Unravelling the Molecular Basis of High Affinity Nanobodies against HIV p24: In Vitro Functional, Structural, and in Silico Insights
Unravelling the Molecular Basis of High Affinity Nanobodies against HIV p24: In Vitro Functional, Structural, and in Silico Insights
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DOI：
10.1021/acsinfecdis.6b00189
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发表时间：
2017-07-01
影响因子：
5.3
通讯作者：
McKendry, Rachel A.
中科院分区：
文献类型：
--
作者：
Gray, Eleanor R.;Brookes, Jennifer C.;McKendry, Rachel A.
预防传染病的传播仍然是世界范围内的一个紧迫优先事项，这推动了先进纳米技术的发展，以在护理点诊断感染。在这里，我们报告了一个新的纳米抗体捕获配体库的创建，以检测p24，HIV感染的最早标志物之一。我们证明，这些纳米抗体，十分之一的传统抗体的大小，表现出高灵敏度和广泛的特异性，全球HIV-1亚型。生物物理表征表明，强690 pM的结合常数和快速的动力学结合速率，比单克隆抗体比较物好1至2个数量级。获得了铅纳米抗体和p24的晶体结构，并与分子动力学模拟一起用于阐明这些增强的性能特征的分子基础。它们表明结合发生在p24的C末端螺旋10和11处，p24的带负电荷的区域由涉及CDR 1、CDR 2和CDR 3环的纳米抗体结合界面的正表面补充。我们的发现对新型抗体的设计和广泛的先进生物医学应用具有广泛的影响。
Preventing the spread of infectious diseases remains an urgent priority worldwide, and this is driving the development of advanced nanotechnology to diagnose infections at the point of care. Herein, we report the creation of a library of novel nanobody capture ligands to detect p24, one of the earliest markers of HIV infection. We demonstrate that these nanobodies, one tenth the size of conventional antibodies, exhibit high sensitivity and broad specificity to global HIV-1 subtypes. Biophysical characterization indicates ,strong 690 pM binding constants and fast kinetic on-rates, 1 to 2 orders of magnitude better than monoclonal antibody comparators. A crystal structure of the lead nanobody and p24 was obtained and used alongside molecular dynamics simulations to elucidate the molecular basis of these enhanced performance characteristics. They indicate that binding occurs at C-terminal helices 10 and 11 of p24, a negatively charged region of p24 complemented by the positive surface of the nanobody binding interface involving CDR1, CDR2, and CDR3 loops. Our findings have broad implications on the design of novel antibodies and a wide range of advanced biomedical applications.