| Biological iron-sulfur cluster assembly in many organisms is mediated by the multi-component ISC system. A transient iron-sulfur cluster is first assembled on a scaffold protein, designated IscU, and is followed by transfer of the cluster to a recipient apo-protein. The efficiency of the second step is increased by the presence of two proteins, designated HscA and HscB, but the mechanistic details of this process are poorly understood. The specific aims of this thesis have been to characterize the solution structure of Escherichia coli HscB, and to identify amino acid residues in its sequence that are required for its interaction with IscU. NMR 1DHN and 1D CalphaHalpha residual dipolar couplings, 15N R1 and R2 values, and 1H-15N heteronuclear NOE data indicated that the solution structure of HscB consists of a series of alpha-helices connected by flexible loops, and that these elements are arranged in an L-shaped fold. NMR chemical shift perturbation data and a thorough sequence analysis of HscB homologues suggested an extensive binding site for apo-IscU on one face of the HscB C-domain. Triple alanine substitutions in a hydrophobic patch (L92, M93, L96, F153) or an acidic cluster (E97, E100, E104) within the binding site decreased the apparent affinity of HscB for apo-IscU, and also affected the ability of these proteins to synergistically stimulate the ATPase activity of HscA. Isothermal titration calorimetry indicated that single alanine substitutions of three binding site residues (L92, L96, and F153) destabilize the complex to a large extent while substitutions at other positions produce only smaller effects or no effects at all. These results will enable researchers to formulate more accurate models of the HscB-IscU complex, which is necessary for a complete understanding of iron-sulfur cluster biosynthesis. |
