Structural and Biochemical Characterization of the Wild Type PCSK9-EGF(AB) Complex and Natural Familial Hypercholesterolemia Mutants
Structural and Biochemical Characterization of the Wild Type PCSK9-EGF(AB) Complex and Natural Familial Hypercholesterolemia Mutants
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DOI：
10.1074/jbc.m808363200
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发表时间：
2009-01-09
影响因子：
4.8
通讯作者：
Carfi, Andrea
中科院分区：
文献类型：
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作者：
Bottomley, Matthew J.;Cirillo, Agostino;Carfi, Andrea
PCSK9调节低密度脂蛋白受体（LDLR）水平，因此是预防动脉粥样硬化和冠心病的靶点。我们研究了LDLR EGF（A/AB）重复序列与PCSK9的相互作用。我们发现PCSK9以ph依赖的方式结合EGF（AB）重复序列。尽管PCSK9 c端结构域不参与LDLR结合，但PCSK9的自切割是必需的。此外，我们报道了中性pH下PCSK9 Delta C-EGF（AB）复合物的x射线结构。与低pH下的PCSK9-EGF(A)结构相比，新结构揭示了EGF(A) His-306侧链的重排和与PCSK9 Asp-374的盐桥断裂，从而表明在低pH下增强相互作用的基础。此外，PCSK9 Delta C结合EGF(AB)（H306Y）的结构，与家族性高胆固醇血症（FH）相关的突变体。结果表明，tyrl -306侧链与PCSK9 Asp-374形成氢键，从而模拟了His-306的低pH构象。与此一致的是，Tyr-306对PCSK9具有更高的亲和力。重要的是，我们发现尽管EGF(AB)(H306Y)-PCSK9的相互作用与pH无关，但LDLRH306Y在低pH下结合PCSK9的效果要好50倍，这表明除了hs -306之外的其他因素也促进了PCSK9- ldlr结合的pH依赖性。此外，我们确定了含有fh相关的D374Y和D374H突变的PCSK9 δ C结合的EGF（AB）的结构，揭示了与Tyr-374/His-374介导的EGF(A)的其他相互作用，并为其疾病表型提供了基本原理。最后，我们报告了EGF重复序列在测量LDL摄取的细胞测定中的抑制特性。
PCSK9 regulates low density lipoprotein receptor (LDLR) levels and consequently is a target for the prevention of atherosclerosis and coronary heart disease. Here we studied the interaction, of LDLR EGF(A/AB) repeats with PCSK9. We show that PCSK9 binds the EGF(AB) repeats in a pH-dependent manner. Although the PCSK9 C-terminal domain is not involved in LDLR binding, PCSK9 autocleavage is required. Moreover, we report the x-ray structure of the PCSK9 Delta C-EGF(AB) complex at neutral pH. Compared with the low pH PCSK9-EGF(A) structure, the new structure revealed rearrangement of the EGF( A) His-306 side chain and disruption of the salt bridge with PCSK9 Asp-374, thus suggesting the basis for enhanced interaction at low pH. In addition, the structure of PCSK9 Delta C bound to EGF(AB)(H306Y), a mutant associated with familial hypercholesterolemia (FH), reveals that the Tyr-306 side chain forms a hydrogen bond with PCSK9 Asp-374, thus mimicking His-306 in the low pH conformation. Consistently, Tyr-306 confers increased affinity for PCSK9. Importantly, we found that although the EGF(AB)(H306Y)-PCSK9 interaction is pH-independent, LDLRH306Y binds PCSK9 50-fold better at low pH, suggesting that factors other than His-306 contribute to the pH dependence of PCSK9-LDLR binding. Further, we determined the structures of EGF(AB) bound to PCSK9 Delta C containing the FH-associated D374Y and D374H mutations, revealing additional interactions with EGF(A) mediated by Tyr-374/His-374 and providing a rationale for their disease phenotypes. Finally, we report the inhibitory properties of EGF repeats in a cellular assay measuring LDL uptake.