Escherichia coli ClpXP is one of the most thoroughly studied AAA+ proteases, but relatively little is known about the reactions that allow it to bind and then engage specific protein substrates before the adenosine triphosphate (ATP)-fueled mechanical unfolding and translocation steps that lead to processive degradation. Here, we employ a fluorescence-quenching assay to study the binding of ssrA-tagged substrates to ClpXP. Polyphasic stopped-flow association and dissociation kinetics support the existence of at least three distinct substrate-bound complexes. These kinetic data fit well to a model in which ClpXP and substrate form an initial recognition complex followed by an intermediate complex and then, an engaged complex that is competent for substrate unfolding. The initial association and dissociation steps do not require ATP hydrolysis, but subsequent forward and reverse kinetic steps are accelerated by faster ATP hydrolysis. Our results, together with recent cryo-EM structures of ClpXP bound to substrates, support a model in which the ssrA degron initially binds in the top portion of the axial channel of the ClpX hexamer and then is translocated deeper into the channel in steps that eventually pull the native portion of the substrate against the channel opening. Reversible initial substrate binding allows ClpXP to check potential substrates for degrons, potentially increasing specificity. Subsequent substrate engagement steps allow ClpXP to grip a wide variety of sequences to ensure efficient unfolding and translocation of almost any native substrate.

大肠杆菌ClpXP是研究最深入的AAA +蛋白酶之一，但是在三磷酸腺苷（ATP）推动的机械解折叠和易位步骤导致进行性降解之前，有关使其结合并随后结合特定蛋白质底物的反应知之甚少。在这里，我们采用荧光猝灭测定来研究ssrA标签底物与ClpXP的结合。多相停止流缔合和解离动力学支持至少三种不同的底物结合复合物的存在。这些动力学数据非常适合模型，其中ClpXP和底物形成一个初始识别复合物，然后是一个中间复合物，然后是一个能胜任底物展开的结合复合物。最初的缔合和解离步骤不需要ATP水解，但是随后的前进和后退动力学步骤会通过更快的ATP水解而加速。我们的研究结果与最近与基质结合的ClpXP低温EM结构一起，支持了以下模型：ssrA degron最初结合在ClpX六聚体轴向通道的顶部，然后逐步转移至更深的通道将基材的原始部分拉向通道开口。可逆的初始底物结合使ClpXP可以检查潜在底物的degrons，从而可能提高特异性。后续的底物接合步骤使ClpXP可以抓握多种序列，以确保几乎任何天然底物的有效展开和易位。与最近与基质结合的ClpXP低温-EM结构一起，支持一种模型，其中ssrA degron最初结合在ClpX六聚体轴向通道的顶部，然后逐步转移到通道的更深处，最终拉动天然衬底部分抵靠通道开口。可逆的初始底物结合使ClpXP可以检查潜在底物的degrons，从而可能提高特异性。后续的底物接合步骤使ClpXP可以抓握多种序列，以确保几乎任何天然底物的有效展开和易位。与最近与基质结合的ClpXP低温-EM结构一起，支持一种模型，其中ssrA degron最初结合在ClpX六聚体轴向通道的顶部，然后逐步转移到通道的更深处，最终拉动天然衬底部分抵靠通道开口。可逆的初始底物结合使ClpXP可以检查潜在底物的degrons，从而可能提高特异性。后续的底物接合步骤使ClpXP可以抓握多种序列，以确保几乎任何天然底物的有效展开和易位。支持一个模型，其中ssrA degron最初结合在ClpX六聚体轴向通道的顶部，然后逐步转移到通道的更深处，最终将基材的天然部分拉向通道开口。可逆的初始底物结合使ClpXP可以检查潜在底物的degrons，从而可能提高特异性。后续的底物接合步骤使ClpXP可以抓握多种序列，以确保几乎任何天然底物的有效展开和易位。支持一个模型，其中ssrA degron最初结合在ClpX六聚体轴向通道的顶部，然后逐步转移到通道的更深处，最终将基材的天然部分拉向通道开口。可逆的初始底物结合使ClpXP可以检查潜在底物的degrons，从而可能提高特异性。后续的底物接合步骤使ClpXP可以抓握多种序列，以确保几乎任何天然底物的有效展开和易位。