Protein synthesis & ribosome biogenesis of pathogenic microbes, & structure-based drug design

Protein synthesis or translation is a vital cellular process that occurs on the ribosome in all cells. It is one of the most energy-consuming cellular processes, which consumes nearly half of the cell's energy, and ~40% of known antibiotics inhibit specific steps of bacterial protein synthesis. A detailed knowledge of the ribosome structure, function, and assembly of pathogenic: bacteria, fungi, and protozoa would reveal the species-specific unique features of their translation machinery, which could be endeavored as a potential drug target. We apply structural biology tools: cryo- electron microscopy (cryo- EM) and X-ray crystallography with molecular biology and biochemistry techniques, as well as structure- based drug design approaches.

Translation regulation in Mycobacterium tuberculosis(Mtb)

Mtb is the causative agent of tuberculosis (TB), one of the most deadly bacterial diseases which remains a major health threat to human race. The MTb becomes dormant, nonreplicating, and phenotypically drug-resistant when it encounters multiple stresses within the host macrophages. This condition is known as latent tuberculosis infection (LTBI) or dormancy. LTBI affects about one-third of the world’s population, with ~10% of those infected developing acute TB infection. During latent TB infection, the nonreplicating persistent mycobacteria slow down all cellular processes, including protein synthesis, making the ribosome-targeting antibiotics less effective in treating tuberculosis. Therefore, the latent Mtb infection serves as a reservoir for TB spread. Our goal is to elucidate the unique features of mycobacterial protein synthesis during dormancy for structure-based inhibitor design.

Protein synthesis in Entamoeba histolytica

Entamoeba histolytica is an anaerobic parasite responsible for amebiasis, an intestinal infection that results in bloody diarrhea and liver abscesses. Amoebiasis is more predominant in tropical areas with poor sanitation conditions, including India. Therefore, amoebiasis puts a huge economic burden on our country. We aim to determine the high- resolution cryo- EM structure of E. histolytica ribosome and identify protozoan- specific unique aspects of its protein synthesis machinery. Exploits these unique features to design protozoan specific inhibitors using a structure-based drug design approach.

Ribosome biogenesis

The ribosomes are the ribonucleoprotein (RNP) complexes of mega Dalton (MDa) sizes that decodes mRNA into protein. Each ribosome comprises two subunits, a large subunit (LSU), which harbors peptidyl transferase centre (PTC) where peptide bond formation takes place, and a small subunit (SSU), on which the decoding of mRNA sequence occurs. Both subunits, LSU and SSU, join to form a functional 70S or 80S ribosome. Ribosome biogenesis involves synthesizing, processing, and modifying individual components: rRNAs and ribosomal proteins (RPs), and their assembly. Defects in ribosome assembly, the ribosomopathy, are associated with various diseases such as cancer, age-related degenerative diseaases and aging. Our goal is to understand the ribosome biogenesis process by depleting the ribosome biogenesis factors and trapping ribosomes in an intermediate assembly stage. Ultimately, determining the cryo-EM structure of assembly intermediates.

Cryo-EM

Cryo-EM is a fast-emerging powerful technique in modern biology for solving the structure of biomolecules at near-atomic resolution. It uses very little sample at cold temperature (liquid nitrogen) to visualize the macromolecular complex in its native state without distorting the molecule's structure. Recent advancements in image-processing algorithms and electron detector technology made it possible to achieve atomic resolution. With respect to other techniques, cryo-EM covers a range of biomolecules and sample conditions. The recent cryo-EM structures of complexes with drug molecules have further proven the power of this technique to visualize the binding sites of drug molecules and its application in structure-based drug design.

X-ray crystallography

X-ray crystallography remains the most powerful technique for the determining the molecular and atomic structure of proteins and biological macromolecules over a century. The high-resolution atomic coordinate obtained from the X-ray crystal structure helps to solve the unanswered mystery and provides a platform for elucidating enzyme mechanisms, specificity of protein-ligand interactions, site-directed mutagenesis, and structure-based drug design.

• DST-SERB Early Career Research Award (2019)
• CSIR Senior Research Fellowship (2005-2008)
• CSIR Junior Research Fellowship (2003-2005)
• Republic day (1996) Prashasti Patr (Citation) recipient form district administration for achievements in the field of Science.
• Outstanding team effort prize winner in reflective type telescope making workshop at CSIO, Chandigarh, 1995.
• Our model on “water purification by solar energy” was displayed in National Science Exhibition 1995 at Pragati Maidan, New Delhi and published by NCERT in 1995.

Highly motivated researchers interested in structural biology, particularly, in Cryo- electron microscopy (Cryo- EM) and X-ray crystallography are requested to contact directly to the PI.

• Niraj Kumar
  Senior Research Fellow

• Ankita Arora
  Senior Research Fellow

• Shivani Sharma
  Junior Research Fellow

• Tajamul Islam
  Integrated MS-PhD student

• Ruchika Kumari
  Junior Research Fellow (Project)

1. Arora A, Kaushal PS (2023). Cryo-EM in the study of ribosome biogenesis. Cryo- Electron Microscopy in Structural Biology. CRC Press Taylor & Francis Group. (Accepted)
2. Kumar N, Sharma S, Kaushal PS (2023). Cryo- EM structure of the mycobacterial 70S ribosome in complex with ribosome hibernation promotion factor RafH, reveals the unique mode of mycobacterial ribosome hibernation. BioRxiv https://doi.org/10.1101/2023.04.18.537051.
3. Pal P, Khan MY, Sharma S, Kumar Y, Mangla N, Kaushal PS, Agarwal N. ResR/McdR-regulated protein translation machinery contributes to drug resilience in Mycobacterium tuberculosis. Commun Biol. 2023 Jul 11;6(1):708. doi: 10.1038/s42003-023-05059-8. PMID: 37433855
4. Agarwal N, Sharma S, Pal P, Kaushal PS, Kumar N. (2022) Era, a GTPase-like protein of the Ras family, does not control ribosome assembly in Mycobacterium tuberculosis. Microbiology (Reading). 2022 Aug;168(8). doi: 10.1099/mic.0.001200. PMID: 35917161.
5. Kumar N, Sharma S & Kaushal PS. (2021) Protein synthesis in Mycobacterium tuberculosis as a potential target for therapeutic interventions. Molecular Aspects of Medicine, https://doi.org/10.1016/j.mam.2021.101002.
6. Koripella RK, Sharma MR, Bhargava K, Datta PP, Kaushal PS, Keshavan P, Spremulli LL, Banavali NK & Agrawal RK. (2020) Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features of mitochondrial translation elongation. Nat Commun 11, 3830 https://doi.org/10.1038/s41467-020-17715-2.
7. Li Y, Sharma MR, Koripella RK, Yong Y, Kaushal PS, Lin Q, Wade JT, Gray TA, Derbyshire KM, Agrawal RK, & Ojha AK. (2018) Zinc depletion induces ribosome hibernation in mycobacteria. Proceedings of the National Academy of Sciences USA (PNAS) 115:8191-6.
8. Qu G *, Kaushal PS*, Wang J*, Shigematsu H, Piazza CL, Agrawal RK, Belfort M & Wang HW. Structure of group II intron complexed with its transcriptase. (2016) Nature Structure and Molecular Biology (NSMB) 23, 549-57. (*equal contributions). Highlighted as the News and Views in NSMB 23, 507-9 and a follow up review in RNA BIOLOGY 13, 1218-22.
9. Kaushal PS, Sharma MR and Agrawal RK. (2015) The 55S mammalian mitochondrial ribosome and its tRNA-exit region. Biochimie. 114, 119-26.
10. Kaushal PS*, Sharma MR*, Booth TM, Haque E, Tung C, Sanbonmatsu KY, Spremulli LL and Agrawal R K. (2014) Cryo-EM structure of the small subunit of the mammalian mitochondrial ribosome. Proceedings of the National Academy of Sciences USA (PNAS) 111(20), 7284-9 (*equal contributions).
11. Sharma MR, Kaushal PS, Gupta M, Banavali N K, & Agrawal RK. (2013) Insights into structural basis of mammalian mitochondrial translation. In Translation in Mitochondria and Other Organelles, Duchene, A.M. ed. (Springer-Verlag, Berlin/ Heidelberg), pp. 1-28
12. Ahmad MF, Kaushal PS, Wan, Q, Wijeratna SR, An X, Huang M & Dealwis CD. (2012) Role of arginine-293 and glutamine-288 in communication between catalytic and allosteric sites in yeast ribonucleotide reductase. Journal of Molecular Biology 419, 315-29.
13. Kaushal PS, Wan Q, Faber C, & Dealwis C. (2012) Crystal dehydration Technique. In Crystallography: Research, Technology and Applications, Hokkaido, M. and Nagano, E. ed. (Nova Science Publishers, Inc.), pp. 91-104.
14. Wijerathna SR, Ahmad MF, Xu H, Fairman JW, Zhang A, Kaushal PS, Wan Q, Kiser J & Dealwis CG. (2011) Targeting the Large Subunit of Human Ribonucleotide Reductase for Cancer Chemotherapy. Pharmaceuticals, 4(10),1328-54.
15. Kaushal PS, Singh P, Sharma A, Muniyappa K & Vijayan M. (2010) X-ray and molecular dynamics studies on Mycobacterium leprae single-stranded DNA binding protein and comparison with other eubacterial SSB structures. Acta Crystallographica D66, 1048-58.
16. Kaushal PS, Talawar RK, Varshney U & Vijayan M. (2010) Structure of uracil-DNA glycosylase from Mycobacterium tuberculosis. Insights into interactions with ligands. Acta Crystallographica F66, 887-92 (cover illustration).
17. Kaushal PS, Talawar RK, Krishna PD, Varshney U & Vijayan M. (2008). Unique features of the structure and interactions of mycobacterial uracil-DNA glycosylase: structure of a complex of the Mycobacterium tuberculosis enzyme in comparison with those from other sources. Acta Crystallographica D64, 551-560.
18. Kaushal PS, Sankaranarayanan R & Vijayan M. (2008) Water-mediated variability in the structure of relaxed-state haemoglobin. Acta Crystallographica F64, 463-9.
19. Singh P, Talawar RK, Krishna PD, Varshney U & Vijayan M. (2006) Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor. Acta Crystallographica F62, 1231-4.