Metal-Linked Dimerization in the Iron-Dependent Regulator from <i>Mycobacterium tuberculosis</i>^†

The iron-dependent regulator (IdeR) is a 230-amino acid transcriptional repressor that regulates iron homeostasis, oxidative stress response and virulence in Mycobacterium tuberculosis. The natural ligand for IdeR is Fe(II), but Ni(II), Co(II), Cd(II), Mn(II), and Zn(II) also bind to and activate the protein in vitro. Protein activation by metal is a complex process involving metal-induced folding of the N-terminal domain, changes in the interaction between the N- and C-terminal domains, and the formation of homodimers. Here, we investigate the energetics of dimerization and metal binding in IdeR. The dimerization energetics were determined as a function of metal binding using equilibrium analytical ultracentrifugation. The equilibrium dimer dissociation constant of apo-IdeR was 4.0 μM at 20 °C. The dissociation constant decreased to 0.5 μM in the presence of one equivalent of Ni(II)Cl2 and decreased further (Kd ≪ 50 nM) in the presence of excess Ni(II). IdeR contains two tryptophan residues. The addition of Ni(II) induced changes in fluorescence intensity and emission maximum of the tryptophan residues that strongly depended on protein concentration. At low IdeR concentration, fluorescence was enhanced at low metal-to-protein ratios but was quenched at high metal-to-protein ratios. At high IdeR concentration, metal binding resulted only in fluorescence quenching. The fluorescence enhancement at low protein concentrations was buffer-dependent and required the presence of both tryptophans. Metal binding affinity was measured quantitatively using equilibrium dialysis. The results showed strongly positive cooperative binding of three equivalents of metal per monomer with an average apparent dissociation constant of 2.2 ± 0.3 μM and a Hill coefficient of 2. Metal binding was not cooperative in an IdeR variant that showed reduced affinity for dimer formation. The results of this study establish the positive cooperative nature of metal binding by IdeR and suggest that dimerization is a major contributor to cooperative binding. The strong coupling between metal binding and dimerization places specific constraints on the activation mechanism

Structure of the Carboxy-Terminal Fragment of the Apo-Biotin Carboxyl Carrier Subunit of <i>Escherichia coli</i> Acetyl-CoA Carboxylase^†

The biotin carboxyl carrier protein (BCCP) is a subunit of acetyl-CoA carboxylase, a biotin-dependent enzyme that catalyzes the first committed step of fatty acid biosynthesis. In its functional cycle the biotin carboxyl carrier protein engages in heterologous protein−protein interactions with three distinct partners, depending on its state of posttranslational modification. Apo-BCCP interacts specifically with the biotin holoenzyme synthetase, BirA, which results in the posttranslational attachment of biotin to an essential lysine residue on BCCP. Holo-BCCP then interacts with the biotin carboxylase subunit, which leads to the addition of the carboxylate group of bicarbonate to biotin. Finally, the carboxybiotinylated form of BCCP interacts with transcarboxylase in the conversion of acetyl-CoA to malonyl-CoA. The determinants of protein−protein interaction specificity in this system are unknown. One hypothesis is that posttranslational modification of BCCP may result in conformational changes that regulate specific protein−protein interactions. To test this hypothesis, we have determined the NMR solution structure of the unbiotinylated form of an 87 residue C-terminal domain fragment of BCCP (apoBCCP87) from Escherichia coli acetyl-CoA carboxylase and compared this structure with the high-resolution structure of the biotinylated form that was recently solved by X-ray crystallographic techniques. Although the overall folding of the two proteins is highly similar, small structural differences are apparent for residues of the biotin-binding loop that may be important for mediating specific protein−protein interactions