Crystal structure of DNA polymerase III β sliding clamp from Mycobacterium tuberculosis

Gui, Wenjun; Lin, Shu‐Fang; Chen, Yuanyuan; Zhang, Xian-En; Bi, Lijun; Jiang, Tao

doi:10.1016/j.bbrc.2011.01.027

Cited by 38 publications

(33 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Not unexpectedly, the structure is similar to that of other clamps from bacteria like the E.coli and S. pyogenes with minor variations ( Figure 5b ). While the manuscript was being prepared another group reported the structure of the Mtbβ-Clamp grown under different conditions and in the monoclinic P2 1 space group [16]. The structural details that have been highlighted in the earlier report will be avoided here to avoid repetitive descriptions.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2012

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

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

et al. 2012

View full text Add to dashboard Cite

show abstract

“…In addition, the HolA peptide shows no binding to Cc DnaN (Fig. C), while the homologous peptide in Mycobacterium tuberculosis for DnaN was revealed to be 50.2 μ m . A comparison of the reported peptide‐binding parameters for Cc DnaN and its homologs is summarized in Table .…”

Section: Resultsmentioning

confidence: 99%

“…Given that high in vitro β‐binding affinity has been previously revealed for many β motif peptides in other species and the primary contributor to the DnaN interaction was found to be contained in the core 9‐mer , we employed isothermal titration calorimetry (ITC) to determine the binding affinity of the three β motif peptides from C. crescentus for Cc DnaN. The results show that the β‐motif peptides of HdaA and DnaE interact with the DnaN protein with an affinity ( K D ) of 15.70 ± 2.61 and 107.8 ± 10.86 μ m , respectively (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Biochemical experiments were subsequently employed to verify our above findings. We constructed eleven Cc DnaN mutants containing both novel and canonical sites: E166A, E186A, K364A, 3‐site mutant E166A‐E186A‐K364A (M3), and ∆ 165–168 were introduced to disrupt the interaction with the novel pocket, while L182A, P248A, V253A, V366A, M368A, and 5‐site mutant L182A‐P248A‐V253A‐V366A‐M368A (M5) were introduced to disrupt binding of the canonical pocket as reported . Pull‐down assays revealed that the novel site mutants suffered dramatic losses in binding to both HdaA and DnaE910C, especially M3 and ∆ 165–168 .…”

Section: Resultsmentioning

confidence: 99%

“…In eubacteria, a canonical DnaN‐associating pattern has been revealed, during which protein partners are tethered into a conserved hydrophobic cleft in DnaN via the β motif. Subsequent studies have revealed that this typical pattern is also observable in some other bacterial species, suggesting that the β motif and the hydrophobic cleft are conserved among species . However, as homologous proteins from evolutionarily remote species show less sequence and structural similarity, it is possible that the DnaN‐binding pattern is different in some species.…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Caulobacter crescentus β sliding clamp employs a noncanonical regulatory model of DNA replication

Jiang

Zhang

et al. 2019

The FEBS Journal

View full text Add to dashboard Cite

The eubacterial b sliding clamp (DnaN) plays a crucial role in DNA metabolism through direct interactions with DNA, polymerases, and a variety of protein factors. A canonical protein-DnaN interaction has been identified in Escherichia coli and some other species, during which protein partners are tethered into the conserved canonical hydrophobic crevice of DnaN via the consensus b-binding motif. Caulobacter crescentus is an excellent research model for use in the investigation of DNA replication and cell-cycle regulation due to its unique asymmetric cell division pattern with restricted replication initiation; however, little is known about the specific features of C. crescentus DnaN (CcDnaN). Here, we report a significant divergence in the association of CcDnaN with proteins based on docking analysis and crystal structures that show that the b-binding motifs of its protein partners bind a novel pocket instead of the canonical site. Pulldown and isothermal titration calorimetry results revealed that mutations within the novel pocket disrupt protein-CcDnaN interactions. It was also shown by replication and regulatory inactivation of DnaA assays that mediation of protein interaction by the novel pocket is closely related to the performance of CcDnaN during replication and the DnaN-mediated regulation process. Moreover, assessments of clamp competition showed that DNA does not compete with protein partners when binding to the novel pocket. Overall, our structural and biochemical analyses provide strong evidence that CcDnaN employs a noncanonical protein association pattern.Abbreviations C. crescentus, Caulobacter crescentus; DnaE, DNA polymerase III subunit alpha; DnaN, b clamp, DNA polymerase III beta sliding clamp subunit; Hda, DnaA regulatory inactivator Hda; HdaA, chromosome replication regulator protein HdaA; HolA, DNA polymerase III subunit delta; ITC, isothermal titration calorimetry; PDB, protein data bank; RIDA, regulatory inactivation of DnaA; b motif, b-binding motif, a consensus short peptide sequence motif for protein-DnaN interaction.

show abstract

New Structural Templates for Clinically Validated and Novel Targets in Antimicrobial Drug Research and Development

Klahn

Brönstrup

2016

Current Topics in Microbiology and Immunology

View full text Add to dashboard Cite

Abstract:The development of bacterial resistance against current antibiotic drugs necessitates a continuous renewal of the arsenal of efficacious drugs. This imperative has not been met by the output of antibiotic research and development of the past decades for various reasons, including the declining efforts of large pharma companies in this area. Moreover, the majority of novel antibiotics are chemical derivatives of existing structures that represent mostly step innovations, implying that the available chemical space may be exhausted. This review negates this impression by showcasing recent achievements in lead finding and optimization of antibiotics that have novel or unexplored chemical structures. Not surprisingly, many of the novel structural templates like teixobactins, lysocin, griselimycin, or the albicidin/cystobactamid pair were discovered from natural sources. Additional compounds were obtained from the screening of synthetic libraries and chemical synthesis, including the gyrase-inhibiting NTBI s a d spiropyrimidinetrione, the tarocin and targocil inhibitors of wall teichoic acid synthesis, or the boronates and diazabicyclo[3.2

show abstract

Crystal structure of DNA polymerase III β sliding clamp from Mycobacterium tuberculosis

Cited by 38 publications

References 33 publications

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

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

Caulobacter crescentus β sliding clamp employs a noncanonical regulatory model of DNA replication

New Structural Templates for Clinically Validated and Novel Targets in Antimicrobial Drug Research and Development

Contact Info

Product

Resources

About