Study of AAA proteins by X-ray protein crystallography

细胞内蛋白降解是一种重要的翻译后调控机制，在许多重要的细胞功能中起着至关重要的作用；它还可以去除受损的、变性的和其他异常的蛋白质。我们选择用晶体学方法研究的Clp蛋白酶在许多生物体中都是必需的，并且高度保守。ClpA属于AAA+蛋白家族，这是一类广泛的蛋白质构象转导atp酶，参与大量重要的细胞功能。Clp和其他依赖ATP的蛋白酶是结构和机制复杂的蛋白质，其结构/功能关系反映了需要在亚分子水平上理解的重要生化原理，并有助于理解其他利用ATP的酶的功能，如ABC转运蛋白。我的团队已经确定了全长ClpA的晶体结构，ClpAP复合体的调节成分。ClpA结构作为首个具有两个AAA+模块的AAA+结构，揭示了两个AAA+模块功能差异的结构基础。我的小组还获得了ClpA与ClpS复合物中分离的n结构域的结构，已知该结构域可以改变ClpA中的底物选择性，就像许多其他AAA+蛋白使用的接头蛋白一样。我们还通过分析其多个蛋白质和肽结合位点的晶体学细节来定义ClpA n端结构域的功能。在获得了ClpAPS体系中所有组分的结构后，我们的研究小组现在正专注于获得由结合核苷酸诱导的各种构象的ClpA结构，以及ClpA与ClpS或ClpP的二元配合物的结晶，以及与各种底物的结晶。为了了解AAA蛋白将ATP水解释放的能量与这些酶所做的机械功耦合的共同机制，我们还研究了其他AAA蛋白（如P97）在不同状态下的结构，P97是一种真核AAA蛋白，参与许多重要的细胞功能，如蛋白质降解和膜融合。我们将比较不同AAA蛋白的结构，以便实现这一目标。

Intracellular protein degradation is a major post-translational regulatory mechanism and plays a crucial role in many vital cellular functions; it also serves to remove damaged, denatured, and other abnormal proteins. The Clp proteases, which we have chosen to study crystallographically are essential in many organisms and highly conserved. ClpA belongs to the family of AAA+ proteins, a broad class of protein conformation-transducing ATPases involved in a plethora of vital cellular functions. Clp and other ATP-dependent proteases are structurally and mechanistically complex proteins, whose structure/function relationships reflect important biochemical principles that need to be understood at the submolecular level and would be beneficial to understanding functions of other ATP utilizing enzymes, such as ABC transporters. My group has determined the crystal structure of the full-length ClpA, the regulatory component of the ClpAP complex. As the first AAA+ structure with two AAA+ modules, the ClpA structure has provided insights into the structural basis for functional difference of the two AAA+ modules. my group has also obtained the structure of the isolated N-domain of ClpA in complex with ClpS that is known to alter the substrate selectivity in ClpA, much like the adaptor proteins used by many other AAA+ proteins. We also defined the function of the ClpA N-terminal domain by analyzing crystallographically details of its multiple protein and peptide binding sites. Having obtained structures of all components in the ClpAPS system, our group is now focusing on obtaining structures of ClpA in various conformations induced by bound nucleotide, and on crystallizing binary complexes of ClpA with either ClpS or ClpP, as well as with various substrates.In order to understand the common mechanism by which AAA proteins couple the energy released from ATP hydrolysis to mechanical work performed by these enzymes, we are also looking at the structures in different states of other AAA proteins such as P97, an eukaryotic AAA protein involved in a number of important cellular functions such as protein degradation and membrane fusion. We will compare structures of different AAA proteins so that such a goal can be achieved.