Fernández-Quintero Monica L, Seidler Clarissa A, Quoika Patrick K, Liedl Klaus R
Department of General, Inorganic and Theoretical Chemistry, Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck, Austria.
Front Mol Biosci. 2021 Apr 20;8:639166. doi: 10.3389/fmolb.2021.639166. eCollection 2021.
Sharks and other cartilaginous fish are the phylogenetically oldest living organisms that have antibodies as part of their adaptive immune system. As part of their humoral adaptive immune response, they produce an immunoglobulin, the so-called immunoglobulin new antigen receptor (IgNAR), a heavy-chain only antibody. The variable domain of an IgNAR, also known as V , binds the antigen as an independent soluble domain. In this study, we structurally and dynamically characterized the affinity maturation mechanism of the germline and somatically matured (PBLA8) V to better understand their function and their applicability as therapeutics. We observed a substantial rigidification upon affinity maturation, which is accompanied by a higher number of contacts, thereby contributing to the decrease in flexibility. Considering the static x-ray structures, the observed rigidification is not obvious, as especially the mutated residues undergo conformational changes during the simulation, resulting in an even stronger network of stabilizing interactions. Additionally, the simulations of the V in complex with the hen egg-white lysozyme show that the V antibodies evidently follow the concept of conformational selection, as the binding-competent state already preexisted even without the presence of the antigen. To have a more detailed description of antibody-antigen recognition, we also present here the binding/unbinding mechanism between the hen egg-white lysozyme and both the germline and matured V s. Upon maturation, we observed a substantial increase in the resulting dissociation-free energy barrier. Furthermore, we were able to kinetically and thermodynamically describe the binding process and did not only identify a two-step binding mechanism, but we also found a strong population shift upon affinity maturation toward the native binding pose.
鲨鱼和其他软骨鱼类是系统发育史上最古老的现存生物，其抗体是适应性免疫系统的一部分。作为其体液适应性免疫反应的一部分，它们产生一种免疫球蛋白，即所谓的免疫球蛋白新抗原受体（IgNAR），一种仅含重链的抗体。IgNAR的可变结构域，也称为V ，作为一个独立的可溶性结构域结合抗原。在本研究中，我们从结构和动力学方面对种系和体细胞成熟（PBLA8）V 的亲和力成熟机制进行了表征，以更好地理解它们的功能及其作为治疗药物的适用性。我们观察到亲和力成熟后有显著的刚性化，伴随着更多的接触，从而导致柔韧性降低。考虑到静态X射线结构，观察到的刚性化并不明显，因为特别是突变残基在模拟过程中会发生构象变化，从而形成更强的稳定相互作用网络。此外，V 与鸡蛋清溶菌酶复合物的模拟表明，V 抗体显然遵循构象选择的概念，因为即使没有抗原存在，具有结合能力的状态也已经预先存在。为了更详细地描述抗体 - 抗原识别，我们在此还展示了鸡蛋清溶菌酶与种系和成熟V 的结合/解离机制。成熟后，我们观察到解离自由能垒大幅增加。此外，我们能够从动力学和热力学方面描述结合过程，不仅确定了两步结合机制，还发现亲和力成熟后向天然结合构象的强烈群体转移。
