Alanine substitution mutations in the DNA binding region of a global staphylococcal virulence regulator affect its structure, function, and stability

Mandal, Sukhendu; Ghosh, Semanti; Sinha, Debabrata; Seal, Soham; Mahapa, Avisek; Polley, Soumitra; Saha, Deeya; Sau, Keya; Bagchi, Angshuman; Sau, Subrata

doi:10.1016/j.ijbiomac.2018.03.045

Cited by 9 publications

(3 citation statements)

References 50 publications

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“…The gel picture, captured by a Gel Documentation System (Bio-Rad, USA), was scanned using (). The amounts of DNA in the shifted bands, estimated from the scanned results, were employed to find out the half-maximal rσ B binding concentration ( K D ) as reported …”

Section: Methodsmentioning

confidence: 99%

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Alternative Sigma Factor of Staphylococcus aureus Interacts with the Cognate Antisigma Factor Primarily Using Its Domain 3

Sinha

Dutta

et al. 2021

Biochemistry

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Metrics & MoreArticle Recommendations * sı Supporting Information ABSTRACT: σ B , an alternative sigma factor, is usually employed to tackle the general stress response in Staphylococcus aureus and other Gram-positive bacteria. This protein, involved in S. aureus-mediated pathogenesis, is typically blocked by RsbW, an antisigma factor having serine kinase activity. σ B , a σ 70 -like sigma factor, harbors three conserved domains designated σ B2 , σ B3 , and σ B4 . To better understand the interaction between RsbW and σ B or its domains, we have studied their recombinant forms, rRsbW, rσ B , rσ B2 , rσ B3 , and rσ B4 , using different probes. The results show that none of the rσ B domains, unlike rσ B , showed binding to a cognate DNA in the presence of a core RNA polymerase. However, both rσ B2 and rσ B3 , like rσ B , interacted with rRsbW, and the order of their rRsbW binding affinity looks like rσ B > rσ B3 > rσ B2 . Furthermore, the reaction between rRsbW and rσ B or rσ B3 was exothermic and occurred spontaneously. rRsbW and rσ B3 also associate with each other at a stoichiometry of 2:1, and different types of noncovalent bonds might be responsible for their interaction. A structural model of the RsbW-σ B3 complex that has supported our experimental results indicated the binding of rσ B3 at the putative dimeric interface of RsbW. A genetic study shows that the tentative dimer-forming region of RsbW is crucial for preserving its rσ B binding ability, serine kinase activity, and dimerization ability. Additionally, a urea-induced equilibrium unfolding study indicated a notable thermodynamic stabilization of σ B3 in the presence of RsbW. Possible implications of the stabilization data in drug discovery were discussed at length.

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Section: Methodsmentioning

confidence: 99%

“…The amounts of DNA in the shifted bands, estimated from the scanned results, were employed to find out the half-maximal rσ B binding concentration (K D ) as reported. 61 Serine Kinase Assay. To obtain clues about the phosphorylation activity of cRsbW or Δ32, we performed a serine kinase assay as previously demonstrated.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Alternative Sigma Factor of Staphylococcus aureus Interacts with the Cognate Antisigma Factor Primarily Using Its Domain 3

Sinha

Dutta

et al. 2021

Biochemistry

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“…The unfolding/refolding study usually indicates whether the folding of a protein occurs by a two-state or by a multi-state mechanism through the generation of no or multiple intermediates. Additionally, unfolding studies intimate about the stability of proteins in the presence of ligands or mutations [26–32]. Furthermore, such studies have greatly influenced many biotechnological fields including drug discovery [33–39].…”

Section: Introductionmentioning

confidence: 99%

A staphylococcal cyclophilin carries a single domain and unfolds via the formation of an intermediate that preserves cyclosporin A binding activity

Seal

Polley

2019

PLoS ONE

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Cyclophilin (Cyp), a peptidyl-prolyl cis - trans isomerase (PPIase), acts as a virulence factor in many bacteria including Staphylococcus aureus . The enzymatic activity of Cyp is inhibited by cyclosporin A (CsA), an immunosuppressive drug. To precisely determine the unfolding mechanism and the domain structure of Cyp, we have investigated a chimeric S . aureus Cyp (rCyp) using various probes. Our limited proteolysis and the consequent analysis of the proteolytic fragments indicate that rCyp is composed of one domain with a short flexible tail at the C-terminal end. We also show that the urea-induced unfolding of both rCyp and rCyp-CsA is completely reversible and proceeds via the synthesis of at least one stable intermediate. Both the secondary structure and the tertiary structure of each intermediate appears very similar to those of the corresponding native protein. Conversely, the hydrophobic surface areas of the intermediates are comparatively less. Further analyses reveal no loss of CsA binding activity in rCyp intermediate. The thermodynamic stability of rCyp was also significantly increased in the presence of CsA, recommending that this protein could be employed to screen new CsA derivatives in the future.

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Removal of an atypical region from a staphylococcal cyclophilin affects its structure, function, stability, and shape

Seal

Chowdhury

Biswas

et al. 2020

International Journal of Biological Macromolecules

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Alanine substitution mutations in the DNA binding region of a global staphylococcal virulence regulator affect its structure, function, and stability

Cited by 9 publications

References 50 publications

Alternative Sigma Factor of Staphylococcus aureus Interacts with the Cognate Antisigma Factor Primarily Using Its Domain 3

Alternative Sigma Factor of Staphylococcus aureus Interacts with the Cognate Antisigma Factor Primarily Using Its Domain 3

A staphylococcal cyclophilin carries a single domain and unfolds via the formation of an intermediate that preserves cyclosporin A binding activity

Removal of an atypical region from a staphylococcal cyclophilin affects its structure, function, stability, and shape

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