Structural and Functional characterization of Eubacterial DNA ligases

Abstract

The work reported in the thesis mainly concerns the structural and biochemical studies involving the M. tuberculosis NAD+-dependent DNA ligaseA (MtuLigA) and its potential partner proteins like the Beta clamp (Mtu -clamp) (the bacterial homolog of eukaryotic PCNA). We have also identified novel classes of inhibitors of MtuLigA that are able to distinguish between the human and mycobacterial proteins several fold and this work is also reported. DNA LigA is the principal and essential ligase in bacteria that is drawing attention as a novel drug target. DNA LigA in bacteria like E. coli are known to interact with protein partners like the Beta clamp while no such studies have been carried out involving the mycobacterial counterparts. MtuLigA is unique because it takes part in HR (Homologous recombination) and NHEJ (Non-homologous end joining) DNA repair processes unlike bacteria like E. coli where NHEJ processes do not operate. Crystal structures of full-length DNA Ligase A and the Beta clamp have not been reported from mycobacterial sources. A brief chapter wise summary of the reported work is given below: Chapter 1 places the current work in the light of already known and studied nucleotidyltransferase family of proteins of which DNA ligases are a member. The focus is mainly on the HR and NHEJ DNA repair strategies and structural and functional description of the ligases that are involved in them. The later part of the chapter deals with structure and functions of potential protein partners of DNA ligases like the Beta sliding clamp and clamp loader. Chapter 2 covers the various techniques and experimental approaches used to clone, purify and characterize the proteins/peptides in the present work. It also deals with the methods used to solve the structures of the MtuLigA domain deletion mutant (MtuLigA1) and that of the Mtu -Clamp. It includes the determination and analysis of the kinetic parameters of different inhibitors as also in silico docking / screening approaches. Chapter 3 The results obtained while cloning, purifying and characterization of the enzyme are detailed here. The preparation of the domain deleted mutants and their functional characterization are also reported. Crystallization, structural solution and analysis of the 68 kDa BRCT domain deleted DNA ligaseA mutant (MtuLigA1) with bound co-factor is also reported. The structure has captured the co-factor in an as yet unobserved interaction state. Chapter 4 deals mainly with the crystallization, structure solution and analysis of the Mtu -Clamp, a potential interacting partner of MtuLigA. The possible interactions with the MtuLigA have been probed using a variety of approaches. The Mtu -Clamp interacts robustly with subunits of the clamp loader while it exhibits differences in the interactions with MtuLigA compared to the other bacteria like E. coli. Chapter 5 deals with the identification of novel classes of inhibitors of MtuLigA through docking/virtual screening techniques. The identified compounds were then tested through in vitro and antibacterial assays reported here. The studies have led to the identification of novel classes of inhibitory compounds, which bind to the co-factor binding region. These compounds inhibit M. tuberculosis NAD+ ligase with higher specificity compared to bacteriophage T4 ATP-dependent DNA ligase as well as Human DNA ligase I and are competitive inhibitors of the enzyme with respective to the co-factor NAD+. A part of the results of this thesis have already been reported in the following publications. PUBLICATIONS & PATENTS 1. Sandeep Kumar Srivastava, Divya Dube, Vandana Kukshal, Ashok Kumar Jha, Kanchan Hajela & Ravishankar Ramachandran. “NAD+-dependent DNA ligase (Rv3014c) from Mycobacterium tuberculosis novel structure-function relationship and identification of a specific inhibitor” Proteins (2007) 69, 97-111. 2. Namrata Dwivedi, Divya Dube, Jyoti Pandey, Biswajit Singh, Vandana Kukshal, Ravishankar Ramachandran & Rama Pati Tripathi “NAD+-dependent DNA ligase- A novel target waiting for right inhibitor” Med Res Rev (2008) 28, 545-68. 3. Divya Dube, Sandeep Kumar Srivastava, Vandana Kukshal & Ravishankar Ramachandran. “NAD+-dependent DNA ligase (Rv3014c) Mycobacterium tuberculosis: Strategies for inhibitor design” Med Chem Res (2008) 17, 189-198. 4. Vandana Kukshal, Mridul Mishra, Divya Dubey, Deepti Chopra, Rama Pati Tripathi, Ravishankar Ramachandran “Hydroxymates can distinguish between NAD+ and ATP dependent DNA ligases” (Manuscript under communication). 5. Vandana Kukshal, Sandeep Srivastava, Ravishankar Ramachandran. “Crystal Structure of a Domain deleted mutant of Mycobacterium tuberculosis NAD+- dependent DNA ligase captures the AMP cofactor in a new interaction state” (Manuscript under communication). 6. Jyoti Pandey#, Vandana Kukshal#, Vinita Chaturvedi, Arya Ajay, Divya Dube, Deepti Chopra, R.P. Tripathi, Ravishankar Ramachandran Synthesis of Dispiro cycloalkanones and their bioevaluation as mycobacterial NAD+-dependent DNA ligase inhibitors and antitubercular action.(Manuscript communicated). 7. Vandana Kukshal, Ravishankar Ramachandran “Crystal Structure of the Beta Clamp of Mycobacterium tuberculosis” (Manuscript under preparation). Patent Title: Novel Dispiro cyclalkanones useful as inhibitors of NAD+-dependent DNA Ligase and antitubercular agents Inventors: Rama Pati Tripathi, Jyoti Pandey, Nimisha Singh, Divya Dube, Vandana Kukshal, Shalini Bhatnagar, Sudhir Sinha, Vinita Chaturvedi, Ravishankar Ramachandran Indian Patent filed: 2009