Recently, Professor Zheng Peng from the School of Chemistry and Chemical Engineering at Nanjing University, in collaboration with Academician Guo Zijian, utilized single-molecule force spectroscopy technology and molecular dynamics simulations to investigate the molecular mechanism of the interaction between the HMGB1 protein and cisplatin-DNA, as well as its regulation by protein phosphorylation. Cisplatin is a classic metal-based drug widely used in cancer treatment. It exerts its cytotoxic effects by binding to DNA and causing damage, which ultimately kills cancer cells. However, the nucleotide excision repair pathways in cells can repair the DNA damage caused by cisplatin, thereby inhibiting its efficacy. For example, the HMGB1 protein can specifically bind to cisplatin-damaged DNA and shield the repair pathways after binding. Therefore, understanding and elucidating the interaction mechanism between HMGB1 and cisplatin-DNA is of significant importance for studying the anti-cancer effects of platinum-based drugs.
First, the authors used a combination of click chemistry and enzymatic reactions to immobilize cisplatinated DNA and HMGB1 protein in the force spectroscopy measurement system with high strength and specificity, thereby achieving precise measurements of the binding strength between DNA and protein molecules. The results of single-molecule force spectroscopy experiments showed that the dissociation force of the complex formed by the two exceeded 100 pN, making it one of the strongest natural DNA-protein complexes known to date (Figure 1). Meanwhile, the stretch molecular dynamics simulations revealed that phenylalanine at position 37 is a critical site in the dissociation process of the complex.
Figure 1. Precise Measurement of the Binding Strength between Cisplatin-DNA and HMGB1 A Domain Using Click Chemistry and Enzymatic Reaction for Immobilization, and Molecular Dynamics Simulation of Their Dissociation
Subsequently, the authors utilized single-molecule force spectroscopy to study the impact of DNA base sequences (Figure 2) and phosphorylation modifications of the full-length HMGB1 protein (Figure 3) on their interaction strength. Based on the measured dissociation forces of the complexes, it was observed that the interaction strength is mainly influenced by the types of bases flanking the two guanine bases to which cisplatin binds. Furthermore, the authors selected three serine sites in HMGB1 for phosphorylation modification based on previous studies. They measured the binding strength of the three modified proteins with cisplatin-DNA using force spectroscopy and determined the kinetic parameters of the complexes at different pulling speeds. By comparing the kinetic parameter koff, the results showed that the phosphorylation of HMGB1 protein increased the binding stability with cisplatin-DNA. Among these sites, the phosphorylation of serine at position 14 had the greatest impact on the stability of the complex, reducing the koff value by approximately 35 times. Molecular dynamics simulations indicated that the phosphorylated serine could form more hydrogen bonds with cisplatin-DNA, thereby enhancing the stability of the complex.
Figure 2. Impact of the Base Sequence of Cisplatin-DNA on the Dissociation Force of the Complex
In summary, this work utilized click chemistry and enzymatic methods to achieve specific immobilization of proteins and DNA, allowing for precise measurement of protein-DNA interactions using single-molecule force spectroscopy. The results showed that the complex formed by HMGB1 and cisplatin-DNA is influenced by the base sequence and revealed that the phosphorylation of serine at position 14 in HMGB1 enhances the stability of its complex with cisplatin-DNA by forming more hydrogen bonds.
Figure 3. Phosphorylation of HMGB1 Enhances the Stability of the Complex Formed with Cisplatin-DNA
The results were recently published in the Journal of the American Chemical Society under the title Click Chemistry-Based Force Spectroscopy Revealed Enhanced Binding Dynamics of Phosphorylated HMGB1 to Cisplatin-DNA (J. Am. Chem. Soc. 2024, 146, 19, 13126–13132). The co-first authors of the paper are Liu Yutong, a direct doctoral student from the 2019 class, Song Dongfan, a doctoral student from the 2018 class, and Li Senmiao, an undergraduate student from the 2020 class. Academician Guo Zijian and Professor Zheng Peng are the corresponding authors of the article. This research was supported by the State Key Laboratory of Coordination Chemistry, the Chemical and Biomedical Innovation Research Institute (ChemBIC) of Nanjing University, and the High-Performance Computing Center of Nanjing University. It also received funding support from the National Natural Science Foundation of China and the Jiangsu Provincial Natural Science Foundation.