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Marie-Alda Gilles-Gonzalez

Publications and source records attributed to Marie-Alda Gilles-Gonzalez.

11 recordsLinked to original sources
PubMed2006

A proximal arginine R206 participates in switching of the Bradyrhizobium japonicum FixL oxygen sensor.

In oxygen-sensing PAS domains, a conserved polar residue on the proximal side of the heme cofactor, usually arginine or histidine, interacts alternately with the protein in the "on-state" or the heme edge in the "off-state" but does not contact the bound ligand directly. We assessed the contributions of this residue in Bradyrhizobium japonicum FixL by determining the effects of an R206A substitution on the heme-PAS structure, ligand affinity, and regulatory capacity. The crystal structures of the unliganded forms of the R206A and wild-type BjFixL heme-PAS domains were similar, except for a more ruffled porphyrin ring in R206A BjFixL and a relaxation of the H214 residue and heme propionate 7 due to their lost interactions. The oxygen affinity of R206A BjFixL (Kd approximately 350 microM) was 2.5 times lower than that of BjFixL, and this was due to a higher off-rate constant for the R206A variant. The enzymatic activities of the unliganded "on-state" forms, either deoxy or met-R206A BjFixL, were comparable to each other and slightly lower (twofold less) than those of the corresponding BjFixL species. The most striking difference between the two proteins was in the enzymatic activities of the liganded "off-state" forms. In particular, saturation with a regulatory ligand (the Fe(III) form with cyanide) caused a >2000-fold inhibition of the BjFixL phosphorylation of BjFixJ, but a 140-fold inhibition of this catalytic activity in R206A BjFixL. Thus, in oxygen-sensing PAS domains, the interactions of polar residues with the heme edge couple the heme-binding domain to a transmitter during signal transduction.

Amino Acid Sequence↗
PubMed2005

Oxygen blocks the reaction of the FixL-FixJ complex with ATP but does not influence binding of FixJ or ATP to FixL.

The RmFixL-RmFixJ oxygen signal transduction system ensures that a cascade of the Sinorhizobium meliloti nitrogen fixation genes is induced as the concentration of O2 drops below 50 microM in symbiotic nodules. Deoxy-RmFixL is a histidine protein kinase that catalyzes a phosphoryl transfer from ATP to the aspartate 54 residue of RmFixJ; RmFixJ is a response regulator that becomes activated as a transcription factor by phosphorylation. Association of O2 with a heme-binding domain in RmFixL triggers a conformational change that inhibits its kinase activity. Here we consider whether this inhibition is achieved by disrupting binding of either of the substrates, i.e., RmFixJ or ATP, to the RmFixL kinase. The ATP affinities of the oxy and deoxy states were compared via competition of ATP against TNP-nucleotide fluorophores. The influence of O2 on formation of the RmFixL-RmFixJ complex was investigated by fluorescence polarization. Oxygen dramatically inhibited the reaction of the RmFixL-RmFixJ complex with ATP but affected neither ATP binding (Kd approximately 100 microM) nor RmFixL-RmFixJ complex formation (Kd approximately 4 microM), indicating that inhibition of the kinase by the oxy-heme in RmFixL is achieved by inactivating the catalytic site, rather than by blocking the association of this enzyme with either of its substrates. An 8-fold enhancement of the rate of reaction of RmFixL with ATP in a deoxy-RmFixL-D54N RmFixJ complex, compared to that in isolated deoxy-RmFixL, exposes the strength of the allosteric effect of RmFixJ on the reaction. These results clarify the mechanistic roles of the signal and regulatory partner in this signal transduction system.

Adenosine Triphosphate↗
PubMed2005

Heme-based sensors: defining characteristics, recent developments, and regulatory hypotheses.

In a great variety of organisms throughout all kingdoms of life, the heme-based-sensor proteins are the key regulators of adaptive responses to fluctuating oxygen, carbon monoxide, and nitric oxide levels. These signal transducers achieve their responses by coupling a regulatory heme-binding domain to a neighboring transmitter. The past decade has witnessed an explosion in the numbers of these modular sensory proteins known, from just two recognized members, FixL and soluble guanylyl cyclase (sGC), to four broad families comprising more than 50 sensors. Heme-based sensors so far feature four different types of heme-binding modules: the heme-binding PAS domain, globin-coupled sensor (GCS), CooA, and heme-NO-binding (HNOB). The transmitters for coupling to such heme-binding domains include histidine protein kinases, cyclic nucleotide phosphodiesterases, chemotaxis methyl-carrier protein receptors, and transcription factors of the basic helix-loop-helix and helix-turn-helix classes. Some well-studied sensors are the FixL, EcDos, AxPDEA1, NPAS2, HemAT-Bs, HemAT-Hs, CooA, and sGC proteins. This review elaborates the defining characteristics of heme-based sensors, examines recent developments on those proteins, and discusses the regulatory hypotheses proposed for those sensors. A general, "helix-swap", model is also proposed here for signal transduction by PAS domains.

Amino Acid Sequence↗
PubMed2004

Ancestral hemoglobins in Archaea.

Hemoglobins are ubiquitous in Eukarya and Bacteria but, until now, have not been found in Archaea. A phylogenetic analysis of the recently revealed microbial family of globin-coupled heme-based sensors suggests that these sensors descended from an ancient globin-only progenitor, or a protoglobin (Pgb). Here, we report the discovery and characterization of two Pgbs from the Archaea: ApPgb from the obligately aerobic hyperthermophile Aeropyrum pernix, and MaPgb from the strictly anaerobic methanogen Methanosarcina acetivorans. Both ApPgb and MaPgb bind molecular oxygen, nitric oxide, and carbon monoxide by means of a heme moiety that is coordinated to the protein through the F8 histidine (histidine 120). We postulate that these archaeal globins are the ancestors of contemporary hemoglobins.

Amino Acid Sequence↗
PubMed2004

Signal transduction by heme-containing PAS-domain proteins.

The most common physiological strategy for detecting the gases oxygen, carbon monoxide, and nitric oxide is signal transduction by heme-based sensors, a broad class of modular proteins in which a heme-binding domain governs the activity of a neighboring transmitter domain. Different structures are possible for the heme-binding domains in these sensors, but, so far, the Per-ARNT-Sim motif, or PAS domain, is the one most commonly encountered. Heme-binding PAS (heme-PAS) domains can accomplish ligand-dependent switching of a variety of partner domains, including histidine kinase, phosphodiesterase, and basic helix-loop-helix (bHLH) DNA-binding modules. Proteins with heme-PAS domains occur in all kingdoms of life and are quite diverse in their physiological roles. Examples include the neuronal bHLH-PAS carbon monoxide sensor NPAS2 that is implicated in the mammalian circadian clock, the acetobacterial oxygen sensor AxPDEA1 that directs cellulose production, and the rhizobial oxygen sensor FixL, which governs nitrogen fixation. What factors determine the range of detection of these sensors? How do they transduce their signal? This review examines the recent advances in answering these questions.

Animals↗
PubMed2003

A distal arginine in oxygen-sensing heme-PAS domains is essential to ligand binding, signal transduction, and structure.

To evaluate the contributions of the G(beta)-2 arginine to signal transduction in oxygen-sensing heme-PAS domains, we replaced this residue with alanine in Bradyrhizobium japonicum FixL and examined the results on heme-domain structure, ligand binding, and kinase regulation. In the isolated R220A BjFixL heme-PAS domain, the iron-histidine bond was increased in length by 0.31 A, the heme flattened even without a ligand, and the interaction of a presumed regulatory loop (the FG loop) with the helix of heme attachment was weakened. Binding of carbon monoxide was similar for ferrous BjFixL and R220A BjFixL. In contrast, the level of binding of oxygen was dramatically lower (K(d) approximately 1.5 mM) for R220A BjFixL, and this was manifested as 60- and 3-fold lower on- and off-rate constants, respectively. Binding of cyanide followed the same pattern as binding of oxygen. The catalytic activity was 3-4-fold higher in the "on-state" unliganded forms of R220A BjFixL than in the corresponding BjFixL species. Cyanide regulation of this activity was strongly impaired, but some inhibition was nevertheless preserved. Carbon monoxide and nitric oxide regulation, although weak in BjFixL, were abolished from R220A BjFixL. We conclude that the G(beta)-2 arginine assists in the binding of oxygen to BjFixL but does not accomplish this by stabilizing the oxy form. This arginine is not absolutely required for regulation, although it is important for shifting a pre-existing kinase equilibrium toward the inactive state on binding of regulatory ligands. These findings support a regulatory model in which the heme-PAS domain operates as an ensemble that couples to the kinase rather than a mechanism driven by a single central switch.

Arginine↗
PubMed2002

NPAS2: a gas-responsive transcription factor.

Neuronal PAS domain protein 2 (NPAS2) is a mammalian transcription factor that binds DNA as an obligate dimeric partner of BMAL1 and is implicated in the regulation of circadian rhythm. Here we show that both PAS domains of NPAS2 bind heme as a prosthetic group and that the heme status controls DNA binding in vitro. NPAS2-BMAL1 heterodimers, existing in either the apo (heme-free) or holo (heme-loaded) state, bound DNA avidly under favorably reducing ratios of the reduced and oxidized forms of nicotinamide adenine dinucleotide phosphate. Low micromolar concentrations of carbon monoxide inhibited the DNA binding activity of holo-NPAS2 but not that of apo-NPAS2. Upon exposure to carbon monoxide, inactive BMAL1 homodimers were formed at the expense of NPAS2-BMAL1 heterodimers. These results indicate that the heterodimerization of NPAS2, and presumably the expression of its target genes, are regulated by a gas through the heme-based sensor described here.

ARNTL Transcription Factors↗
PubMed2002

Nature of the displaceable heme-axial residue in the EcDos protein, a heme-based sensor from Escherichia coli.

The EcDos protein belongs to a group of heme-based sensors that detect their ligands with a heme-binding PAS domain. Among these various heme-PAS proteins, EcDos is unique in having its heme iron coordinated at both axial positions to residues of the protein. To achieve its high affinities for ligands, one of the axial heme-iron residues in EcDos must be readily displaceable. Here we present evidence from mutagenesis, ligand-binding measurements, and magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopies about the nature of the displaceable residue in the heme-PAS domain of EcDos, i.e., EcDosH. The magnetic circular dichroism spectra in the near-infrared region establish histidine-methionine coordination in met-EcDos. To determine whether in deoxy-EcDos coordination of the sixth axial position is also to methionine, methionine 95 was substituted with isoleucine. This substitution caused the ferrous heme iron to change from an exclusively hexacoordinate low-spin form (EcDosH) to an exclusively pentacoordinate high-spin form (M95I EcDosH). This was accompanied by a modest acceleration of the dissociation rates of ligands but a dramatic increase (60-1300-fold) in the association rate constants for binding of O(2), CO, and NO. As a result, the affinity for O(2) was enhanced 10-fold in M95I EcDosH, but the partition constant M = [K(d)(O(2))/K(d)(CO)] between CO and O(2) was raised to about 30 from the extraordinarily low EcDosH value of 1. Thus a major consequence of the increased O(2) affinity of this sensor was the loss of its unusually strong ligand discrimination.

Amino Acid Sequence↗
PubMed2002

Ligand and oxidation-state specific regulation of the heme-based oxygen sensor FixL from Sinorhizobium meliloti.

Phosphorylation of the transcription factor RmFixJ is the key step in the hypoxic induction of Sinorhizobium meliloti nitrogen fixation genes. Oxygen regulates this process by binding reversibly to RmFixL, a heme protein kinase whose deoxy form catalyzes the phosphoryl transfer from ATP to RmFixJ. Here we present the first quantitative measure of the extent by which various heme ligands inhibit the turnover of RmFixJ to phospho-RmFixJ. We also quantitate the inhibition by ligands of the reaction of RmFixL with ATP, in the absence of RmFixJ, to form phospho-RmFixL, i.e., the "autophosphorylation". Phospho-RmFixL formed from autophosphorylation will transfer its phosphoryl group to RmFixJ in an oxygen-independent "phosphotransfer." Here we show that the mode of substrate presentation, i.e., simultaneous versus sequential, influences the regulation of phosphoryl transfer by heme status. Inhibition factors for O(2), CO, NO, CN(-), and imidazole in the presence of RmFixJ are drastically different from the inhibition of autophosphorylation by the same ligands. Oxidation of the heme iron in unliganded RmFixL is known to have no effect on either of the sequential reactions; yet oxidation causes a 100-fold decrease in RmFixJ turnover when ATP and RmFixJ are presented simultaneously. The profound difference between the regulation of isolated RmFixL versus the complex of RmFixL with RmFixJ shows that interaction of a response regulator with its histidine-kinase partner need not be limited to the enzymatic regions of the histidine kinase, but can extend also to its sensory domain.

Adenosine Triphosphate↗
PubMed2002

A comparative resonance Raman analysis of heme-binding PAS domains: heme iron coordination structures of the BjFixL, AxPDEA1, EcDos, and MtDos proteins.

The heme-PAS is a specialized domain with which a broad class of signal-transducing heme proteins detect physiological heme ligands. Such domains exhibit a wide range of ligand binding parameters, yet they are all expected to feature an alpha-beta heme binding fold and a predominantly hydrophobic heme distal pocket without a distal histidine. We have compared, for the first time, the resonance Raman spectra of several heme-PASs: the heme-binding domains of Bradyrhizobium japonicum FixL, Escherichia coli Dos, Acetobacter xylinum PDEA1, and Methanobacterium thermoautotrophicum Dos. In all cases, the nu(Fe)-(CO) and nu(C-O) values of the carbonmonoxy forms were consistent with coordination of the heme iron to histidine on the proximal side and binding of the CO without electrostatic interaction with the heme distal pocket. EcDos was unusual in having predominantly hexacoordinate heme iron in the deoxy and met forms. Despite an evident lack of CO interaction with the EcDos heme pocket, relatively low Fe-O(2) (562 cm(-1)) and N-O (1576 cm(-1)) stretching frequencies indicated that strong polar interactions with that heme distal pocket are possible for highly bent ligands such as O(2) or NO. None of the newly studied NO adducts exhibited evidence of the Fe-His rupture and pentacoordination previously noted for Sinorhizobium meliloti FixL. A low Fe-His stretching frequency, formerly interpreted as a strained Fe-His bond, and the slow association of O(2) with S. meliloti FixL failed to correlate with the newly studied proteins having low association rate or low equilibrium association constants for binding of O(2). We conclude that although heme-PASs share some features, they represent distinct signal transduction mechanisms.

Acetobacter↗