Biomolecular interaction of purified recombinant Arabidopsis thaliana's alternative oxidase 1A with TCA cycle metabolites: Biophysical and molecular docking studies.

In higher plants, the mitochondrial alternative oxidase (AOX) pathway plays an essential role in maintaining the TCA cycle/cellular carbon and energy balance under various physiological and stress conditions. Though the activation of AOX pathway upon exogenous addition of α-ketoacids/TCA cycle metabolites [pyruvate, α-ketoglutarate (α-KG), oxaloacetic acid (OAA), succinate and malic acid] to isolated mitochondria is known, the molecular mechanism of interaction of these metabolites with AOX protein is limited. The present study is designed to understand the biomolecular interaction of pure recombinant Arabidopsis thaliana AOX1A with TCA cycle metabolites under in vitro conditions using various biophysical and molecular docking studies. The binding of α-KG, fumaric acid and OAA to rAtAOX1A caused conformational change in the microenvironment of tryptophan residues as evidenced by red shift in the synchronous fluorescence spectra (∆λ = 60 nm). Besides, a decrease in conventional fluorescence emission spectra, tyrosine specific synchronous fluorescence spectra (∆λ = 15 nm) and α-helical content of CD spectra revealed the conformation changes in rAtAOX1A structure associated with binding of various TCA cycle metabolites. Further, surface plasmon resonance (SPR) and microscale thermophoresis (MST) studies revealed the binding affinity, while docking studies identified binding pocket residues, respectively, for these metabolites on rAtAOX1A.

Structural insights into the substrate binding mechanism of novel ArgA from Mycobacterium tuberculosis.

The Mycobacterium tuberculosis (Mtb) Rv2747 gene encodes for a functional protein known as ArgA, which plays an important role in the first step of the l-arginine biosynthesis pathway. ArgA transfers the acetyl group from the acetyl-CoA to either l-glutamate or l-glutamine, which are the known substrates. Here, we present two crystal structures of ArgA: one complexed with CoA and product bound N-acetylglutamine and the other complexed with acetyl-CoA and the inhibitor l-arginine at 2.3 and 3.0 Šresolution respectively. The Mtb ArgA protomer was found to have a "V" cleft and a "ß" bulge, archetypal of a classical GCN5-related N-acetyltransferase superfamily of proteins. The product bound form implies that ArgA can also acetylate l-glutamine like l-glutamate. The active site is strongly inhibited by l-arginine resulting in a closed conformation of ArgA and both l-arginine and N-acetylglutamine were found to occupy at the same active site. Together with structural analysis, molecular docking studies, microscale thermophoresis and enzyme inhibition assays, we conclude that l-glutamine, l-glutamate and l-arginine, all occupy at the same active site of ArgA. Furthermore in case of Mtb ArgA, l-arginine does not act as an allosteric inhibitor unlike other N-acetylglutamate synthase family of proteins.

Lnc-EPB41-Protein Interactions Associated with Congenital Pouch Colon.

Congenital Pouch Colon (CPC) is a rare anorectal anomaly common to northwestern India, specifically Rajasthan. Despite efforts to understand the clinical genetic makeup of CPC, no attempt on identifying non-coding RNAs was done. We have earlier reported CPC's rare variants from whole exome sequencing (WES) across 18 affected samples in a total of 64 subjects. A Smith⁻Waterman algorithm was used to infer a couple of lncRNAs from WES samples of CPC with predictions from the Noncode database. Further screening and quantification using polymerase chain reaction (PCR), we ascertained interactions using Micro Scale Thermophoresis (MST). We report the role of lnc-EPB41-1-1 shown to be promiscuously interacting with KIF13A substantiating their role in regulation.