Balogh Dóra, Dahmen Maria, Stahl Matthias, Poreba Marcin, Gersch Malte, Drag Marcin, Sieber Stephan A
Center for Integrated Protein Science at the Department of Chemistry , Technische Universität München , Lichtenbergstraße 4 , Garching bei München , D-85747 , Germany . Email:
Department of Bioorganic Chemistry , Faculty of Chemistry , Wrocław University of Technology , Wybrzeże Wyspiańskiego 27 , 50-370 Wrocław , Poland.
Chem Sci. 2017 Feb 1;8(2):1592-1600. doi: 10.1039/c6sc03438a. Epub 2016 Oct 28.
Caseinolytic proteases (ClpP) are important for recognition and controlled degradation of damaged proteins. While the majority of bacterial organisms utilize only a single ClpP, expresses two isoforms (LmClpP1 and LmClpP2). LmClpPs assemble into either a LmClpP2 homocomplex or a LmClpP1/2 heterooligomeric complex. The heterocomplex in association with the chaperone ClpX, exhibits a boost in proteolytic activity for unknown reasons. Here, we use a combined chemical and biochemical strategy to unravel two activation principles of LmClpPs. First, determination of apparent affinity constants revealed a 7-fold elevated binding affinity between the LmClpP1/2 heterocomplex and ClpX, compared to homooligomeric LmClpP2. This tighter interaction favors the formation of the proteolytically active complex between LmClpX and LmClpP1/2 and thereby accelerating the overall turnover. Second, screening a diverse library of fluorescent labeled peptides and proteins with various ClpP mutants allowed the individual analysis of substrate preferences for both isoforms within the heterocomplex. In addition to Leu and Met, LmClpP2 preferred a long aliphatic chain (2-Aoc) in the P1 position for cleavage. Strikingly, design and synthesis of a corresponding 2-Aoc chloromethyl ketone inhibitor resulted in stimulation of proteolysis by 160% when LmClpP2 was partially alkylated on 20% of the active sites. Determination of apparent affinity constants also revealed an elevated complex stability between partially modified LmClpP2 and the cognate chaperone LmClpX. Thus, the stimulation of proteolysis through enhanced binding to the chaperone seems to be a characteristic feature of LmClpPs.
酪蛋白水解蛋白酶（ClpP）对于识别和控制受损蛋白质的降解非常重要。虽然大多数细菌仅利用一种ClpP，但[某种细菌名称未给出]表达两种异构体（LmClpP1和LmClpP2）。LmClpP可组装成LmClpP2同聚体或LmClpP1/2异源寡聚体复合物。与伴侣蛋白ClpX结合的异源复合物，其蛋白水解活性不知为何会增强。在此，我们采用化学和生化相结合的策略来揭示LmClpP两种激活原理。首先，表观亲和常数的测定表明，与同聚体LmClpP2相比，LmClpP1/2异源复合物与ClpX之间的结合亲和力提高了7倍。这种更紧密的相互作用有利于LmClpX与LmClpP1/2之间形成蛋白水解活性复合物，并由此加速整体周转。其次，用各种ClpP突变体筛选一个多样化的荧光标记肽和蛋白质文库，使得能够单独分析异源复合物中两种异构体的底物偏好。除了亮氨酸和甲硫氨酸外，LmClpP2在P1位置更倾向于长脂肪链（2 - Aoc）进行切割。引人注目的是，当LmClpP2的20%活性位点被部分烷基化时，相应的2 - Aoc氯甲基酮抑制剂的设计和合成导致蛋白水解增加了160%。表观亲和常数的测定还揭示了部分修饰的LmClpP2与同源伴侣蛋白LmClpX之间的复合物稳定性提高。因此，通过增强与伴侣蛋白的结合来刺激蛋白水解似乎是LmClpP的一个特征。
