Calcium-Induced Folding of a Beta Roll Motif Requires C-Terminal Entropic Stabilization

Beta roll motifs are associated with several proteins secreted by the type 1 secretion system (T1SS). Located just upstream of the C-terminal T1SS secretion signal, they are believed to act as calcium-induced switches that prevent folding before secretion. Bordetella pertussis adenylate cyclase (CyaA) toxin has five blocks of beta roll motifs (or repeats-in-toxin motifs) separated by linkers. The block V motif on its own has been reported to be non-responsive to calcium. Only when the N- and C-terminal linkers, or flanking groups, were fused did the motif bind calcium and fold. In an effort to understand the requirements for beta roll folding, we have truncated the N- and C-terminal flanks at several locations to determine the minimal essential sequences. Calcium-responsive beta roll folding occurred even in the absence of the natural N-terminal flank. The natural C-terminal flank could not be truncated without decreased calcium affinity and only partially truncated before losing calcium-responsiveness. Globular protein fusion at the C-terminus likewise enabled calcium-induced folding but fusions solely at the N-terminus failed. This demonstrates that calcium-induced folding is an inherent property of the beta roll motif rather than the flanking groups. Given the disparate nature of the observed functional flanking groups, C-terminal fusions appear to confer calcium-responsiveness to the beta roll motif via a non-specific mechanism, suggesting that entropic stabilization of the unstructured C-terminus can enable beta roll folding. Increased calcium affinity was observed when the natural C-terminal flank was used to enable calcium-induced folding, pointing to its cooperative participation in beta roll formation. This work indicates that a general principle of C-terminal entropic stabilization can enable stimulus-responsive repeat protein folding, while the C-terminal flank has a specific role in tuning calcium-responsive beta roll formation. These observations are in stark contrast to what has been reported for other repeat proteins.     

Order-disorder-order transitions mediate the activation of cholera toxin

Cholera toxin (CT) holotoxin must be activated to intoxicate host cells. This process requires the intracellular dissociation of the enzymatic CTA1 domain from the holotoxin components CTA2 and B5, followed by subsequent interaction with the host factor ADP ribosylation factor 6 (ARF6)-GTP. We report the first NMR-based solution structural data for the CT enzymatic domain (CTA1). We show that this free enzymatic domain partially unfolds at the C-terminus and binds its protein partners at both the beginning and the end of this activation process. Deviations from random coil chemical shifts (Δδ_coil) indicate helix formation in the activation loop, which is essential to open the toxin's active site and occurs prior to its association with human protein ARF6. We performed NMR titrations of both free CTA1 and an active CTA1:ARF6-GTP complex with NAD⁺, which revealed that the formation of the complex does not significantly enhance NAD⁺ binding. Partial unfolding of CTA1 is further illustrated by using 4,4′-bis(1-anilinonaphthalene 8-sulfonate) fluorescence as an indicator of the exposed hydrophobic character of the free enzyme, which is substantially reduced when bound to ARF6-GTP. We propose that the primary role of ARF6's allostery is to induce refolding of the C-terminus of CTA1. Thus, as a folded globular toxin complex, CTA1 escapes the chaperone and proteasomal components of the endoplasmic reticulum associated degradation pathway in the cytosol and then proceeds to ADP ribosylate its target G_sα, triggering the downstream events associated with the pathophysiology of cholera.     

The Non-Core Regions of Human Lysozyme Amyloid Fibrils Influence Cytotoxicity

Identifying the cause of the cytotoxicity of species populated during amyloid formation is crucial to understand the molecular basis of protein deposition diseases. We have examined different types of aggregates formed by lysozyme, a protein found as fibrillar deposits in patients with familial systemic amyloidosis, by infrared spectroscopy, transmission electron microscopy, and depolymerization experiments, and analyzed how they affect cell viability. We have characterized two types of human lysozyme amyloid structures formed in vitro that differ in morphology, molecular structure, stability, and size of the cross-β core. Of particular interest is that the fibrils with a smaller core generate a significant cytotoxic effect. These findings indicate that protein aggregation can give rise to species with different degree of cytotoxicity due to intrinsic differences in their physicochemical properties.     

The role of a disulfide bridge in the stability and folding kinetics of Arabidopsis thaliana cytochrome c6A

Cytochrome c_6A is a eukaryotic member of the Class I cytochrome c family possessing a high structural homology with photosynthetic cytochrome c₆ from cyanobacteria, but structurally and functionally distinct through the presence of a disulfide bond and a heme mid-point redox potential of + 71 mV (vs normal hydrogen electrode). The disulfide bond is part of a loop insertion peptide that forms a cap-like structure on top of the core α-helical fold. We have investigated the contribution of the disulfide bond to thermodynamic stability and (un)folding kinetics in cytochrome c_6A from Arabidopsis thaliana by making comparison with a photosynthetic cytochrome c₆ from Phormidium laminosum and through a mutant in which the Cys residues have been replaced with Ser residues (C67/73S). We find that the disulfide bond makes a significant contribution to overall stability in both the ferric and ferrous heme states. Both cytochromes c_6A and c₆ fold rapidly at neutral pH through an on-pathway intermediate. The unfolding rate for the C67/73S variant is significantly increased indicating that the formation of this region occurs late in the folding pathway. We conclude that the disulfide bridge in cytochrome c_6A acts as a conformational restraint in both the folding intermediate and native state of the protein and that it likely serves a structural rather than a previously proposed catalytic role.     

Resistance to ciprofloxacin by enhancement of antioxidant defenses in biofilm and planktonic Proteus mirabilis

Antibiotic resistance and antioxidant defense were induced by ciprofloxacin in planktonic Proteus mirabilis and compared with the natural antibiotic resistance of biofilm. Resistant variants (1X and 1Y) were obtained from cultures of the sensitive wild type “wt” strain 1 in the presence of the antibiotic. Planktonic strain 1 exhibited oxidative stress with increases in the reactive oxygen species (ROS) and consumption of NO in the presence of ciprofloxacin, whereas 1X and 1Y suffered non-significant rises in ROS generation, but produced and consumed more NO than sensitive strain 1. The two resistant variants were more resistant to telluride than wt and showed increased levels of intracellular superoxide dismutase (SOD) and glutathione (GSH). However, ciprofloxacin did not stimulate oxidative stress in biofilm. The production of ROS and NO with or without ciprofloxacin was less significant in biofilms than in an equivalent number of planktonic bacteria; sensitive and resistant strains did not present differences. On the other hand, SOD and GSH were more elevated in the biofilm than in planktonic bacteria. In summary, these results indicate that ciprofloxacin can induce resistance by the enhancement of antioxidant defense in planktonic bacteria, similar to the natural resistance occurring in biofilm. This feature may be added to the factors that regulate the susceptibility to this antibiotic.     

Signaling by the human serotonin1A receptor is impaired in cellular model of Smith-Lemli-Opitz Syndrome

The Smith-Lemli-Opitz Syndrome (SLOS) is a congenital and developmental malformation syndrome associated with defective cholesterol biosynthesis. SLOS is clinically diagnosed by reduced plasma levels of cholesterol along with elevated levels of 7-dehydrocholesterol (and its positional isomer 8-dehydrocholesterol) and the ratio of their concentrations to that of cholesterol. Since SLOS is associated with neurological deformities and malfunction, exploring the function of neuronal receptors and their interaction with membrane cholesterol under these conditions assumes significance. We have earlier shown the requirement of membrane cholesterol for the ligand binding function of an important neurotransmitter G-protein coupled receptor, the serotonin_1A receptor. In the present work, we have generated a cellular model of SLOS using CHO cells stably expressing the human serotonin_1A receptor. This was achieved by metabolically inhibiting the biosynthesis of cholesterol, utilizing a specific inhibitor (AY 9944) of the enzyme required in the final step of cholesterol biosynthesis. We utilized this cellular model to monitor the function of the human serotonin_1A receptor under SLOS-like condition. Our results show that ligand binding activity, G-protein coupling and downstream signaling of serotonin_1A receptors are impaired in SLOS-like condition, although the membrane receptor level does not exhibit any reduction. Importantly, metabolic replenishment of cholesterol using serum partially restored the ligand binding activity of the serotonin_1A receptor. These results are potentially useful in developing strategies for the future treatment of the disease since intake of dietary cholesterol is the only feasible treatment for SLOS patients.     

NAD(P)H- and superoxide-dependent nitric oxide degradation by rat liver mitochondria

Mitochondria consume nitric oxide (NO) mainly through reaction with superoxide anion (). Here, we analyzed the sources for NO degradation by isolated rat liver mitochondria. Electron leakage from complex III and reverse electron transport to complex I accounted for -dependent NO degradation by mitochondria in the presence of a protonmotive force. Mitochondria incubated with NAD(P)H also presented intense generation and NO degradation rates that were insensitive to respiratory inhibitors and abolished after proteinase treatment. These results suggest that an outer membrane-localized NAD(P)H oxidase activity, in addition to the electron leakage from the respiratory chain, promotes -dependent NO degradation in rat liver mitochondria.     

Binding of bivalent ions to actinomycete mannanase is accompanied by conformational change and is a key factor in its thermal stability

The study aimed to define the key factors involved in the modulation of actinomycete mannanases. We focused on the roles of carbohydrate-binding modules (CBMs) and bivalent ions. To investigate the effects of these factors, two actinomycete mannanase genes were cloned from Streptomyces thermoluteus (StManII) and Streptomyces lividans (SlMan). CBMs fused to mannanase catalytic domains do not affect the thermal stability of the proteins. CBM2 of StManII increased the catalytic efficiency toward soluble-mannan and insoluble-mannan by 25%–36%, and CBM10 of SlMan increased the catalytic efficiency toward soluble-mannan by 40%–50%. Thermal stability of wild-type and mutant enzymes was enhanced by calcium and manganese. Thermal stability of SlMandC was also slightly enhanced by magnesium. These results indicated that bivalent ion-binding site responsible for thermal stability was in the catalytic domains. Thermal stability of mannanase differed in the kinds of bivalent ions. Isothermal titration calorimetry revealed that the catalytic domain of StManII bound bivalent ions with a K_a of 5.39 ± 0.45 × 10³–7.56 ± 1.47 × 10³ M^− 1, and the catalytic domain of SlMan bound bivalent ions with a K_a of 1.06 ± 0.34 × 10³–3.86 ± 0.94 × 10³ M^− 1. The stoichiometry of these bindings was consistent with one bivalent ion-binding site per molecule of enzyme. Circular dichroism spectrum revealed that the presence of bivalent ions induced changes in the secondary structures of the enzymes. The binding of certain bivalent ion responsible for thermal stability was accompanied by a different conformational change by each bivalent ion. Actinomycete mannanases belong to GHF5 which contained various hemicellulases; therefore, the information obtained from mannanases applies to the other enzymes.     

Expression, purification and preliminary biochemical and structural characterization of the leucine rich repeat namesake domain of leucine rich repeat kinase 2

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. Much research effort has been directed towards the catalytic core region of LRRK2 composed of GTPase (ROC, Ras of complex proteins) and kinase domains and a connecting COR (C-terminus of ROC) domain. In contrast, the precise functions of the protein-protein interaction domains, such as the leucine-rich repeat (LRR) domain, are not known. In the present study, we modeled the LRRK2 LRR domain (LRR_LRRK2) using a template assembly approach, revealing the presence of 14 LRRs. Next, we focused on the expression and purification of LRR_LRRK2 in Escherichia coli. Buffer optimization revealed that the protein requires the presence of a zwitterionic detergent, namely Empigen BB, during solubilization and the subsequent purification and characterization steps. This indicates that the detergent captures the hydrophobic surface patches of LRR_LRRK2 thereby suppressing its aggregation. Circular dichroism (CD) spectroscopy measured 18% α-helices and 21% β-sheets, consistent with predictions from the homology model. Size exclusion chromatography (SEC) and dynamic light scattering measurements showed the presence of a single species, with a Stokes radius corresponding to the model dimensions of a protein monomer. Furthermore, no obvious LRR_LRRK2 multimerization was detected via cross-linking studies. Finally, the LRR_LRRK2 clinical mutations did not influence LRR_LRRK2 secondary, tertiary or quaternary structure as determined via SEC and CD spectroscopy. We therefore conclude that these mutations are likely to affect putative LRR_LRRK2 inter- and intramolecular interactions.     

Effects of precipitation on photosynthesis and water potential in Andropogon gerardii and Schizachyrium scoparium in a southern mixed grass prairie

In grassland ecosystems, spatial and temporal variability in precipitation is a key driver of species distributions and population dynamics. We experimentally manipulated precipitation to understand the physiological basis for differences in responses of species to water availability in a southern mixed grass prairie. We focused on the performance of two dominant C₄ grasses, Andropogon gerardii Vitman and Schizachyrium scoparium (Michx.) Nash, in treatments that received ambient rainfall, half of ambient rainfall (“drought” treatment), or approximately double ambient rainfall (“irrigated” treatment). Water potentials of S. scoparium were lower than A. gerardii, suggesting superior ability to adjust to water deficit in S. scoparium. Additionally, drought reduced photosynthesis to a greater extent in A. gerardii compared to S. scoparium. Leaf-level photosynthesis rates were similar in ambient and irrigated treatments, but were significantly lower in the drought treatment. Although stomatal conductance was reduced by drought, this was not limiting for photosynthesis. Leaf δ¹³C values were decreased by drought, caused by an increase in C_i/C_a. Chlorophyll fluorescence measures indicated light-harvesting rates were highest in irrigated treatments, and were lower in ambient and drought treatments. Moreover, drought resulted in a greater proportion of absorbed photon energy being lost via thermal pathways. Reductions in photosynthesis came as a result of non-stomatal limitations in the C₄ cycle. Our results provide mechanistic support for the hypothesis that S. scoparium is more drought tolerant than A. gerardii.