M. tuberculosis Sliding β-Clamp Does Not Interact Directly with the NAD+ -Dependent DNA Ligase

Abstract

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 C2221 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 Kd 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.

The β2 clamp in the Mycobacterium tuberculosis DNA polymerase III αβ2ε replicase promotes polymerization and reduces exonuclease activity

“…A 3-D structural model of Mtb α (MtbDnaE1) in complex with a small molecule inhibitor confirmed its structural differences from the human genomic replicase, and thus its promise as a drug target 10 . The crystal structure of the β 2 clamp, the classical processive factor of DNA pol III, has been solved in Mtb at resolutions of 2.89 Å 11 and 3.00 Å 12 , likewise confirming its close homology, including binding sites for α and other subunits, with the β 2 clamp of E. coli . Little, however, is known about the structure and function of the other subunits of Mtb DNA pol III.…”

Structural insight into β-Clamp and its interaction with DNA Ligase in Helicobacter pylori

“…They have also identified MutL & MutS will also bind to β-clamp in E. coli . However Vandana Kukshal et al 11 reported that M. tuberculosis DNA ligase do not interact with β-clamp and they hypothesized that some other factor may be involved that aids the interaction between these two proteins. Such findings made us curious to determine whether an interaction between HpDNA ligase and Hpβ-clamp exists or some other factor is involved that mediates the interaction between these two proteins.…”

“…Coordinates of the β-clamp structures from different organisms used for comparison were obtained from the PDB. These structures were from E. coli (PDB code: 1MMI, 3BEP) 38 , Thermotoga maritima (PDB code: 1VPK), Streptococcus pyogenes (PDB code: 2AVT) 39 , Streptococcus pneumonia (PDB code: 2AWA), Mycobacterium tuberculosis (PDB code: 3RB9) 11 , Mycobacterium smegmatis (PDB code: 5AH2) 17 , Bacillus subtilis (PDB code: 4TR6) 6 , Pseudomonas aeruginosa (PDB code: 4TR8) 6 and Deinococcus radiodurans (PDB code: 4TRT) 40 . PromalS3D was used to create the structure-based multiple sequence alignment.…”

“…Therefore, studying the interaction between these two components is of great importance. In case of prokaryotes nothing much is known about β-clamp – DNA ligase interaction except for the existence of binding in case of E. coli 10 and absent in M. tuberculosis 11 . Despite of having similar catalytic mechanism, the structure of prokaryotic DNA ligase is little bit different from that of eukaryotic in having four domains – an adenylation domain, oligomer-binding (OB) fold, Zinc finger & helix-hairpin-helix and the last one BRCT (breast cancer carboxy-terminal) domain.…”

Replisome Assembly at Bacterial Chromosomes and Iteron Plasmids

“…β-clamp crystal structures were obtained from various organisms i.e., E. coli (Oakley et al, 2003 ; Burnouf et al, 2004 ), P. aeruginosa (Wolff et al, 2014 ), Streptococcus pyogenes (Argiriadi et al, 2006 ), M. tuberculosis (Gui et al, 2011 ; Kukshal et al, 2012 ; Wolff et al, 2014 ), B. subtillis (Wolff et al, 2014 ), T. maritima (structure 1VPK), Eubacterium rectale (structure 3T0P) , Streptococcus pneumoniae (Argiriadi et al, 2006 ). The crystal structures of β-clamp homologs—Proliferating Cell Nuclear Antigen (PCNA)—are also available from Eukaryotes and Archea (to name a few: Homo sapiens (Punchihewa et al, 2012 ), S. cerevisiae (Krishna et al, 1994 ), Sulfolobus solfaraticus (Williams et al, 2006 ).…”