DEPTOR is an inhibitor of the mTOR kinase which controls cell growth. DEPTOR consists of two DEP domains and a PDZ domain connected by an unstructured linker, and its stability is tightly regulated through post-translational modifications of its linker region that contains the 286SSGYFS291 degron. Based on the mTORC1 complex, our modelling suggests a possible spatial arrangement of DEPTOR which is characterised to form a dimer. Our model shows that the two PDZ domains of a DEPTOR dimer bind separately to the dimeric mTOR's FAT domains ~130 Å apart, while each of the two extended linkers is sufficiently long to span from the FAT domain to the kinase domain of mTOR and beyond to join a shared dimer of the DEP domains. This places the linker's S299 closest to the kinase's catalytic site, indicating that phosphorylation would start with it and successively upstream towards DEPTOR's degron. The CK1α kinase is reportedly responsible for the phosphorylation of the degron, and our docking analysis further reveals that CK1α contains sites to bind DEPTOR's pS286, pS287 and pT295, which may act as priming phosphates for the phosphorylation of the degron's S291. DEPTOR's linker can also be ubiquitylated by the UbcH5A-SCFβ-TrCP complex without its PDZ dissociating from mTOR according to the modelling. As the catalytic cleft of mTOR's kinase is restricted, interactions between the kinase's unstructured segment surrounding the cleft and DEPTOR's linker, which may involve S293 and S299, may be critical to controlling DEPTOR's access to the catalytic cleft and hence its phosphorylation by mTOR in a manner dependent on mTOR's activation.
Carbapenem resistance in Gram-negative pathogens has become a global concern for health workers worldwide. In one of our earlier studies, a Klebsiella pneumoniae-carbapenemase-2 producing strain was induced with meropenem to explore differentially expressed proteins under induced and uninduced conditions. There is, LysM domain BON family protein, was found over 12-fold expressed under the induced state. A hypothesis was proposed that LysM domain protein might have an affinity towards carbapenem antibiotics making them unavailable to bind with their target. Hence, we initiated a study to understand the binding mode of carbapenem with LysM domain protein. MICs of imipenem and meropenem against LysM clone were increased by several folds as compared to NP-6 clinical strain as well as DH5 α (PET-28a KPC-2) clone. This study further revealed a strong binding of both antibiotics to LysM domain protein. Molecular simulation studies of LysM domain protein with meropenem and imipenem for 80 ns has also showed stable structure. We concluded that overexpressed LysM domain under induced condition interacted with carbapenems, leading to enhanced resistance as proved by high MIC values. Hence, the study proved the proposed hypothesis that the LysM domain plays a significant role in the putative mechanism of antibiotics resistance.