Crystal structure of HEL4, a soluble, refoldable Human VH single domain with a germ-line scaffold
Crystal structure of HEL4, a soluble, refoldable Human VH single domain with a germ-line scaffold
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DOI：
10.1016/j.jmb.2004.02.013
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发表时间：
2004-04-02
影响因子：
5.6
通讯作者：
Winter, G
中科院分区：
文献类型：
--
作者：
Jespers, L;Schon, O;Winter, G
抗体的抗原结合位点通常包含相关的重链（V-H）和轻链（V-L）可变结构域，但在骆驼和美洲驼中，结合位点通常仅包含重链可变结构域（称为V-HH）。与报道的人类 V-H 结构域相比，V-HH 结构域在细菌和酵母中得到良好表达，易于以可溶形式纯化，并在热变性后可逆地重折叠。这些理想的特性归因于 V-H 结构域疏水残基的高度保守取代，这些残基通常与互补的 V-L 结构域相互作用。在这里，我们描述了分离的人类 V-H 结构域 (HEL4) 的发现和表征，其特性与 V-HH 结构域相似。 HEL4 在大于或等于 3 mM 的浓度下高度可溶，基本上是单体，并且在浓度高达 56 mM 时热变性时不会聚集。然而，与 V-HH 结构域相反，VH:VL 界面的疏水框架残基得以保留，并且与相应人种系片段 (V3-23/DP-47) 的唯一序列变化位于包含互补决定区 (CDR) 的环中。 HEL4 的晶体结构揭示了一个不寻常的特征：将框架残基（Trp47）的侧链翻转到CDR1的Gly35形成的空腔中，从而增加VH:VL界面的亲水性。为了评估 Gly35 对结构域性质的具体贡献，将 Gly35 引入溶液性质较差的 V-H 结构域中。这大大提高了突变体从凝胶过滤基质中的回收率，但对其热变性后可逆重折叠的能力几乎没有影响。我们的结果证实了亲水性 V-H:V-L 界面对于纯化分离的 V-H 结构域的重要性，并朝着设计具有免疫治疗实用特性的分离的人 V-H 结构域迈出了一步。 (C) 2004 Elsevier Ltd. 保留所有权利。
The antigen binding site of antibodies usually comprises associated heavy (V-H) and light (V-L) chain variable domains, but in camels and llamas, the binding site frequently comprises the heavy chain variable domain only (referred to as V-HH). In contrast to reported human V-H domains, V-HH domains are well expressed from bacteria and yeast, are readily purified in soluble form and refold reversibly after heat-denaturation. These desirable properties have been attributed to highly conserved substitutions of the hydrophobic residues of V-H domains, which normally interact with complementary V-L domains. Here, we describe the discovery and characterisation of an isolated human V-H domain (HEL4) with properties similar to those of V-HH domains. HEL4 is highly soluble at concentrations of greater than or equal to3 mM, essentially monomeric and resistant to aggregation upon thermodenaturation at concentrations as high as 56 muM. However, in contrast to V-HH domains, the hydrophobic framework residues of the VH:VL interface are maintained and the only sequence changes from the corresponding human germ-line segment (V3-23/DP-47) are located in the loops comprising the complementarity determining regions (CDRs). The crystallographic structure of HEL4 reveals an unusual feature; the side-chain of a framework residue (Trp47) is flipped into a cavity formed by Gly35 of CDR1, thereby increasing the hydrophilicity of the VH:VL interface. To evaluate the specific contribution of Gly35 to domain properties, Gly35 was introduced into a V-H domain with poor solution properties. This greatly enhanced the recovery of the mutant from a gel filtration matrix, but had little effect on its ability to refold reversibly after heat denaturation. Our results confirm the importance of a hydrophilic V-H:V-L interface for purification of isolated V-H domains, and constitute a step towards the design of isolated human V-H domains with practical properties for immunotherapy. (C) 2004 Elsevier Ltd. All rights reserved.