Denysenko Olena, Horn Anselm H C, Sticht Heinrich
Bioinformatics, Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany.
Erlangen National High Performance Computing Center (NHR@FAU), Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 91058 Erlangen, Germany.
Antibodies (Basel). 2025 Aug 13;14(3):70. doi: 10.3390/antib14030070.
: Cows produce antibodies with ultralong CDRH3 segments (ulCABs) that contain a disulfide-stabilized knob domain. This domain is connected to the globular core of the antibody by a β-strand stalk. In the crystal structures, the stalk protrudes from the core in an extended conformation and presents the knob at its distal end. However, the rigidity of this topology has been questioned due to the extensive crystal packing present in most ulCAB crystal structures. To gain more insight into the dynamics of ultralong CDRH3s, we performed a comparative molecular dynamics (MD) study of 19 unique ulCABs. : For all 19 systems, one-microsecond MD simulations were performed in explicit solvent. The analyses included an investigation of the systems' conformational stability and the dynamics of the knob domain as well as an energetic analysis of the intramolecular knob interactions. : The simulations show that the extended stalk-knob conformation observed in the crystal structures is not preserved in solution. There are significant differences in the degree of knob dynamics, the orientations of the knobs, the number of flexible stalk residues, and the frequency of the motions. Furthermore, interactions between the knob and the light chain (LC) of the ulCABs were observed in about half of the systems. : The study reveals that pronounced knob dynamics is a general feature of ulCABs rather than an exception. The magnitude of knob motions depends on the system, thus reflecting the high sequence diversity of the CDRH3s in ulCABs. The observed knob-LC interactions might play a role in stabilizing distinct knob orientations. The MD simulations of ulCABs could also help to identify suitable knob fragments as mini-antibodies by suggesting appropriate truncation points based on flexible sites in the stalks.
奶牛产生具有超长互补决定区3（CDRH3）片段的抗体（超长互补决定区抗体，ulCABs），这些片段包含一个二硫键稳定的球状结构域。该结构域通过一条β链茎与抗体的球状核心相连。在晶体结构中，茎以伸展构象从核心突出，并在其远端呈现球状结构域。然而，由于大多数超长互补决定区抗体晶体结构中存在广泛的晶体堆积，这种拓扑结构的刚性受到了质疑。为了更深入地了解超长CDRH3的动力学，我们对19种独特的超长互补决定区抗体进行了比较分子动力学（MD）研究。
对于所有19个系统，在显式溶剂中进行了1微秒的分子动力学模拟。分析包括对系统构象稳定性、球状结构域动力学的研究以及分子内球状结构域相互作用的能量分析。
模拟结果表明，晶体结构中观察到的伸展茎 - 球状结构域构象在溶液中无法保持。在球状结构域动力学程度、球状结构域方向、柔性茎残基数量以及运动频率方面存在显著差异。此外，在大约一半的系统中观察到了超长互补决定区抗体的球状结构域与轻链（LC）之间的相互作用。
该研究表明，明显的球状结构域动力学是超长互补决定区抗体的普遍特征而非例外。球状结构域运动的幅度取决于系统，从而反映了超长互补决定区抗体中CDRH3的高度序列多样性。观察到的球状结构域 - 轻链相互作用可能在稳定不同的球状结构域方向中发挥作用。超长互补决定区抗体的分子动力学模拟还可以通过根据茎中的柔性位点建议合适的截断点，帮助识别合适的球状结构域片段作为微型抗体。
