Study of AAA proteins by X-ray protein crystallography

我们最近的工作主要集中在人类AAA蛋白p97，主要的细胞质AAA伴侣蛋白。p97基因突变导致与骨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。最近，我们研究了IBMPFD突变如何影响控制p97功能的分子机制，但仍不清楚。我们发现，在突变体p97的六聚体环内，D1结构域无法调节各自的核苷酸结合状态，这表现为ADP的预结合量较低，ADP的结合亲和力较弱，腺苷- 5_o -（3-硫代三磷酸）的结合完全占据，以及总体atp酶活性升高，表明亚基之间的通信丧失。残基ph -360侧链的构象改变进一步说明了亚基之间的通信缺陷，该残基从邻近亚基探针到核苷酸结合口袋。因此，突变体p97的六聚体环n结构域的构象变得不协调，从而影响其处理底物的能力。我们对分子内通讯途径的研究还发现，在人类AAA+蛋白p97的N-D1截尾末端存在一个22个氨基酸的肽，称为D1- d2连接体，已被证明可以激活D1结构域的ATP水解，但其激活机制尚不清楚。我们确定了D1-D2连接体的n端一半，从人类到真菌都普遍保守，对atp酶的激活至关重要。通过对所有可用p97结构的分析，我们观察到D1-D2连接体的存在影响了p97亚基结合形成六聚环的方式，这表现在晶体对称性上。连接子的存在导致较低的晶体对称性，这一观察结果被两种新的晶体结构所加强，一种是野生型N-D1截断带连接子，另一种是L198W突变型N-D1截断不带连接子。在D1- d2连接体缺失的情况下，D1 atp酶结构域缺乏活性意味着不对称亚基排列的功能重要性，我们建议通过不对称指数来定量估计。结构比较将D1-D2连接体的构象与邻近亚基的Arg-finger的构象联系起来，表明d1结构域对d2结构域的构象具有调节作用。

Our recent work has been focusing on the human AAA protein p97, the major cytosolic AAA chaperone. Mutations in p97 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. More recently, we investigated how IBMPFD mutations affect the molecular mechanism that governs the function of p97 remains unclear. We showed that within the hexameric ring of a mutant p97, D1 domains fail to regulate their respective nucleotide-binding states, as evidenced by the lower amount of prebound ADP, weaker ADP binding affinity, full occupancy of adenosine-5_-O-(3-thiotriphosphate) binding, and elevated overall ATPase activity, indicating a loss of communication among subunits. Defective communication between subunits is further illustrated by altered conformation in the side chain of residue Phe-360 that probes into the nucleotide-binding pocket from a neighboring subunit. Consequently, conformations of N-domains in a hexameric ring of a mutant p97 become uncoordinated, thus impacting its ability to process substrate. Our investigation into the intra-molecular communication pathway also led to the discovery that the presence of a 22 amino acid peptide at the end of N-D1 truncate, named D1-D2 linker, of the human AAA+ protein p97 has been shown to activate ATP hydrolysis of the D1 domain, but the mechanism of activation remains unclear. We identified the N-terminal half of this D1-D2 linker, which is ubiquitously conserved from human to fungi, is essential for the activation of the ATPase. Based on the analysis of all available p97 structures, we observed that the presence of the D1-D2 linker affects the way subunits of p97 associate to form hexameric rings, which was manifested in the crystal symmetry. The presence of the linker leads to lower crystal symmetry, an observation that is reinforced by the two new crystal structures, a wild-type N-D1 truncate with the linker and a L198W mutant N-D1 truncate without the linker, determined in the present work. The lack of activity of the D1 ATPase domain in the absence of D1-D2 linker implies the functional importance of asymmetric subunit arrangement, which we suggest to be estimated quantitatively by the metrics Asymmetirc Index. Structure comparison correlates the conformation of the D1-D2 linker to conformation of the Arg-finger from a neighboring subunit, suggesting a regulatory role of the D1-domain in the conformation of D2-domain.