The FixL protein is a biological sensor of O2. Dissociation of O2 from a heme-binding domain is required for FixL to autophosphorylate at a conserved histidine with a gamma-phosphoryl group from ATP. Transfer of the phosphoryl group from FixL to the transcription factor FixJ enables FixJ to activate a cascade of gene expression. The kinase domain of FixL belongs to a large class of sensory histidine kinases that occur in plants, fungi, Archaea, and every bacterium that has been examined. The structure of such a kinase domain has recently become available. Crystal structures were also recently solved for the heme-binding domain of FixL with and without an inhibitory ligand. These FixL structures have led to a new model for heme-driven conformational changes. Although the alpha/beta fold of the FixL, heme-binding domain represents a novel hemoglobin fold, this fold closely matches the structures of the HERG voltage sensory in humans and the PYP light sensory in halophilic bacterium. As such, the heme-binding domain of FixL provides a prototype for a large family of sensory proteins called the PAS proteins. Based on sequence alignments, over 200 PAS proteins have been identified so far in Archaea, Bacteria, and Eukarya, where they transduce key regulatory signals. The direct coupling in FixL of a histidine kinase to an O2-detecting PAS domain provides an ideal system for study of signal transduction. The proposed work will combined mutagenesis with biochemical, biophysical, and structural approaches to: test the regulatory mechanism indicated by the FixL """"""""on"""""""" and """"""""off"""""""" structures, examine the role of conserved PAS residues, determine factors that govern affinity for ligands, and identify key contacts between the heme-binding and kinase domains of FixL. This research is expected to extend knowledge of signal transduction mechanisms and sensing of physiological heme ligands. Some possible applications are the development of novel antibiotics directed at specific sensors in bacteria and anti-cancer drugs targeting O2 sensors that are implicated in angiogenesis.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
7R01HL064038-04
Application #
6627543
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Peterson, Charles M
Project Start
2000-02-07
Project End
2004-12-31
Budget Start
2003-01-01
Budget End
2004-12-31
Support Year
4
Fiscal Year
2003
Total Cost
$169,613
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Gilles-Gonzalez, Marie-Alda; Gonzalez, Gonzalo (2005) Heme-based sensors: defining characteristics, recent developments, and regulatory hypotheses. J Inorg Biochem 99:1-22
Freitas, Tracey Allen K; Hou, Shaobin; Dioum, Elhadji M et al. (2004) Ancestral hemoglobins in Archaea. Proc Natl Acad Sci U S A 101:6675-80
Gilles-Gonzalez, Marie-Alda; Gonzalez, Gonzalo (2004) Signal transduction by heme-containing PAS-domain proteins. J Appl Physiol 96:774-83
Dunham, Christine M; Dioum, Elhadji M; Tuckerman, Jason R et al. (2003) A distal arginine in oxygen-sensing heme-PAS domains is essential to ligand binding, signal transduction, and structure. Biochemistry 42:7701-8
Tuckerman, Jason R; Gonzalez, Gonzalo; Dioum, Elhadji M et al. (2002) Ligand and oxidation-state specific regulation of the heme-based oxygen sensor FixL from Sinorhizobium meliloti. Biochemistry 41:6170-7
Tomita, Takeshi; Gonzalez, Gonzalo; Chang, Alan L et al. (2002) A comparative resonance Raman analysis of heme-binding PAS domains: heme iron coordination structures of the BjFixL, AxPDEA1, EcDos, and MtDos proteins. Biochemistry 41:4819-26
Gonzalez, Gonzalo; Dioum, Elhadji M; Bertolucci, Craig M et al. (2002) Nature of the displaceable heme-axial residue in the EcDos protein, a heme-based sensor from Escherichia coli. Biochemistry 41:8414-21
Chang, A L; Tuckerman, J R; Gonzalez, G et al. (2001) Phosphodiesterase A1, a regulator of cellulose synthesis in Acetobacter xylinum, is a heme-based sensor. Biochemistry 40:3420-6
Tuckerman, J R; Gonzalez, G; Gilles-Gonzalez, M A (2001) Complexation precedes phosphorylation for two-component regulatory system FixL/FixJ of Sinorhizobium meliloti. J Mol Biol 308:449-55