Reassessment of Protein Stability, DNA Binding, and Protection of Mycobacterium smegmatis Dps

Ceci, Pierpaolo; Ilari, Andrea; Falvo, Elisabetta; Giangiacomo, Laura; Chiancone, Emilia

doi:10.1074/jbc.m502343200

Cited by 56 publications

(70 citation statements)

References 32 publications

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“…S3 in the supplemental material), suggesting assembly of a dodecameric particle like that expected for DPS or DPSL, as opposed to the 24-mer common to ferritin and bacterioferritin. While members of the ferritin superfamily are often found as stable oligomers, their assembly states can also be dependent upon changes in pH, salt concentration, metal ions, and temperature (8,12,14,20,30). However, despite surveying a variety of protein concentrations and buffer conditions, we were unable to identify conditions that give a stable dodecamer on the SEC column.…”

Section: Resultsmentioning

confidence: 67%

“…In this light, we note that dimeric forms of DPS from two other organisms have demonstrated DNA protection activity. In the first example, the dimeric form of DPS from Mycobacterium smegmatis protects DNA without apparent DNA binding (14). Ceci et al (14) attributed the DNA protection to iron oxidation at the two ferroxidase sites which remain intact at the subunit interface of dimeric Mycobacterium DPS.…”

Section: Discussionmentioning

confidence: 99%

“…In the first example, the dimeric form of DPS from Mycobacterium smegmatis protects DNA without apparent DNA binding (14). Ceci et al (14) attributed the DNA protection to iron oxidation at the two ferroxidase sites which remain intact at the subunit interface of dimeric Mycobacterium DPS. Similarly, the intact ferroxidase sites within each subunit of the dimeric form of BfDPSL may provide protection in our assay.…”

Section: Discussionmentioning

confidence: 99%

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Characterization of the Bacteroides fragilis bfr Gene Product Identifies a Bacterial DPS-Like Protein and Suggests Evolutionary Links in the Ferritin Superfamily

Gauss¹,

Reott²,

Rocha³

et al. 2011

Journal of Bacteriology

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A factor contributing to the pathogenicity of Bacteroides fragilis, the most common anaerobic species isolated from clinical infections, is the bacterium's extreme aerotolerance, which allows survival in oxygenated tissues prior to anaerobic abscess formation. We investigated the role of the bacterioferritin-related (bfr) gene in the B. fragilis oxidative stress response. The bfr mRNA levels are increased in stationary phase or in response to O 2 or iron. In addition, bfr null mutants exhibit reduced aerotolerance, and the bfr gene product protects DNA from hydroxyl radical cleavage in vitro. Crystallographic studies revealed a protein with a dodecameric structure and greater similarity to an archaeal DNA protection in starved cells (DPS)-like protein than to the 24-subunit bacterioferritins. Similarity to the DPS-like (DPSL) protein extends to the subunit and includes a pair of conserved cysteine residues juxtaposed to a buried dimetal binding site within the four-helix bundle. Compared to archaeal DPSLs, however, this bacterial DPSL protein contains several unique features, including a significantly different conformation in the C-terminal tail that alters the number and location of pores leading to the central cavity and a conserved metal binding site on the interior surface of the dodecamer. Combined, these characteristics confirm this new class of miniferritin in the bacterial domain, delineate the similarities and differences between bacterial DPSL proteins and their archaeal homologs, allow corrected annotations for B. fragilis bfr and other dpsl genes within the bacterial domain, and suggest an evolutionary link within the ferritin superfamily that connects dodecameric DPS to the (bacterio)ferritin 24-mer. Bacteroides fragilis is a strict anaerobe that comprises approximately 1 to 2% of the normal intestinal flora in humans. While normally present as a commensal bacterium in this reducing environment, it is also the pathogenic anaerobe most frequently isolated from intra-abdominal infections, abscesses, and blood (27,57,58). Factors contributing to the pathogenesis of B. fragilis include resistance to oxidative stress and extreme aerotolerance, each of which is an important virulence factor for extraintestinal infections (61).A significant pathway for the production of reactive oxygen species (ROS) is dependent upon the presence of free Fe 2ϩ , where Fe 2ϩ and O 2 react to form the toxic superoxide anion (19, 37). Biologically, superoxide is then converted to H 2 O 2 and O 2 by the action of superoxide dismutase (69) or superoxide reductase (43). Then, via the Fenton reaction, the hydrogen peroxide may react with remaining Fe 2ϩ to produce the hydroxyl radical (HO·), the most toxic of all ROSs. Management of the intracellular iron pool is thus an important facet in the control of oxidative stress in virtually all organisms.One strategy for limiting the availability of free iron is adopted by ferritin, bacterioferritin, and DNA protection in nutrientstarved cells (DPS) protein (31,37,64). These members of...

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Section: Resultsmentioning

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Characterization of the Bacteroides fragilis bfr Gene Product Identifies a Bacterial DPS-Like Protein and Suggests Evolutionary Links in the Ferritin Superfamily

Gauss¹,

Reott²,

Rocha³

et al. 2011

Journal of Bacteriology

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“…31 Moreover, Dps from Mycobacterium smegmatis dissociates into lower order oligomers at low temperature (4 C). 32,33 These examples all involve changing solution conditions, and while they may be providing relevant information that can be extrapolated, ways to alter the oligomerization state in standard conditions with minimal perturbation are necessary for delivery and materials applications and for example, as controls to understand the role of oligomerization in DNA binding. The few mutants that have been discovered to alter the Dps oligomerization state involved deletions of relatively large subdomains that have been implicated in other roles such as making interactions with DNA or in iron transport.…”

Section: Discussionmentioning

confidence: 99%

Rational disruption of the oligomerization of the mini‐ferritin E. coli DPS through protein‐protein interface mutation

Zhang

Chee

et al. 2011

Protein Science

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DNA-binding protein from starved cells (DPS), a mini-ferritin capable of self-assembling into a 12-meric nano-cage, was chosen as the basis for an alanine-shaving mutagenesis study to investigate the importance of key amino acid residues, located at symmetry-related protein-protein interfaces, in controlling protein stability and self-assembly. Nine mutants were designed through simple inspection, synthesized, and subjected to transmission electron microscopy, circular dichroism, size exclusion chromatography, and ''virtual alanine scanning'' computational analysis. The data indicate that many of these residues may be hot spot residues. Most remarkably, two residues, R83 and R133, were observed to shift the oligomerization state to~50% dimer. Based on the hypothesis that these two residues constitute a ''hot strip,'' located at the ferritin-like threefold axis, the double mutant was generated which completely shuts down detectable formation of 12-mer in solution, favoring a cooperatively folded dimer. The fact that this effect logically builds upon the single mutants emphasizes that complex self-assembly has the potential to be manipulated rationally. This study should have an impact on the fundamental understanding of the assembly of DPS protein cages specifically and protein quaternary structure in general. In addition, as there is much interest in applying these and similar systems to the templation of nano-materials and drug delivery, the ability to control this ferritin's oligomerization state and stability could prove especially valuable.

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“…Using comparative modelling it was possible to demonstrate that Dps from Mycobacterium smegmatis could form a dodecamer structure similar to the Dps from Escherichia coli (Gupta et al, 2002). The intriguing properties related to protein stability, DNA binding property and protection was further revisited to consolidate the structure function attribute of this unique protein (Ceci et al, 2005). These studies put emphasis on the need of global proteome analysis not only for actively growing cells but also for cells under dormant conditions.…”

Section: The Vendetta In Light Of Protein Turnovermentioning

confidence: 99%

Mycobacterium tuberculosis: Dormancy, Persistence and Survival in the Light of Protein Synthesis

Kumar¹,

Sanyal²

2012

Understanding Tuberculosis - Deciphering the Secret Life of the Bacilli

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Reassessment of Protein Stability, DNA Binding, and Protection of Mycobacterium smegmatis Dps

Cited by 56 publications

References 32 publications

Characterization of the Bacteroides fragilis bfr Gene Product Identifies a Bacterial DPS-Like Protein and Suggests Evolutionary Links in the Ferritin Superfamily

Characterization of the Bacteroides fragilis bfr Gene Product Identifies a Bacterial DPS-Like Protein and Suggests Evolutionary Links in the Ferritin Superfamily

Rational disruption of the oligomerization of the mini‐ferritin E. coli DPS through protein‐protein interface mutation

Mycobacterium tuberculosis: Dormancy, Persistence and Survival in the Light of Protein Synthesis

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