Our study aims to investigate the physiological role of organophosphate hydrolase (OPH), hitherto known for its involvement in the degradation of organophosphate insecticides and nerve agents in Sphingobium fuliginis. We find that OPH exists as part of the TonB‐dependent Transport system that is involved in nutrient transport across the bacterial outer membrane. OPH interacts physically with the Ton complex components ExbD and TonB. The surface‐exposed arginine residues (R91 and R96) of OPH facilitate its interaction with ExbD. OPH is targeted to the inner membrane of Escherichia coli only when it is co‐expressed with either ExbD or the ExbB/ExbD complex. In the absence of ExbD, OPH remains in the cytoplasm. Our findings suggest a role for OPH in outer membrane transport.
Our previous studies have shown the existence of organophosphate hydrolase (OPH) as a part of the inner membrane associated TonB complex (ExbB/ExbD and TonB) of Sphingobium fuliginis. We now show its involvement in iron uptake by establishing direct interactions with ferric-enterobactin. The interactions between OPH and ferric-enterobactin were not affected even when the active site architecture is altered by substituting active site aspartate with either alanine or asparagine. Protein docking studies further substantiated these findings and predicted the existence of ferric-enterobactin binding site that is different from the catalytic site of OPH. A lysine residue (82 K) found at the predicted ferric-enterobactin binding site facilitated interactions between OPH and ferric-enterobactin. Substitution of lysine with alanine did not affect triesterase activity, but it abrogated OPH ability to interact with both ferric-enterobactin and ExbD, strengthening further the fact that the catalytic site is not the site for binding of these ligands. In the absence of interactions between OPHK82A and ExbD, OPHK82A failed to target membrane in E. coli cells. The Sphingobium fuliginis TonB dependent transport (SfTonBDT) system was reconstituted in E. coli GS027 cells generated by deleting the exbD and tonB genes. The E. coli GS030 cells having SfTonBDT system with OPH showed increased iron uptake. Such an increase was not seen in E. coli GS029, cells having SfTonBDT system generated either by omitting OPH or by including its variants, OPHD301A, OPHD301N suggesting a role for OPH in enhanced iron uptake.
This study proposes a novel model for SARS-CoV-2 viral integration into the host cell via endocytosis pathway and hence a target for preventive treatment of COVID-19 infection. SARS-CoV-2 spike protein undergoes proteolytic cleavage at S1-S2 cleavage site and the cleaved out S2 domain is further cleaved by the activated serine protease domain of humanTMPRSS2 to become S2'. The activated serine protease domain of TMPRSS2 is formed after it undergoes autocatalysis by which the catalytic domain is cleaved from the membrane bound non-catalytic ectodomain (hNECD) comprising of LDLRA CLASS-I repeat and a SRCR domain. It is known that the SRCR domains as well as LDLRA repeat harboring proteins mediate endocytosis of viruses and certain ligands. Based on this, we put forward a hypothesis that the exposed hNECD binds to the S2' as both are at an interaction proximity soon after S2' is processed by the serine protease domain and this interaction may lead to the endocytosis of virus as an alternate mechanism to the direct fusion model. Based on this hypothesis we have modelled the hNECD structure, followed by protein-protein docking studies with the known 3D structure of S2'. The interaction interface of hNECD discerned from the modelled complex structure was used for virtual screening of known FDA-approved drug molecules and also some of the Indian medicinal plant-based compounds. We also mapped the known mutations of concern and mutations of interest on interaction interface of S2' and found that none of the known mutations map onto the interaction interface. This indicates that targeting the probable interaction between the hNECD of TMPRSS2 and S2' may serve as an attractive potential therapeutic target which is variant independent.
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