Ventilatory responses to acute and chronic hypoxia are altered in female but not male Paskin-deficient mice

Proteins harboring a Per-Arnt-Sim (PAS) domain are versatile and allow archaea, bacteria, and plants to sense oxygen partial pressure, as well as light intensity and redox potential. A PAS domain associated with a histidine kinase domain is found in FixL, the oxygen sensor molecule of Rhizobium species. PASKIN is the mammalian homolog of FixL, but its function is far from being understood. Using whole body plethysmography, we evaluated the ventilatory response to acute and chronic hypoxia of homozygous deficient male and female PASKIN mice (Paskin-/-). Although only slight ventilatory differences were found in males, female Paskin-/- mice increased ventilatory response to acute hypoxia. Unexpectedly, females had an impaired ability to reach ventilatory acclimatization in response to chronic hypoxia. Central control of ventilation occurs in the brain stem respiratory centers and is modulated by catecholamines via tyrosine hydroxylase (TH) activity. We observed that TH activity was altered in male and female Paskin-/- mice. Peripheral chemoreceptor effects on ventilation were evaluated by exposing animals to hyperoxia (Dejours test) and domperidone, a peripheral ventilatory stimulant drug directly affecting the carotid sinus nerve discharge. Male and female Paskin-/- had normal peripheral chemosensory (carotid bodies) responses. In summary, our observations suggest that PASKIN is involved in the central control of hypoxic ventilation, modulating ventilation in a gender-dependent manner.     

Crystal structures of deoxy and CO-bound bjFixLH reveal details of ligand recognition and signaling

Rhizobia directly regulate the expression of genes required for symbiotic nitrogen fixation in response to oxygen concentration via the sensor protein FixL. The N-terminal PAS domain of FixL contains a histidine-coordinated heme and regulates the activity of its effector domain, a C-terminal histidine kinase, in response to binding of oxygen and other ligands at the heme. To further investigate ligand-induced inhibition of FixL, we have determined the crystal structures of the heme domain in both the deoxy state and bound to carbon monoxide, a weak inhibitor of FixL kinase activity. Structures collected at room temperature are presented in each state from two crystallographic space groups at 1.8 and 2 A resolution. These structures reveal displacement of the residues of the H(beta) and I(beta) strands by Leu236 upon CO binding, and this structural change propagates more than 15 A to a region of the structure implicated in signal transduction in PAS proteins. Displacement of residues Ile215, Ile216, and Gly217 in the FG loop is also evident, accompanied by the movement of heme propionate 6 upon change in iron ligation. CO binding increases the temperature factors in the FG loop of the protein and disorders the side chain of Arg206, a conserved residue involved in the FG loop switch mechanism. We relate these results to structural changes in other PAS sensor domains and their involvement in catalytic control.     

Changes in quaternary structure in the signaling mechanisms of PAS domains

FixL from Bradyrhizobium japonicum is a PAS sensor protein in which two PAS domains covalently linked to a histidine kinase domain are responsible for regulating nitrogen fixation in an oxygen-dependent manner. The more C-terminal PAS domain, denoted bjFixLH, contains a heme cofactor that binds diatomic molecules such as carbon monoxide and oxygen and regulates the activity of the FixL histidine kinase as part of a two-component signaling system. We present the structures of ferric, deoxy, and carbon monoxide-bound bjFixLH in a new space group ( P1) and at resolutions (1.5-1.8 A) higher than the resolutions of those previously obtained. Interestingly, bjFixLH can form two different dimers (in P1 and R32 crystal forms) in the same crystallization solution, where the monomers in one dimer are rotated approximately 175 degrees relative to the second. This suggests that PAS monomers are plastic and that two quite distinct quaternary structures are closely similar in free energy. We use screw rotation analysis to carry out a quantitative pairwise comparison of PAS quaternary structures, which identifies five different relative orientations adopted by isolated PAS monomers. We conclude that PAS monomer arrangement is context-dependent and could differ depending on whether the PAS domains are isolated or are part of a full-length protein. Structurally homologous residues comprise a conserved dimer interface. Using network analysis, we find that the architecture of the PAS dimer interface is continuous rather than modular; the network of residues comprising the interface is strongly connected. A continuous dimer interface is consistent with the low dimer-monomer dissociation equilibrium constant. Finally, we quantitate quaternary structural changes induced by carbon monoxide binding to a bjFixLH dimer, in which monomers rotate by up to approximately 2 degrees relative to each other. We relate these changes to those in other dimeric PAS domains and discuss the role of quaternary structural changes in the signaling mechanisms of PAS sensor proteins.     

Substrate preference and phosphatidylinositol monophosphate inhibition of the catalytic domain of the Per-Arnt-Sim domain kinase PASKIN

The Per-Arnt-Sim (PAS) domain serine/threonine kinase PASKIN, or PAS kinase, links energy flux and protein synthesis in yeast, regulates glycogen synthesis and protein translation in mammals, and might be involved in insulin regulation in the pancreas. According to the current model, binding of a putative ligand to the PAS domain disinhibits the kinase domain, leading to PASKIN autophosphorylation and increased kinase activity. To date, only synthetic but no endogenous PASKIN ligands have been reported. In the present study, we identified a number of novel PASKIN kinase targets, including ribosomal protein S6. Together with our previous identification of eukaryotic elongation factor 1A1, this suggests a role for PASKIN in the regulation of mammalian protein translation. When searching for endogenous PASKIN ligands, we found that various phospholipids can bind PASKIN and stimulate its autophosphorylation. Interestingly, the strongest binding and autophosphorylation was achieved with monophosphorylated phosphatidylinositols. However, stimulated PASKIN autophosphorylation did not correlate with ribosomal protein S6 and eukaryotic elongation factor 1A1 target phosphorylation. Although autophosphorylation was enhanced by monophosphorylated phosphatidylinositols, di- and tri-phosphorylated phosphatidylinositols inhibited autophosphorylation. By contrast, target phosphorylation was always inhibited, with the highest efficiency for di- and tri-phosphorylated phosphatidylinositols. Because phosphatidylinositol monophosphates were found to interact with the kinase rather than with the PAS domain, these data suggest a multiligand regulation of PASKIN activity, including a still unknown PAS domain binding/activating ligand and kinase domain binding modulatory phosphatidylinositol phosphates.     

The hypoxic testis and post-meiotic expression of PAS domain proteins

Spermatogenesis in the seminiferous tubuli of the testis occurs under a high proliferation rate, suggesting considerable oxygen consumption. Because of the lack of blood vessels, the oxygen partial pressure in the lumen of the tubuli is very low. However, the consequences of these environmental conditions on spermatogenesis are unknown. The PAS domain is found in environmental protein sensors involved in the perception of oxygen partial pressure, light intensity, redox potentials, voltage and certain ligands. We previously identified two PAS proteins highly expressed in the testis: a novel isoform of the hypoxia-inducible factor (HIF)-1alpha and PASKIN, a PAS-Ser/Thr kinase related to bacterial oxygen sensing PAS-domain proteins.     

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.     

MHYT, a new integral membrane sensor domain

MHYT, a new conserved protein domain with a likely signaling function, is described. This domain consists of six transmembrane segments, three of which contain conserved methionine, histidine, and tyrosine residues that are projected to lie near the outer face of the cytoplasmic membrane. In Synechocystis sp. PCC6803, this domain forms the N-terminus of the sensor histidine kinase Slr2098. In Pseudomonas aeruginosa and several other organisms, the MHYT domain forms the N-terminal part of a three-domain protein together with previously described GGDEF and EAL domains, both of which have been associated with signal transduction due to their presence in likely signaling proteins. In Bacillus subtilis YkoW protein, an additional PAS domain is found between the MHYT and GGDEF domains. A ykoW null mutant of B. subtilis did not exhibit any growth alterations, consistent with a non-essential, signaling role of this protein. A model of the membrane topology of the MHYT domain indicates that its conserved residues could coordinate one or two copper ions, suggesting a role in sensing oxygen, CO, or NO.     

Dos, a heme-binding PAS protein from Escherichia coli, is a direct oxygen sensor

A direct sensor of O(2), the Dos protein, has been found in Escherichia coli. Previously, the only biological sensors known to respond to O(2) by direct and reversible binding were the FixL proteins of Rhizobia. A heme-binding region in Dos is 60% homologous to the O(2)-sensing PAS domain of the FixL protein, but the remainder of Dos does not resemble FixL. Specifically, the C-terminal domain of Dos, presumed to be a regulatory partner that couples to its heme-binding domain, is not a histidine kinase but more closely resembles a phosphodiesterase. The absorption spectra of Dos indicate that both axial positions of the heme iron are coordinated to side chains of the protein. Nevertheless, O(2) and CO bind to Dos with K(d) values of 13 and 10 microM, respectively, indicating a strong discrimination against CO binding. Association rate constants for binding of O(2) (3 mM(-)(1) s(-)(1)), CO (1 mM(-)(1) s(-)(1)) and even NO (2 mM(-)(1) s(-)(1)) are extraordinarily low and very similar. Displacement of an endogenous ligand, probably Met 95, from the heme iron in Dos triggers a conformational change that alters the activity of the enzymatic domain. This sensing mechanism differs from that of FixL but resembles that of the CO sensor CooA of Rhodospirillum rubrum. Overall the results provide evidence for a heme-binding subgroup of PAS-domain proteins whose working range, signaling mechanisms, and regulatory partners can vary considerably.     

Epac and PKA: a tale of two intracellular cAMP receptors

cAMP-mediated signaling pathways regulate a multitude of important biological processes under both physiological and pathological conditions, including diabetes, heart failure and cancer. In eukaryotic cells, the effects of cAMP are mediated by two ubiquitously expressed intracellular cAMP receptors, the classic protein kinase A (PKA)/cAMP-dependent protein kinase and the recently discovered exchange protein directly activated by cAMP (Epac)/cAMP-regulated guanine nucleotide exchange factors. Like PKA, Epac contains an evolutionally conserved cAMP binding domain that acts as a molecular switch for sensing intracellular second messenger cAMP levels to control diverse biological functions. The existence of two families of cAMP effectors provides a mechanism for a more precise and integrated control of the cAMP signaling pathways in a spatial and temporal manner. Depending upon the specific cellular environments as well as their relative abundance, distribution and localization, Epac and PKA may act independently, converge synergistically or oppose each other in regulating a specific cellular function.     

Sensory mechanism of oxygen sensor FixL from Rhizobium meliloti: crystallographic, mutagenesis and resonance Raman spectroscopic studies

FixL of Rhizobium meliloti (RmFixL) is a sensor histidine kinase of the two-component system, which regulates the expression of the genes related to nitrogen fixation in the root nodule in response to the O(2) levels. The crystal structure of the sensor domain of FixL (RmFixLH), which contains a heme (Fe-porphyrin) as a sensing site, was determined at 1.4 A resolution. Based on the structural and spectroscopic analyses, we propose the O(2) sensing mechanism that differs from the case proposed in BjFixLH as follows; conformational changes in the F/G loop, which are induced by steric repulsion between the bent-bound O(2) and the Ile209 side-chain, would be transmitted to the histidine kinase domain. Interaction between the iron-bound O(2) and Ile209 was also observed in the resonance Raman spectra of RmFixLH as evidenced by the fact that the Fe-O(2) and Fe-CN stretching frequencies were shifted from 575 to 570 cm(-1) (Fe-O(2)), and 504 to 499 cm(-1), respectively, as the result of the replacement of Ile209 with an Ala residue. In the I209A mutant of RmFixL, the O(2) sensing activity was destroyed, thus confirming our proposed mechanism.     

Rhizobium leguminosarum bv. viciae contains a second fnr/fixK-like gene and an unusual fixL homologue

Genes of Rhizobium leguminosarum bv. viciae VF39 coding for the regulatory elements NifA, FixL and FixK were isolated, sequenced and genetically analysed. The fixK–fixL region is located upstream of the fixNOQP operon on the non-nodulation plasmid pRleVF39c. The deduced amino acid sequence of FixL revealed an unusual structure in that it contains a receiver module (homologous to the N-terminal domain of response regulators) fused to its transmitter domain. An oxygen-sensing haem-binding domain, found in other FixL proteins, is conserved in R. leguminosarum bv. viciae FixL. R. leguminosarum bv. viciae possesses a second fnr -like gene, designated fixK , whose encoded gene product is very similar to Rhizobium meliloti and Azorhizobium caulinodans FixK. Individual R. leguminosarum bv. viciae fixK and fixL insertion mutants displayed a Fix⁺ phenotype. A reduced nitrogen-fixation activity was found for a R. leguminosarum bv. viciae fnrN -deletion mutant, whereas no nitrogen-fixation activity was detectable for a fixK / fnrN double mutant. The R. leguminosarum bv. viciae nifA gene is expressed independently of FixL and FixK under aerobic and microaerobic conditions, whereas fixL gene expression is induced under microaerobiosis. Another orf was identified downstream of fixK–fixL and encodes a product which has homology to pseudoazurins from different species. Mutation of this azu gene showed that it is dispensable for nitrogen fixation.     

Functional implications of the propionate 7-arginine 220 interaction in the FixLH oxygen sensor from Bradyrhizobium japonicum

BjFixL from Bradyrhizobium japonicum is a heme-based oxygen sensor implicated in the signaling cascade that enables the bacterium to adapt to fluctuating oxygen levels. Signal transduction is initiated by the binding of O(2) to the heme domain of BjFixL, resulting in protein conformational changes that are transmitted to a histidine kinase domain. We report structural changes of the heme and its binding pocket in the Fe(II) deoxy and Fe(III) met states of the wild-type BjFixLH oxygen sensor domain and four mutants of the highly conserved residue arginine 220. UV-visible, electron paramagnetic resonance, and resonance Raman spectroscopies all showed that the heme iron of the R220H mutant is unexpectedly six-coordinated at physiological pH in the Fe(III) state but undergoes pH- and redox-dependent coordination changes. This behavior is unprecedented for FixL proteins, but is reminiscent of another oxygen sensor from E. coli, EcDos. All mutants in their deoxy states are five-coordinated Fe(II), although we report rupture of the residue 220-propionate 7 interaction and structural modifications of the heme conformation as well as propionate geometry and flexibility. In this work, we conclude that part of the structural reorganization usually attributed to O(2) binding in the wild-type protein is in fact due to rupture of the Arg220-P7 interaction. Moreover, we correlate the structural modifications of the deoxy Fe(II) states with k(on) values and conclude that the Arg220-P7 interaction is responsible for the lower O(2) and CO k(on) values reported for the wild-type protein.