title: 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 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.
authors.affiliation: Molecular and Structural Biology Division, Central Drug Research Institute (Council of Scientific and Industrial Research), Lucknow, Uttar Pradesh, India.
authors.forename: Vandna
authors.initials: V
authors.lastname: Kukshal
authors.forename: Taran
authors.initials: T
authors.lastname: Khanam
authors.forename: Deepti
authors.initials: D
authors.lastname: Chopra
authors.forename: Nidhi
authors.initials: N
authors.lastname: Singh
authors.forename: Sabyasachi
authors.initials: S
authors.lastname: Sanyal
authors.forename: Ravishankar
authors.initials: R
authors.lastname: Ramachandran
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D011088:DNA Ligases / Q000378:metabolism*; D004258:DNA Polymerase III / Q000737:chemistry / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C101139:beta subunit, DNA polymerase III; D004258:DNA Polymerase III; D011088:DNA Ligases; C120640:DNA ligase (NAD)