Papadopoulos Athanasios, Busch Max, Reiners Jens, Hachani Eymen, Baeumers Miriam, Berger Julia, Schmitt Lutz, Jaeger Karl-Erich, Kovacic Filip, Smits Sander H J, Kedrov Alexej
Synthetic Membrane Systems, Institute of Biochemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Center for Structural Studies, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Front Mol Biosci. 2022 Oct 24;9:1026724. doi: 10.3389/fmolb.2022.1026724. eCollection 2022.
is a wide-spread opportunistic human pathogen and a high-risk factor for immunodeficient people and patients with cystic fibrosis. The extracellular lipase A belongs to the virulence factors of . Prior to the secretion, the lipase undergoes folding and activation by the periplasmic foldase LipH. At this stage, the enzyme is highly prone to aggregation in mild and high salt concentrations typical for the sputum of cystic fibrosis patients. Here, we demonstrate that the periplasmic chaperone Skp of efficiently prevents misfolding of the lipase A experiments in show that the lipase secretion is nearly abolished in absence of the endogenous Skp. Small-angle X-ray scattering elucidates the trimeric architecture of Skp and identifies two primary conformations of the chaperone, a compact and a widely open. We describe two binding modes of Skp to the lipase, with affinities of 20 nM and 2 μM, which correspond to 1:1 and 1:2 stoichiometry of the lipase:Skp complex. Two Skp trimers are required to stabilize the lipase via the apolar interactions, which are not affected by elevated salt concentrations. We propose that Skp is a crucial chaperone along the lipase maturation and secretion pathway that ensures stabilization and carry-over of the client to LipH.
是一种广泛传播的机会性人类病原体，是免疫缺陷人群和囊性纤维化患者的高风险因素。细胞外脂肪酶A属于其毒力因子。在分泌之前，脂肪酶通过周质折叠酶LipH进行折叠和激活。在此阶段，该酶在囊性纤维化患者痰液典型的低盐和高盐浓度下极易聚集。在这里，我们证明了的周质伴侣蛋白Skp能有效防止脂肪酶A错误折叠。体外实验表明，在没有内源性Skp的情况下，脂肪酶分泌几乎被消除。小角X射线散射阐明了Skp的三聚体结构，并确定了伴侣蛋白的两种主要构象，一种紧凑的和一种广泛开放的。我们描述了Skp与脂肪酶的两种结合模式，亲和力分别为20 nM和2 μM，这对应于脂肪酶：Skp复合物的1:1和1:2化学计量比。需要两个Skp三聚体通过非极性相互作用来稳定脂肪酶，这种相互作用不受盐浓度升高的影响。我们提出Skp是脂肪酶成熟和分泌途径中的关键伴侣蛋白，可确保底物稳定并传递给LipH。
