Study of AAA proteins by X-ray protein crystallography

我们最近的工作一直集中在人类AAA蛋白p97。主要的胞质AAA分子伴侣p97的突变导致与骨佩吉特病和额颞叶痴呆（IBMPFD）相关的包涵体肌病。IBMPFD突变体在N-末端结构域（N-结构域）和相邻AAA结构域（D1）之间的界面处具有单个氨基酸取代，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Mg 2 +-ATPgS存在下采用非典型的N-结构域构象，这是ADP可逆的，首次证明了N-结构域的核苷酸依赖性构象变化。从ADP-到ATP-结合状态的转变伴随着N-D1接头中的环到螺旋的转换和p97的N-末端区域中的明显重新排序。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-结构域构象变化。我们建议，IBMPFD突变，通过破坏ADP结合形式的稳定，改变核苷酸状态之间的过渡的时间，从而干扰N-结构域和它们的底物之间的相互作用。野生型和突变型N-D1片段也通过SAXS在ATPgS或ADP存在下进行了研究。与ADP结合的形式相比，ATPgS结合的N-D1片段的回转半径（Rg）始终小3-5 A。N-D1在溶液中的构象变化也可以通过距离分布函数p（r）来证明，其中对于ATPgS结合的N-D1片段，观察到分布向较短载体的显著偏移。p（r）的这种偏移在载体长度超过90 A时最明显，与大规模N结构域构象变化一致。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，表明晶体学观察到的N-结构域构象的差异不仅存在于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），我们测定了野生型N-D1对ADP的Kd值为0.88 μ M，化学计量为0.35，表明6个位点中只有2个可用于结合，这与先前报道的值一致。相比之下，突变型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，R155 H突变体显示最大降低，Kd为4.25 μ M。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。与先前的发现一致，野生型p97显示出与ADP相似的0.89 μ M的ATPgS的Kd值。出乎意料的是，突变体的ATPgS滴定曲线是双相的，只能拟合到两个位点模型。高亲和力位点的Kd值被很好地确定，并且对于所有突变体接近0.1 μ M，而低亲和力位点的Kd值与显著误差相关。同样，突变体p97在ATPgS滴定实验中显示出比野生型更高的化学计量。提出了一个D1结构域ATP循环的四核苷酸结合态模型。首先，存在ATP状态，其中ATP结合并且N结构域处于上构象。在野生型p97六聚体中，由于不可交换的、预结合的ADP，即使在溶液中存在过量的ATP，也不是所有亚基的N结构域都处于Up-conformation。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结合ADP，并且N结构域处于向下构象。这种状态似乎对野生型p97功能很重要，并且预先结合的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开放，ADP是结合的，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，并推测其与ADP锁定状态平衡。具有结合ADP的R155 H的结构代表这种构象。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。我们提出，在野生型蛋白质中，这种转变是严格控制的，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开放状态的浓度低，而在IBMPFD突变体中，这种控制机制被改变，导致ADP开放状态的亚基浓度高。

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.