X-ray Characterization of Conformational Changes of Human Apo- and Holo-Transferrin.
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ABSTRACT:
SUBMITTER: Campos-Escamilla C
PROVIDER: S-EPMC8705962 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Biological function of proteins is frequently associated with the formation of complexes with small-molecule ligands. Experimental structure determination of such complexes at atomic resolution, however, can be time-consuming and costly. Computational methods for structure prediction of protein/ligand complexes, particularly docking, are as yet restricted by their limited consideration of receptor flexibility, rendering them not applicable for predicting protein/ligand complexes if large conformational changes of the receptor upon ligand binding are involved. Accurate receptor models in the ligand-bound state (holo structures), however, are a prerequisite for successful structure-based drug design. Hence, if only an unbound (apo) structure is available distinct from the ligand-bound conformation, structure-based drug design is severely limited. We present a method to predict the structure of protein/ligand complexes based solely on the apo structure, the ligand and the radius of gyration of the holo structure. The method is applied to ten cases in which proteins undergo structural rearrangements of up to 7.1 A backbone RMSD upon ligand binding. In all cases, receptor models within 1.6 A backbone RMSD to the target were predicted and close-to-native ligand binding poses were obtained for 8 of 10 cases in the top-ranked complex models. A protocol is presented that is expected to enable structure modeling of protein/ligand complexes and structure-based drug design for cases where crystal structures of ligand-bound conformations are not available.
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Project description:Understanding the effect of glycation on the function of transferrin, the systemic iron transporter, is fundamental to fully grasp the mechanisms leading to the loss of iron homeostasis observed in diabetes mellitus (DM). The spontaneous reaction with protein amino groups is one of the main causes of glucose toxicity, but the site specificity of this reaction is still poorly understood. Unbalance of iron metabolism has long been described in DM patients, and one of the main features is the occurrence of non-transferrin-bound iron in the blood serum. The presence of these toxic iron species is observed, despite low transferrin saturation levels. Here in, an in vitro approach was used to study human holo-transferrin glycation in detail. Holo-transferrin was incubated with increasing concentration of glucose (10; 20; 100 and 500 mM) and glycation sites were identified using nano-reverse phase – liquid chromatography coupled to high resolution mass spectrometry (nLC-MS). Overall 21 glycation hotspots were identified with lysine residues 103, 312 and 380 proving to be the most reactive sites. Glycation specificity was found to be remarkably different from that described for apo-transferrin.
2018-05-30 | PXD009345 | Pride
Project description:Flavodoxins are a family of small FMN-binding proteins that commonly exist in prokaryotes. They utilize a non-covalently bound FMN molecule to act as the redox center during the electron transfer processes in various important biological pathways. Although extensive investigations were performed, detailed molecular mechanisms of cofactor binding and electron transfer remain elusive. Herein we report the solution NMR studies on Escherichia coli flavodoxins FldA and YqcA, belonging to the long-chain and short-chain flavodoxin subfamilies respectively. Our structural studies demonstrate that both proteins show the typical flavodoxin fold, with extensive conformational exchanges observed near the FMN binding pocket in their apo-forms. Cofactor binding significantly stabilizes both proteins as revealed by the extension of secondary structures in the holo-forms, and the overall rigidity shown by the backbone dynamics data. However, the 50 s loops of both proteins in the holo-form still show conformational exchanges on the µs-ms timescales, which appears to be a common feature in the flavodoxin family, and might play an important role in structural fine-tuning during the electron transfer reactions.
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Project description:BackgroundApo- (iron free) and holo- (iron bound) transferrin (Tf) participate in precise regulation of brain iron uptake at endothelial cells of the blood-brain barrier. Apo-Tf indicates an iron deficient environment and stimulates iron release, while holo-Tf indicates an iron sufficient environment and suppresses additional iron release. Free iron is exported through ferroportin, with hephaestin as an aid to the process. Until now, the molecular mechanism of apo- and holo-Tf's influence on iron release was largely unknown.MethodsHere we use a variety of cell culture techniques, including co-immunoprecipitation and proximity ligation assay, in iPSC-derived endothelial cells and HEK 293 cells to investigate the mechanism of apo- and holo-Tf's influence over iron release. We placed our findings in physiological context by further deciphering how hepcidin played a role in this mechanism as well.ResultsWe demonstrate that holo-Tf induces the internalization of ferroportin through the established ferroportin degradation pathway. Furthermore, holo-Tf directly binds to ferroportin, whereas apo-Tf directly binds to hephaestin. Only pathological levels of hepcidin disrupt the interaction between holo-Tf and ferroportin, and no amount of hepcidin disrupts the interaction between apo-Tf and hephaestin. The disruption of the holo-Tf and ferroportin interaction by hepcidin is due to hepcidin's ability to rapidly internalize ferroportin compared to holo-Tf.ConclusionsThese novel findings provide a molecular mechanism for apo- and holo-Tf regulation of iron release from endothelial cells. They further demonstrate how hepcidin impacts these protein-protein interactions, and offer a model for how holo-Tf and hepcidin corporate to suppress iron release. We have established a more thorough understanding of the mechanisms behind iron release regulation with great clinical impact for a variety of neurological conditions in which iron release is dysregulated.
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Project description:BackgroundApo- (iron free) and holo- (iron bound) transferrin (Tf) participate in precise regulation of brain iron uptake at endothelial cells of the blood-brain barrier. Apo-Tf indicates an iron-deficient environment and stimulates iron release, while holo-Tf indicates an iron sufficient environment and suppresses additional iron release. Free iron is exported through ferroportin, with hephaestin as an aid to the process. Until now, the molecular mechanisms of apo- and holo-Tf influence on iron release was largely unknown.MethodsHere we use a variety of cell culture techniques, including co-immunoprecipitation and proximity ligation assay, in iPSC-derived endothelial cells and HEK 293 cells to investigate the mechanism by which apo- and holo-Tf influence cellular iron release. Given the established role of hepcidin in regulating cellular iron release, we further explored the relationship of hepcidin to transferrin in this model.ResultsWe demonstrate that holo-Tf induces the internalization of ferroportin through the established ferroportin degradation pathway. Furthermore, holo-Tf directly interacts with ferroportin, whereas apo-Tf directly interacts with hephaestin. Only pathophysiological levels of hepcidin disrupt the interaction between holo-Tf and ferroportin, but similar hepcidin levels are unable to interfere with the interaction between apo-Tf and hephaestin. The disruption of the holo-Tf and ferroportin interaction by hepcidin is due to hepcidin's ability to more rapidly internalize ferroportin compared to holo-Tf.ConclusionsThese novel findings provide a molecular mechanism for apo- and holo-Tf regulation of iron release from endothelial cells. They further demonstrate how hepcidin impacts these protein-protein interactions, and offer a model for how holo-Tf and hepcidin cooperate to suppress iron release. These results expand on our previous reports on mechanisms mediating regulation of brain iron uptake to provide a more thorough understanding of the regulatory mechanisms mediating cellular iron release in general.
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