the ATPgS-bound N-D1 fragment as compared to the ADP-bound form. The conformational change of N-D1 in solution can also be demonstrated by the distance distribution functions, p(r), in which a significant shift in the distribution towards shorter vectors was observed for the ATPgS-bound N-D1 fragments, This shift in p(r) is most obvious at vector lengths beyond 90 A, consistent with the large-scale N-domain conformational change. Furthermore, calculated changes in the distribution function based on crystal structures are in agreement with the experimentally obtained distribution functions, suggesting that the crystallographically observed differences in conformation of the N-domain exist in solution not only for p97 mutants but also for wild type p97. 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 reported values. By contrast, mutant p97 N-D1 fragments displayed reduced binding affinities for ADP and the level of reduction is site dependent. For example, the R155H mutant showed a maximum reduction with a Kd of 4.25 uM. Notably, the changes in the binding stoichiometry are correlated with the changes in binding affinities for the mutants. Consistent with the previous findings, wild type p97 showed a Kd value for ATPgS of 0.89 uM, similar to that for ADP. 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.

我们最近的工作重点是人类 AAA 蛋白 p97。 p97（主要的胞质 AAA 伴侣）的突变会导致与佩吉特骨病和额颞叶痴呆 (IBMPFD) 相关的包涵体肌病。 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，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 存在下采用非典型的 N 结构域构象，该构象可被 ADP 可逆，这首次证明了 N 结构域的核苷酸依赖性构象变化。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 ATPgS 或 ADP 存在的情况下研究了野生型和突变型 N-D1 片段。与 ADP 结合形式相比，ATPgS 结合的 N-D1 片段的回转半径 (Rg) 始终小 3-5 A。溶液中 N-D1 的构象变化也可以通过距离分布函数 p(r) 来证明，其中观察到 ATPgS 结合的 N-D1 片段的分布向较短载体的显着变化。 p(r) 在向量长度超过 90 A 时最明显，与大规模 N 域构象变化一致。此外，基于晶体结构计算的分布函数的变化与实验获得的分布函数一致，这表明晶体学观察到的N结构域构象差异不仅存在于p97突变体的溶液中，而且存在于野生型p97的溶液中。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，这与之前报道的值一致。相比之下，突变的p97 N-D1片段显示出对ADP的结合亲和力降低，并且降低的水平是位点依赖性的。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，结合化学计量的变化与突变体的结合亲和力的变化相关。与之前的发现一致，野生型 p97 显示 ATPgS 的 Kd 值为 0.89 uM，与 ADP 相似。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 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。