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

Anna K Füzéry,Dennis T Ta,Jenny J Oh,Larry E Vickery,John L Markley

doi:10.1186/1471-2091-12-3

Abstract

BackgroundGeneral 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.ResultsIn 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.ConclusionsOur 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.

Highlights

General iron-sulfur cluster biosynthesis proceeds through assembly of a transient cluster on IscU followed by its transfer to a recipient apo-protein
Sequence analysis of HscB homologs identified a series of highly conserved residues (L92, M93, L96, E97, E100, E104, and F153 in E. coli HscB) that form an extensive, surfaceexposed patch on one face of the C-terminal domain and it was hypothesized that this region participates in functionally relevant protein-protein interactions [13]
The most dramatic effects were observed for three hydrophobic residues in the center of the proposed IscU binding site

Summary

Introduction

General iron-sulfur cluster biosynthesis proceeds through assembly of a transient cluster on IscU followed by its transfer to a recipient apo-protein. 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. Extensive biochemical and genetic studies [3,4,5,6,7,8,9] have shown that this process occurs through the assembly of a cluster on the scaffold protein IscU (Isu in yeast) followed by its transfer to a recipient apo-protein. Sequence analysis of HscB homologs identified a series of highly conserved residues (L92, M93, L96, E97, E100, E104, and F153 in E. coli HscB) that form an extensive, surfaceexposed patch on one face of the C-terminal domain and it was hypothesized that this region participates in functionally relevant protein-protein interactions [13]

Methods

Results

Discussion

Conclusion