Molecular Dynamics Investigation on the Effects of Protonation and Lysyl Hydroxylation on Sulfilimine Cross-links in Collagen IV

Collagen IV networks are an essential component of basement membranes that are important for their structural integrity and thus that of an organism’s tissues. Improper functioning of these networks has been associated with several diseases. Cross-links, such as sulfilimine bonds interconnecting NC1 domains, are critical for forming and mechanically stabilizing these collagen IV networks. More specifically, the sulfilimine cross-links form between methionine (Met93) and lysine/hydroxylsine (Lys211/Hyl211) residues of NC1 domains. Therefore, the dynamic nature of the sulfilimine bond in collagen IV is crucial for network formation. To understand the dynamic nature of a neutral and protonated sulfilimine bond in collagen IV, we performed molecular dynamics (MD) simulations on four sulfilimine cross-linked systems (i.e., Met93S–NLys211, Met93S–NHLys211+, Met93S–NHyl211, and Met93S–NHHyl211+) of collagen IV. The MD results showed that the neutral Met93S–NLys211 system has the smallest protein backbone and showed the cross-linked residues’ RMSD value. The conformational change analyses showed that the conformations of the sulfilimine cross-linked residues take on a U-shape for the Met93S–NHyl211 and Met93S–HNHyl211+ systems, whereas the conformations of the sulfilimine cross-linked residues are more open for the Met93S–NLys211, and Met93S–NHLys211+ systems. Protonation is a crucial biochemical process to stabilize the protein structure or the biological cross-links. Furthermore, the protonation of the sulfilimine bond could potentially influence hydrogen bond interaction with near amino acid residues, and according to water distribution analyses, the sulfilimine bond can potentially exist in one or more protonation states.

IV型胶原（Collagen IV）网络是基底膜（basement membranes）的核心组成部分，对维持基底膜乃至机体组织的结构完整性至关重要。此类网络功能异常与多种疾病密切相关。连接NC1结构域（NC1 domains）的硫亚胺键（sulfilimine bond）等交联结构，对于IV型胶原网络的组装与力学稳定具有关键作用。具体而言，硫亚胺交联形成于NC1结构域的甲硫氨酸（methionine, Met93）残基与赖氨酸/羟赖氨酸（lysine/hydroxylysine, Lys211/Hyl211）残基之间。因此，IV型胶原中硫亚胺键的动态特性对网络形成至关重要。为探究IV型胶原中中性与质子化硫亚胺键的动态特性，我们针对四类硫亚胺交联的IV型胶原体系开展了分子动力学（molecular dynamics, MD）模拟，分别为Met93S–NLys211、Met93S–NHLys211+、Met93S–NHyl211以及Met93S–HNHyl211+。分子动力学模拟结果显示，中性体系Met93S–NLys211的蛋白质主链均方根偏差（RMSD）最小，且交联残基的RMSD值最低。构象变化分析表明，Met93S–NHyl211与Met93S–HNHyl211+体系中的硫亚胺交联残基呈U型构象，而Met93S–NLys211与Met93S–NHLys211+体系中的交联残基构象则更为舒展。质子化是稳定蛋白质结构或生物交联的重要生化过程。此外，硫亚胺键的质子化可影响其与邻近氨基酸残基的氢键相互作用；结合水分子分布分析可知，硫亚胺键可能以一种或多种质子化状态存在。