Polyketide synthases (PKSs) share a subset of biosynthetic steps in construction of a polyketide, and the offload from the PKS main module of specific product release is most often catalyzed by a thioesterase (TE). In spite of the fact that various PKS systems have been discovered in polyketide biosynthesis, the molecular basis of TE-catalyzed macrocyclization remains challenging. In this study, MD simulations and QM/MM methods were combined to investigate the catalytic mechanism and substrate diversity of pikromycin (PIK) TE with two systems (PIK-TE-1 and PIK-TE-2), where substrates 1 and 2 correspond to TE-catalyzed precursors of 10-deoxymethynolide and narbonolide, respectively. The results showed that, in comparison with PIK-TE-2, system PIK-TE-1 exhibited a greater tendency to form a stable prereaction state, which is critical to macrocyclization. In addition, the structural characteristics of prereaction states were uncovered through analyses of hydrogen-bonding and hydrophobic interactions, which were found to play a key role in substrate recognition and product release. Furthermore, potential energy surfaces were calculated to study the molecular mechanism of macrocyclization, including the formation of tetrahedral intermediates from re- and si-face nucleophilic attacks and the release of products. The energy barrier of macrocyclization from re-face attack was calculated to be 16.3 kcal/mol in PIK-TE-1, 3.6 kcal/mol lower than that from si-face attack and 4.1 kcal/mol lower than that from re-face attack in PIK-TE-2. These results are in agreement with experimental observations that the yield of 10-deoxymethynolide is superior to that of narbonolide in PIK TE catalyzed macrocyclization. Our findings elucidate the catalytic mechanism of PIK TE and provide a better understanding of type I PKS TEs in protein engineering.

Hepatitis B virus surface antigen (HBsAg) carriers are highly susceptible to liver injury triggered by environmental biochemical stimulation. Previously, we have reported an inverse correlation between γδ T cells and liver damage in patients with hepatitis B virus (HBV). However, whether γδ T cells play a role in regulating the hypersensitivity of HBsAg carriers to biochemical stimulation-induced hepatitis is unknown. In this study, using HBV transgenic (HBs-Tg) and HBs-Tg T-cell receptor-δ-deficient (TCR-δ ) mice, we found that mice genetically deficient in γδ T cells exhibited more severe liver damage upon Concanavalin A (Con A) treatment, as indicated by substantially higher serum alanine aminotransferase levels, further elevated interferon-γ (IFN-γ) levels and more extensive necrosis. γδ T-cell deficiency resulted in elevated IFN-γ in CD4 T cells but not in natural killer or natural killer T cells. The depletion of CD4 T cells and neutralization of IFN-γ reduced liver damage in HBs-Tg and HBs-Tg-TCR-δ mice to a similar extent. Further investigation revealed that HBs-Tg mice showed an enhanced interleukin-17 (IL-17) signature. The administration of exogenous IL-23 enhanced IL-17A production from Vγ4 γδ T cells and ameliorated liver damage in HBs-Tg mice, but not in HBs-Tg-TCR-δ mice. In summary, our results demonstrated that γδ T cells played a protective role in restraining Con A-induced hepatitis by inhibiting IFN-γ production from CD4 T cells and are indispensable for IL-23-mediated protection against Con A-induced hepatitis in HBs-Tg mice. These results provided a potential therapeutic approach for treating the hypersensitivity of HBV carriers to biochemical stimulation-induced liver damage.

Akt (known as protein kinase B or PKB) is a serine/threonine kinase that regulates multiple biological processes, including cell growth, survival, and differentiation. Akt plays a critical role in the intracellular signaling network through conformational changes responsive to diverse signal inputs, and dysregulation of Akt activity could give rise to a number of diseases. However, understanding of Akt's dynamic structures and conformational transitions between active and inactive states remains unclear. In this work, classical MD simulations and QM/MM calculations were carried out to unveil the structural characteristics of Akt1, especially in its active state. The doubly protonated H194 was investigated, and both ATP−Akt1 and ADP−Akt1 complexes were constructed to demonstrate the significance of ATP in maintaining the ATP−K179−E198 salt bridge and the corresponding allosteric pathway. Besides, conformational transitions from the inactive state to the active state showed different permeation patterns of water molecules in the ATP pocket. The coordination modes of Mg 2+ in the dominant representative conformations (I and I′) are presented. Unlike the water-free conformation I′, three water molecules appear around Mg 2+ in the water-occupied conformation I, which can finally exert an influence on the catalytic mechanism of Akt1. Furthermore, QM/MM calculations were performed to study the phosphoryl-transfer reaction of Akt1. The transfer of ATP γ-phosphate was achieved through a reversible conformational change from the reactant to a critical prereaction state, with a water molecule moving into the reaction center to coordinate with Mg 2+ , after which the γ-phosphate group was transferred from ATP to the substrate. Taken together, our results elucidate the structural characteristics of the Akt1 active state and shed new light on the catalytic mechanism of Akt kinases.

As a polyene antibiotic of great pharmaceutical significance, pimaricin has been extensively studied to enhance its productivity and effectiveness. In our previous studies, pre-reaction state (PRS) has been validated as one of the significant conformational categories before macrocyclization, and is critical to mutual recognition and catalytic preparation in thioesterase (TE)-catalyzed systems. In our study, molecular dynamics (MD) simulations were conducted on pimaricin TE-polyketide complex and PRS, as well as pre-organization state (POS), a molecular conformation possessing a pivotal intra-molecular hydrogen bond, were detected. Conformational transition between POS and PRS was observed in one of the simulations, and POS was calculated to be energetically more stable than PRS by 4.58 kcal/mol. The structural characteristics of PRS and POS-based hydrogen-bonding, and hydrophobic interactions were uncovered, and additional simulations were carried out to rationalize the functions of several key residues (Q29, M210, and R186). Binding energies, obtained from MM/PBSA calculations, were further decomposed to residues, in order to reveal their roles in product release. Our study advanced a comprehensive understanding of pimaricin TE-catalyzed macrocyclization from the perspectives of conformational change, protein-polyketide recognition, and product release, and provided potential residues for rational modification of pimaricin TE.