Study of AAA proteins by X-ray protein crystallography

我们确定了全长ClpA结构，这是第一个2型AAA蛋白。ClpA是一种Hsp100/Clp伴侣，是atp依赖性ClpAP蛋白酶的组成部分，参与调节蛋白质降解以及蛋白质聚集体的溶解和降解。ClpA亚基的晶体结构显示一个具有伪双重对称的n端结构域和两个AAA+模块（D1和D2），每个模块由一个大的和一个小的子结构域组成，ADP结合在子结构域交界处。n结构域与D1结构域的相互作用方式类似于其他AAA+蛋白的适配器结合结构域。D1和D2头尾相连，符合蛋白质底物的协同和矢量易位。在ClpA的平面六聚体模型中，将ClpA D1和D2组装成AAA+模块已知结构的同六聚环，D1-D1和D2-D2界面的差异与其各自对六聚体稳定性和atp酶活性的贡献有关。我们还确定了ClpA与其调节蛋白ClpS的复杂结构，该蛋白参与了底物选择。大肠杆菌atp依赖性蛋白酶（ClpAP）的底物选择性和蛋白水解活性是由接合蛋白（ClpS）调节的。ClpS与ClpAP复合物的调节成分ClpA结合。在2.3 A和3.3 A的分辨率下，我们报道了ClpS与ClpA分离的n端结构配合物在两种不同晶体形式下的晶体结构。ClpS结构形成一个α / β三明治，在拓扑结构上类似于核糖体蛋白L7/L12的c端结构域。在两种晶体形式中，ClpS都在n端区域上接触两个表面；蛋白酶保护实验表明，更广泛的界面在溶液中更有利。ClpS的n端残基在晶体中不可见；去除前17个残基产生ClpS/N， ClpS/N与ClpA N结构域结合，但不再抑制ClpA活性。在ClpA的n端结构域中发现了两个His和一个Glu残基的锌结合位点。在ClpS与六聚体ClpA结合的模型中，ClpS的n端指向ClpA的远端表面，这表明ClpS的n端区域可能影响顶端表面的生产底物相互作用或底物进入ClpA易位通道。我们最近的工作集中在人类AAA蛋白p97上。主要的胞浆AAA伴侣蛋白p97的突变可导致与骨Pagets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）和相邻的AAA结构域（D1）之间的界面上有单个氨基酸取代，导致对ADP的亲和力降低。携带IBMPFD突变的p97 N-D1片段在Mg2+- atpg存在下，结构呈非典型n结构域，ADP可逆转，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连接体的环向螺旋转换以及p97 n端区域的明显重排序。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结构域构象变化。我们认为，IBMPFD突变通过破坏ADP结合形式的稳定性，改变了核苷酸状态之间转变的时间，从而干扰了n结构域与其底物之间的相互作用。在atpg或ADP存在的情况下，SAXS还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3-5 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atpgs结合的N-D1片段的分布向较短的向量方向明显移动，这种p(r)的移动在向量长度超过90 a时最为明显，与大规模的n域构象变化相一致。此外，基于晶体结构计算的分布函数变化与实验得到的分布函数一致，表明晶体学观察到的溶液中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。使用等温滴定量热法（ITC），我们确定了野生型N-D1对ADP的Kd值为0.88 uM，化学计量量为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，突变体p97 N-D1片段对ADP的结合亲和力降低，并且降低的程度取决于位点。例如，R155H突变体的Kd最大降低为4.25 uM。值得注意的是，突变体的结合化学计量的变化与结合亲和力的变化相关。与之前的研究结果一致，野生型p97对atpg的Kd值为0.89 uM，与ADP相似。出乎意料的是，突变体的atpg滴定谱是双相的，只能适用于两个位点的模型。高亲和位点的Kd值确定得很好，所有突变体的Kd值都接近0.1 uM，而低亲和位点的Kd值存在显著误差。在atpg滴定实验中，突变体p97再次显示出比野生型更高的化学计量学。提出了d1结构域ATP循环的四种核苷酸结合状态模型。首先是ATP状态，ATP结合，n结构域呈上构象。在野生型p97六聚体中，由于不可交换的预结合ADP，即使溶液中有过量的ATP，也不是所有亚基的n结构域都呈上构象。因此，我们假设存在ADP锁定状态，D1位点具有不可交换的预结合ADP，而n结构域位于down构象中。这种状态似乎对野生型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。在第三种状态下，称为ADP开放状态，ADP是绑定的，但可以交换。这种状态在突变体p97的双相ITC滴定谱中观察到，可能与adp锁定状态相平衡。R155H结合ADP的结构就是这种构象。第四种状态是空状态，核苷酸结合位点未被占用，n结构域处于未知位置。然而，野生型和突变型的区别在于adp锁定状态和adp开放状态之间的过渡。我们提出，在野生型蛋白中，这种转变受到严格控制，其特征是不同亚基d1结构域核苷酸结合状态的不对称，导致adp开放状态的浓度低，而在IBMPFD突变体中，这种控制机制发生改变，导致adp开放状态的亚基浓度高。

We have determined the full-length ClpA structure, which was the first type-2 AAA protein. ClpA, an Hsp100/Clp chaperone and an integral component of the ATP-dependent ClpAP protease, participates in regulatory protein degradation and the dissolution and degradation of protein aggregates. The crystal structure of the ClpA subunit reveals an N-terminal domain with pseudo two-fold symmetry and two AAA+ modules (D1 and D2) each consisting of a large and a small sub-domain with ADP bound in the sub-domain junction. The N-domain interacts with the D1 domain in a manner similar to adaptor-binding domains of other AAA+ proteins. D1 and D2 are connected head-to-tail consistent with a cooperative and vectorial translocation of protein substrates. In a planar hexamer model of ClpA, built by assembling ClpA D1 and D2 into homohexameric rings of known structures of AAA+ modules, the differences in D1-D1 and D2-D2 interfaces correlate with their respective contributions to hexamer stability and ATPase activity. We also determined the complex structure of ClpA with its regulator protein ClpS that involves in substrate selection. Substrate selectivity and proteolytic activity for the E. coli ATP-dependent protease, ClpAP, is modulated by an adaptor protein, ClpS. ClpS binds to ClpA, the regulatory component of the ClpAP complex. We report the crystal structure of ClpS in complex with the isolated N-terminal domain of ClpA in two different crystal forms at 2.3 A and 3.3 A resolution. The ClpS structure forms a alfa/beta-sandwich and is topologically analogous to the C-terminal domain of the ribosomal protein L7/L12. ClpS contacts two surfaces on the N-terminal domain in both crystal forms; the more extensive interface was shown to be favored in solution by protease protection experiments. The N-terminal 20 residues of ClpS are not visible in crystal; removal of the first 17 residues produces ClpS/N, which binds to the ClpA N-domain but no longer inhibits ClpA activity. A zinc-binding site involving two His and one Glu residue was identified crystallographically in the N-terminal domain of ClpA. In a model of ClpS bound to hexameric ClpA, ClpS is oriented with its N-terminus directed toward the distal surface of ClpA, suggesting that the N-terminal region of ClpS may affect productive substrate interactions at the apical surface or substrate entry into the ClpA translocation channel.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.