Ishikawa Takashi, Maurizi Michael R, Steven Alasdair C
Laboratory of Structural Biology Research, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, MD 20892-8025, USA.
J Struct Biol. 2004 Apr-May;146(1-2):180-8. doi: 10.1016/j.jsb.2003.11.018.
ClpAP is a barrel-like complex consisting of hexameric rings of the ClpA ATPase stacked on the double heptameric ring of ClpP peptidase. ClpA has two AAA+ domains (Dl and D2) and a 153-residue N-domain. Substrate proteins bind to the distal surface of ClpA and are unfolded and translocated axially into ClpP. To gain insight into the functional architecture of ClpA in the ATPgammaS state, we have determined its structure at 12A resolution by cryo-electron microscopy. The resulting model has two tiers, corresponding to rings of Dl and D2 domains: oddly, there is no sign of the N-domains in the density map. However, they were detected as faint diffuse density distal to the Dl tier in a difference image between wild-type ClpAP and a mutant lacking the N-domain. This region is also accentuated in a variance map of ClpAP and in a difference imaging experiment with ClpAP complexed with ClpS, a 12kDa protein that binds to the N-domain. These observations demonstrate that the N-domains are highly mobile. From molecular modeling, we identify their median position and estimate that they undergo fluctuations of at least 30A. We discuss the implications of these observations for the role of N-domains in substrate binding: either they effect an initial transient binding, relaying substrate to a second site on the Dl tier where unfolding ensues, or they may serve as an entropic brush to clear the latter site of non-specifically bound ligands or substrates bound in non-productive complexes.
ClpAP是一种桶状复合物，由ClpA ATP酶的六聚体环堆叠在ClpP肽酶的双七聚体环上组成。ClpA有两个AAA+结构域（D1和D2）和一个153个残基的N结构域。底物蛋白与ClpA的远端表面结合，然后被展开并轴向转运到ClpP中。为了深入了解处于ATPγS状态的ClpA的功能结构，我们通过冷冻电子显微镜在12埃分辨率下确定了其结构。所得模型有两层，对应于D1和D2结构域的环：奇怪的是，在密度图中没有N结构域的迹象。然而，在野生型ClpAP与缺乏N结构域的突变体之间的差异图像中，它们被检测为D1层远端微弱的弥散密度。在ClpAP的方差图以及与ClpS（一种与N结构域结合的12kDa蛋白质）复合的ClpAP的差异成像实验中，该区域也很突出。这些观察结果表明N结构域具有高度的流动性。通过分子建模，我们确定了它们的中间位置，并估计它们经历至少30埃的波动。我们讨论了这些观察结果对N结构域在底物结合中作用的影响：要么它们实现初始的瞬时结合，将底物传递到D1层上随后发生展开的第二个位点，要么它们可能作为一种熵刷，清除该位点上非特异性结合的配体或在非生产性复合物中结合的底物。
