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The receptor-interacting serine/threonine protein kinase 1 (RIPK1) is tightly related to Digestive System Neoplasms genesis. Therefore, inhibitors that target RIPK1 have gained popularity in today's anti-cancer therapy. Molecular docking, molecular dynamics simulations, and molecular mechanics/Poisson-Boltzmann surface area calculations were used to investigate the binding mode and inhibition mechanism of five 4,5-dihydropyrazole derivatives with RIPK1 in the present work. The results showed that the five inhibitors mainly interacted with RIPK1 through van der Waals interaction and were stably present in the hydrophobic pocket next to the ATP-binding pocket. During the simulation, the inhibitor 2R with opposite chirality displayed a significant flip-flop in the binding pose, causing the key residue Asp156 to not easily maintain the inactive conformation, affecting its inhibitory ability. Inhibitors with identical chirality but different 1-substituents could exert non-competitive inhibition via three pathways. First of all, the inhibitor interacted directly with ATP, influencing its γ-phosphate position. Second, it altered the conformation of Lys45, which was crucial for both ATP binding and γ-phosphate transfer. And then it affected the conformation of the P-loop and β1 sheet, resulting in differences in substrate peptide recognition. The results can provide a theoretical basis for the design of such inhibitors in the future.

E. coli thioesterase ‘TesA is an important enzyme in fatty acid production. Medium-chain fatty acids (MCFAs, C6-C10) are of great interest due to their similar physicochemical properties to petroleum-based oleo-chemicals. It has been shown that wild-type ‘TesA had better selectivity for long-chain acyl substrates (≥C16), while the two mutants ‘TesAE142D/Y145G and ‘TesAM141L/E142D/Y145G had better selectivity for medium-chain acyl substrates. However, it is difficult to obtain the selectivity mechanism of substrates for proteins by traditional experimental methods. In this study, in order to obtain more MCFAs, we analyzed the binding mode of proteins (‘TesA, ‘TesAE142D/Y145G and ‘TesAM141L/E142D/Y145G) and substrates (C16/C8-N-acetylcysteamine analogs, C16/C8-SNAC), the key residues and catalytic mechanisms through molecular docking, molecular dynamics simulations and the molecular mechanics Poisson–Boltzmann surface area (MM/PBSA). The results showed that several main residues related to catalysis, including Ser10, Asn73 and His157, had a strong hydrogen bond interaction with the substrates. The mutant region (Met141-Tyr146) and loop107–113 were mainly dominated by Van der Waals contributions to the substrates. For C16-SNAC, except for ‘TesAM141L/E142D/Y145G with large conformational changes, there were strong interactions at both head and tail ends that distorted the substrate into a more favorable high-energy conformation for the catalytic reaction. For C8-SNAC, the head and tail found it difficult to bind to the enzyme at the same time due to insufficient chain length, which made the substrate binding sites more variable, so ‘TesAM141L/E142D/Y145G with better binding sites had the strongest activity, and ‘TesA had the weakest activity, conversely. In short, the matching substrate chain and binding pocket length are the key factors affecting selectivity. This will be helpful for the further improvement of thioesterases.

Thrombin is the main target of antithrombotic and anticoagulant therapy. Although recent studies have shown that surface-modified fullerene derivatives can be used as novel nano-anticoagulants to effectively inhibit thrombin, the specific mechanism remains unclear. In this study, we investigated the binding mode and inhibition mechanism of fullerenes with thrombin by using various molecular modelling approaches. The results showed that owing to the hydrophobicity of the fullerene skeleton, the four existing fullerene inhibitors mainly combined with the active site of thrombin through van der Waals interaction and tended to make the surrounding environment hydrophobic. This made the inhibitors with hydrophobic side chains, especially those with large hydrophobic side chains containing benzene rings, have greater binding free energy with protein. However, as more hydrophilic groups can change the surrounding hydrophilic–hydrophobic environment and form more hydrogen bonds, the electrostatic contribution of the polyhydroxy side chain designed in this study was increased further. At the same time, the van der Waals interaction between the fullerene carbon skeleton and protein did not decrease significantly, resulting in a significantly increased total binding free energy and an enhanced inhibition effect. These findings provide a theoretical basis for the improvement of highly effective fullerene anticoagulants.

Cellobiohydrolase A from Ruminiclostridium thermocellum (Cbh9A) is a processive exoglucanase from family 9 and is an important cellobiohydrolase that hydrolyzes cello-oligosaccharide into cellobiose. Residues Tyr555 and Trp678 considerably affect catalytic activity, but their mechanisms are still unknown. To investigate how the Tyr555 and Trp678 affect the processivity of Cbh9A, conventional molecular dynamics, steered molecular dynamics, and free energy calculation were performed to simulate the processive process of wild type (WT)-Cbh9A, Y555S mutant, and W678G mutant. Analysis of simulation results suggests that the binding free energies between the substrate and WT-Cbh9A are lower than those of Y555S and W678G mutants. The pull forces and energy barrier in Y555S and W678G mutants also reduced significantly during the steered molecular dynamics (SMD) simulation compared with that of the WT-Cbh9A. And the potential mean force calculations showed that the pulling energy barrier of Y555S and W678G mutants is much lower than that of WT-Cbh9A.