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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 results mapped onto the crystal structure of E. coli HscB. Alanine substitution of L92, L96, or F153 (red) had the most detrimental effects on the affinity of HscB for apo-IscU, while alanine substitution at positions 87, 99, or 100 (green) resulted in smaller effects. Alanine substitution at positions 89, 93, 97, 103, 104, 152, 156, or 160 (dark grey) left the affinity of HscB for apo-IscU unchanged. This image was prepared with PyMol [30].
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Figure 4: ITC results mapped onto the crystal structure of E. coli HscB. Alanine substitution of L92, L96, or F153 (red) had the most detrimental effects on the affinity of HscB for apo-IscU, while alanine substitution at positions 87, 99, or 100 (green) resulted in smaller effects. Alanine substitution at positions 89, 93, 97, 103, 104, 152, 156, or 160 (dark grey) left the affinity of HscB for apo-IscU unchanged. This image was prepared with PyMol [30].

Mentions: The experiments described herein continue previous work from our laboratory on the HscB-IscU interaction. We individually replaced with alanine fourteen surface-exposed residues in the C-terminal domain of E. coli HscB, and evaluated their effects using ITC and NMR spectroscopy. Of the three highly conserved acidic residues (E97, E100, E104), only the E100A substitution perturbed the affinity of HscB for IscU (Figure 4). The resultant change in the free energy of binding (ΔΔGb ≅ 0.9 kcal/mol) was close to the experimentally determined value for a mutant with alanine substitutions at all three acidic positions, suggesting that the effects of the substitutions are additive and that the slightly decreased function of the triple mutant observed in our previous work [21] is caused predominantly by the E100A substitution.


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 results mapped onto the crystal structure of E. coli HscB. Alanine substitution of L92, L96, or F153 (red) had the most detrimental effects on the affinity of HscB for apo-IscU, while alanine substitution at positions 87, 99, or 100 (green) resulted in smaller effects. Alanine substitution at positions 89, 93, 97, 103, 104, 152, 156, or 160 (dark grey) left the affinity of HscB for apo-IscU unchanged. This image was prepared with PyMol [30].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: ITC results mapped onto the crystal structure of E. coli HscB. Alanine substitution of L92, L96, or F153 (red) had the most detrimental effects on the affinity of HscB for apo-IscU, while alanine substitution at positions 87, 99, or 100 (green) resulted in smaller effects. Alanine substitution at positions 89, 93, 97, 103, 104, 152, 156, or 160 (dark grey) left the affinity of HscB for apo-IscU unchanged. This image was prepared with PyMol [30].
Mentions: The experiments described herein continue previous work from our laboratory on the HscB-IscU interaction. We individually replaced with alanine fourteen surface-exposed residues in the C-terminal domain of E. coli HscB, and evaluated their effects using ITC and NMR spectroscopy. Of the three highly conserved acidic residues (E97, E100, E104), only the E100A substitution perturbed the affinity of HscB for IscU (Figure 4). The resultant change in the free energy of binding (ΔΔGb ≅ 0.9 kcal/mol) was close to the experimentally determined value for a mutant with alanine substitutions at all three acidic positions, suggesting that the effects of the substitutions are additive and that the slightly decreased function of the triple mutant observed in our previous work [21] is caused predominantly by the E100A substitution.

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