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

Pandey, Preeti; Tarique, K.F.; Mazumder, Mohit; Rehman, S.A. Abdul; Kumari, Nilima; Gourinath, S.

doi:10.1038/srep31181

Cited by 17 publications

(25 citation statements)

References 59 publications

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“…Second, the in vitro affinity between DnaN and the core 9‐mer β‐motif peptide is much weaker or absent in C. crescentus . While the core 9‐mer peptide makes a significant contribution to the DnaN interaction in E. coli , and many types of research that have investigated the peptide–DnaN interaction have demonstrated their high in vitro affinity , our ITC results indicated a considerable reduction in affinity of this interaction in C. crescentus (Fig. C, Tables and ).…”

Section: Discussionmentioning

confidence: 67%

“…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%

“…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%

“…The canonical hydrophobic crevice that is located in the DnaN C‐terminal region is a conserved target of the β motif in many eubacterial species . Their interaction involves two subsites on the surface (Fig.…”

Section: Discussionmentioning

confidence: 99%

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

Jiang

Zhang

et al. 2019

The FEBS Journal

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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.

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Section: Discussionmentioning

confidence: 67%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

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“…Kinetic rate constants: K a and K d (association and dissociation rate constants, respectively) as well as affinity constant, K D were measured at RT. SPR analysis was performed by following previously described protocols (95)(96)(97)(98)(99). Briefly, 5 μM of recombinant 14-3-3I protein was immobilized on the surface (self-assembled monolayer of 11-Mercapto-Undecanoic Acid, MUA on gold surface) of SPR sensor chip by the mechanism of covalent amine coupling.…”

Section: Surface Plasmon Resonance Analysis Of Binding Affinity Betwementioning

confidence: 99%

Interaction of 14-3-3I and CDPK1 mediates the growth of human malaria parasite

Jain

Dey

Gupta

et al. 2020

Preprint

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Scaffold proteins play pivotal role as modulators of cellular processes by operating as multipurpose conformation clamps. 14-3-3 proteins are gold-standard scaffold modules that recognize phosphoSer/Thr (pS/pT) containing conserved motifs of target proteins and confer conformational changes leading to modulation of their functional parameters. Modulation in functional activity of kinases has been attributed to their interaction with 14-3-3 proteins. Herein, we have characterized Plasmodium falciparum 14-3-3 and its interaction with key kinase of the parasite, Calcium-Dependent Protein Kinase 1 (CDPK1) by performing various analytical biochemistry and biophysical assays. Towards this, we annotated PF3D7_0818200 as 14-3-3 isoform I through extensive phylogenetic and comparative sequence analysis. Molecular dynamics simulation studies indicated that phosphoSer64 present in CDPK1 polypeptide sequence (61KLGpS64) behaves as canonical Mode I-type (RXXpS/pT) consensus 14-3-3 binding motif, mediating the interaction. The protein-protein interaction was validated in vitro with ELISA and SPR, which confirmed that CDPK1 interacts with 14-3-3I in a phosphorylation dependent manner, with binding affinity constant of 670 ± 3.6 nM. The interaction of 14-3-3I with CDPK1 was validated with well characterized optimal 14-3-3 recognition motifs: ARSHpSYPA and RLYHpSLPA as CDPK1 mimetics, by simulation studies and ITC. Further, interaction antagonizing peptidomimetics showed growth inhibitory impact on the parasite indicating crucial physiological role of 14-3-3/CDPK1 interaction. Overall, this study characterizes 14-3-3I as a scaffold protein in the malaria parasite and unveils CDPK1 as its previously unidentified target. This sets a precedent for the rational design of 14-3-3 based PPI inhibitors by utilizing 14-3-3 recognition motif peptides, as a potential antimalarial strategy.

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Targeting the β‐clamp in Helicobacter pylori with FDA‐approved drugs reveals micromolar inhibition by diflunisal

et al. 2017

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The β-clamp is the processivity-promoting factor for most of the enzymes in prokaryotic DNA replication; hence, it is a crucial drug target. In the present study, we investigated the β-clamp from Helicobacter pylori, aiming to seek potential drug molecules against this gastric-cancer-causing bacterium. An in silico screening of Food and Drug Administration (FDA) approved drugs against the H. pylori β-clamp, followed by its in vitro inhibition using a surface competition approach, yielded the drug diflunisal as a positive initial hit. Diflunisal inhibits the growth of H. pylori in the micromolar range. We determined the structure of diflunisal in complex with the β-clamp to show that the drug binds at subsite I, which is a protein-protein interaction site. Successful identification of FDA-approved molecules against H. pylori may lead to better and faster drug development.

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

Cited by 17 publications

References 59 publications

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

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

Interaction of 14-3-3I and CDPK1 mediates the growth of human malaria parasite

Targeting the β‐clamp in Helicobacter pylori with FDA‐approved drugs reveals micromolar inhibition by diflunisal

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