The major function of human transferrin is to deliver iron from the bloodstream to actively dividing cells. Upon iron release, the protein changes its conformation from 'closed' to 'open'. Extensive studies in vitro indicate that iron release from transferrin is very complex and involves many factors, including pH, the chelator used, an anion effect, temperature, receptor binding and intra-lobe interactions. Our earlier work [He, Mason and Woodworth (1997) Biochem. J. 328, 439-445] using the isolated transferrin N-lobe (recombinant N-lobe of human transferrin comprising residues 1-337; hTF/2N) has shown that anions and pH modulate iron release from hTF/2N in an interdependent manner: chloride retards iron release at neutral pH, but accelerates the reaction at acidic pH. The present study supports this idea and further details the nature of the dual effect of chloride: the anion effect on iron release is closely related to the strength of anion binding to the apoprotein. The negative effect seems to originate from competition between chloride and the chelator for an anion-binding site(s) near the metal centre. With decreasing pH, the strength of anion binding to hTF/2N increases linearly, decreasing the contribution of competition with the chelator. In the meantime, the 'open' or 'loose' conformation of hTF/2N, induced by the protonation of critical residues such as the Lys-206/Lys-296 pair at low pH, enables chloride to enter the cleft and bind to exposed side chains, thereby promoting cleft opening and synergistically allowing removal of iron by the chelator, leading to a positive anion effect. Disabling one or more of the primary anion-binding residues, namely Arg-124, Lys-206 and Lys-296, substantially decreases the anion-binding ability of the resulting mutant proteins. In these cases, the competition for the remaining binding residue(s) is increased, leading to a negative chloride effect or, at most, a very small positive effect, even at low pH.

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Human serum transferrin N-lobe (hTF\2N) contains three conserved tryptophan residues, Trp), Trp"#) and Trp#'%, located in three different environments. The present report addresses the different contributions of the three tryptophan residues to the UV-visible, fluorescence and NMR spectra of hTF\2N and the effect of the mutations at each tryptophan residue on the iron-binding properties of the protein. Trp) resides in a hydrophobic box containing a cluster of three phenylalanine side chains and is H bonded through the indole N to an adjacent water cluster lying between two β-sheets containing Trp) and Lys#*' respectively. The fluorescence of Trp) may be quenched by the benzene rings. The apparent increase in the rate of iron release from the Trp) Tyr mutant could be due to the interference of the mutation with the H-bond linkage resulting in an effect on the second shell network. The partial quenching in the fluorescence

The major function of human transferrin is to deliver iron from the bloodstream to actively dividing cells. Upon iron release, the protein changes its conformation from 'closed' to 'open'. Extensive studies in vitro indicate that iron release from transferrin is very complex and involves many factors, including pH, the chelator used, an anion effect, temperature, receptor binding and intra-lobe interactions. Our earlier work [He, Mason and Woodworth (1997) Biochem. J. 328, 439-445] using the isolated transferrin N-lobe (recombinant N-lobe of human transferrin comprising residues 1-337; hTF/2N) has shown that anions and pH modulate iron release from hTF/2N in an interdependent manner: chloride retards iron release at neutral pH, but accelerates the reaction at acidic pH. The present study supports this idea and further details the nature of the dual effect of chloride: the anion effect on iron release is closely related to the strength of anion binding to the apoprotein. The negative effect seems to originate from competition between chloride and the chelator for an anion-binding site(s) near the metal centre. With decreasing pH, the strength of anion binding to hTF/2N increases linearly, decreasing the contribution of competition with the chelator. In the meantime, the 'open' or 'loose' conformation of hTF/2N, induced by the protonation of critical residues such as the Lys-206/Lys-296 pair at low pH, enables chloride to enter the cleft and bind to exposed side chains, thereby promoting cleft opening and synergistically allowing removal of iron by the chelator, leading to a positive anion effect. Disabling one or more of the primary anion-binding residues, namely Arg-124, Lys-206 and Lys-296, substantially decreases the anion-binding ability of the resulting mutant proteins. In these cases, the competition for the remaining binding residue(s) is increased, leading to a negative chloride effect or, at most, a very small positive effect, even at low pH.

ATP‐citrate lyase (ACLY) catalyzes production of acetyl‐CoA and oxaloacetate from CoA and citrate using ATP. In humans, this cytoplasmic enzyme connects energy metabolism from carbohydrates to the production of lipids. In certain bacteria, ACLY is used to fix carbon in the reductive tricarboxylic acid cycle. The carboxy(C)‐terminal portion of ACLY shows sequence similarity to citrate synthase of the tricarboxylic acid cycle. To investigate the roles of residues of ACLY equivalent to active site residues of citrate synthase, these residues in ACLY from Chlorobium limicola were mutated, and the proteins were investigated using kinetics assays and biophysical techniques. To obtain the crystal structure of the C‐terminal portion of ACLY, full‐length C. limicola ACLY was cleaved, first non‐specifically with chymotrypsin and subsequently with Tobacco Etch Virus protease. Crystals of the C‐terminal portion diffracted to high resolution, providing structures that show the positions of active site residues and how ACLY tetramerizes.

The transferrins (TF) are a group of metal-binding proteins that are involved in iron homeostasis [1]. Structural studies have revealed that the TFs consist of a single polypeptide chain of M r 80000 that folds into two halves called the N-and C-lobes, each of approximately 330 amino acids. In human transferrin (hTF), the lobes are connected by a short peptide of seven residues. Each lobe itself can be further subdivided into two domains separated by a deep cleft that forms the iron-binding site [2][3][4]. The N1 domain (residues 1-93 and 247-315), C1 domain (residues 340-424 and 583-679), N2 domain (residues 94-246) and C2 domain (residues 425-582) are composed of a similar a ⁄ b fold in which a number of helices are packed against a central mixed b-sheet [5]. These domains are connected by two extended b-strands running antiparallel to each other forming a 'hinge' that allows the domains to open and close upon metal binding and release [6]. Iron is bound in a distorted octahedral coordination involving four amino acid ligands and two oxygen atoms from a synergistically bound carbonate ion.The iron-TF complex enters the cell by binding with high affinity (K d 1-10 nm) to a specific TF receptor (TFR), a type-II membrane protein consisting of two identical M r 95000 subunits covalently linked by two disulfide bonds [7]. The N-terminal region of the receptor projects into the cytoplasm of the cell and is joined via the transmembrane region to a 671-residue extracellular domain that binds TF. A soluble form of the receptor can be released by trypsin [8] or produced by recombinant techniques [9,10]. Although these forms lack the two disulfide linkages, strong noncovalent The molecular basis of the transferrin (TF)-transferrin receptor (TFR) interaction is not known. The C-lobe of TF is required to facilitate binding to the TFR and both the N-and C-lobes are necessary for maximal binding. Several mAb have been raised against human transferrin (hTF). One of these, designated F11, is specific to the C-lobe of hTF and does not recognize mouse or pig TF. Furthermore, mAb F11 inhibits the binding of TF to TFR on HeLa cells. To map the epitope for mAb F11, constructs spanning various regions of hTF were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The recombinant fusion proteins were analysed in an iterative fashion by immunoblotting using mAb F11 as the probe. This process resulted in the localization of the F11 epitope to the C1 domain (residues 365-401) of hTF. Subsequent computer modelling suggested that the epitope is probably restricted to a surface patch of hTF consisting of residues 365-385. Mutagenesis of the F11 epitope of hTF to the sequence of either mouse or pig TF confirmed the identity of the epitope as immunoreactivity was diminished or lost. In agreement with other studies, these epitope mapping studies support a role for residues in the C1 domain of hTF in receptor binding.Abbreviations TF, transferrin; TFR, transferrin receptor; GST, glutathione S-transferase; hTF(R),...

Succinyl-CoA synthetase (SCS) catalyzes the only step of the tricarboxylic acid cycle that leads to substrate-level phosphorylation. Some forms of SCS are specific for ADP/ATP or for GDP/GTP, while others can bind all of these nucleotides, generally with different affinities. The theory of 'gatekeeper' residues has been proposed to explain the nucleotide-specificity. Gatekeeper residues lie outside the binding site and create specific electrostatic interactions with incoming nucleotides to determine whether the nucleotides can enter the binding site. To test this theory, the crystal structure of the nucleotide-binding domain in complex with Mg 2+ -ADP was determined, as well as the structures of four proteins with single mutations, K46E, K114D, V113L and L227F, and one with two mutations, K46E/K114D. The crystal structures show that the enzyme is specific for ADP/ATP because of interactions between the nucleotide and the binding site. Nucleotide-specificity is provided by hydrogen-bonding interactions between the adenine base and Gln20, Gly111 and Val113. The O atom of the side chain of Gln20 interacts with N6 of ADP, while the sidechain N atom interacts with the carbonyl O atom of Gly111. It is the different conformations of the backbone at Gln20, of the side chain of Gln20 and of the linker that make the enzyme ATP-specific. This linker connects the two subdomains of the ATP-grasp fold and interacts differently with adenine and guanine bases. The mutant proteins have similar conformations, although the L227F mutant shows structural changes that disrupt the binding site for the magnesium ion. Although the K46E/K114D double mutant of Blastocystis hominis SCS binds GTP better than ATP according to kinetic assays, only the complex with Mg 2+ -ADP was obtained.

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