Teh, A.-H. | Search bioRxiv

bioRxiv2024

Loop modelling provides insights into regulation of mTORC1 activity via DEPTOR dimerisation

mTOR regulates cell growth by forming the mTORC1 and mTORC2 complexes. DEPTOR partially inhibits mTORC1, which in turn phosphorylates and inactivates it. Despite the mTORC1-DEPTOR structures, the exact mechanism remains unclear largely because functionally flexible key elements, DEPTORs linker in particular, are unresolved. By taking DEPTORs dimerisation into consideration, our modelling of these missing loops suggests that monomeric DEPTOR bound to mTORC1 in a non-inhibitory mode, while the domain-swapped dimeric DEPTOR could interact with mTORC1s FRB domain and block the kinases catalytic site with its linker. These two states indicate that linker phosphorylation inactivates DEPTOR possibly by disrupting its dimerisation, which could tether the linker to the kinase domain to enhance mTORC1 inhibition. In addition to DEPTOR, mTORs k9b-k10 loop, which harbours the S2481 autophosphorylation site, and mSIN1s flexible domains in mTORC2 might act as inhibitory elements too.

bioinformatics↗

bioRxiv2017

Characterisation and modelling of a polyhydroxyalkanoate synthase from Aquitalea sp. USM4 reveals a mechanism for polymer elongation

Polyhydroxyalkanoate synthase, PhaC, is a key enzyme in the biosynthesis of PHA, a type of bioplastics with huge potential to replace conventional petroleum-based plastics. While two PhaC structures have been determined recently, the exact mechanism remains unclear partly due to the absence of a tunnel for product passage. The PhaC from Aquitalea sp. USM4, PhaCAq, was characterised and showed a Km of 394 {micro}M and a kcat of 476.4 s-1 on the 3HB-CoA substrate. A model based on the structure of the closely related PhaC from Cupriavidus necator, PhaCCn revealed a three-branched tunnel at the dimeric interface. Two of the branches open to the solvent and serve as the putative routes for substrate entrance and product exit, while the third branch is elongated in a PhaC1 model from Pseudomonas aeruginosa, indicating a function of accommodating the hydroxyalkanoate (HA) moiety of the HA-CoA substrate. Docking of the two tetrahedral intermediates formed during catalysis suggests a PHA elongation mechanism that requires the HA moiety of the ligand to rotate ~180{degrees}. Both classes I and II PhaCs share a common mechanism for polymer elongation, and substrate specificity is determined in part by a bulky Phe/Tyr/Trp residue in the third branch in class I, which is conserved as Ala in class II to create room for longer substrates. The PhaCAq model provides fresh insights into a general PhaC mechanism, pinpointing key residues for potential engineering of PhaCs with desirable characteristics.

molecular biology↗