M. tuberculosis sliding β-clamp does not interact directly with the NAD+-dependent DNA ligase

• Published in: PloS one 2012-04, Vol.7 (4), p.e35702
• Main Authors: Kukshal, Vandna, Khanam, Taran, Chopra, Deepti, Singh, Nidhi, Sanyal, Sabyasachi, Ramachandran, Ravishankar
• Format: Article
• Language: English
• Subjects: Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Binding sites; Biology; Chromatography; Clamps; Cloning; Collaboration; Conserved sequence; Councils; Crystal structure; Crystallization; Crystallography, X-Ray; Deoxyribonucleic acid; DNA; DNA biosynthesis; DNA Ligases - metabolism; DNA Polymerase III - chemistry; DNA Polymerase III - metabolism; DNA repair; DNA, Bacterial - chemistry; DNA, Bacterial - metabolism; E coli; Escherichia coli - chemistry; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Experiments; Ligands; Medicine; Models, Molecular; Mycobacterium tuberculosis - chemistry; Mycobacterium tuberculosis - enzymology; Mycobacterium tuberculosis - metabolism; NAD; Protein Multimerization; Protein Structure, Tertiary; Proteins; Salts; Size exclusion chromatography; Sliding; Substrates; Tuberculosis; Yeast
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0035702

The sliding β-clamp, an important component of the DNA replication and repair machinery, is drawing increasing attention as a therapeutic target. We report the crystal structure of the M. tuberculosis β-clamp (Mtbβ-clamp) to 3.0 Å resolution. The protein crystallized in the space group C222(1) with cell-dimensions a = 72.7, b = 234.9 & c = 125.1 Å respectively. Mtbβ-clamp is a dimer, and exhibits head-to-tail association similar to other bacterial clamps. Each monomer folds into three domains with similar structures respectively and associates with its dimeric partner through 6 salt-bridges and about 21 polar interactions. Affinity experiments involving a blunt DNA duplex, primed-DNA and nicked DNA respectively show that Mtbβ-clamp binds specifically to primed DNA about 1.8 times stronger compared to the other two substrates and with an apparent K(d) of 300 nM. In bacteria like E. coli, the β-clamp is known to interact with subunits of the clamp loader, NAD(+)-dependent DNA ligase (LigA) and other partners. We tested the interactions of the Mtbβ-clamp with MtbLigA and the γ-clamp loader subunit through radioactive gel shift assays, size exclusion chromatography, yeast-two hybrid experiments and also functionally. Intriguingly while Mtbβ-clamp interacts in vitro with the γ-clamp loader, it does not interact with MtbLigA unlike in bacteria like E. coli where it does. Modeling studies involving earlier peptide complexes reveal that the peptide-binding site is largely conserved despite lower sequence identity between bacterial clamps. Overall the results suggest that other as-yet-unidentified factors may mediate interactions between the clamp, LigA and DNA in mycobacteria.