Miaomiao Li

The action of abscisic acid (ABA) is closely related to its level in plant tissues. Uridine diphosphate-glycosyltransferase71c5 (UGT71C5) was characterized as a major UGT enzyme to catalyze the formation of the ABA-glucose ester (ABA-GE), a reversible inactive form of free ABA in Arabidopsis thaliana (thale cress). UGTs function in a mode where the catalytic base deprotonates an acceptor to allow a nucleophilic attack at the anomeric center of the donor, achieving the transfer of a glucose moiety. The proteomic data revealed that UGT71C5 can be persulfidated. Herein, an experimental method was employed to detect the persulfidation site of UGT71C5, and the computational methods were further used to identify the yet unknown molecular basis of ABA glycosylation as well as the regulatory role of persulfidation in this process. Our results suggest that the linker and the U-shaped loop are regulatory structural elements: the linker is associated with the binding of uridine diphosphate glucose (UPG) and the U-shaped loop is involved in binding both UPG and ABA.It was also found that it is through tuning the dynamics of the U-shaped loop that is accompanied by the movement of tyrosine (Y388) that the persulfidation of cysteine (C311) leads to the catalytic residue histidine (H16) being in place, preparing for the deprotonation of ABA, and then reorientates UPG and deprotonated ABA closer to the ‘Michaelis’ complex, facilitating the transfer of a glucose moiety. Ultimately, the persulfidation of UGT71C5 is in favor of ABA glycosylation. Our results provide insights into the molecular details of UGT71C5 recognizing substrates and insights concerning persulfidation as a possible mechanism for hydrogen sulfide (H₂S) to modulate the content of ABA, which helps us understand how modulating ABA level strengthens plant tolerance.

SnRK2.6 (SUCROSE NONFERMENTING 1-RELATED PROTEIN KINASE2.6) has been characterized as a molecular switch for the intracellular abscisic acid (ABA) signal-transduction pathway. Normally, SnRK2.6 is kept in an “off” state, forming a binary complex with protein phosphatase type 2Cs (PP2Cs). Upon stressful conditions, SnRK2.6 turns into an “on” state by its release from PP2Cs and then phosphorylation at Ser175. However, how the ”on” and “off” states for SnRK2.6 are fine-tuned, thereby controlling the initiation and braking processes of ABA signaling, is still largely unclear. SnRK2.6 activity was tightly regulated through protein post-translational modifications (PTM), such as persulfidation and phosphorylation. Taking advantage of molecular dynamics simulations, our results showed that Cys131/137 persulfidation on SnRK2.6 induces destabilized binding and weakened interactions between SnRK2.6 and HAB1 (HYPERSENSITIVE TO ABA1), an important PP2C family protein. This unfavorable effect on the association of the SnRK2.6–HAB1 complex suggests that persulfidation functions are a positive regulator of ABA signaling initiation. In addition, Ser267 phosphorylation in persulfidated SnRK2.6 renders a stable physical association between SnRK2.6 and HAB1, a key characterization for SnRK2.6 inhibition. Rather than Ser175, HAB1 cannot dephosphorylate Ser267 in SnRK2.6, which implies that the retained phosphorylation status of Ser267 could ensure that the activated SnRK2.6 reforms the binary complex to cease ABA signaling. Taken together, our findings expand current knowledge concerning the regulation of persulfidation and phosphorylation on the state transition of SnRK2.6 and provide insights into the fine-tuned mechanism of ABA signaling.

Epidermal growth factor receptor (EGFR) is an intensively focused target for anti-tumor compounds used in non-small cell lung cancer (NSCLC) therapy. Compared to the classical activating mutations, there are still many uncommon EGFR mutations associated with poorer responses to EGFR inhibitors. A detailed understanding of the molecular basis for multiple EGFR mutants exhibiting diverse responses to inhibitors is of critical importance for related drug development. Herein, we explored the molecular determinants contributing to the distinct responses of EGFR with a single rare mutation (G719S) or combined mutations (G719S/L858R and G719S/l861Q) to Gefitinib (IRE). Our results indicated that interactions, formed within the tetrad of residues S768 (in the αC-helix), D770 (in the αC-β4 loop), Y827 (in the αE-helix), and R831 (in the catalytic loop), play an important role in the stability of αC-helix and the maintenance of K745–E762 salt bridge in the absence of IRE, which are weakened in the EGFR^G719S system and enhanced in the EGFR^G719S/L858R system upon IRE binding. Besides, the introduced hydrogen bonds by the co-occurring mutation partner also contribute to the stability of αC-helix. The work done for inhibitor dissociation suggests that IRE exhibits a stronger binding affinity to EGFR^G719S/L858R mutant. Together, these findings provide a deeper understanding of minor mutations, which is essential for drug development targeting EGFR with less common mutations.

Parathyroid hormone (PTH) is an endogenous ligand that activates the PTH type 1 receptor (PTH1R) signaling. Ca²⁺, a common second messenger, acts as an allosteric regulator for prolonging the activation of PTH1R. However, a clear picture of the underlying allosteric mechanism is still missing. Herein, extensive molecular dynamics (MD) simulations are performed for PTH1R-PTH complexes with and without Ca²⁺ ions, allowing us to delineate the molecular details of calcium-induced allostery. Our results indicate that acidic residues in the extracellular loop 1 (ECL1) (D251, E252, E254, and E258–E260) and PTH (E19 and E22) serve as key determinants for local Ca²⁺-coupling structures and rigidity of ECL1. Moreover, the binding of Ca²⁺ induces conformational changes of transmembrane domain 6/7 (TM6/7) that are related to PTH1R activation and strengthens the residue–residue communication within PTH and TMD allosterically. Moreover, our results demonstrate that the presence of Ca²⁺ ions potentiates the interaction between PTH and PTH1R via steered molecular dynamics (SMD) simulations, while the point mutation in the PTH (PTH^R25C) weakens the binding of PTH and PTH1R. These results support that Ca²⁺ ions might further prolong the residence time of PTH on PTH1R and facilitate the positive allostery of PTH1R. Together, the present work provides new insights into the allosteric regulation mechanism of GPCRs induced by ions and related drug design targeting the PTH1R allosteric pathway.