Study of AAA proteins by X-ray protein crystallography

我们最近的工作集中在人类AAA蛋白p97上。P97是一种主要的胞浆AAA伴侣蛋白，它的突变会导致与Pagets病和额颞性痴呆(IBMPFD)相关的包涵体肌病。IBMPFD突变体在N-末端结构域(N-结构域)和相邻的AAA结构域(D1)之间的界面上有单一的氨基酸取代，导致与ADP的亲和力降低。携带IBMPFD突变的p97N-D1片段在Mg2+-ATPgS存在下呈非典型的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的N-末端区域的明显的重新排序。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的N结构域的构象变化。我们认为，IBMPFD突变通过破坏ADP结合形式的稳定，改变了核苷酸状态之间的转换时间，从而干扰了N-结构域与其底物之间的相互作用。在ATPgS或ADP存在下，用SAXS研究了野生型和突变型N-D1片段。与ADP结合的形式相比，ATPgS结合的N-D1片段的旋转半径(Rg)始终小3-5A。N-d1在溶液中的构象变化也可以用距离分布函数p(R)来表示，其中ATPgS结合的N-d1片段的分布显著向较短的载体移动，这种移动在载体长度超过90A时最为明显，这与大规模的N-结构域的构象变化相一致。此外，计算的基于晶体结构的分布函数的变化与实验获得的分布函数相一致，表明在溶液中观察到的N-结构域的构象差异不仅存在于p97突变体中，也存在于野生型p97中。利用等温滴定热法(ITC)，我们测定了野生型N-d1对ADP的Kd值为0.88um，化学计量比为0.35，表明6个位点中只有2个可结合，这与以前报道的值一致。相反，突变型p97N-d1片段与ADP的结合亲和力降低，且降低的程度与位点有关。例如，R155H突变体的Kd值最大，为4.25um。值得注意的是，结合化学计量比的变化与突变体结合亲和力的变化相关。与以前的发现一致，野生型p97的ATPgS的Kd值为0.89um，与ADP的Kd值相似。出乎意料的是，突变体的ATPgS滴定曲线是两相的，只能适用于两个位点的模型。高亲和力位点的Kd值被很好地测定，并且所有突变体的Kd值都接近0.1um，而低亲和力位点的Kd值与显著误差有关。在ATPgS滴定实验中，突变体p97的化学计量比再次高于野生型。提出了一个具有四个核苷酸结合态的D_1结构域的ATP循环模型。首先，存在一个ATP状态，结合了ATP，N-结构域在上构象中。在野生型p97六聚体中，由于不可交换的、预先结合的ADP，即使在溶液中有过量的ATP，也不是所有的亚基都会有N-结构域在上构象中。因此，我们假设存在ADP锁定状态，在D1位具有不可交换的预先结合的ADP，N-结构域处于向下构象。这种状态似乎对野生型p97的功能很重要，并且预先结合的ADP特别难交换。野生型p97的N-D1片段的结构可能代表了这种构象。在称为ADP开放的第三种状态下，ADP是结合但可交换的。突变体p97的双相ITC滴定曲线观察到了这种状态，并推测与ADP锁定状态是平衡的。与ADP结合的R155H的结构代表了这种构象。第四种状态是空态，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区别在于ADP锁定状态和ADP开放状态之间的转换。我们认为，在野生型蛋白中，这一转变受到严格控制，其特征是不同亚基的D1域的核苷酸结合状态不对称，导致低浓度的ADP-开放状态，而在IBMPFD突变体中，这种控制机制发生改变，导致高浓度的亚基处于ADP-开放状态。

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.