Structural and Biophysical Characterization of the Mycobacterium tuberculosis Protein Rv0577, a Protein Associated with Neutral Red Staining of Virulent Tuberculosis Strains and Homologue of the Streptomyces coelicolor Protein KbpA

Abstract

Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the component responsible for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has an amino acid sequence that is 36% identical to that of Streptomyces coelicolor AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomyces genus. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Å, binding properties with neutral red and deoxyadenosine were surveyed, backbone dynamics were measured, and thermal stability was assayed by circular dichroism spectroscopy. The protein is composed of four approximate repeats with a βαβββ topology arranged radially in consecutive pairs to form two continuous eight-strand β-sheets capped on both ends with an α-helix. The two β-sheets intersect in the center at roughly a right angle and form two asymmetric deep "saddles" that may serve to bind ligands. Nuclear magnetic resonance chemical shift perturbation experiments show that neutral red and deoxyadenosine bind to Rv0577. Binding to deoxyadenosine is weaker with an estimated dissociation constants of 4.1 ± 0.3 mM for saddle 1. Heteronuclear steady-state {H}-N nuclear Overhauser effect, T, and T values were generally uniform throughout the sequence with only a few modest pockets of differences. Circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C.

BCG Substrains Change Their Outermost Surface as a Function of Growth Media

“…Accordingly, M. tuberculosis overproducing CFP32 multiplied 25% faster than the parental strain grown in glycerol-containing medium, suggesting that CFP32 might participate in the detoxification of glycerol metabolism by-products [26]. In our experiments, BCG cells grown on A60 showed the highest red intensity and higher Rv0577 gene expression, which could be related to the abundance of the encoded protein CFP32 [27]. Interestingly, CFP32 regulates the immune response by interacting with TLR2 [28], which is associated with BCG immunogenicity.…”

Drug-Repurposing Screening Identifies a Gallic Acid Binding Site on SARS-CoV-2 Non-structural Protein 7

“…Solution NMR structures have been determined for SARS-CoV nsp7 at pH 7.5 and at pH 6.5. , More recently, the NMR chemicals shifts for SARS-CoV-2 nsp7 have also been reported . To corroborate the binding of gallic acid to SARS-CoV-2 nsp7 and to map the ligand-binding surface of gallic acid on nsp7, a chemical shift perturbation study was performed by titrating gallic acid into 15 N-labeled SARS-CoV-2 nsp7. , A summary of this experiment, shown in Figure A, indicates that the major chemical shift perturbations (Δ ave > 0.1 ppm) are clustered in α1, the C-terminal of α2, and the N-terminal of α3. These major chemical shift perturbations are mapped onto the solution structure of SARS-CoV nsp7 in Figure B (yellow) and show that, except for the two resonances in α3, all the perturbations are along a continuous linear surface spanning α1 and α2 centered roughly at H38, the most perturbed amide resonance overall.…”

Application of endobronchial ultrasound-guided transbronchial needle aspiration in the diagnosis and treatment of mediastinal lymph node tuberculous abscess: a case report and literature review

“…3 ). Polymerase chain reaction (PCR) assay showed the pus sample was positive for the detection of prokaryotic 16S RNA gene, RV0577 coding an important antigen RV0577 in TB patients [ 11 , 12 ], and IS6110 fragment, an insert fragment within TB [ 13 , 14 ], indicating the presence of featured DNA fragments of TB. The smear examination of pus showed positive for acid bacillus.…”