AAA Proteins, Their Functions and Related Diseases

我们最近的工作主要集中在两种哺乳动物的AAA蛋白：人类AAA蛋白p97和小鼠线粒体AAA蛋白bcs1。人类p97是一种主要的细胞质AAA伴侣蛋白。虽然已知p97的D2环对p97的整体atp酶活性贡献最大，但D1环的功能尚不清楚。我们的工作为我们理解D1环的功能做出了重大贡献，D1环是p97的调控区域。我们的研究重点是一种引起IBMPFD或MSP1的p97突变体。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，我们的研究表明突变导致对ADP的亲和力降低。携带IBMPFD突变的p97 N-D1片段的结构在Mg2+- atpg存在时呈上向n结构域构象或上向构象，这是ADP（下向构象）可逆的，首次证明了n结构域的核苷酸依赖性构象变化。我们进一步发现野生型p97在溶液中也发生核苷酸依赖的上下n域构象变化。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，化学计量学为0.35，表明6个位点中只有2个可用于结合，这与先前报道的野生型p97中ADP被阻断的数量一致。相比之下，突变体p97 N-D1片段对ADP的结合亲和力降低。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p97中被阻断的ADP数量显著减少。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。我们还研究了IBMPFD突变如何影响控制p97功能的分子机制。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，这可以从ADP的预结合量较低、ADP的结合亲和力较弱、ATP-gS的结合完全占据以及总体atp酶活性升高中得到证明，这表明亚基之间失去了通信。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。最近，我们研究了胞质AAA蛋白p97与细胞膜的关联，这对于包括内质网（ER）相关降解在内的各种细胞过程至关重要。众所周知，p97的n结构域会发生大的核苷酸依赖性构象变化，但这种构象变化的生理相关性尚未确定。我们发现p97主要通过与内质网驻留蛋白VIMP相互作用被募集到内质网膜上。在野生型p97中，这种募集可以通过核苷酸依赖的n结构域构象开关来调节，而这种调节在致病性突变体中被消除。p97、VIMP及其复合物的一系列结构揭示了调控的分子机制，从而提示p97的n结构域核苷酸依赖性构象变化的生理作用。此外，当AMP-PNP占据d1结构域时，可以看到n结构域的中间位置，从而可以构建n结构域运动的轨迹。我们的研究结果表明，依赖于核苷酸的膜相互作用周期可能适用于其他依赖于p97的事件。另一种在实验室中被积极研究的AAA蛋白被称为bcs1，它与迄今为止已知的大多数AAA蛋白的功能不同，它参与折叠蛋白跨膜的易位。在确定了不同核苷酸状态和构象的小鼠Bcs1 （mBcs1）的结构后，我们现在已经获得了一个结构框架，可以从中更详细地了解Bcs1的转运机制。目前，我们的研究重点是揭示Bcs1如何识别和结合折叠的ISP-ED，捕捉其在跨膜转运底物中的作用，以及可视化其如何将底物释放到膜中。为了实现这些目标，必须采用各种研究方法的结合。从结构的角度来看，为了解决底物结合如何触发Bcs1的变化以及底物结合是否足以诱导核苷酸交换等问题，有必要获得Bcs1与底物ISP复合物的结构。还需要结构来确定Bcs1的亚单位是按顺序还是以协调的方式起作用。前者是许多六聚体AAA蛋白所显示的手拉手或分裂洗涤易位机制的标志。在载脂蛋白和ADP结合结构中，未知密度堵塞bcs1特异性结构域中心的小孔也应进行研究。生物化学方面，对不同核苷酸状态生命周期的动力学研究将为反应过程中的限速步骤提供线索。将这些研究与诱变结合起来，可能在验证各种机制假设方面发挥重要作用。例如，为了防止质子在易位过程中泄漏，提出了一种类似气闸的机制。然而，如何控制密封孔的开闭还有待进一步研究。诱变研究将允许许多记录的疾病相关突变的功能和结构特征。这些结构也有助于开发调节Bcs1功能的药物。

Our recent work has been focusing on two mammalian AAA proteins: the human AAA protein p97 and the mouse mitochondrial AAA protein bcs1. The human p97 is a major cytosolic AAA chaperone. Although it has been known that D2 ring of p97 contributes most to the overall ATPase activity of p97, the function of the D1 ring is not clear. Our work has contributed significantly to our understanding the function of the D1 ring, which is the regulatory domain of p97. We focus our study on one type of p97 mutants that cause IBMPFD or MSP1. IBMPFD mutants have single amino acid substitutions at the interface between the N-terminal domain (N-domain) and the adjacent AAA domain (D1) and our work suggests that the mutations result in a reduced affinity for ADP. The structures of p97 N-D1 fragments bearing IBMPFD mutations adopt an Up N-domain conformation or Up-conformation in the presence of Mg2+-ATPgS, which is reversible by ADP (Down-conformation), demonstrating for the first time the nucleotide-dependent conformational change of the N-domain. We further found that wild type p97 also undergoes nucleotide-dependent Up- and Down-N-domain conformational change in solution. 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 reports of the number of occluded ADP in wild-type p97. By contrast, mutant p97 N-D1 fragments displayed reduced binding affinities for ADP. For example, the R155H mutant showed a maximum reduction with a Kd of 4.25 uM. Notably, the number of occluded ADP in mutant p97 is dramatically reduced. 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. We also investigated how IBMPFD mutations affect the molecular mechanism that governs the function of p97. 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 ATP-gS 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. More recently, we studied the association of cytosolic AAA protein p97 to membranes, which is essential for various cellular processes including the endoplasmic reticulum (ER)-associated degradation. The N-domain of p97 is known for undergoing large nucleotide-dependent conformational change but the physiological relevance this conformational change has not been established. We showed p97 is recruited to the ER membrane predominantly by interacting with VIMP, an ER resident protein. The recruitment can be regulated through a nucleotide-dependent conformation switch of the N-domain in wild-type p97 and this regulation is obliterated in pathogenic mutants. The molecular mechanism of the regulation is revealed by a series of structures of p97, VIMP and their complex, thus suggesting a physiological role of the nucleotide-dependent conformational change of the N-domain of p97. In addition, intermediate positions of the N-domain are seen when AMP-PNP occupies the D1-domain, allowing construction of a trajectory for the N-domain movement. Our findings suggest the nucleotide-dependent membrane interaction cycle may be applicable to other p97-dependent events. Another AAA protein that are being actively pursued in the lab is called bcs1 that, unlike the functions of most AAA proteins known to date, involves in folded protein translocation across the membrane. Having determined the structures of mouse Bcs1 (mBcs1) in different nucleotide states and conformations, we now have acquired a structural framework from which more detailed mechanistic insights into the transport mechanism of Bcs1 can be expected. Currently, we focus on studies that will likely reveal how Bcs1 recognizes and binds the folded ISP-ED, capture its action in translocating the substrate across the membrane, and visualize how it releases the substrate into the membrane. To achieve these goals, a combination of various research approaches will have to be employed. From a structural point of view, it is necessary to obtain the structure of Bcs1 in complex with the substrate ISP in order to address the questions on how substrate binding trigger changes in Bcs1 and whether binding of substrate is sufficient to induce nucleotide exchange. Structures are also needed to determine whether subunits of Bcs1 functions in a sequential fashion or in a concerted manner. The former is the hallmark of the hand-over-hand or split wash mechanism of translocation displayed by many hexameric AAA proteins. In the apo and ADP bound structures, the unknown density plugging the small pore in the center of the Bcs1-specific domains should also be investigated. Biochemically, kinetic study of the life span of different nucleotide states will provide clues on the rate limiting steps in the reaction landscape. Coupling these studies with mutagenesis will likely play a major role in verifying various mechanistic hypotheses. For example, to prevent proton leakage during translocation, an airlock-like mechanism was proposed. However, how the opening and closure of the seal pore is controlled requires further elucidation. Mutagenesis studies will allow functional and structural characterizations of many documented disease-related mutants. The structures should also facilitate development of drugs to modulate function of Bcs1.