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Structural and functional characterization of a frataxin from a thermophilic organism.

ABSTRACT: Frataxins form an interesting family of iron-binding proteins with an almost unique fold and are highly conserved from bacteria to primates. They have a pivotal role in iron-sulfur cluster biogenesis as regulators of the rates of cluster formation, as it is testified by the fact that frataxin absence is incompatible with life and reduced levels of the protein lead to the recessive neurodegenerative disease Friedreich's ataxia. Despite its importance, the structure of frataxin has been solved only from relatively few species. Here, we discuss the X-ray structure of frataxin from the thermophilic fungus Chaetomium thermophilum, and the characterization of its interactions and dynamics in solution. We show that this eukaryotic frataxin has an unusual variation in the classical frataxin fold: the last helix is shorter than in other frataxins which results in a less symmetrical and compact structure. The stability of this protein is comparable to that of human frataxin, currently the most stable among the frataxin orthologues. We also characterized the iron-binding mode of Ct frataxin and demonstrated that it binds it through a semiconserved negatively charged ridge on the first helix and beta-strand. Moreover, this frataxin is also able to bind the bacterial ortholog of the desulfurase, which is central in iron-sulfur cluster synthesis, and act as its inhibitor.

SUBMITTER: Rasheed M

PROVIDER: S-EPMC6506826 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Structural and functional characterization of a frataxin from a thermophilic organism.

Rasheed Masooma M Jamshidiha Mostafa M Puglisi Rita R Yan Robert R Cota Ernesto E Pastore Annalisa A

The FEBS journal 20190130 3

Frataxins form an interesting family of iron-binding proteins with an almost unique fold and are highly conserved from bacteria to primates. They have a pivotal role in iron-sulfur cluster biogenesis as regulators of the rates of cluster formation, as it is testified by the fact that frataxin absence is incompatible with life and reduced levels of the protein lead to the recessive neurodegenerative disease Friedreich's ataxia. Despite its importance, the structure of frataxin has been solved onl ...[more]

PMID: 30636112

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Project description:Proteases play an essential role in living organisms and represent one of the largest groups of industrial enzymes. The aim of this work was recombinant production and characterization of a newly identified thermostable protease 1147 from thermophilum indigenous Cohnella sp. A01. Phylogenetic tree analysis showed that protease 1147 is closely related to the cysteine proteases from DJ-1/ThiJ/PfpI superfamily, with the conserved catalytic tetrad. Structural prediction using MODELLER 9v7 indicated that protease 1147 has an overall α/β sandwich tertiary structure. The gene of protease 1147 was cloned and expressed in Escherichia coli (E. coli) BL21. The recombinant protease 1147 appeared as a homogenous band of 18 kDa in SDS-PAGE, which was verified by western blot and zymography. The recombinant protein was purified with a yield of approximately 88% in a single step using Ni-NTA affinity chromatography. Furthermore, a rapid one-step thermal shock procedure was successfully implemented to purify the protein with a yield of 73%. Using casein as the substrate, Km, and kcat, kcat/Km values of 13.72 mM, 3.143 × 10-3 (s-1), and 0.381 (M-1 S-1) were obtained, respectively. The maximum protease activity was detected at pH = 7 and 60°C with the inactivation rate constant (kin) of 2.10 × 10-3 (m-1), and half-life (t1/2) of 330.07 min. Protease 1147 exhibited excellent stability to organic solvent, metal ions, and 1% SDS. The protease activity was significantly enhanced by Tween 20 and Tween 80 and suppressed by cysteine protease specific inhibitors. Docking results and molecular dynamics (MD) simulation revealed that Tween 20 interacted with protease 1147 via hydrogen bonds and made the structure more stable. CD and fluorescence spectra indicated structural changes taking place at 100°C, very basic and acidic pH, and in the presence of Tween 20. These properties make this newly characterized protease a potential candidate for various biotechnological applications.

Dataset Information

Structural and functional characterization of a frataxin from a thermophilic organism.

Publications

Structural and functional characterization of a frataxin from a thermophilic organism.

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