Rv2231c, a unique histidinol phosphate aminotransferase from Mycobacterium tuberculosis, supports virulence by inhibiting host-directed defense.

Nitrogen metabolism of M. tuberculosis is critical for its survival in infected host cells. M. tuberculosis has evolved sophisticated strategies to switch between de novo synthesis and uptake of various amino acids from host cells for metabolic demands. Pyridoxal phosphate-dependent histidinol phosphate aminotransferase-HspAT enzyme is critically required for histidine biosynthesis. HspAT is involved in metabolic synthesis of histidine, phenylalanine, tyrosine, tryptophan, and novobiocin. We showed that M. tuberculosis Rv2231c is a conserved enzyme with HspAT activity. Rv2231c is a monomeric globular protein that contains α-helices and ß-sheets. It is a secretory and cell wall-localized protein that regulates critical pathogenic attributes. Rv2231c enhances the survival and virulence of recombinant M. smegmatis in infected RAW264.7 macrophage cells. Rv2231c is recognized by the TLR4 innate immune receptor and modulates the host immune response by suppressing the secretion of the antibacterial pro-inflammatory cytokines TNF, IL-12, and IL-6. It also inhibits the expression of co-stimulatory molecules CD80 and CD86 along with antigen presenting molecule MHC-I on macrophage and suppresses reactive nitrogen species formation, thereby promoting M2 macrophage polarization. Recombinant M. smegmatis expressing Rv2231c inhibited apoptosis in macrophages, promoting efficient bacterial survival and proliferation, thereby increasing virulence. Our results indicate that Rv2231c is a moonlighting protein that regulates multiple functions of M. tuberculosis pathophysiology to increase its virulence. These mechanistic insights can be used to better understand the pathogenesis of M. tuberculosis and to design strategies for tuberculosis mitigation.

Structural and Biophysical properties of therapeutically important proteins Rv1509 and Rv2231A of Mycobacterium tuberculosis.

Through comparative analyses using BLASTp and BLASTn of the 25 target sequences, our research identified two unique post-transcriptional modifiers, Rv1509 and Rv2231A, which serve as distinctive and characteristic proteins of M.tb - the Signature Proteins. Here, we have characterized these two signature proteins associated with pathophysiology of M.tb which may prove to be therapeutically important targets. Dynamic Light Scattering and Analytical Gel Filtration Chromatography exhibited that Rv1509 exists as a monomer while Rv2231A as a dimer in solution. Secondary structures were determined using Circular Dichroism and further validated through Fourier Transform Infrared spectroscopy. Both the proteins are capable of withstanding a wide range of temperature and pH variations. Fluorescence spectroscopy based binding affinity experiments showed that Rv1509 binds to iron and may promote organism growth by chelating iron. In the case of Rv2231A, a high affinity for its substrate RNA was observed, which is facilitated in presence of Mg2+ suggesting it might have RNAse activity, supporting the prediction through in-silico studies. This is the first study on biophysical characterization of these two therapeutically important proteins, Rv1509 and Rv2231A, providing important insights into their structure -function correlations which are crucial for development of new drugs/ early diagnostics tools targeting these proteins.

Binding and structural studies of the complexes of type 1 ribosome inactivating protein from Momordica balsamina with uracil and uridine.

Ribosome inactivating protein (RIP) catalyzes the cleavage of glycosidic bond formed between adenine and ribose sugar of ribosomal RNA to inactivate ribosomes. Previous structural studies have shown that RNA bases, adenine, guanine, and cytosine tend to bind to RIP in the substrate binding site. However, the mode of binding of uracil with RIP was not yet known. Here, we report crystal structures of two complexes of type 1 RIP from Momordica balsamina (MbRIP1) with base, uracil and nucleoside, uridine. The binding studies of MbRIP1 with uracil and uridine as estimated using fluorescence spectroscopy showed that the equilibrium dissociation constants (KD ) were 1.2 × 10-6 M and 1.4 × 10-7 M respectively. The corresponding values obtained using surface plasmon resonance (SPR) were found to be 1.4 × 10-6 M and 1.1 × 10-7 M, respectively. Structures of the complexes of MbRIP1 with uracil (Structure-1) and uridine (Structure-2) were determined at 1.70 and 1.98 Å resolutions respectively. Structure-1 showed that uracil bound to MbRIP1 at the substrate binding site but its mode of binding was significantly different from those of adenine, guanine and cytosine. However, the mode of binding of uridine was found to be similar to those of cytidine. As a result of binding of uracil to MbRIP1 at the substrate binding site, three water molecules were expelled while eight water molecules were expelled when uridine bound to MbRIP1.

Structure of iron saturated C-lobe of bovine lactoferrin at pH 6.8 indicates a weakening of iron coordination.

The bilobal lactoferrin is an approximately 76 kDa glycoprotein. It sequesters two Fe(3+) ions together with two CO(3)(2-) ions. The C-terminal half (residues, Tyr342-Arg689, C-lobe) of bovine lactoferrin (BLF) (residues Ala1-Arg689) was prepared by limited proteolysis using trypsin. Both C-lobe and intact BLF were saturated to 100%. Both of them retained up to nearly 85% of iron at pH 6.5. At pH 5.0, C-lobe retained 75% of iron whereas intact protein could retain only slightly more than 60%. At pH 4.0 both contained 25% iron and at pH 2.0 they were left with iron concentration of only 10%. The structure of iron saturated C-lobe was determined at 2.79 Å resolution and refined to R(cryst) and R(free) factors of 0.205 and 0.273, respectively. The structure contains two crystallographically independent molecules, A and B. They were found to have identical structures with an r.m.s. shift of 0.5 Å for their C(α) atoms. A high solvent content of 66% was observed in the crystals. The average value of an overall B-factor was 68.0 Å(2). The distance of 2.9 Å observed for the coordination bond between Fe(3+) ion and N(e2) of His595 appeared to be considerably longer than the normally observed values of 1.9-2.2 Å. This indicated that the coordination bond involving His595 may be absent. Other coordination distances were observed in the range of 2.1-2.3 Å. Based on the present structure of iron saturated C-lobe, it may be stated that His595 is the first residue to dissociate from ferric ion when the pH is lowered.

Structure of the iron-free true C-terminal half of bovine lactoferrin produced by tryptic digestion and its functional significance in the gut.

Bovine lactoferrin, a 76-kDa glycoprotein (Ala1-Arg689) consists of two similar N- and C-terminal molecular halves with the ability to bind two Fe(3+) ions. The N-terminal half, designated as the N-lobe (Ala1-Arg341) and the C-terminal half designated as the C-lobe (Tyr342-Arg689) have similar iron-binding properties, but the resistant C-lobe prolongs the physiological role of bovine lactoferrin in the digestive tract. Here, we report the crystal structure of true C-lobe, which was produced by limited proteolysis of bovine lactoferrin using trypsin. In the first proteolysis step, two fragments of 21 kDa (Glu86-Lys282) and 45 kDa (Ser283-Arg689) were generated because two lysine residues, Lys85 and Lys282, in the structure of iron-saturated bovine lactoferrin were fully exposed. The 45-kDa fragment was further digested at the newly exposed side chain of Arg341, generating a 38-kDa perfect C-lobe (Tyr342-Arg689). By contrast, the apo-lactoferrin was cut by trypsin only at Arg341, which was exposed in the structure of apo-lactoferrin, whereas the other two sites with Lys85 and Lys282 are inaccessible. The purified iron-saturated C-lobe was crystallized at pH 4.0. The structure was determined by the molecular replacement method using coordinates of the C-terminal half (Arg342-Arg689) of intact camel apo-lactoferrin. The structure determination revealed that the iron atom was absent and the iron-binding cleft was found in a wide-open conformation, whereas in the previously determined structure of iron-saturated C-lobe of bovine lactoferrin, the iron atom was present and the iron-binding site was in the closed confirmation.

Preparation and antimicrobial action of three tryptic digested functional molecules of bovine lactoferrin.

Lactoferrin is an 80 kDa bilobal, iron binding glycoprotein which is primarily antimicrobial in nature. The hydrolysis of lactoferrin by various proteases in the gut produces several functional fragments of lactoferrin which have varying molecular sizes and properties. Here, bovine lactoferrin has been hydrolyzed by trypsin, the major enzyme present in the gut, to produce three functional molecules of sizes approximately 21 kDa, 38 kDa and 45 kDa. The molecules have been purified using ion exchange and gel filtration chromatography and identified using N-terminal sequencing, which reveals that while the 21 kDa molecule corresponds to the N2 domain (21LF), the 38 kDa represents the whole C-lobe (38LF) and the 45 kDa is a portion of N1 domain of N-lobe attached to the C-lobe (45LF). The iron binding and release properties of 21LF, 38LF and 45LF have been studied and compared. The sequence and structure analysis of the portions of the excision sites of LF from various species have been done. The antibacterial properties of these three molecules against bacterial strains, Streptococcus pyogenes, Escherichia coli, Yersinia enterocolitica and Listeria monocytogenes were investigated. The antifungal action of the molecules was also evaluated against Candida albicans. This is the first report on the antimicrobial actions of the trypsin cleaved functional molecules of lactoferrin from any species.