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: 8937777
• 负责人: di s xia
• 金额: $ 18.39万
• 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/8937777
• 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.

我们最近的工作一直集中在人类AAA蛋白p97上。 p97的突变是主要的胞质AAA伴侣伴侣，导致包容体肌病与骨骼和额颞痴呆症（IBMPFD）相关的肌病。 IBMPFD突变体在N末端结构域（N-域）和相邻AAA结构域（D1）之间的界面上具有单个氨基酸取代（D1），从而降低了对ADP的亲和力。在MG2+-ATPG的存在下，带有IBMPFD突变的P97 N-D1片段的结构采用了非典型的N域构象，ADP可逆转，这是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。还研究了在ATPGS或SAXS存在ADP的情况下，还研究了野生型和突变的N-D1片段。与ADP结合形式相比，对于ATPGS结合的N-D1片段而言，回旋的半径（RG）始终为3-5 A较小。溶液中N-D1的构象变化也可以通过距离分布函数P（R）证明，其中在ATPGS结合的N-D1片段中观察到分布向较短矢量的显着转移，P（R）中的这种转移在90 A以上的矢量长度最明显，一致，一致，符合大尺度的N-尺度构态构态变化。此外，基于晶体结构的分布函数的计算变化与实验获得的分布函数一致，这表明晶体学上观察到的N-域构型的差异在溶液中不仅对于P97突变体而言，而且对于野生型P97。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个可用于结合，这与先前报道的值一致。相比之下，突变体p97 n-d1片段显示出降低的ADP结合亲和力，而还原水平取决于位点。例如，R155H突变体显示最大降低，KD为4.25 um。值得注意的是，结合化学计量学的变化与突变体的结合亲和力的变化相关。与先前的发现一致，野生型P97显示ATPG的KD值为0.89 UM，类似于ADP。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。