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. 2020 Sep:210:111162.
doi: 10.1016/j.jinorgbio.2020.111162. Epub 2020 Jun 23.

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

Sambuddha Banerjee  1 , Ryan J Garrigues  2 , Mina N Chanakira  3 , Jacob J Negron-Olivo  3 , Yasmene H Odeh  3 , Anne M Spuches  3 , R Martin Roop 2nd  2 , Joshua Edison Pitzer  2 , Daniel W Martin  2 , Saumya Dasgupta  4
Affiliations

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

Sambuddha Banerjee et al. J Inorg Biochem. 2020 Sep.

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.

Keywords: Brucella; Cu(2+)-binding mutant; Differential scanning calorimetry; FtrA; Homology model; Isothermal titration calorimetry.

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Figures

Figure 1.
Figure 1.
A schematic representation of the ferrous ion transporter, FtrABCD, from Brucella spp. which is a bacterial homolog of the ferroxidase-dependent eukaryotic Ftr1p-Fet3P system (39, 40, 45). The operons responsible for expressing the individual components of the FtrABCD system are presented at the top as blue arrows. It is hypothesized that Fe2+enters the periplasm through an unassigned outer membrane receptor where it is sequestered by the periplasmic FtrA in a Cu2+-dependent manner, like P19-type proteins. FtrB, the CupII-type multicopper ferroxidase catalyzes the conversion of this bound Fe2+to Fe3+ (shown by the arrows with electron, e-, flow), whereas FtrC, the Ftr1p homolog is proposed to transport this oxidized metal, and FtrD acts as the electron sink and resets the ferrous uptake system (39, 40, 45).
Figure 2.
Figure 2.
a) Homology modeled structure of FtrA (Template 3lzl with GMQE 0.8, QSQE 0.98, and sequence similarity of 0.48). The Cu2+-coordinating residues from chain A and chain B are indicated from this model (–48). b) Cartoon representation of the coordination of Cu2+in the FtrA model structure between chain A (His65, Glu67, and His118) and chain B (His151). The Cu2+ interacting amino acids are indicated in ball and stick model. c) Cartoon diagram of Cu coordination in FtrA showing the N-H…O interaction between H118 of chain A and Gly116 of chain A.
Figure 3.
Figure 3.
Representative ITC isotherms for Cu2+ titrations into solutions of wild-type FtrA at a) pH 6.3, [FtrA] = 20 μM, [Cu2+] in the syringe = 150 μM and b) pH 7.3, [FtrA] = 47 μM, [Cu2+] in the syringe = 350 μM. The upper panels show exothermic heat peaks per injection as a function of time, whereas the dots on the lower panel are the integrated heat data for each Cu2+ addition. The solid red line represents the best fit to the integrated heat data using independent one site binding model. All experiments were performed in 25 mM ACES buffer at 25 °C. See Table 2 for thermodynamic binding parameters.
Figure 4.
Figure 4.
Representative ITC isotherms for the Fe2+ mimic (Mn2+) titrations into solutions of wild-type as-isolated FtrA saturated with Cu2+, at a) pH 6.3, [FtrA] = 45 μM, [Mn2+] in the syringe = 550 μM and b) pH 7.3, [FtrA] = 58 μM, [Mn2+] in the syringe = 550 μM. The upper panels show exothermic heat peaks per injection as a function of time, whereas the dots on the lower panel are the integrated heat data for each Cu2+ addition. The solid red line represents the best fit to the integrated heat data using independent one site binding model. All experiments were performed in 25 mM ACES buffer at 25 °C. The inset in figure b) shows Mn2+ titration into wild-type FtrA without adding any Cu2+ ions, indicating no binding between the Fe2+ mimic and the wild-type protein under these experimental conditions.
Figure 5.
Figure 5.
Representative ITC isotherms for Cu2+ titrations into solutions of a) [H65A] =70 μM, [Cu2+] in the syringe = 600 μM and b) [E67A] = 75 μM, [Cu2+] in the syringe = 650 μM at pH 7.3. The upper panels show exothermic heat peaks per injection as a function of time, whereas the dots on the lower panel are the integrated heat data for each Cu2+ addition. The solid red line represents the best fit to the integrated heat data using independent one site binding model. All experiments were performed in 25 mM ACES buffer at 25 °C. See Table 2 for thermodynamic binding parameters.
Figure 6.
Figure 6.
Representative DSC thermograms for wild-type FtrA at a) pH 6.3 and b) pH 7.3 in 25 mM ACES buffer and indicating pH invariant folding for the wild-type protein. Temperature scans were done between 40–98 °C under 3 atmosphere pressure at a scan rate of 1°C/min. The solid thick lines represent the raw melting data, the dashed lines represent the model sum obtained by fitting the raw data using two Gaussian models, and the thin solid lines represent individual two-state model fits (Table 3).
Figure 7.
Figure 7.
Representative DSC thermograms for wild-type FtrA + 5X Cu2+ at a) pH 6.3 and b) pH 7.3 in 25 mM ACES buffer and indicating similar folding for the wild-type protein in the presence of excess Cu2+ at two pH tested. Temperature scans were done between 40–98 °C under 3 atmosphere pressure at a scan rate of 1°C/min. The solid thick lines represent the raw melting data, the dashed lines represent the model sum obtained by fitting the raw data using two Gaussian models, and the thin solid lines represent individual two-state model fits (Table 3).
Figure 8.
Figure 8.
Representative DSC thermograms for wild-type FtrA + 5X Cu2++ Mn2+ at a) pH 6.3 and b) pH 7.3 in 25 mM ACES buffer indicating similar folding stability of the wild-type protein in the presence of excess Mn2+ and Cu2+ at both tested pH’s. Temperature scans were done between 40–98 °C under 3 atmosphere pressure at a scan rate of 1°C/min. The solid thick lines represent the raw melting data, the dashed lines represent the model sum obtained by fitting the raw data using two Gaussian models, and the thin solid lines represent individual two-state model fits (Table 3).
Figure 9.
Figure 9.
Representative DSC thermograms for a) H151A and b) H118A mutants at pH 7.3 in 25 mM ACES buffer, showing the effect of mutation of the metal-binding mutant on the folding stability of the protein. All mutants tested in this work show reduced thermal stability compared to the wild-type data. Temperature scans were done between 40–98 °C under 3 atmosphere pressure at a scan rate of 1°C/min. The solid thick lines represent the raw melting data, the dashed lines represent the model sum obtained by fitting the raw data using Gaussian models, and the thin solid lines represent individual two-state model fits (Table 3).
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