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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 [U-15N]-labeled HscB in the presence of a six-fold molar excess of apo-IscU. Combined chemical shift changes (ΔδHNIscU) are plotted for wild-type HscB (black), HscB(D103A) (yellow), HscB(E100A) (green), and HscB(L96A) (red). ΔδHNIscU values are reported as the combination of changes in the proton (ΔδHIscU) 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. Residues whose peaks disappear in the presence of apo-IscU for at least one form of HscB are marked with an asterisk and are also listed in Additional Files 5 to 8. Values are not shown for residues that have an unassigned 1HN-15N cross-peak in free HscB, that lack an observable 1H-15N cross-peak in free and/or bound HscB, have an overlapped 1H-15N cross-peak in free and/or bound HscB, or are prolines (P10, P33, and P64). The diagram at the top of each panel shows the location of secondary structural elements in E. coli HscB.
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Figure 3: NMR data for [U-15N]-labeled HscB in the presence of a six-fold molar excess of apo-IscU. Combined chemical shift changes (ΔδHNIscU) are plotted for wild-type HscB (black), HscB(D103A) (yellow), HscB(E100A) (green), and HscB(L96A) (red). ΔδHNIscU values are reported as the combination of changes in the proton (ΔδHIscU) 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. Residues whose peaks disappear in the presence of apo-IscU for at least one form of HscB are marked with an asterisk and are also listed in Additional Files 5 to 8. Values are not shown for residues that have an unassigned 1HN-15N cross-peak in free HscB, that lack an observable 1H-15N cross-peak in free and/or bound HscB, have an overlapped 1H-15N cross-peak in free and/or bound HscB, or are prolines (P10, P33, and P64). The diagram at the top of each panel shows the location of secondary structural elements in E. coli HscB.

Mentions: Figure 3 shows the combined chemical shift changes observed for all residues of wild-type HscB and of the three single alanine HscB mutants in the presence of a six-fold molar excess of apo-IscU. The pattern of chemical shift changes parallels that observed for F77, V133, and E166: residues of HscB(D103A) behave similarly to wild-type HscB (yellow versus black bars), residues of HscB(E100A) show changes that are ≅ 10-25% smaller than for wild-type HscB (green versus black bars), and residues of HscB(L96A) show changes that are over 50% smaller than for wild-type HscB (red versus black bars). Therefore, these results also support the conclusion that the D103A substitution has the smallest effect on the affinity of HscB for IscU whereas the L96A substitution has the largest effect. Taken together, the NMR results are fully consistent with the ITC data and confirm that alanine substitutions at different positions within the proposed binding site affect the affinity of HscB for IscU to different degrees.


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 [U-15N]-labeled HscB in the presence of a six-fold molar excess of apo-IscU. Combined chemical shift changes (ΔδHNIscU) are plotted for wild-type HscB (black), HscB(D103A) (yellow), HscB(E100A) (green), and HscB(L96A) (red). ΔδHNIscU values are reported as the combination of changes in the proton (ΔδHIscU) 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. Residues whose peaks disappear in the presence of apo-IscU for at least one form of HscB are marked with an asterisk and are also listed in Additional Files 5 to 8. Values are not shown for residues that have an unassigned 1HN-15N cross-peak in free HscB, that lack an observable 1H-15N cross-peak in free and/or bound HscB, have an overlapped 1H-15N cross-peak in free and/or bound HscB, or are prolines (P10, P33, and P64). The diagram at the top of each panel shows the location of secondary structural elements in E. coli HscB.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 3: NMR data for [U-15N]-labeled HscB in the presence of a six-fold molar excess of apo-IscU. Combined chemical shift changes (ΔδHNIscU) are plotted for wild-type HscB (black), HscB(D103A) (yellow), HscB(E100A) (green), and HscB(L96A) (red). ΔδHNIscU values are reported as the combination of changes in the proton (ΔδHIscU) 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. Residues whose peaks disappear in the presence of apo-IscU for at least one form of HscB are marked with an asterisk and are also listed in Additional Files 5 to 8. Values are not shown for residues that have an unassigned 1HN-15N cross-peak in free HscB, that lack an observable 1H-15N cross-peak in free and/or bound HscB, have an overlapped 1H-15N cross-peak in free and/or bound HscB, or are prolines (P10, P33, and P64). The diagram at the top of each panel shows the location of secondary structural elements in E. coli HscB.
Mentions: Figure 3 shows the combined chemical shift changes observed for all residues of wild-type HscB and of the three single alanine HscB mutants in the presence of a six-fold molar excess of apo-IscU. The pattern of chemical shift changes parallels that observed for F77, V133, and E166: residues of HscB(D103A) behave similarly to wild-type HscB (yellow versus black bars), residues of HscB(E100A) show changes that are ≅ 10-25% smaller than for wild-type HscB (green versus black bars), and residues of HscB(L96A) show changes that are over 50% smaller than for wild-type HscB (red versus black bars). Therefore, these results also support the conclusion that the D103A substitution has the smallest effect on the affinity of HscB for IscU whereas the L96A substitution has the largest effect. Taken together, the NMR results are fully consistent with the ITC data and confirm that alanine substitutions at different positions within the proposed binding site affect the affinity of HscB for IscU to different degrees.

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