Structural Catalytic Core of the Members of the Superfamily of Acid Proteases.

The superfamily of acid proteases has two catalytic aspartates for proteolysis of their peptide substrates. Here, we show a minimal structural scaffold, the structural catalytic core (SCC), which is conserved within each family of acid proteases, but varies between families, and thus can serve as a structural marker of four individual protease families. The SCC is a dimer of several structural blocks, such as the DD-link, D-loop, and G-loop, around two catalytic aspartates in each protease subunit or an individual chain. A dimer made of two (D-loop + DD-link) structural elements makes a DD-zone, and the D-loop + G-loop combination makes a psi-loop. These structural markers are useful for protein comparison, structure identification, protein family separation, and protein engineering.

Oxygen-Induced Conformational Changes in the PAS-Heme Domain of the Pseudomonas aeruginosa Aer2 Receptor.

The Aer2 receptor from Pseudomonas aeruginosa has an O2-binding PAS-heme domain that stabilizes O2 via a Trp residue in the distal heme pocket. Trp rotates ∼90° to bond with the ligand and initiate signaling. Although the isolated PAS domain is monomeric, both in solution and in a cyanide-bound crystal structure, an unliganded structure forms a dimer. An overlay of the two structures suggests possible signaling motions but also predicts implausible clashes at the dimer interface when the ligand is bound. Moreover, in a full-length Aer2 dimer, PAS is sandwiched between multiple N- and C-terminal HAMP domains, which would feasibly restrict PAS motions. To explore the PAS dimer interface and signal-induced motions in full-length Aer2, we introduced Cys substitutions and used thiol-reactive probes to examine in vivo accessibility and residue proximities under both aerobic and anaerobic conditions. In vivo, PAS dimers were retained in full-length Aer2 in the presence and absence of O2, and the dimer interface was consistent with the isolated PAS dimer structure. O2-mediated changes were also consistent with structural predictions in which the PAS N-terminal caps move apart and the C-terminal DxT region moves closer together. The DxT motif links PAS to the C-terminal HAMP domains and was critical for PAS-HAMP signaling. Removing the N-terminal HAMP domains altered the distal PAS dimer interface and prevented signaling, even after signal-on lesions were introduced into PAS. The N-terminal HAMP domains thus facilitate the O2-dependent shift of PAS to the signal-on conformation, clarifying their role upstream of the PAS-sensing domain.

Using Evidence Integration Techniques in the Development of Health-Based Occupational Exposure Levels.

Development of toxicology-based criteria such as occupational exposure levels (OELs) are rarely straightforward. This process requires a rigorous review of the literature, searching for patterns in toxicity, biological plausibility, coherence, and dose-response relationships. Despite the direct applicability, human data are rarely used primarily because of imprecise exposure estimates, unknown influence of assumptions, and confounding factors. As a result, high reliance is often placed on laboratory animal data. Often, data from a single study is typically used to represent an entire database to extrapolate an OEL, even for data-rich compounds. Here we present a holistic framework for evaluating epidemiological, controlled in vivo, mechanistic/in vitro, and computational evidence that can be useful in deriving OELs. It begins with describing a documented review process of the literature, followed by sorting of data into either controlled laboratory in vivo, in silico/read-across, mechanistic/in vitro, or epidemiological/field data categories. Studies are then evaluated and qualified based on rigor, risk of bias, and applicability for point of departure development. Other data (eg, in vitro, in silico estimates, read-across data and mechanistic information, and data that failed to meet the former criteria) are used alongside qualified epidemiological exposure estimates to help inform points of departure or human-equivalent concentrations that are based on toxic end points. Bayesian benchmark dose methods are used to estimate points of departure and for estimating uncertainty factors (UFs) to develop preliminary OELs. These are then compared with epidemiological data to support the OEL and the use and magnitude of UFs, when appropriate.

An unbiased in vitro screen for activating epidermal growth factor receptor mutations.

Cancer tissues harbor thousands of mutations, and a given oncogene may be mutated at hundreds of sites, yet only a few of these mutations have been functionally tested. Here, we describe an unbiased platform for the functional characterization of thousands of variants of a single receptor tyrosine kinase (RTK) gene in a single assay. Our in vitroscreen for activating mutations (iSCREAM) platform enabled rapid analysis of mutations conferring gain-of-function RTK activity promoting clonal growth. The screening strategy included a somatic model of cancer evolution and utilized a library of 7,216 randomly mutated epidermal growth factor receptor (EGFR) single-nucleotide variants that were tested in murine lymphoid Ba/F3 cells. These cells depend on exogenous interleukin-3 (IL-3) for growth, but this dependence can be compensated by ectopic EGFR overexpression, enabling selection for gain-of-function EGFR mutants. Analysis of the enriched mutants revealed EGFR A702V, a novel activating variant that structurally stabilized the EGFR kinase dimer interface and conferred sensitivity to kinase inhibition by afatinib. As proof of concept for our approach, we recapitulated clinical observations and identified the EGFR L858R as the major enriched EGFR variant. Altogether, iSCREAM enabled robust enrichment of 21 variants from a total of 7,216 EGFR mutations. These findings indicate the power of this screening platform for unbiased identification of activating RTK variants that are enriched under selection pressure in a model of cancer heterogeneity and evolution.

Gap-filling approaches for eddy covariance methane fluxes: A comparison of three machine learning algorithms and a traditional method with principal component analysis.

Methane flux (FCH4 ) measurements using the eddy covariance technique have increased over the past decade. FCH4 measurements commonly include data gaps, as is the case with CO2 and energy fluxes. However, gap-filling FCH4 data are more challenging than other fluxes due to its unique characteristics including multidriver dependency, variabilities across multiple timescales, nonstationarity, spatial heterogeneity of flux footprints, and lagged influence of biophysical drivers. Some researchers have applied a marginal distribution sampling (MDS) algorithm, a standard gap-filling method for other fluxes, to FCH4 datasets, and others have applied artificial neural networks (ANN) to resolve the challenging characteristics of FCH4 . However, there is still no consensus regarding FCH4 gap-filling methods due to limited comparative research. We are not aware of the applications of machine learning (ML) algorithms beyond ANN to FCH4 datasets. Here, we compare the performance of MDS and three ML algorithms (ANN, random forest [RF], and support vector machine [SVM]) using multiple combinations of ancillary variables. In addition, we applied principal component analysis (PCA) as an input to the algorithms to address multidriver dependency of FCH4 and reduce the internal complexity of the algorithmic structures. We applied this approach to five benchmark FCH4 datasets from both natural and managed systems located in temperate and tropical wetlands and rice paddies. Results indicate that PCA improved the performance of MDS compared to traditional inputs. ML algorithms performed better when using all available biophysical variables compared to using PCA-derived inputs. Overall, RF was found to outperform other techniques for all sites. We found gap-filling uncertainty is much larger than measurement uncertainty in accumulated CH4 budget. Therefore, the approach used for FCH4 gap filling can have important implications for characterizing annual ecosystem-scale methane budgets, the accuracy of which is important for evaluating natural and managed systems and their interactions with global change processes.

Chronic oral toxicity of 3-nitro-1,2,4-triazol-5-one (NTO) in rats.

To evaluate the effects of chronic exposure to 3-nitro-1,2,4-triazol-5-one (nitrotriazolone, NTO), male and female rats were given ad libitum access to NTO in drinking water at concentrations of 0, 36, 110, 360, 1100, and 3600 mg/L for one year. NTO did not affect body weight, body weight gain, or food consumption in either sex. No treatment-related effects were observed in clinical chemistry and hematology parameters at the 6 month or one year sampling. At both the interim and final sampling, males and females from the 3600 mg/L group produced smaller volumes of urine that was darker, more concentrated, and contained more bilirubin than the controls. Total and motile sperm counts were not affected by NTO treatment. Absolute and relative organ weights did not differ between control and NTO treated groups for either sex. Spontaneous age-related neoplasms occurred in controls and NTO groups at rates consistent with published historic controls. NTO was generally non-toxic in females at the doses tested. Toxicity in males was limited to testicular toxicity as demonstrated in previous studies. Chronic exposure did not result in testicular toxicity at lower doses and the toxicity observed only in the high dose group in this study is less severe than that observed in shorter exposures of previous studies, suggesting differences may be associated with influences of study design on kinetics. A Benchmark Dose (BMD) of 1604 mg/L (76 mg/kg-day) and a Benchmark Dose Lower Bound (BMDL10) of 921 mg/L (44 mg/kg-day) were determined for chronic effects of NTO in male rats.

The Aer2 receptor from Vibrio cholerae is a dual PAS-heme oxygen sensor.

The diarrheal pathogen Vibrio cholerae navigates complex environments using three chemosensory systems and 44-45 chemoreceptors. Chemosensory cluster II modulates chemotaxis, whereas clusters I and III have unknown functions. Ligands have been identified for only five V. cholerae chemoreceptors. Here, we report that the cluster III receptor, VcAer2, binds and responds to O2 . VcAer2 is an ortholog of Pseudomonas aeruginosa Aer2 (PaAer2) but differs in that VcAer2 has two, rather than one, N-terminal PAS domain. We have determined that both PAS1 and PAS2 form homodimers and bind penta-coordinate b-type heme via an Eη-His residue. Heme binding to PAS1 required the entire PAS core, but receptor function also required the N-terminal cap. PAS2 functioned as an O2 -sensor [ K d( O 2 ) , 19 µM], utilizing the same Iß Trp (W276) as PaAer2 to stabilize O2 . The crystal structure of PAS2-W276L was similar to that of PaAer2-PAS but resided in an active conformation mimicking the ligand-bound state, consistent with its signal-on phenotype. PAS1 also bound O2 [ K d( O 2 ) , 12 µM], although O2 binding was stabilized by either a Trp residue or Tyr residue. Moreover, PAS1 appeared to function as a signal modulator, regulating O2 -mediated signaling from PAS2 and resulting in activation of the cluster III chemosensory pathway.

Evolution and functional classification of mammalian copper amine oxidases.

Mammalian copper-containing amine oxidases (CAOs), encoded by four genes (AOC1-4) and catalyzing the oxidation of primary amines to aldehydes, regulate many biological processes and are linked to various diseases including inflammatory conditions and histamine intolerance. Despite the known differences in their substrate preferences, CAOs are currently classified based on their preference for either primary monoamines (EC 1.4.3.21) or diamines (EC 1.4.3.22). Here, we present the first extensive phylogenetic study of CAOs that, combined with structural analyses of the CAO active sites, provides in-depth knowledge of their relationships and guidelines for classification of mammalian CAOs into AOC1-4 sub-families. The phylogenetic results show that CAOs can be classified based on two residues, X1 and X2, from the active site motif: T/S-X1-X2-N-Y-D. Residue X2 discriminates among the AOC1 (Tyr), AOC2 (Gly), and AOC3/AOC4 (Leu) proteins, while residue X1 further classifies the AOC3 (Leu) and AOC4 (Met) proteins that so far have been poorly identified and annotated. Residues X1 and X2 conserved within each sub-family and located in the catalytic site seem to be the key determinants for the unique substrate preference of each CAO sub-family. Furthermore, one residue located at 10 Šdistance from the catalytic site is different between the sub-families but highly conserved within each sub-family (Asp in AOC1, His in AOC2, Thr in AOC3 and Asn in AOC4) and likely contributes to substrate selectivity. Altogether, our results will benefit the design of new sub-family specific inhibitors and the design of in vitro tests to detect individual CAO levels for diagnostic purposes.

Radiative forcing of methane fluxes offsets net carbon dioxide uptake for a tropical flooded forest.

Wetlands are important sources of methane (CH4 ) and sinks of carbon dioxide (CO2 ). However, little is known about CH4 and CO2 fluxes and dynamics of seasonally flooded tropical forests of South America in relation to local carbon (C) balances and atmospheric exchange. We measured net ecosystem fluxes of CH4 and CO2 in the Pantanal over 2014-2017 using tower-based eddy covariance along with C measurements in soil, biomass and water. Our data indicate that seasonally flooded tropical forests are potentially large sinks for CO2 but strong sources of CH4 , particularly during inundation when reducing conditions in soils increase CH4 production and limit CO2 release. During inundation when soils were anaerobic, the flooded forest emitted 0.11 ± 0.002 g CH4 -C m-2  d-1 and absorbed 1.6 ± 0.2 g CO2 -C m-2  d-1 (mean ± 95% confidence interval for the entire study period). Following the recession of floodwaters, soils rapidly became aerobic and CH4 emissions decreased significantly (0.002 ± 0.001 g CH4 -C m-2  d-1 ) but remained a net source, while the net CO2 flux flipped from being a net sink during anaerobic periods to acting as a source during aerobic periods. CH4 fluxes were 50 times higher in the wet season; DOC was a minor component in the net ecosystem carbon balance. Daily fluxes of CO2 and CH4 were similar in all years for each season, but annual net fluxes varied primarily in relation to flood duration. While the ecosystem was a net C sink on an annual basis (absorbing 218 g C m-2 (as CH4 -C + CO2 -C) in anaerobic phases and emitting 76 g C m-2 in aerobic phases), high CH4 effluxes during the anaerobic flooded phase and modest CH4 effluxes during the aerobic phase indicate that seasonally flooded tropical forests can be a net source of radiative forcings on an annual basis, thus acting as an amplifying feedback on global warming.

Water quality and greenhouse gas fluxes for stormwater detained in a constructed wetland.

Constructed wetlands (CWs) have been extensively used to deal with stormwater runoff and prevent flooding, as well as to improve water quality. However, some studies indicate that runoff from CWs can be a source of greenhouse gas (GHG) emissions to the atmosphere, as well as have increased levels of organic matter and nitrogen that may affect the environment and water quality of receiving waterways. We collected water samples at five different locations within a constructed stormwater detention wetland and determined dissolved organic carbon (DOC) and nitrate (NO3-), as well as carbon dioxide (CO2), methane (CH4) and nitrous oxide (N2O) concentrations and fluxes. Results showed that the water had high levels of DOC and NO3-. Analysis of DOC quality showed that the organic matter is mainly derived from soil and plant litter, but it has some contribution from microbial sources as well. Additionally, the water was supersaturated with CO2 and CH4 with respect to the atmosphere, which resulted in high evasion fluxes of 4.1 g CO2-C m-2 d-1 and 45 mg CH4-C m-2 d-1. N2O flux was low, 36.6 µg N2O-N m-2 d-1, and varied between uptake and emission throughout the study period. Consideration of water quality and GHG emissions is key to evaluate the environmental performance of stormwater management systems.

Top surface blade residues and the central channel water molecules are conserved in every repeat of the integrin-like ß-propeller structures.

An integrin-like ß-propeller domain contains seven repeats of a four-stranded antiparallel ß-sheet motif (blades). Previously we described a 3D structural motif within each blade of the integrin-type ß-propeller. Here, we show unique structural links that join different blades of the ß-propeller structure, which together with the structural motif for a single blade are repeated in a ß-propeller to provide the functional top face of the barrel, found to be involved in protein-protein interactions and substrate recognition. We compare functional top face diagrams of the integrin-type ß-propeller domain and two non-integrin type ß-propeller domains of virginiamycin B lyase and WD Repeat-Containing Protein 5.

SUMOylation regulates nuclear accumulation and signaling activity of the soluble intracellular domain of the ErbB4 receptor tyrosine kinase.

Erb-B2 receptor tyrosine kinase 4 (ErbB4) is a kinase that can signal via a proteolytically released intracellular domain (ICD) in addition to classical receptor tyrosine kinase-activated signaling cascades. Previously, we have demonstrated that ErbB4 ICD is posttranslationally modified by the small ubiquitin-like modifier (SUMO) and functionally interacts with the PIAS3 SUMO E3 ligase. However, direct evidence of SUMO modification in ErbB4 signaling has remained elusive. Here, we report that the conserved lysine residue 714 in the ErbB4 ICD undergoes SUMO modification, which was reversed by sentrin-specific proteases (SENPs) 1, 2, and 5. Although ErbB4 kinase activity was not necessary for the SUMOylation, the SUMOylated ErbB4 ICD was tyrosine phosphorylated to a higher extent than unmodified ErbB4 ICD. Mutation of the SUMOylation site compromised neither ErbB4-induced phosphorylation of the canonical signaling pathway effectors Erk1/2, Akt, or STAT5 nor ErbB4 stability. In contrast, SUMOylation was required for nuclear accumulation of the ErbB4 ICD. We also found that Lys-714 was located within a leucine-rich stretch, which resembles a nuclear export signal, and could be inactivated by site-directed mutagenesis. Furthermore, SUMOylation modulated the interaction of ErbB4 with chromosomal region maintenance 1 (CRM1), the major nuclear export receptor for proteins. Finally, the SUMO acceptor lysine was functionally required for ErbB4 ICD-mediated inhibition of mammary epithelial cell differentiation in a three-dimensional cell culture model. Our findings indicate that a SUMOylation-mediated mechanism regulates nuclear localization and function of the ICD of ErbB4 receptor tyrosine kinase.

Multiple environmental stressors induce complex transcriptomic responses indicative of phenotypic outcomes in Western fence lizard.

BACKGROUND: The health and resilience of species in natural environments is increasingly challenged by complex anthropogenic stressor combinations including climate change, habitat encroachment, and chemical contamination. To better understand impacts of these stressors we examined the individual- and combined-stressor impacts of malaria infection, food limitation, and 2,4,6-trinitrotoluene (TNT) exposures on gene expression in livers of Western fence lizards (WFL, Sceloporus occidentalis) using custom WFL transcriptome-based microarrays. RESULTS: Computational analysis including annotation enrichment and correlation analysis identified putative functional mechanisms linking transcript expression and toxicological phenotypes. TNT exposure increased transcript expression for genes involved in erythropoiesis, potentially in response to TNT-induced anemia and/or methemoglobinemia and caused dose-specific effects on genes involved in lipid and overall energy metabolism consistent with a hormesis response of growth stimulation at low doses and adverse decreases in lizard growth at high doses. Functional enrichment results were indicative of inhibited potential for lipid mobilization and catabolism in TNT exposures which corresponded with increased inguinal fat weights and was suggestive of a decreased overall energy budget. Malaria infection elicited enriched expression of multiple immune-related functions likely corresponding to increased white blood cell (WBC) counts. Food limitation alone enriched functions related to cellular energy production and decreased expression of immune responses consistent with a decrease in WBC levels. CONCLUSIONS: Despite these findings, the lizards demonstrated immune resilience to malaria infection under food limitation with transcriptional results indicating a fully competent immune response to malaria, even under bio-energetic constraints. Interestingly, both TNT and malaria individually increased transcriptional expression of immune-related genes and increased overall WBC concentrations in blood; responses that were retained in the TNT x malaria combined exposure. The results demonstrate complex and sometimes unexpected responses to multiple stressors where the lizards displayed remarkable resiliency to the stressor combinations investigated.

Concept for maritime near-surface surveillance using water Raman scattering.

We discuss a maritime surveillance and detection concept based on Raman scattering of water molecules. Using a range-gated scanning lidar that detects Raman scattered photons from water, the absence or change of signal indicates the presence of a non-water object. With sufficient spatial resolution, a two-dimensional outline of the object can be generated by the scanning lidar. Because Raman scattering is an inelastic process with a relatively large wavelength shift for water, this concept avoids the often problematic elastic scattering for objects at or very close to the water surface or from the bottom surface for shallow waters. The maximum detection depth for this concept is limited by the attenuation of the excitation and return Raman light in water. If excitation in the UV is used, fluorescence can be used for discrimination between organic and non-organic objects. In this paper, we present a lidar model for this concept and discuss results of proof-of-concept measurements. Using published cross section values, the model and measurements are in reasonable agreement and show that a sufficient number of Raman photons can be generated for modest lidar parameters to make this concept useful for near-surface detection.

Neuromuscular anomalies following oral exposure to 3-nitro-1,2,4-triazol-5-one (NTO) in a one-generation study with Japanese quail (Coturnix japonica).

Substances used as explosives in munitions by the military often result in environmental releases through manufacturing, testing, training, and combat activities. The toxicity of 3-nitro-1,2,4-triazol-5-one (nitrotriazolone or NTO) was evaluated following oral exposure in Japanese quail (Coturnix japonica) to determine if environmental releases result in unacceptable risks to avian populations. In an acute test at the limit dose (2000 mg/kg), one female was ataxic, exhibited tremors, and showed signs of neurological toxicity approximately 24 h after dosing. In a subsequent one-generation study, parental generation (F0) birds were exposed orally to 1000, 500, 100, or 20mg/kg-day NTO suspended in corn oil. After 5 consecutive days of dosing, 2-week-old birds receiving 1000 mg/kg-day displayed ataxia, convulsions, backward arching of the neck (opisthotonos), and alternated between prostrate inactivity and ataxic wing activity. Birds in the 500 mg/kg-day group exhibited neuromuscular anomalies after 17 days exposure. Ultimately, all of the 1000 mg/kg-day birds and all but one of the 500 mg/kg-day birds met euthanasia criteria and were humanely euthanized prior to behavioral and reproductive evaluation. As such, first-generation (F1) birds were exposed to 100 or 20 mg/kg-day NTO. Mild neuromuscular anomalies occurred in 10% of F1 birds from the 100 mg/kg-day group, but not in birds from 20 mg/kg-day or controls in either generation. Vacuolization of cerebellum and/or the brainstem was observed on histopathologic examination in a dose-dependent manner. Therefore, brain vacuoles and neuromuscular anomalies were identified as critical endpoints in this study. A mean Benchmark Dose (BMD) for brain vacuoles of 62 mg/kg-day was derived for male and female F0-generation quail, which corresponded to a Benchmark Dose Low (BMDL10) of 35 mg/kg-day.

Status of acute systemic toxicity testing requirements and data uses by U.S. regulatory agencies.

Acute systemic toxicity data are used by a number of U.S. federal agencies, most commonly for hazard classification and labeling and/or risk assessment for acute chemical exposures. To identify opportunities for the implementation of non-animal approaches to produce these data, the regulatory needs and uses for acute systemic toxicity information must first be clarified. Thus, we reviewed acute systemic toxicity testing requirements for six U.S. agencies (Consumer Product Safety Commission, Department of Defense, Department of Transportation, Environmental Protection Agency, Food and Drug Administration, Occupational Safety and Health Administration) and noted whether there is flexibility in satisfying data needs with methods that replace or reduce animal use. Understanding the current regulatory use and acceptance of non-animal data is a necessary starting point for future method development, optimization, and validation efforts. The current review will inform the development of a national strategy and roadmap for implementing non-animal approaches to assess potential hazards associated with acute exposures to industrial chemicals and medical products. The Acute Toxicity Workgroup of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), U.S. agencies, non-governmental organizations, and other stakeholders will work to execute this strategy.

Biochar feedstock and pyrolysis temperature effects on leachate: DOC characteristics and nitrate losses from a Brazilian Cerrado Arenosol mixed with agricultural waste biochars.

Dissolved organic carbon (DOC) leached from Brazilian Cerrado Arenosols can lead to carbon (C) losses and lower soil fertility, while excessive nutrient, e.g. nitrate (NO3-), leaching can potentially cause water contamination. As biochar has been shown to stabilize C and retain soil nutrients, a greenhouse experiment was conducted to test different biochars' contributions to DOC and NO3- leaching from a sandy soil. Biochars were made from four local agricultural waste feedstocks (cotton residue, swine manure, eucalyptus sawmill residue, sugarcane filtercake) pyrolysed at 400, 500 and 600 °C. Biochar was mixed with soil at 5% weight in pots and maize seeds planted. Leachate was collected weekly for six weeks and analyzed for DOC and NO3- concentrations, while fluorescence spectroscopy with parallel factor analysis (PARAFAC) was used to interpret DOC characteristics. Cotton and swine manure biochar treatments had higher DOC and NO3- losses than eucalyptus biochar, filtercake biochar, and control treatments. Cotton and swine manure biochar treatments at high temperatures lost mostly terrestrial, humified DOC, while swine manure, filtercake, and eucalyptus biochars at low temperatures lost mostly labile, microbially-derived DOC. Through the practical use of fluorescence spectroscopy, our study identified filtercake and eucalyptus biochars as most promising for retaining DOC and NO3- in a Cerrado Arenosol, potentially reducing stable C and nutrient losses.

Gas Sensing and Signaling in the PAS-Heme Domain of the Pseudomonas aeruginosa Aer2 Receptor.

The Aer2 chemoreceptor from Pseudomonas aeruginosa contains a PAS sensing domain that coordinates b-type heme and signals in response to the binding of O2, CO, or NO. PAS-heme structures suggest that Aer2 uniquely coordinates heme via a His residue on a 310 helix (H234 on Eη), stabilizes O2 binding via a Trp residue (W283), and signals via both W283 and an adjacent Leu residue (L264). Ligand binding may displace L264 and reorient W283 for hydrogen bonding to the ligand. Here, we clarified the mechanisms by which Aer2-PAS binds heme, regulates ligand binding, and initiates conformational signaling. H234 coordinated heme, but additional hydrophobic residues in the heme cleft were also critical for stable heme binding. O2 appeared to be the native Aer2 ligand (dissociation constant [Kd ] of 16 µM). With one exception, mutants that bound O2 could signal, whereas many mutants that bound CO could not. W283 stabilized O2 binding but not CO binding, and it was required for signal initiation; W283 mutants that could not stabilize O2 were rapidly oxidized to Fe(III). W283F was the only Trp mutant that bound O2 with wild-type affinity. The size and nature of residue 264 was important for gas binding and signaling: L264W blocked O2 binding, L264A and L264G caused O2-mediated oxidation, and L264K formed a hexacoordinate heme. Our data suggest that when O2 binds to Aer2, L264 moves concomitantly with W283 to initiate the conformational signal. The signal then propagates from the PAS domain to regulate the C-terminal HAMP and kinase control domains, ultimately modulating a cellular response.IMPORTANCEPseudomonas aeruginosa is a ubiquitous environmental bacterium and opportunistic pathogen that infects multiple body sites, including the lungs of cystic fibrosis patients. P. aeruginosa senses and responds to its environment via four chemosensory systems. Three of these systems regulate biofilm formation, twitching motility, and chemotaxis. The role of the fourth system, Che2, is unclear but has been implicated in virulence. The Che2 system contains a chemoreceptor called Aer2, which contains a PAS sensing domain that binds heme and senses oxygen. Here, we show that Aer2 uses unprecedented mechanisms to bind O2 and initiate signaling. These studies provide both the first functional corroboration of the Aer2-PAS signaling mechanism previously proposed from structure as well as a signaling model for Aer2-PAS receptors.

Alpha/beta-hydrolases: A unique structural motif coordinates catalytic acid residue in 40 protein fold families.

The alpha/beta-hydrolases are a family of acid-base-nucleophile catalytic triad enzymes with a common fold, but using a wide variety of substrates, having different pH optima, catalyzing unique catalytic reactions and often showing improved chemical and thermo stability. The ABH enzymes are prime targets for protein engineering. Here, we have classified active sites from 51 representative members of 40 structural ABH fold families into eight distinct conserved geometries. We demonstrate the occurrence of a common structural motif, the catalytic acid zone, at the catalytic triad acid turn. We show that binding of an external ligand does not change the structure of the catalytic acid zone and both the ligand-free and ligand-bound forms of the protein belong to the same catalytic acid zone subgroup. We also show that the catalytic acid zone coordinates the position of the catalytic histidine loop directly above its plane, and consequently, fixes the catalytic histidine in a proper position near the catalytic acid. Finally, we demonstrate that the catalytic acid zone plays a key role in multi-subunit complex formation in ABH enzymes, and is involved in interactions with other proteins. As a result, we speculate that each of the catalytic triad residues has its own supporting structural scaffold, similar to the catalytic acid zone, described above, which together form the extended catalytic triad motif. Each scaffold coordinates the function of its respective catalytic residue, and can even compensate for the loss of protein function, if the catalytic amino acid is mutated.

Delineating PAS-HAMP interaction surfaces and signalling-associated changes in the aerotaxis receptor Aer.

The Escherichia coli aerotaxis receptor, Aer, monitors cellular oxygen and redox potential via FAD bound to a cytosolic PAS domain. Here, we show that Aer-PAS controls aerotaxis through direct, lateral interactions with a HAMP domain. This contrasts with most chemoreceptors where signals propagate along the protein backbone from an N-terminal sensor to HAMP. We mapped the interaction surfaces of the Aer PAS, HAMP and proximal signalling domains in the kinase-off state by probing the solvent accessibility of 129 cysteine substitutions. Inaccessible PAS-HAMP surfaces overlapped with a cluster of PAS kinase-on lesions and with cysteine substitutions that crosslinked the PAS ß-scaffold to the HAMP AS-2 helix. A refined Aer PAS-HAMP interaction model is presented. Compared to the kinase-off state, the kinase-on state increased the accessibility of HAMP residues (apparently relaxing PAS-HAMP interactions), but decreased the accessibility of proximal signalling domain residues. These data are consistent with an alternating static-dynamic model in which oxidized Aer-PAS interacts directly with HAMP AS-2, enforcing a static HAMP domain that in turn promotes a dynamic proximal signalling domain, resulting in a kinase-off output. When PAS-FAD is reduced, PAS interaction with HAMP is relaxed and a dynamic HAMP and static proximal signalling domain convey a kinase-on output.