Biology subjects

Bukhdruker, S.

Publications and source records attributed to Bukhdruker, S..

4 recordsLinked to original sources

bioRxiv2020

NanoFAST: Structure-based design of a small fluorogen-activating protein with only 98 amino acids

One of the essential characteristics of any tag used in bioscience and medical applications is its size. The larger the label, the more it may affect the studied object, and the more it may distort its behavior. In this paper, using NMR spectroscopy and X-ray crystallography, we have studied the structure of fluorogen-activating protein FAST both in the apo form and in complex with the fluorogen. We shown that significant change in the protein occurs upon interaction with the ligand. While the protein is completely ordered in the complex, its apo form is characterized by higher mobility and disordering of its N-terminus. We used structural information to design the shortened FAST (which we named nanoFAST) by truncating 26 N-terminal residues. Thus, we created the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, which is composed of only 98 amino acids.

biochemistry↗

bioRxiv2020

A new twist of rubredoxin function in M. tuberculosis

Electron transfer mediated by metalloproteins drives many biological processes. Rubredoxins are ubiquitous iron-containing electron carriers that play important roles in bacterial adaptation to changing environmental conditions. In Mycobacterium tuberculosis, oxidative and acidic stresses as well as iron starvation induce rubredoxin expression. However, their functions during M. tuberculosis infection is unknown. In the present work, we show that rubredoxin B (RubB) supports catalytic activity of mycobacterial cytochrome P450s, CYP124, CYP125, and CYP142, which are important for bacterial viability and pathogenicity. We solved the crystal structure of RubB and characterized the interaction between RubB and CYPs using site-directed mutagenesis. Mutations that neutralized single charge on the surface of RubB did not dramatically decrease activity of studied CYPs, and isothermal calorimetry (ITC) experiments indicated that interactions are transient and not highly specific. Our findings suggest that a switch from ferredoxins to rubredoxins support CYP activity in M. tuberculosis-infected macrophages. Our electrochemical experiments suggest potential applications of RubB in biotechnology.

biochemistry↗

bioRxiv2020

Hydroxylation of antitubercular drug candidate, SQ109, by mycobacterial cytochrome P450

Spreading of the multidrug-resistant (MDR) strains of the deadliest pathogen Mycobacterium tuberculosis (Mtb) generates the need for new effective drugs. SQ109 showed activity against resistant Mtb and already advanced to Phase II/III clinical trials. Fast SQ109 degradation is attributed to the human liver Cytochrome P450s (CYPs). However, no information is available about interactions of the drug with Mtb CYPs. Here, we show that Mtb CYP124, previously assigned as a methyl-branched lipid monooxygenase, binds and hydroxylates SQ109 in vitro. A 1.25 [A]-resolution crystal structure of the CYP124-SQ109 complex unambiguously shows two conformations of the drug, both positioned for hydroxylation of the {omega}-methyl group in the trans position. The hydroxylated SQ109 presumably forms stabilizing H-bonds with its target, the Mycobacterial membrane protein Large 3 (MmpL3). We anticipate that Mtb CYPs could function as analogs of drug-metabolizing human CYPs affecting pharmacokinetics and pharmacodynamics of antitubercular (anti-TB) drugs.

biochemistry↗

bioRxiv2020

Metabolic fate of human immunoactive sterols in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) infection is among top ten causes of death worldwide, and the number of drug-resistant strains is increasing. The direct interception of human immune signaling molecules by Mtb remains elusive, limiting drug discovery. Oxysterols and secosteroids regulate both innate and adaptive immune responses. Here we report a functional, structural, and bioinformatics study of Mtb enzymes initiating cholesterol catabolism and demonstrated their interrelation with human immunity. We show that these enzymes metabolize human immune oxysterol messengers. Rv2266, the most potent among them, can also metabolize vitamin D3 (VD3) derivatives. High-resolution structures show common patterns of sterols binding and reveal a site for oxidative attack during catalysis. Finally, we designed a compound that binds and inhibits three studied proteins. The compound shows activity against Mtb H37Rv residing in macrophages. Our findings contribute to molecular understanding of suppression of immunity and suggest that Mtb has its own transformation system resembling the human phase I drug-metabolizing system.Competing Interest StatementA.Gi. and E.S. are employees of MT-Medicals LLC. The other authors declare no competing interests.View Full Text

biochemistry↗