RESEARCH @ THE NATIONAL INSTITUTE OF IMMUNOLOGY, NEW DELHI, INDIA
The NiRAN domain of SARS-CoV-2 RNA Polymerase
This study started as a fun project following the COVID-19 pandemic, where we computationally predicted the GTP binding pocket in the active site of the SARS-CoV-2 RNA polymerase and predicted the binding for a few small molecule inhibitors originally designed against the GTP binding pocket of the Hepatitis C RNA polymerase (Read the full text here).
Figure: A computational Model of the SARS-CoV-2 Initiation complex displaying the GTP biding site and a lead compound.
What caught our curiosity were the two additional domains present in the SARS-CoV-2 RNA polymerase, in addition to the canonical thumb, palm and finger domains. These additional domains, known as the NiRAN (Nidovirus RNA-polymerase Associated Nucleotidyltransferase) and interface domains are unique only to the RNA polymerases of nidoviruses. However, information on the functional aspects of these domains remains scanty. Earlier reports suggest structural similarities between the NiRAN domain and the pseudokinase SelO. We performed a combination of computational analyses which suggested that the NiRAN domain assumes a structural fold similar to the N-terminal lobe of kinases. Further, biochemical assays combined with mass spectrometry and ex vivo infection indicated that the SARS-CoV-2 RNA polymerase exhibits a kinase/phosphotransferase like activity. Interestingly, we also observed a loss of this activity in presence of broad spectrum kinase inhibitors and FDA approved anti-cancer drugs. (Read the full text here)
Figure: Left Panel- Structural superimposition of the SARS-CoV-2 NiRAN domain (yellow) with the N-terminal lobes of kinases (cyan). Kinases from top left to bottom right- Lym2, Syk kinase, O-mannosyl kinase, IRAK4, FGFR2 and Insulin receptor kinase. Right panel- Anti-cancer drug Sorafenib, which effectively inhibits the kinase like activity of SARS-CoV-2 RNA polymerase, significantly reduces viral load in SARS-CoV-2 infected cells both independently and in combination with the anti-viral drug Remdesivir.
The essentiality of histidine biosynthesis for mycobacterial survival in a murine model of tuberculosis
Histidine, in addition to its proteinogenic and catalytic roles, is known to regulate a variety of cellular processes. In course of an active infection, the host often employs a variety of immuno-metabolic strategies to prevent the usage of its cellular resources, such as lipid, amino acids, iron etc by the invading pathogens. For example, the host uses IDO (Indoleamine 2,3-dioxygenase) to sequester the available free tryptophan within its cells by converting tryptophan to kynurenine, thereby starving the pathogen of this key amino acid. While the proliferation of pathogens that are natural auxotroph for tryptophan, such as Chlamydia and Leishmania is readily hindered, pathogens like Mycobacterium tuberculosis that harbor a de novo biosynthetic pathway for tryptophan, evade this strategy and continue to multiply. We investigated the probable mechanisms Mycobacterium tuberculosis employs to fulfil its histidine requirements in course of murine model of tubercular infection. We showed that following the onset of the adaptive immune response, an interferon gamma mediated mechanism up-regulates the expressions of two histidine catabolizing enzymes- Histidine ammonia lyase and Histidine decarboxylase. This results in a sharp decline in the levels of free histidine within the mice lungs. Concurrently, we also observed a rise in the levels of histidine catabolites- urocanate, histamine and methyl-histidine. Interestingly, a simultaneous increase in the expressions of the mycobacterial histidine biosynthesis enzymes was also noted, suggesting that the bacilli no longer depended on the host resources to meet its histidine demands. (Read the full text here)
Figure: A cartoon representation of the host immune response mediated free histidine sequestration and the mycobacterial counter-response.
The accidental discovery of the Achromobacter Dh1f Ferritin 3D structure
Bacterioferritins are ferritin like molecules present exclusively in bacteria and archaea. The presence of a heme molecule sandwiched between two bacterioferritin dimers is what gives them a unique identity. In addition to being a critical molecule for iron metabolism, ferritins find wide variety of applications in drug discovery and delivery, nanotechnology, biomedical research, and surface chemistry. The bacterioferritin from Achromobacter Dh1f (identity confirmed post structure solution utilizing mass spectrometry) was accidentally co-purified and crystallized during an attempt to determine the structure of an unrelated mycobacterial protein. The bacterioferritin expression was so high that the overexpression culture that would normally look grey-white turned brick red in color. Achromobacter are opportunistic pathogens causing respiratory tract infections and are well-known contaminants of the laboratory cultures. The functional unit of the Achromobacter bacterioferritin is a 24-mer hollow spherical complex bearing 22 entry pores, similar to all known bacterioferritins. Interestingly, the di-nuclear/ferroxidase center of Achromobacter bacterioferritin contained a single iron (II), as opposed to two iron (II) observed in other bacterioferritin structures, hinting at the possibility that at a given instance, only one of the two catalytic sites of the di-nuclear center is enzymatically active. (Read the full text here)
Figure: Top Panel- Achromobacter bacterioferritin crystals obtained in various conditions. Bottom Left Panel- The biologically functional unit of Achromobacter bacterioferritin is a 24-mer complex displaying a 432 symmetry and assumes a hollow spherical structure. Bottom Right Panel- The Achromobacter bacterioferritin 24-mer complex has a total of 22 entry pores- 6 at the four fold axis (hexagon), 8 at the 3 fold axis (ellipses) and 8 canonical B-pores (circles).