Department of General, Inorganic and Theoretical Chemistry, Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, 6020 Innsbruck, Austria.
Biomolecules. 2023 Feb 17;13(2):380. doi: 10.3390/biom13020380.
Camelid heavy-chain antibody variable domains (VH), nanobodies, are the smallest-known functional antibody fragments with high therapeutic potential. In this study, we investigate a VH binding to hen egg-white lysozyme (HEL). We structurally and dynamically characterized the conformational diversity of four VH variants to elucidate the antigen-binding process. For two of these antibodies, not only are the dissociation constants known, but also the experimentally determined crystal structures of the VH in complex with HEL are available. We performed well-tempered metadynamics simulations in combination with molecular dynamics simulations to capture a broad conformational space and to reconstruct the thermodynamics and kinetics of conformational transitions in the antigen-binding site, the paratope. By kinetically characterizing the loop movements of the paratope, we found that, with an increase in affinity, the state populations shift towards the binding competent conformation. The contacts contributing to antigen binding, and those who contribute to the overall stability, show a clear trend towards less variable but more intense contacts. Additionally, these investigated nanobodies clearly follow the conformational selection paradigm, as the binding competent conformation pre-exists within the structural ensembles without the presence of the antigen.
骆驼重链抗体可变区（VH），即纳米抗体，是目前已知的最小的具有高治疗潜力的功能性抗体片段。在这项研究中，我们研究了一种与鸡卵清溶菌酶（HEL）结合的 VH。我们对四个 VH 变体的结构和动态特征进行了表征，以阐明抗原结合过程。对于其中两种抗体，不仅已知解离常数，而且还可获得 VH 与 HEL 复合物的实验确定的晶体结构。我们进行了经过良好调整的元动力学模拟与分子动力学模拟相结合，以捕获广泛的构象空间，并重建抗原结合部位（表位）中构象转变的热力学和动力学。通过对表位环运动的动力学特征进行分析，我们发现随着亲和力的增加，状态群体向结合能力构象转移。对抗原结合有贡献的接触，以及对整体稳定性有贡献的接触，表现出接触逐渐变得不那么可变但更强烈的明显趋势。此外，这些被研究的纳米抗体显然遵循构象选择范例，因为在没有抗原存在的情况下，结合能力构象预先存在于结构集合体中。
