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Three hydrophobic amino acids in Escherichia coli HscB make the greatest contribution to the stability of the HscB-IscU complex.

Füzéry AK, Oh JJ, Ta DT, Vickery LE, Markley JL - BMC Biochem. (2011)

Bottom Line: However, the individual contribution of each substitution to the observed effect remains to be determined as well as the possible involvement of other residues in the proposed binding site.Our results suggest that the triple alanine substitution at HscB positions 92, 96, and 153 will destabilize the HscB-IscU complex by ΔΔGb≅ 5.7 kcal/mol, equivalent to a ≅ 15000-fold reduction in the affinity of HscB for IscU.We propose that this triple mutant could provide a more definitive test of the functional importance of the HscB-IscU interaction in vivo than those used previously that yielded inconclusive results.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.

ABSTRACT

Background: General iron-sulfur cluster biosynthesis proceeds through assembly of a transient cluster on IscU followed by its transfer to a recipient apo-protein. The efficiency of the second step is increased by the presence of HscA and HscB, but the reason behind this is poorly understood. To shed light on the function of HscB, we began a study on the nature of its interaction with IscU. Our work suggested that the binding site of IscU is in the C-terminal domain of HscB, and two different triple alanine substitutions ([L92A, M93A, F153A] and [E97A, E100A, E104A]) involving predicted binding site residues had detrimental effects on this interaction. However, the individual contribution of each substitution to the observed effect remains to be determined as well as the possible involvement of other residues in the proposed binding site.

Results: In the work reported here, we used isothermal titration calorimetry to characterize the affinity of single alanine HscB mutants for IscU, and subsequently confirmed our results with nuclear magnetic resonance spectroscopy. Alanine substitutions of L92, L96, and F153 severely impaired the ability of HscB to form a complex with IscU; substitutions of R87, R99, and E100 had more modest effects; and substitutions of T89, M93, E97, D103, E104, R152, K156, and S160 had only minor or no detectable effects.

Conclusions: Our results show that the residues of HscB most important for strong interaction with IscU include three hydrophobic residues (L92, L96, and F153); in addition, we identified a number of other residues whose side chains contribute to a lesser extent to the interaction. Our results suggest that the triple alanine substitution at HscB positions 92, 96, and 153 will destabilize the HscB-IscU complex by ΔΔGb≅ 5.7 kcal/mol, equivalent to a ≅ 15000-fold reduction in the affinity of HscB for IscU. We propose that this triple mutant could provide a more definitive test of the functional importance of the HscB-IscU interaction in vivo than those used previously that yielded inconclusive results.

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ITC data for titration of wild-type or selected alanine-substituted forms of HscB with apo-IscU. Titrations were performed in 50 mM HEPES pH 7.5, 150 mM NaCl, 4 mM TCEP at 25 °C. The concentrations of reactants were 0.25-0.4 mM HscB and 2.5-4 mM apo-IscU. Additional File 4 contains similar data for the remaining alanine-substituted forms of HscB.
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Figure 1: ITC data for titration of wild-type or selected alanine-substituted forms of HscB with apo-IscU. Titrations were performed in 50 mM HEPES pH 7.5, 150 mM NaCl, 4 mM TCEP at 25 °C. The concentrations of reactants were 0.25-0.4 mM HscB and 2.5-4 mM apo-IscU. Additional File 4 contains similar data for the remaining alanine-substituted forms of HscB.

Mentions: To investigate whether any of the alanine substitutions perturbed the stability of the HscB-IscU complex, we used ITC to determine the binding affinity of each alanine mutant for apo-IscU (Table 1, Figure 1, and Additional File 4). Under the conditions of the experiment, wild-type HscB bound IscU with an affinity of 9 ± 2 μM which approximates the previously reported value of 13 μM [19]. Alanine substitutions at position 89, 93, 97, 103, 104, 152, 156, or 160 had no or only minor effects on the binding of HscB to IscU (Kd ≅ 4-17 μM). In contrast, substitutions R87A (Kd ≅ 36 μM), R99A (Kd ≅ 53 μM), and E100A (Kd ≅ 42 μM) decreased the affinity of HscB for IscU ≅ 4- to 6-fold. The most dramatic effects were observed for three hydrophobic residues in the center of the proposed IscU binding site. Substitutions L92A (Kd ≅ 200 μM), L96A (Kd ≅ 200 μM), and F153A (Kd ≅ 200 μM) decreased the affinity of HscB for IscU greater than 20-fold compared to that of wild-type protein.


Three hydrophobic amino acids in Escherichia coli HscB make the greatest contribution to the stability of the HscB-IscU complex.

Füzéry AK, Oh JJ, Ta DT, Vickery LE, Markley JL - BMC Biochem. (2011)

ITC data for titration of wild-type or selected alanine-substituted forms of HscB with apo-IscU. Titrations were performed in 50 mM HEPES pH 7.5, 150 mM NaCl, 4 mM TCEP at 25 °C. The concentrations of reactants were 0.25-0.4 mM HscB and 2.5-4 mM apo-IscU. Additional File 4 contains similar data for the remaining alanine-substituted forms of HscB.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040723&req=5

Figure 1: ITC data for titration of wild-type or selected alanine-substituted forms of HscB with apo-IscU. Titrations were performed in 50 mM HEPES pH 7.5, 150 mM NaCl, 4 mM TCEP at 25 °C. The concentrations of reactants were 0.25-0.4 mM HscB and 2.5-4 mM apo-IscU. Additional File 4 contains similar data for the remaining alanine-substituted forms of HscB.
Mentions: To investigate whether any of the alanine substitutions perturbed the stability of the HscB-IscU complex, we used ITC to determine the binding affinity of each alanine mutant for apo-IscU (Table 1, Figure 1, and Additional File 4). Under the conditions of the experiment, wild-type HscB bound IscU with an affinity of 9 ± 2 μM which approximates the previously reported value of 13 μM [19]. Alanine substitutions at position 89, 93, 97, 103, 104, 152, 156, or 160 had no or only minor effects on the binding of HscB to IscU (Kd ≅ 4-17 μM). In contrast, substitutions R87A (Kd ≅ 36 μM), R99A (Kd ≅ 53 μM), and E100A (Kd ≅ 42 μM) decreased the affinity of HscB for IscU ≅ 4- to 6-fold. The most dramatic effects were observed for three hydrophobic residues in the center of the proposed IscU binding site. Substitutions L92A (Kd ≅ 200 μM), L96A (Kd ≅ 200 μM), and F153A (Kd ≅ 200 μM) decreased the affinity of HscB for IscU greater than 20-fold compared to that of wild-type protein.

Bottom Line: However, the individual contribution of each substitution to the observed effect remains to be determined as well as the possible involvement of other residues in the proposed binding site.Our results suggest that the triple alanine substitution at HscB positions 92, 96, and 153 will destabilize the HscB-IscU complex by ΔΔGb≅ 5.7 kcal/mol, equivalent to a ≅ 15000-fold reduction in the affinity of HscB for IscU.We propose that this triple mutant could provide a more definitive test of the functional importance of the HscB-IscU interaction in vivo than those used previously that yielded inconclusive results.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.

ABSTRACT

Background: General iron-sulfur cluster biosynthesis proceeds through assembly of a transient cluster on IscU followed by its transfer to a recipient apo-protein. The efficiency of the second step is increased by the presence of HscA and HscB, but the reason behind this is poorly understood. To shed light on the function of HscB, we began a study on the nature of its interaction with IscU. Our work suggested that the binding site of IscU is in the C-terminal domain of HscB, and two different triple alanine substitutions ([L92A, M93A, F153A] and [E97A, E100A, E104A]) involving predicted binding site residues had detrimental effects on this interaction. However, the individual contribution of each substitution to the observed effect remains to be determined as well as the possible involvement of other residues in the proposed binding site.

Results: In the work reported here, we used isothermal titration calorimetry to characterize the affinity of single alanine HscB mutants for IscU, and subsequently confirmed our results with nuclear magnetic resonance spectroscopy. Alanine substitutions of L92, L96, and F153 severely impaired the ability of HscB to form a complex with IscU; substitutions of R87, R99, and E100 had more modest effects; and substitutions of T89, M93, E97, D103, E104, R152, K156, and S160 had only minor or no detectable effects.

Conclusions: Our results show that the residues of HscB most important for strong interaction with IscU include three hydrophobic residues (L92, L96, and F153); in addition, we identified a number of other residues whose side chains contribute to a lesser extent to the interaction. Our results suggest that the triple alanine substitution at HscB positions 92, 96, and 153 will destabilize the HscB-IscU complex by ΔΔGb≅ 5.7 kcal/mol, equivalent to a ≅ 15000-fold reduction in the affinity of HscB for IscU. We propose that this triple mutant could provide a more definitive test of the functional importance of the HscB-IscU interaction in vivo than those used previously that yielded inconclusive results.

Show MeSH
Related in: MedlinePlus