3RB9

Crystal structure of the M. tuberculosis beta clamp

Summary for 3RB9

• Entry DOI: 10.2210/pdb3rb9/pdb
• Descriptor: DNA polymerase III subunit beta (1 entity in total)
• Functional Keywords: mycobacterium tuberculosis, dna sliding beta clamp, beta clamp, clamp loader, transferase
• Biological source: Mycobacterium tuberculosis
• Cellular location: Cytoplasm (By similarity): Q50790
• Total number of polymer chains: 2
• Total formula weight: 82806.05

Authors

Kukshal, V.,Ramachandran, R. (deposition date: 2011-03-29, release date: 2012-04-04, Last modification date: 2024-03-20)

Primary citation

Kukshal, V.,Khanam, T.,Chopra, D.,Singh, N.,Sanyal, S.,Ramachandran, R.
M. tuberculosis Sliding beta-Clamp Does Not Interact Directly with the NAD(+)-Dependent DNA Ligase
Plos One, 7:e35702-e35702, 2012

PubMed 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 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.

PubMed: 22545130
DOI: 10.1371/journal.pone.0035702

Experimental method

X-RAY DIFFRACTION (3 Å)