Dataset Information

Investigating the roles of the conserved Cu²⁺-binding residues on Brucella FtrA in producing conformational stability and functionality.

ABSTRACT: Brucella is a zoonotic pathogen requiring iron for its survival and acquires this metal through the expression of several high-affinity uptake systems. Of these, the newly discovered ferrous iron transporter, FtrABCD, is proposed to take part in ferrous iron uptake. Sequence homology shows that, FtrA, the proposed periplasmic ferrous-binding component, is a P19-type protein (a periplasmic protein from C. jejuni which shows Cu²⁺ dependent iron affinity). Previous structural and biochemical studies on other P19 systems have established a Cu²⁺ dependent Mn²⁺ affinity as well as formation of homodimers for these systems. The Cu²⁺ coordinating amino acids from these proteins are conserved in Brucella FtrA, hinting towards similar properties. However, there has been no experimental evidence, till date, establishing metal affinities and the possibility of dimer formation by Brucella FtrA. Using wild-type FtrA and Cu²⁺-binding mutants (H65A, E67A, H118A, and H151A) we investigated the metal affinities, folding stabilities, dimer forming abilities, and the molecular basis of the Cu²⁺ dependence for this P19-type protein employing homology modeling, analytical gel filtration, calorimetric, and spectroscopic methods. The data reported here confirm a Cu²⁺-dependent, low-μM Mn²⁺ (Fe²⁺ mimic) affinity for the wild-type FtrA. In addition, our data clearly show the loss of Mn²⁺ affinity, and the formation of less stable protein conformations as a result of mutating these conserved Cu²⁺-binding residues, indicating the important roles these residues play in producing a native and functional fold of Brucella FtrA.

SUBMITTER: Banerjee S

PROVIDER: S-EPMC7484176 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Investigating the roles of the conserved Cu<sup>2+</sup>-binding residues on Brucella FtrA in producing conformational stability and functionality.

Banerjee Sambuddha S Garrigues Ryan J RJ Chanakira Mina N MN Negron-Olivo Jacob J JJ Odeh Yasmene H YH Spuches Anne M AM Martin Roop R R Pitzer Joshua Edison JE Martin Daniel W DW Dasgupta Saumya S

Journal of inorganic biochemistry 20200623

Brucella is a zoonotic pathogen requiring iron for its survival and acquires this metal through the expression of several high-affinity uptake systems. Of these, the newly discovered ferrous iron transporter, FtrABCD, is proposed to take part in ferrous iron uptake. Sequence homology shows that, FtrA, the proposed periplasmic ferrous-binding component, is a P19-type protein (a periplasmic protein from C. jejuni which shows Cu<sup>2+</sup> dependent iron affinity). Previous structural and biochem ...[more]

PMID: 32623149

Similar Datasets

Project description:Escherichia coli cysteine desulfurase (CD), IscS, modifies basal metabolism by transferring sulphur (S) from L-cysteine to numerous cellular pathways, whereas NFS1, a human CD, is active only in the formation of the [Acp]2:[ISD11]2:[NFS1]2 complex. Despite the accumulation of red-coloured IscS in E. coli cells as a result of the deficiency of accessible iron, as revealed in our previous studies, the mechanism of the potential enzymatic reaction remains unclear. In this study, the N-terminus of IscS was fused with the C-terminus of NFS1, which was reported to be almost fully active as IscS and exhibits a pyridoxal 5'-phosphate (PLP) absorption peak at 395 nm. Moreover, SUMO-EH-IscS exhibited significant growth recovery and NADH-dehydrogenase I activity in the iscS mutant cells. Furthermore, through in vitro and in vivo experiments combined with high-performance liquid chromatography and ultra-performance liquid chromatography-tandem mass spectrometry, it was shown that the new absorption peaks of the IscS H104Q, IscS Q183E, IscS K206A, and IscS K206A&C328S variants at 340 and 350 nm may correspond to the enzyme reaction intermediates, Cys-ketimine and Cys-aldimine, respectively. However, after mutation of the conserved active-site residues, additional absorption peaks at 420 and 430 nm were associated with PLP migration in the active-site pocket. Additionally, the corresponding absorption peaks of Cys-quinonoid, Ala-ketimine, and Ala-aldimine intermediates in IscS were 510, 325, and 345 nm, respectively, as determined by site-directed mutagenesis and substrate/product-binding analyses during the CD reaction process. Notably, red IscS formed in vitro by incubating IscS variants (Q183E and K206A) with excess L-alanine and sulphide under aerobic conditions produced an absorption peak similar to the wild-type IscS, at 510 nm. Interestingly, site-directed mutation of IscS with hydrogen bonds to PLP at Asp180 and Gln183 resulted in a loss of enzymatic activity followed by an absorption peak consistent with NFS1 (420 nm). Furthermore, mutations at Asp180 or Lys206 inhibited the reaction of IscS in vitro with L-cysteine (substrate) and L-alanine (product). These results suggest that the conserved active site residues (His104, Asp180, and Gln183) and their hydrogen bond with PLP in the N-terminus of IscS play a key role in determining whether the L-cysteine substrate can enter the active-site pocket and regulate the enzymatic reaction process. Therefore, our findings provide a framework for evaluating the roles of conserved active-site residues, motifs, and domains in CDs.

Dataset Information

Investigating the roles of the conserved Cu²⁺-binding residues on Brucella FtrA in producing conformational stability and functionality.

Publications

Investigating the roles of the conserved Cu<sup>2+</sup>-binding residues on Brucella FtrA in producing conformational stability and functionality.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Investigating the roles of the conserved Cu2+-binding residues on Brucella FtrA in producing conformational stability and functionality.

Publications

Investigating the roles of the conserved Cu<sup>2+</sup>-binding residues on Brucella FtrA in producing conformational stability and functionality.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Investigating the roles of the conserved Cu²⁺-binding residues on Brucella FtrA in producing conformational stability and functionality.