PKC-related serine/threonine protein kinase N1 (PKN1) is a protease/lipid-activated protein kinase that acts downstream of the RhoA and Rac1 pathways. PKN1 comprises unique regulatory, hinge region, and PKC homologous catalytic domains. The regulatory domain harbors two homologous regions, i.e., HR1 and C2-like. HR1 consists of three heptad repeats (HR1a, HR1b, and HR1c), with PKN1-(HR1a) hosting an amphipathic high-affinity cardiolipin-binding site for phospholipid interactions. Cardiolipin and C18:1 oleic acid are the most potent lipid activators of PKN1. PKN1-(C2) contains a pseudosubstrate sequence overlapping that of C20:4 arachidonic acid. However, the cardiolipin-binding site(s) within PKN1-(C2) and the respective binding properties remain unclear. Herein, we reveal (i) that the primary PKN1-(C2) sequence contains conserved amphipathic cardiolipin-binding motif(s); (ii) that trimeric PKN1-(C2) predominantly adopts a β-stranded conformation; (iii) that two distinct types of cardiolipin (or phosphatidic acid) binding occur, with the hydrophobic component playing a key role at higher salt levels; (iv) the multiplicity of C18 fatty acid binding to PKN1-(C2); and (v) the relevance of our lipid-binding parameters for PKN1-(C2) in terms of kinetic parameters previously determined for the full-length PKN1 enzyme. Thus, our discoveries create opportunities to design specific mammalian cell inhibitors that disrupt the localization of membrane-associated PKN1 signaling molecules.

中文翻译：

---

PKC 相关丝氨酸/苏氨酸蛋白激酶 N1 (PKN1) 内的脂质结合区域是其调节所必需的

PKC 相关丝氨酸/苏氨酸蛋白激酶 N1 (PKN1) 是一种蛋白酶/脂质激活蛋白激酶，作用于 RhoA 和 Rac1 通路的下游。PKN1 包含独特的调节区、铰链区和 PKC 同源催化结构域。调控域包含两个同源区域，即HR1 和C2 样区域。HR1 由三个七肽重复序列（HR1a、HR1b 和 HR1c）组成，其中 PKN1-(HR1a) 具有用于磷脂相互作用的两亲性高亲和力心磷脂结合位点。心磷脂和 C18:1 油酸是 PKN1 最有效的脂质激活剂。PKN1-(C2) 包含与 C20:4 花生四烯酸重叠的假底物序列。然而，PKN1-(C2) 内的心磷脂结合位点以及各自的结合特性仍不清楚。在此，我们揭示 (i) 初级 PKN1-(C2) 序列包含保守的两亲性心磷脂结合基序；(ii) 三聚体 PKN1-(C2) 主要采用 β 链构象；(iii) 发生两种不同类型的心磷脂（或磷脂酸）结合，其中疏水性成分在较高盐水平下发挥关键作用；(iv) C18脂肪酸与PKN1-(C2)结合的多重性；(v) PKN1-(C2) 的脂质结合参数与先前为全长 PKN1 酶确定的动力学参数的相关性。因此，我们的发现为设计特定的哺乳动物细胞抑制剂创造了机会，这些抑制剂可以破坏膜相关 PKN1 信号分子的定位。