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首页> 外文期刊>Journal of Molecular Biology >Calcium-independent inhibition of PCSK9 by affinity-improved variants of the LDL receptor EGF(A) domain
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Calcium-independent inhibition of PCSK9 by affinity-improved variants of the LDL receptor EGF(A) domain

机译：LDL受体EGF（A）结构域的亲和力改良变异对PCSK9的钙依赖性抑制

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摘要

LDL (low-density lipoprotein) receptor (LDLR) binds to its negative regulator proprotein convertase subtilisin/kexin type 9 (PCSK9) through the first EGF (epidermal growth factor-like) domain [EGF(A)]. The isolated EGF(A) domain is a poor antagonist due to its low affinity for PCSK9. To improve binding affinity, we used a phage display approach by randomizing seven PCSK9 contact residues of EGF(A), including the Ca 2 +-coordinating Asp310. The library was panned in Ca 2 +-free solution, and 26 unique clones that bind to PCSK9 were identified. Four selected variants demonstrated improved inhibitory activities in a PCSK9-LDLR competition binding ELISA. The Fc fusion protein of variant EGF66 bound to PCSK9 with a K d value of 71 nM versus 935 nM of wild type [EGF(A)-Fc] and showed significantly improved potency in inhibiting LDLR degradation in vitro and in vivo. The five mutations in EGF66 could be modeled in the EGF(A) structure without perturbation of the EGF domain fold, and their contribution to affinity improvement could be rationalized. The most intriguing change was the substitution of the Ca 2 +-coordinating Asp310 by a Lys residue, whose side-chain amine may have functionally replaced Ca 2 +. EGF66-Fc and other EGF variants having the Asp310Lys change bound to PCSK9 in a Ca 2 +-independent fashion. The findings indicate that randomization of an important Ca 2 +-chelating residue in conjunction with selection pressure applied by Ca 2 +-free phage selection conditions can yield variants with an alternatively stabilized Ca 2 + loop and with increased binding affinities. This approach may provide a new paradigm for the use of diversity libraries to improve affinities of members of the Ca 2 +-binding EGF domain subfamily.
机译：LDL（低密度脂蛋白）受体（LDLR）通过第一个EGF（表皮生长因子样）结构域[EGF（A）]与其负调节剂原蛋白转化酶枯草杆菌蛋白酶/ kexin型9（PCSK9）结合。分离的EGF（A）域由于对PCSK9的亲和力低，因此是较差的拮抗剂。为了提高结合亲和力，我们通过随机化EGF（A）的七个PCSK9接触残基（包括Ca 2 +配位的Asp310）来使用噬菌体展示方法。在无Ca 2+溶液中淘选该文库，并鉴定了26个与PCSK9结合的独特克隆。四个选定的变体在PCSK9-LDLR竞争结合ELISA中表现出改善的抑制活性。突变体EGF66的Fc融合蛋白与PCSK9结合，其K值为71 nM，而野生型[EGF（A）-Fc]为935 nM，在体外和体内抑制LDLR降解方面显示出显着提高的效力。可以在EGF（A）结构中模拟EGF66中的五个突变，而不会干扰EGF结构域折叠，并且可以合理化它们对亲和力改善的贡献。最引人入胜的变化是，Lys残基取代了配位于Ca 2 +的Asp310，后者的侧链胺可能已在功能上取代了Ca 2 +。具有Asp310Lys的EGF66-Fc和其他EGF变体以独立于Ca 2 +的方式与PCSK9结合。该发现表明，重要的Ca 2+螯合残基的随机化与无Ca 2+的噬菌体选择条件施加的选择压力相结合，可以产生具有稳定的Ca 2+环和结合亲和力增加的变体。这种方法可能为使用多样性文库改善Ca 2 +结合EGF域亚家族成员的亲和力提供了新的范例。

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