Structural and molecular characterization of iron-sensing hemerythrin-like domain within F-box and leucine-rich repeat protein 5 (FBXL5).
ABSTRACT Mammalian cells maintain iron homeostasis by sensing changes in bioavailable iron levels and promoting adaptive responses. FBXL5 is a subunit of an E3 ubiquitin ligase complex that mediates the stability of iron regulatory protein 2, an important posttranscriptional regulator of several genes involved in iron metabolism. The stability of FBXL5 is regulated in an iron- and oxygen-responsive manner, contingent upon the presence of its N-terminal domain. Here we present the atomic structure of the FBXL5 N terminus, a hemerythrin-like α-helical bundle fold not previously observed in mammalian proteins. The core of this domain employs an unusual assortment of amino acids necessary for the assembly and sensing properties of its diiron center. These regulatory features govern the accessibility of a mapped sequence required for proteasomal degradation of FBXL5. Detailed molecular and structural characterization of the ligand-responsive hemerythrin domain provides insights into the mechanisms by which FBXL5 serves as a unique mammalian metabolic sensor.
[show abstract] [hide abstract]
ABSTRACT: The majority of 3D structures of macromolecules are currently determined by macromolecular crystallography, which employs the diffraction of X-rays on single crystals. However, during diffraction experiments, the X-rays can damage the protein crystals by ionization processes, especially when powerful X-ray sources at synchrotron facilities are used. This process of radiation damage generates photo-electrons that can get trapped in protein moieties. The 3D structure derived from such experiments can differ remarkably from the structure of the native molecule. Recently, the crystal structures of different oxidation states of horseradish peroxidase and nickel-containing superoxide dismutase were determined using crystallographic redox titration performed during the exposure of the crystals to the incident X-ray beam. Previous crystallographic analyses have not shown the distinct structures of the active sites associated with the redox state of the structural features of these enzymes. These new studies show that, for protein moieties that are susceptible to radiation damage and prone to reduction by photo-electrons, care is required in both the design of the diffraction experiment and the analysis and interpretation.Trends in Biochemical Sciences 05/2005; 30(4):213-9. · 10.85 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Eukaryotic cells require iron for survival and have developed regulatory mechanisms for maintaining appropriate intracellular iron concentrations. The degradation of iron regulatory protein 2 (IRP2) in iron-replete cells is a key event in this pathway, but the E3 ubiquitin ligase responsible for its proteolysis has remained elusive. We found that a SKP1-CUL1-FBXL5 ubiquitin ligase protein complex associates with and promotes the iron-dependent ubiquitination and degradation of IRP2. The F-box substrate adaptor protein FBXL5 was degraded upon iron and oxygen depletion in a process that required an iron-binding hemerythrin-like domain in its N terminus. Thus, iron homeostasis is regulated by a proteolytic pathway that couples IRP2 degradation to intracellular iron levels through the stability and activity of FBXL5.Science 09/2009; 326(5953):718-21. · 31.20 Impact Factor
Article: An E3 ligase possessing an iron-responsive hemerythrin domain is a regulator of iron homeostasis.[show abstract] [hide abstract]
ABSTRACT: Cellular iron homeostasis is maintained by the coordinate posttranscriptional regulation of genes responsible for iron uptake, release, use, and storage through the actions of the iron regulatory proteins IRP1 and IRP2. However, the manner in which iron levels are sensed to affect IRP2 activity is poorly understood. We found that an E3 ubiquitin ligase complex containing the FBXL5 protein targets IRP2 for proteasomal degradation. The stability of FBXL5 itself was regulated, accumulating under iron- and oxygen-replete conditions and degraded upon iron depletion. FBXL5 contains an iron- and oxygen-binding hemerythrin domain that acted as a ligand-dependent regulatory switch mediating FBXL5's differential stability. These observations suggest a mechanistic link between iron sensing via the FBXL5 hemerythrin domain, IRP2 regulation, and cellular responses to maintain mammalian iron homeostasis.Science 09/2009; 326(5953):722-6. · 31.20 Impact Factor