Structures and solution properties of two novel periplasmic sensor domains with c-type heme from chemotaxis proteins of Geobacter sulfurreducens: implications for signal transduction

Periplasmic sensor domains from two methyl-accepting chemotaxis proteins from Geobacter sulfurreducens (encoded by genes GSU0935 and GSU0582) were expressed in Escherichia coli. The sensor domains were isolated, purified, characterized in solution, and their crystal structures were determined. In the crystal, both sensor domains form swapped dimers and show a PAS-type fold. The swapped segment consists of two helices of about 45 residues at the N terminus with the hemes located between the two monomers. In the case of the GSU0582 sensor, the dimer contains a crystallographic 2-fold symmetry and the heme is coordinated by an axial His and a water molecule. In the case of the GSU0935 sensor, the crystals contain a non-crystallographic dimer, and surprisingly, the coordination of the heme in each monomer is different; monomer A heme has His-Met ligation and monomer B heme has His-water ligation as found in the GSU0582 sensor. The structures of these sensor domains are the first structures of PAS domains containing covalently bound heme. Optical absorption, electron paramagnetic resonance and NMR spectroscopy have revealed that the heme groups of both sensor domains are high-spin and low-spin in the oxidized and reduced forms, respectively, and that the spin-state interconversion involves a heme axial ligand replacement. Both sensor domains bind NO in their ferric and ferrous forms but bind CO only in the reduced form. The binding of both NO and CO occurs via an axial ligand exchange process, and is fully reversible. The reduction potentials of the sensor domains differ by 95 mV (− 156 mV and − 251 mV for sensors GSU0582 and GSU0935, respectively). The swapped dimerization of these sensor domains and redox-linked ligand switch might be related to the mechanism of signal transduction by these chemotaxis proteins.     

Predicting loop–helix tertiary structural contacts in RNA pseudoknots

Tertiary interactions between loops and helical stems play critical roles in the biological function of many RNA pseudoknots. However, quantitative predictions for RNA tertiary interactions remain elusive. Here we report a statistical mechanical model for the prediction of noncanonical loop–stem base-pairing interactions in RNA pseudoknots. Central to the model is the evaluation of the conformational entropy for the pseudoknotted folds with defined loop–stem tertiary structural contacts. We develop an RNA virtual bond-based conformational model (Vfold model), which permits a rigorous computation of the conformational entropy for a given fold that contains loop–stem tertiary contacts. With the entropy parameters predicted from the Vfold model and the energy parameters for the tertiary contacts as inserted parameters, we can then predict the RNA folding thermodynamics, from which we can extract the tertiary contact thermodynamic parameters from theory–experimental comparisons. These comparisons reveal a contact enthalpy (ΔH) of −14 kcal/mol and a contact entropy (ΔS) of −38 cal/mol/K for a protonated C⁺•(G–C) base triple at pH 7.0, and (ΔH = −7 kcal/mol, ΔS = −19 cal/mol/K) for an unprotonated base triple. Tests of the model for a series of pseudoknots show good theory–experiment agreement. Based on the extracted energy parameters for the tertiary structural contacts, the model enables predictions for the structure, stability, and folding pathways for RNA pseudoknots with known or postulated loop–stem tertiary contacts from the nucleotide sequence alone.     

Requirement for canonical base pairing in the short pseudoknot structure of genomic hepatitis delta virus ribozyme

The tertiary structure of the 3′-cleaved product of the genomic hepatitis delta virus (HDV) ribozyme was solved by X-ray crystallographic analysis. In this structure, three single-stranded regions (SSrA, -B and -C) interact intricately with one another via hydrogen bonds between nucleotide bases, phosphate oxygens and 2′-OHs to form a nested double pseudoknot structure. Among these interactions, two Watson–Crick (W–C) base pairs, 726G–710C and 727G–709C, that form between SSrA and SSrC (P1.1) seem to be especially important for compact folding. To characterize the importance of these base pairs, ribozymes were subjected to in vitro selection from a pool of RNA molecules randomly substituted at positions 709, 710, 726 and 727. The results establish the importance of the two W–C base pairs for activity, although some mutants are active with one G–C base pair. In addition, the kinetic parameters were analyzed in all 16 combinations with two canonical base pairs. Comparison of variant ribozymes with the wild-type ribozyme reveals that the difference in reaction rates for these variants (ΔΔG^‡) is not simply accounted for by the differences in the stability of P1.1 (ΔΔG⁰₃₇). The role played by Mg²⁺ ions in formation of the P1.1 structure is also discussed.     

Relationship between Intracellular Phosphate, Proton Motive Force, and Rate of Nongrowth Energy Dissipation (Energy Spilling) in Streptococcus bovis JB1

When the rate of glucose addition to nongrowing Streptococcus bovis cell suspensions was increased, the fermentation was homolactic, fructose-1,6-diphosphate (FDP) increased, intracellular inorganic phosphate (P_i) declined, and the energy-spilling rate increased. ATP and ADP were not significantly affected by glucose consumption rate, but the decrease in P_i was sufficient to cause an increase in the free energy of ATP hydrolysis (ΔG′p). The increase in ΔG′p was correlated with an increase in proton motive force (Δp). S. bovis continuous cultures (dilution rate of 0.65 h⁻¹) that were provided with ammonia as the sole nitrogen source also had high rates of lactate production and energy spilling. When Trypticase was added as a source of amino acids, lactate production decreased; a greater fraction of the glucose was converted to acetate, formate, and ethanol; and the energy-spilling rate decreased. Trypticase also caused a decrease in FDP, an increase in P_i, and a decrease in Δp. The change in Δp could be explained by P_i-dependent changes in the ΔG′p. When P_i declined, ΔG′p and Δp increased. The ratio of ΔG′p to Δp (millivolt per millivolt) was always high (>4) at low rates of energy spilling but declined when the energy-spilling rate increased. Based on these results, it appears that Δp and the energy-spilling rate are responsive to fluctuations in the intracellular P_i concentration.     

Heat capacity changes in RNA folding: application of perturbation theory to hammerhead ribozyme cold denaturation

In proteins, empirical correlations have shown that changes in heat capacity (ΔC_P) scale linearly with the hydrophobic surface area buried upon folding. The influence of ΔC_P on RNA folding has been widely overlooked and is poorly understood. In addition to considerations of solvent reorganization, electrostatic effects might contribute to ΔC_Ps of folding in polyanionic species such as RNAs. Here, we employ a perturbation method based on electrostatic theory to probe the hot and cold denaturation behavior of the hammerhead ribozyme. This treatment avoids much of the error associated with imposing two-state folding models on non-two-state systems. Ribozyme stability is perturbed across a matrix of solvent conditions by varying the concentration of NaCl and methanol co-solvent. Temperature-dependent unfolding is then monitored by circular dichroism spectroscopy. The resulting array of unfolding transitions can be used to calculate a ΔC_P of folding that accurately predicts the observed cold denaturation temperature. We confirm the accuracy of the calculated ΔC_P by using isothermal titration calorimetry, and also demonstrate a methanol-dependence of the ΔC_P. We weigh the strengths and limitations of this method for determining ΔC_P values. Finally, we discuss the data in light of the physical origins of the ΔC_Ps for RNA folding and consider their impact on biological function.     

Increased Circulating Endothelial Apoptotic Microparticle to Endothelial Progenitor Cell Ratio Is Associated with Subsequent Decline in Glomerular Filtration Rate in Hypertensive Patients

Background

Recent research indicates hypertensive patients with microalbuminuria have decreased endothelial progenitor cells (EPCs) and increased levels of endothelial apoptotic microparticles (EMP). However, whether these changes are related to a subsequent decline in glomerular filtration rate (GFR) remains unclear.

Methods and Results

We enrolled totally 100 hypertensive out-patients with eGFR ≥30 mL/min/1.73 m². The mean annual rate of GFR decline (△GFR/y) was −1.49±3.26 mL/min/1.73 m² per year during the follow-up period (34±6 months). Flow cytometry was used to assess circulating EPC (CD34⁺/KDR⁺) and EMP levels (CD31⁺/annexin V⁺) in peripheral blood. The △GFR/y was correlated with the EMP to EPC ratio (r = −0.465, p<0.001), microalbuminuria (r = −0.329, p = 0.001), and the Framingham risk score (r = −0.245, p = 0.013). When we divided the patients into 4 groups according to the EMP to EPC ratio, there was an association between the EMP to EPC ratio and the ΔGFR/y (mean ΔGFR/y: 0.08±3.04 vs. −0.50±2.84 vs. −1.25±2.49 vs. −4.42±2.82, p<0.001). Multivariate analysis indicated that increased EMP to EPC ratio is an independent predictor of ΔeGFR/y.

Conclusions

An increased circulating EMP to EPC ratio is associated with subsequent decline in GFR in hypertensive patients, which suggests endothelial damage with reduced vascular repair capacity may contribute to further deterioration of renal function in patients with hypertension.     

Probing the Interaction of a Therapeutic Flavonoid,Pinostrobin with Human Serum Albumin: Multiple Spectroscopic and Molecular Modeling Investigations

Interaction of a pharmacologically important flavonoid, pinostrobin (PS) with the major transport protein of human blood circulation, human serum albumin (HSA) has been examined using a multitude of spectroscopic techniques and molecular docking studies. Analysis of the fluorescence quenching data showed a moderate binding affinity (1.03 × 10⁵ M⁻¹ at 25°C) between PS and HSA with a 1∶1 stoichiometry. Thermodynamic analysis of the binding data (ΔS = +44.06 J mol⁻¹ K⁻¹ and ΔH = −15.48 kJ mol⁻¹) and molecular simulation results suggested the involvement of hydrophobic and van der Waals forces, as well as hydrogen bonding in the complex formation. Both secondary and tertiary structural perturbations in HSA were observed upon PS binding, as revealed by intrinsic, synchronous, and three-dimensional fluorescence results. Far-UV circular dichroism data revealed increased thermal stability of the protein upon complexation with PS. Competitive drug displacement results suggested the binding site of PS on HSA as Sudlow’s site I, located at subdomain IIA, and was well supported by the molecular modelling data.     

Pathway Thermodynamics Highlights Kinetic Obstacles in Central Metabolism

In metabolism research, thermodynamics is usually used to determine the directionality of a reaction or the feasibility of a pathway. However, the relationship between thermodynamic potentials and fluxes is not limited to questions of directionality: thermodynamics also affects the kinetics of reactions through the flux-force relationship, which states that the logarithm of the ratio between the forward and reverse fluxes is directly proportional to the change in Gibbs energy due to a reaction (Δ_rG′). Accordingly, if an enzyme catalyzes a reaction with a Δ_rG′ of -5.7 kJ/mol then the forward flux will be roughly ten times the reverse flux. As Δ_rG′ approaches equilibrium (Δ_rG′ = 0 kJ/mol), exponentially more enzyme counterproductively catalyzes the reverse reaction, reducing the net rate at which the reaction proceeds. Thus, the enzyme level required to achieve a given flux increases dramatically near equilibrium. Here, we develop a framework for quantifying the degree to which pathways suffer these thermodynamic limitations on flux. For each pathway, we calculate a single thermodynamically-derived metric (the Max-min Driving Force, MDF), which enables objective ranking of pathways by the degree to which their flux is constrained by low thermodynamic driving force. Our framework accounts for the effect of pH, ionic strength and metabolite concentration ranges and allows us to quantify how alterations to the pathway structure affect the pathway''s thermodynamics. Applying this methodology to pathways of central metabolism sheds light on some of their features, including metabolic bypasses (e.g., fermentation pathways bypassing substrate-level phosphorylation), substrate channeling (e.g., of oxaloacetate from malate dehydrogenase to citrate synthase), and use of alternative cofactors (e.g., quinone as an electron acceptor instead of NAD). The methods presented here place another arrow in metabolic engineers'' quiver, providing a simple means of evaluating the thermodynamic and kinetic quality of different pathway chemistries that produce the same molecules.     

Truncation of a β-Barrel Scaffold Dissociates Intrinsic Stability from Its Propensity to Aggregation

Δ98Δ is a functional all-β sheet variant of intestinal fatty acid binding protein (IFABP) that was generated by controlled proteolysis. This framework is useful to study the molecular determinants related to aggregation of β-barrel proteins. Albeit displaying increased conformational plasticity, Δ98Δ exhibits a nativelike β-barrel topology and is able to support a cooperative folding behavior. Here we present a comparative study of IFABP and Δ98Δ regarding their conformational perturbation and aggregation propensity triggered by trifluoroethanol. Both proteins share a common nucleation-elongation mechanism, whereby the rate-limiting step is the formation of stable dimeric nuclei followed by the association of monomers to the growing aggregates. Despite leading to a less stable structure, the extensive truncation of IFABP yields a form exhibiting a somewhat lower tendency to aggregate. This finding appears at odds with the established notion that a perturbation of the native compact fold should necessarily favor the population of aggregation-prone species. In addition to the aggregation propensity dictated by a given amino-acid sequence, our contention holds that long-range interactions might also play a major role in determining the overall aggregation propensity.     

Interplay between ER Exit Code and Domain Conformation in CFTR Misprocessing and Rescue

Multiple mutations in cystic fibrosis transmembrane conductance regulator (CFTR) impair its exit from the endoplasmic reticulum (ER). We compared two processing mutants: ΔF508 and the ER exit code mutant DAA. Although both have severe kinetic processing defect, DAA but not ΔF508 has substantial accumulation in its mature form, leading to higher level of processing at the steady state. DAA has much less profound conformational abnormalities. It has lower Hsp70 association and higher post-ER stability than ΔF508. The ER exit code is necessary for ΔF508 residual export and rescue. R555K, a mutation that rescues ΔF508 misprocessing, improves Sec24 association and enhances its post-ER stability. Using in situ limited proteolysis, we demonstrated a clear change in trypsin sensitivity in ΔF508 NBD1, which is reversed, together with that of other domains, by low temperature, R555K or both. We observed a conversion of the proteolytic pattern of DAA from the one resembling ΔF508 to the one similar to wild-type CFTR during its maturation. Low temperature and R555K are additive in improving ΔF508 conformational maturation and processing. Our data reveal a dual contribution of ER exit code and domain conformation to CFTR misprocessing and underscore the importance of conformational repair in effective rescue of ΔF508.     

Heat Shock Protein 90 Associates with the Per-Arnt-Sim Domain of Heme-free Soluble Guanylate Cyclase: IMPLICATIONS FOR ENZYME MATURATION*

Heat shock protein 90 (hsp90) drives heme insertion into the β1 subunit of soluble guanylate cyclase (sGC) β1, which enables it to associate with a partner sGCα1 subunit and mature into a nitric oxide (NO)-responsive active form. We utilized fluorescence polarization measurements and hydrogen-deuterium exchange mass spectrometry to define molecular interactions between the specific human isoforms hsp90β and apo-sGCβ1. hsp90β and its isolated M domain, but not its isolated N and C domains, bind with low micromolar affinity to a heme-free, truncated version of sGCβ1 (sGCβ1(1–359)-H105F). Surprisingly, hsp90β and its M domain bound to the Per-Arnt-Sim (PAS) domain of apo-sGC-β1(1–359), which lies adjacent to its heme-binding (H-NOX) domain. The interaction specifically involved solvent-exposed regions in the hsp90β M domain that are largely distinct from sites utilized by other hsp90 clients. The interaction strongly protected two regions of the sGCβ1 PAS domain and caused local structural relaxation in other regions, including a PAS dimerization interface and a segment in the H-NOX domain. Our results suggest a means by which the hsp90β interaction could prevent apo-sGCβ1 from associating with its partner sGCα1 subunit while enabling structural changes to assist heme insertion into the H-NOX domain. This mechanism would parallel that in other clients like the aryl hydrocarbon receptor and HIF1α, which also interact with hsp90 through their PAS domains to control protein partner and small ligand binding interactions.     

Dynamics and Sources of Soil Organic C Following Afforestation of Croplands with Poplar in a Semi-Arid Region in Northeast China

Afforestation of former croplands has been proposed as a promising way to mitigate rising atmospheric CO₂ concentration in view of the commitment to the Kyoto Protocol. Central to this C sequestration is the dynamics of soil organic C (SOC) storage and stability with the development of afforested plantations. Our previous study showed that SOC storage was not changed after afforestation except for the 0–10 cm layer in a semi-arid region of Keerqin Sandy Lands, northeast China. In this study, soil organic C was further separated into light and heavy fractions using the density fractionation method, and their organic C concentration and ¹³C signature were analyzed to investigate the turnover of old vs. new SOC in the afforested soils. Surface layer (0–10 cm) soil samples were collected from 14 paired plots of poplar (Populus × xiaozhuanica W. Y. Hsu & Liang) plantations with different stand basal areas (the sum of the cross-sectional area of all live trees in a stand), ranging from 0.2 to 32.6 m² ha⁻¹, and reference maize (Zea mays L.) croplands at the same sites as our previous study. Soil Δ_C stocks (Δ_C refers to the difference in SOC content between a poplar plantation and the paired cropland) in bulk soil and light fraction were positively correlated with stand basal area (R ² = 0.48, p<0.01 and R ² = 0.40, p = 0.02, respectively), but not for the heavy fraction. SOC_crop (SOC derived from crops) contents in the light and heavy fractions in poplar plantations were significantly lower as compared with SOC contents in croplands, but tree-derived C in bulk soil, light and heavy fraction pools increased gradually with increasing stand basal area after afforestation. Our study indicated that cropland afforestation could sequester new C derived from trees into surface mineral soil, but did not enhance the stability of SOC due to a fast turnover of SOC in this semi-arid region.     

Timing and Intensity of Light Correlate with Body Weight in Adults

Light exposure can influence sleep and circadian timing, both of which have been shown to influence weight regulation. The goal of this study was to evaluate the relationship between ambient light, sleep and body mass index. Participants included 54 individuals (26 males, mean age 30.6, SD = 11.7 years). Light levels, sleep midpoint and duration were measured with wrist actigraphy (Actiwatch-L) for 7 days. BMI was derived from self-reported height and weight. Caloric intake was determined from 7 days of food logs. For each participant, light and activity data were output in 2 minute epochs, smoothed using a 5 point (10 minute) moving average and then aggregated over 24 hours. The mean light timing above 500 lux (MLiT⁵⁰⁰) was defined as the average clock time of all aggregated data points above 500 lux. MLiT⁵⁰⁰ was positively correlated with BMI (r = 0.51, p<0.001), and midpoint of sleep (r = 0.47, p<0.01). In a multivariable linear regression model including MLiT⁵⁰⁰ and midpoint of sleep, MLiT⁵⁰⁰ was a significant predictor of BMI (B = 1.26 SE = 0.34, β = 0.53 p = 0.001, r ² _Δ = 0.22). Adjusting for covariates, MLiT⁵⁰⁰ remained an independent predictor of BMI (B = 1.28 SE = 0.36, β = 0.54, p = 0.002, r ² _Δ = 0.20). The full model accounted for 34.7% of the variance in BMI (p = 0.01). Exposure to moderate levels of light at biologically appropriate times can influence weight, independent of sleep timing and duration.     

Studies on a Novel Serine Protease of a ΔhapAΔprtV Vibrio cholerae O1 Strain and Its Role in Hemorrhagic Response in the Rabbit Ileal Loop Model

Background

Two well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP) and V. cholerae protease (PrtV). The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL).

Methodology/Principal Findings

We partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/−0.3 n = 3), CHA6.8 (FA ratio 1.08+/−0.2 n = 3), CHA6.8ΔprtV (FA ratio 1.02+/−0.2 n = 3) and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/−0.3 n = 3) induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/−0.005 n = 3) and with protease incubated with PMSF and EDTA (FA ratio 0.3+/−0.05 n = 3) induced a significantly reduced FA ratio with almost complete normal villus structure.

Conclusion

Our results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model.     

Biophysical Analysis of the MHR Motif in Folding and Domain Swapping of the HIV Capsid Protein C-Terminal Domain

Infection by human immunodeficiency virus (HIV) depends on the function, in virion morphogenesis and other stages of the viral cycle, of a highly conserved structural element, the major homology region (MHR), within the carboxyterminal domain (CTD) of the capsid protein. In a modified CTD dimer, MHR is swapped between monomers. While no evidence for MHR swapping has been provided by structural models of retroviral capsids, it is unknown whether it may occur transiently along the virus assembly pathway. Whatever the case, the MHR-swapped dimer does provide a novel target for the development of anti-HIV drugs based on the concept of trapping a nonnative capsid protein conformation. We have carried out a thermodynamic and kinetic characterization of the domain-swapped CTD dimer in solution. The analysis includes a dissection of the role of conserved MHR residues and other amino acids at the dimerization interface in CTD folding, stability, and dimerization by domain swapping. The results revealed some energetic hotspots at the domain-swapped interface. In addition, many MHR residues that are not in the protein hydrophobic core were nevertheless found to be critical for folding and stability of the CTD monomer, which may dramatically slow down the swapping reaction. Conservation of MHR residues in retroviruses did not correlate with their contribution to domain swapping, but it did correlate with their importance for stable CTD folding. Because folding is required for capsid protein function, this remarkable MHR-mediated conformational stabilization of CTD may help to explain the functional roles of MHR not only during immature capsid assembly but in other processes associated with retrovirus infection. This energetic dissection of the dimerization interface in MHR-swapped CTD may also facilitate the design of anti-HIV compounds that inhibit capsid assembly by conformational trapping of swapped CTD dimers.     

Influence of PAS Domain Flanking Regions on Oligomerisation and Redox Signalling By NifL

Per-ARNT-Sim (PAS) domains constitute a typically dimeric, conserved α/β tertiary fold of approximately 110 amino acids that perform signalling roles in diverse proteins from all kingdoms of life. The amino terminal PAS1 domain of NifL from Azotobacter vinelandii accommodates a redox-active FAD group; elevation of cytosolic oxygen concentrations result in FAD oxidation and a concomitant conformational re-arrangement that is relayed via a short downstream linker to a second PAS domain, PAS2. At PAS2, the signal is amplified and passed on to effector domains generating the ‘on’ (inhibitory) state of the protein. Although the crystal structure of oxidised PAS1 reveals regions that contribute to the dimerisation interface, 21 amino acids at the extreme N-terminus of NifL, are unresolved. Furthermore, the structure and function of the linker between the two PAS domains has not been determined. In this study we have investigated the importance to signalling of residues extending beyond the core PAS fold. Our results implicate the N-terminus of PAS1 and the helical linker connecting the two PAS domains in redox signal transduction and demonstrate a role for these flanking regions in controlling the oligomerisation state of PAS1 in solution.