scholarly journals Conservation of the structural and functional architecture of encapsulated ferritins in bacteria and archaea

2019 ◽  
Vol 476 (6) ◽  
pp. 975-989 ◽  
Author(s):  
Didi He ◽  
Cecilia Piergentili ◽  
Jennifer Ross ◽  
Emma Tarrant ◽  
Laura R. Tuck ◽  
...  
Keyword(s):  
Metal Binding ◽  
Rhodospirillum Rubrum ◽  
Quaternary Structure ◽  
Pyrococcus Furiosus ◽  
Large Family ◽  
Native Mass Spectrometry ◽  
Wide Distribution ◽  
Iron Store ◽  
Unusual Structure ◽  
Ferroxidase Activity

Abstract Ferritins are a large family of intracellular proteins that protect the cell from oxidative stress by catalytically converting Fe(II) into less toxic Fe(III) and storing iron minerals within their core. Encapsulated ferritins (EncFtn) are a sub-family of ferritin-like proteins, which are widely distributed in all bacterial and archaeal phyla. The recently characterized Rhodospirillum rubrum EncFtn displays an unusual structure when compared with classical ferritins, with an open decameric structure that is enzymatically active, but unable to store iron. This EncFtn must be associated with an encapsulin nanocage in order to act as an iron store. Given the wide distribution of the EncFtn family in organisms with diverse environmental niches, a question arises as to whether this unusual structure is conserved across the family. Here, we characterize EncFtn proteins from the halophile Haliangium ochraceum and the thermophile Pyrococcus furiosus, which show the conserved annular pentamer of dimers topology. Key structural differences are apparent between the homologues, particularly in the centre of the ring and the secondary metal-binding site, which is not conserved across the homologues. Solution and native mass spectrometry analyses highlight that the stability of the protein quaternary structure differs between EncFtn proteins from different species. The ferroxidase activity of EncFtn proteins was confirmed, and we show that while the quaternary structure around the ferroxidase centre is distinct from classical ferritins, the ferroxidase activity is still inhibited by Zn(II). Our results highlight the common structural organization and activity of EncFtn proteins, despite diverse host environments and contexts within encapsulins.

scholarly journals Conservation of the structural and functional architecture of encapsulated ferritins in bacteria and archaea

2018 ◽  
Author(s):  
Didi He ◽  
Cecilia Piergentili ◽  
Jennifer Ross ◽  
Emma Tarrant ◽  
Laura R. Tuck ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Rhodospirillum Rubrum ◽  
Quaternary Structure ◽  
Pyrococcus Furiosus ◽  
Essential Element ◽  
Wide Distribution ◽  
High Reactivity ◽  
Iron Store ◽  
Fenton Chemistry ◽  
Ferroxidase Activity

ABSTRACTIron is an essential element for many biological processes; however, due to its high reactivity iron can also be very toxic, producing reactive oxygen species through Fenton chemistry. Ferritins protect the cell from oxidative stress by catalytically converting Fe(II) into less toxic Fe(III) and storing the resulting iron minerals within their core. Encapsulated ferritins (EncFtn) are a sub-family of ferritin-like proteins, which are widely distributed in all bacterial and archaeal phyla. We recently characterised the Rhodospirillum rubrum EncFtn, showing that although enzymatically active, due to its open structure it requires the association with an encapsulin nanocage in order to act as an iron store. Given the wide distribution of the EncFtn family in organisms with diverse environmental niches, a question arises as to whether the structure and catalytic activity is conserved across the family. Here we structurally characterise two EncFtn members from the halophile Haliangium ochraceum and the thermophile Pyrococcus furiosus, which show the same distinct annular decamer topology observed in R. rubrum EncFtn, with the ferroxidase centre (FOC) formed between one of the dimer interfaces. Solution and native mass spectrometr analyses show that the stability of the protein quaternary structure differs between EncFtn proteins from different species. The catalytic role of the EncFtn proteins was confirmed by biochemical assays, and we show that Zn(II) ions inhibit the ferroxidase activity of the EncFtn proteins to varying degrees. Our results represent a further step in the characterisation of the recently discovered EncFtn ferritin-like sub-family, indicating a common structural organisation and catalytic activity, despite diverse host environments.

scholarly journals Dissecting the structural and functional roles of a vicinal iron-binding site in encapsulated ferritins

2019 ◽  
Author(s):  
Cecilia Piergentili ◽  
Jennifer Ross ◽  
Didi He ◽  
Kelly J. Gallagher ◽  
Will A. Stanley ◽  
...  
Keyword(s):  
Binding Site ◽  
Metal Binding ◽  
Rhodospirillum Rubrum ◽  
Catalytic Mechanism ◽  
Iron Oxidation ◽  
Functional Study ◽  
Secondary Site ◽  
Iron Store ◽  
Entry Site ◽  
Ferroxidase Center

AbstractEncapsulated ferritins belong to the universally distributed ferritin superfamily, which function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question, due to differences in organization of the ferroxidase catalytic site and secondary metal binding sites vicinal to this. We have previously identified a metal binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this proposed iron entry site by means of enzymatic assays, mass-spectrometry, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, while enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site.

scholarly journals Dissecting the structural and functional roles of a putative metal entry site in encapsulated ferritins

2020 ◽  
Vol 295 (46) ◽  
pp. 15511-15526
Author(s):  
Cecilia Piergentili ◽  
Jennifer Ross ◽  
Didi He ◽  
Kelly J. Gallagher ◽  
Will A. Stanley ◽  
...  
Keyword(s):  
Metal Binding ◽  
Rhodospirillum Rubrum ◽  
Catalytic Mechanism ◽  
Iron Oxidation ◽  
Functional Study ◽  
Secondary Site ◽  
Iron Store ◽  
Entry Site ◽  
X Ray Crystallography ◽  
Ferroxidase Center

Encapsulated ferritins belong to the universally distributed ferritin superfamily, whose members function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question because of the differences in organization of the ferroxidase catalytic site and neighboring secondary metal-binding sites. We have previously identified a putative metal-binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this putative iron-entry site by means of enzymatic assays, MS, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, although enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site. Our study demonstrates a novel molecular mechanism by which substrate flux to the ferroxidase center is controlled, potentially to ensure that iron oxidation is productively coupled to mineralization.

scholarly journals Metal ions and redox balance regulate distinct amyloid-like aggregation pathways of GAPR-1

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jie Sheng ◽  
Nick K. Olrichs ◽  
Willie J. Geerts ◽  
Dora V. Kaloyanova ◽  
J. Bernd Helms
Keyword(s):  
Metal Ions ◽  
Metal Binding ◽  
Molecular Mechanisms ◽  
Quaternary Structure ◽  
Copper Ions ◽  
Redox Homeostasis ◽  
Secretory Proteins ◽  
Pathogenesis Related Protein ◽  
Pathogenesis Related ◽  
Induced Aggregation

Abstract Members of the CAP superfamily (Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-Related 1 proteins) are characterized by the presence of a structurally conserved CAP domain. The common structure-function relationship of this domain is still poorly understood. In this study, we unravel specific molecular mechanisms modulating the quaternary structure of the mammalian CAP protein GAPR-1 (Golgi-Associated plant Pathogenesis-Related protein 1). Copper ions are shown to induce a distinct amyloid-like aggregation pathway of GAPR-1 in the presence of heparin. This involves an immediate shift from native multimers to monomers which are prone to form amyloid-like fibrils. The Cu2+-induced aggregation pathway is independent of a conserved metal-binding site and involves the formation of disulfide bonds during the nucleation process. The elongation process occurs independently of the presence of Cu2+ ions, and amyloid-like aggregation can proceed under oxidative conditions. In contrast, the Zn2+-dependent aggregation pathway was found to be independent of cysteines and was reversible upon removal of Zn2+ ions. Together, our results provide insight into the regulation of the quaternary structure of GAPR-1 by metal ions and redox homeostasis with potential implications for regulatory mechanisms of other CAP proteins.

scholarly journals Stoichiometry of Nucleotide Binding to Proteasome AAA+ ATPase Hexamer Established by Native Mass Spectrometry

2020 ◽  
Vol 19 (12) ◽  
pp. 1997-2014
Author(s):  
Yadong Yu ◽  
Haichuan Liu ◽  
Zanlin Yu ◽  
H. Ewa Witkowska ◽  
Yifan Cheng
Keyword(s):  
Protein Unfolding ◽  
Atp Hydrolysis ◽  
Mass Measurement ◽  
Site Occupancy ◽  
Internal Standard ◽  
Large Family ◽  
Nucleotide Binding ◽  
Native Mass Spectrometry ◽  
Aaa Atpase ◽  
Aaa Atpases

AAA+ ATPases constitute a large family of proteins that are involved in a plethora of cellular processes including DNA disassembly, protein degradation and protein complex disassembly. They typically form a hexametric ring-shaped structure with six subunits in a (pseudo) 6-fold symmetry. In a subset of AAA+ ATPases that facilitate protein unfolding and degradation, six subunits cooperate to translocate protein substrates through a central pore in the ring. The number and type of nucleotides in an AAA+ ATPase hexamer is inherently linked to the mechanism that underlies cooperation among subunits and couples ATP hydrolysis with substrate translocation. We conducted a native MS study of a monodispersed form of PAN, an archaeal proteasome AAA+ ATPase, to determine the number of nucleotides bound to each hexamer of the WT protein. We utilized ADP and its analogs (TNP-ADP and mant-ADP), and a nonhydrolyzable ATP analog (AMP-PNP) to study nucleotide site occupancy within the PAN hexamer in ADP- and ATP-binding states, respectively. Throughout all experiments we used a Walker A mutant (PANK217A) that is impaired in nucleotide binding as an internal standard to mitigate the effects of residual solvation on mass measurement accuracy and to serve as a reference protein to control for nonspecific nucleotide binding. This approach led to the unambiguous finding that a WT PAN hexamer carried – from expression host – six tightly bound ADP molecules that could be exchanged for ADP and ATP analogs. Although the Walker A mutant did not bind ADP analogs, it did bind AMP-PNP, albeit at multiple stoichiometries. We observed variable levels of hexamer dissociation and an appearance of multimeric species with the over-charged molecular ion distributions across repeated experiments. We posit that these phenomena originated during ESI process at the final stages of ESI droplet evolution.

Purification and Mn2+ activation of Rhodospirillum rubrum nitrogenase activating enzyme

1982 ◽  
Vol 152 (2) ◽  
pp. 714-721
Author(s):  
J W Gotto ◽  
D C Yoch
Keyword(s):  
Metal Binding ◽  
Metal Ion ◽  
Rhodospirillum Rubrum ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Maximum Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Fe Protein ◽  
Single Polypeptide ◽  
Sparing Effect

The Fe protein activating enzyme for Rhodospirillum rubrum nitrogenase was purified to approximately 90% homogeneity, using DE52-cellulose chromatography and sucrose density gradient centrifugation. Activating enzyme consists of a single polypeptide of molecular weight approximately 24,000. ATP was required for catalytic activity, but was relatively ineffective in the absence of Mg2+. When the concentration of MgATP2- was held in excess, there was an additional requirement for a free divalent metal ion (Mn2+) for enzyme activity. Kinetic experiments showed that the presence of Mg2+ influenced the apparent binding of Mn2+ by the enzyme, resulting in a lowering of the concentration of Mn2+ required to give half-maximum activity (K alpha) as the free Mg2+ concentration was increased. A low concentration of Mn2+ had a sparing effect on the requirement for free Mg2+. There is apparently a single metal-binding site on activating enzyme which preferentially binds Mn2+ as a positive effector, and free Mg2+ can compete for this site.

Native mass spectrometry-based metabolomics identifies metal-binding compounds

2021 ◽  
Author(s):  
Allegra T. Aron ◽  
Daniel Petras ◽  
Robin Schmid ◽  
Julia M. Gauglitz ◽  
Isabell Büttel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Metal Binding ◽  
Native Mass Spectrometry

scholarly journals Chemical modifications of metallothionein. Preparation and characterization of polymers

1984 ◽  
Vol 221 (3) ◽  
pp. 569-575 ◽  
Author(s):  
D M Templeton ◽  
M G Cherian
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Quaternary Structure ◽  
Biological Significance ◽  
Cross Linking ◽  
Metal Binding Sites ◽  
Polymeric Species ◽  
Lysine Residues ◽  
Dimethyl Suberimidate ◽  
High Yields

Reaction of rat liver metallothionein-II with two bifunctional cross-linking reagents, glutaraldehyde and dimethyl suberimidate, produces high yields of polymeric species. It is argued that cross-linking is trapping preformed aggregates of the protein, which therefore represent a stabilized quaternary structure of metallothionein. The two polymeric species differ in a number of respects. With dimethyl suberimidate, the polymer retains all metal-binding sites of the monomer, and has an unaltered isoelectric point. Reaction with glutaraldehyde causes loss of one or two Cd2+/Zn2+-binding sites and elevates the pI. Both species are nearly spherical aggregates, in contrast with the highly asymmetrical metallothionein. Both polymers are linked through lysine residues, and the thiol groups remain reduced. The biological significance of these aggregates is discussed.

Sign in / Sign up

Export Citation Format

Share Document