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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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NMR data for the titration of [U-15N]-labeled HscB with apo-IscU. Combined chemical shift changes (ΔδHNIscU) of selected residues are plotted as a function of IscU/HscB molar ratio for wild-type HscB (black diamonds), HscB(D103A) (yellow circles), HscB(E100A) (green triangles), and HscB(L96A) (red squares). ΔδHNIscU values are reported as the combination of changes in the proton (HIscU) and nitrogen (ΔδNIscU) dimensions according to ΔδHNIscU = [(ΔδHIscU)2 + ΔδNIscU/6)2]1/2 [29]. ΔδHIscU and ΔδNIscU are calculated relative to the free form of each protein.
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Figure 2: NMR data for the titration of [U-15N]-labeled HscB with apo-IscU. Combined chemical shift changes (ΔδHNIscU) of selected residues are plotted as a function of IscU/HscB molar ratio for wild-type HscB (black diamonds), HscB(D103A) (yellow circles), HscB(E100A) (green triangles), and HscB(L96A) (red squares). ΔδHNIscU values are reported as the combination of changes in the proton (HIscU) and nitrogen (ΔδNIscU) dimensions according to ΔδHNIscU = [(ΔδHIscU)2 + ΔδNIscU/6)2]1/2 [29]. ΔδHIscU and ΔδNIscU are calculated relative to the free form of each protein.

Mentions: The 15N-HSQC spectral series for each mutant was first examined to identify all peaks showing chemical shift changes during the titration; subsequently, the observed chemical shift changes were plotted as a function of IscU/HscB molar ratio, and the results were compared to those obtained for wild-type HscB. Figure 2 shows examples of such a comparison for three well resolved peaks in the HSQC spectral series: F77, V133, and E166. The behaviour of F77 is representative of the majority of peaks that experienced large chemical shift changes, while the behaviours of V133 and E166 are representative of the majority of peaks that experienced moderate and small chemical shift changes, respectively.


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)

NMR data for the titration of [U-15N]-labeled HscB with apo-IscU. Combined chemical shift changes (ΔδHNIscU) of selected residues are plotted as a function of IscU/HscB molar ratio for wild-type HscB (black diamonds), HscB(D103A) (yellow circles), HscB(E100A) (green triangles), and HscB(L96A) (red squares). ΔδHNIscU values are reported as the combination of changes in the proton (HIscU) and nitrogen (ΔδNIscU) dimensions according to ΔδHNIscU = [(ΔδHIscU)2 + ΔδNIscU/6)2]1/2 [29]. ΔδHIscU and ΔδNIscU are calculated relative to the free form of each protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: NMR data for the titration of [U-15N]-labeled HscB with apo-IscU. Combined chemical shift changes (ΔδHNIscU) of selected residues are plotted as a function of IscU/HscB molar ratio for wild-type HscB (black diamonds), HscB(D103A) (yellow circles), HscB(E100A) (green triangles), and HscB(L96A) (red squares). ΔδHNIscU values are reported as the combination of changes in the proton (HIscU) and nitrogen (ΔδNIscU) dimensions according to ΔδHNIscU = [(ΔδHIscU)2 + ΔδNIscU/6)2]1/2 [29]. ΔδHIscU and ΔδNIscU are calculated relative to the free form of each protein.
Mentions: The 15N-HSQC spectral series for each mutant was first examined to identify all peaks showing chemical shift changes during the titration; subsequently, the observed chemical shift changes were plotted as a function of IscU/HscB molar ratio, and the results were compared to those obtained for wild-type HscB. Figure 2 shows examples of such a comparison for three well resolved peaks in the HSQC spectral series: F77, V133, and E166. The behaviour of F77 is representative of the majority of peaks that experienced large chemical shift changes, while the behaviours of V133 and E166 are representative of the majority of peaks that experienced moderate and small chemical shift changes, respectively.

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