Study of the DnaB:DciA interplay reveals insights into the primary mode of loading of the bacterial replicative helicase.

Replicative helicases are essential proteins that unwind DNA in front of replication forks. Their loading depends on accessory proteins and in bacteria, DnaC and DnaI are well characterized loaders. However, most bacteria do not express either of these two proteins. Instead, they are proposed to rely on DciA, an ancestral protein unrelated to DnaC/I. While the DciA structure from Vibrio cholerae shares no homology with DnaC, it reveals similarities with DnaA and DnaX, two proteins involved during replication initiation. As other bacterial replicative helicases, VcDnaB adopts a toroid-shaped homo-hexameric structure, but with a slightly open dynamic conformation in the free state. We show that VcDnaB can load itself on DNA in vitro and that VcDciA stimulates this function, resulting in an increased DNA unwinding. VcDciA interacts with VcDnaB with a 3/6 stoichiometry and we show that a determinant residue, which discriminates DciA- and DnaC/I-helicases, is critical in vivo. Our work is the first step toward the understanding of the ancestral mode of loading of bacterial replicative helicases on DNA. It sheds light on the strategy employed by phage helicase loaders to hijack bacterial replicative helicases and may explain the recurrent domestication of dnaC/I through evolution in bacteria.

Dynamics and structural changes of calmodulin upon interaction with the antagonist calmidazolium.

BACKGROUND: Calmodulin (CaM) is an evolutionarily conserved eukaryotic multifunctional protein that functions as the major sensor of intracellular calcium signaling. Its calcium-modulated function regulates the activity of numerous effector proteins involved in a variety of physiological processes in diverse organs, from proliferation and apoptosis, to memory and immune responses. Due to the pleiotropic roles of CaM in normal and pathological cell functions, CaM antagonists are needed for fundamental studies as well as for potential therapeutic applications. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Yet, CDZ, as all other CaM antagonists described thus far, also affects additional cellular targets and its lack of selectivity hinders its application for dissecting calcium/CaM signaling. A better understanding of CaM:CDZ interaction is key to design analogs with improved selectivity. Here, we report a molecular characterization of CaM:CDZ complexes using an integrative structural biology approach combining SEC-SAXS, X-ray crystallography, HDX-MS, and NMR. RESULTS: We provide evidence that binding of a single molecule of CDZ induces an open-to-closed conformational reorientation of the two domains of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from physiological protein targets, despite their distinct chemical natures. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified. CONCLUSION: Our results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists. Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists.

Low-frequency stimulation of a fiber tract in bilateral temporal lobe epilepsy.

OBJECTIVE: Pharmacoresistant bilateral mesial temporal lobe epilepsy often implies poor resective surgical candidacy. Low-frequency stimulation of a fiber tract connected to bilateral hippocampi, the fornicodorsocommissural tract, has been shown to be safe and efficacious in reducing seizures in a previous short-term study. Here, we report a single-blinded, within-subject control, long-term deep-brain stimulation trial of low-frequency stimulation of the fornicodorsocommissural tract in bilateral mesial temporal lobe epilepsy. Outcomes of interest included safety with respect to verbal memory scores and reduction of seizure frequency. METHODS: Our enrollment goal was 16 adult subjects to be randomized to 2-Hz or 5-Hz low-frequency stimulation of the fornicodorsocommissural tract starting at 2 mA. The study design consisted of four two-month blocks of stimulation with a 50%-duty cycle, alternating with two-month blocks of no stimulation. RESULTS: We terminated the study after enrollment of five subjects due to slow accrual. Fornicodorsocommissural tract stimulation elicited bilateral hippocampal evoked responses in all subjects. Three subjects underwent implantation of pulse generators and long-term low-frequency stimulation with mean monthly seizures of 3.14 ±â€¯2.67 (median 3.0 [IQR 1-4.0]) during stimulation-off blocks, compared with 0.96 ±â€¯1.23 (median 1.0 [IQR 0-1.0]) during stimulation-on blocks (p = 0.0005) during the blinded phase. Generalized Estimating Equations showed that low-frequency stimulation reduced monthly seizure-frequency by 0.71 per mA (p < 0.001). Verbal memory scores were stable with no psychiatric complications or other adverse events. SIGNIFICANCE: The results demonstrate feasibility of stimulating both hippocampi using a single deep-brain stimulation electrode in the fornicodorsocommissural tract, efficacy of low-frequency stimulation in reducing seizures, and safety as regards verbal memory.

Pulse-frequency-dependent resonance in a population of pyramidal neuron models.

Stochastic resonance is known as a phenomenon whereby information transmission of weak signal or subthreshold stimuli can be enhanced by additive random noise with a suitable intensity. Another phenomenon induced by applying deterministic pulsatile electric stimuli with a pulse frequency, commonly used for deep brain stimulation (DBS), was also shown to improve signal-to-noise ratio in neuron models. The objective of this study was to test the hypothesis that pulsatile high-frequency stimulation could improve the detection of both sub- and suprathreshold synaptic stimuli by tuning the frequency of the stimulation in a population of pyramidal neuron models. Computer simulations showed that mutual information estimated from a population of neural spike trains displayed a typical resonance curve with a peak value of the pulse frequency at 80-120 Hz, similar to those utilized for DBS in clinical situations. It is concluded that a "pulse-frequency-dependent resonance" (PFDR) can enhance information transmission over a broad range of synaptically connected networks. Since the resonance frequency matches that used clinically, PFDR could contribute to the mechanism of the therapeutic effect of DBS.

Multiple cases of sensitization to an antiseptic containing chlorhexidine digluconate/benzalkonium chloride/benzyl alcohol with different profiles of sensitization in adults and children.

BACKGROUND: An aqueous antiseptic containing "chlorhexidine digluconate/benzalkonium chloride/benzyl alcohol" (CBB) is widely used in France. The only previous documented study dealing with allergic contact dermatitis (ACD) to this antiseptic is one small case series in children. The French Vigilance Network for Dermatology and Allergy (REVIDAL-GERDA) has collected many cases in the last few years. OBJECTIVES: To evaluate the clinical and sensitization profiles of patients diagnosed with ACD to CBB. METHODS: We performed a retrospective study of patients with contact dermatitis to CBB and positive tests to CBB and/or at least one of its components. All patients had to be tested with all components of CBB. RESULTS: A total of 102 patients (71 adults and 31 children) were included. The lesions were extensive in 63% of patients and 55% had delayed time to diagnosis. CBB patch tests were positive in 93.8% of cases. The allergen was identified in 97% of patients, mainly benzyl alcohol in adults (81.7%) and chlorhexidine digluconate in children (54.8%). About 32.4% of the patients were sensitized to several components. CONCLUSION: CBB is a cause of ACD at all ages. The components of the antiseptic should be tested. The sensitization profile seems to be different between adults and children.

Structural and dynamic characterization of the C-terminal tail of ErbB2: Disordered but not random.

ErbB2 (or HER2) is a receptor tyrosine kinase overexpressed in some breast cancers and associated with poor prognosis. Treatments targeting the receptor extracellular and kinase domains have greatly improved disease outcome in the last 20 years. In parallel, the structures of these domains have been described, enabling better mechanistic understanding of the receptor function and targeted inhibition. However, the ErbB2 disordered C-terminal cytoplasmic tail (CtErbB2) remains very poorly characterized in terms of structure, dynamics, and detailed functional mechanism. Yet, it is where signal transduction is triggered via phosphorylation of tyrosine residues and carried out via interaction with adaptor proteins. Here, we report the first description, to our knowledge, of the ErbB2 disordered tail at atomic resolution using NMR, complemented by small-angle x-ray scattering. We show that although no part of CtErbB2 has any fully populated secondary or tertiary structure, it contains several transient α-helices and numerous transient polyproline II helices, populated up to 20 and 40%, respectively, and low but significant compaction. The presence of some structural elements suggests, along the lines of the results obtained for EGFR (ErbB1), that they may have a functional role in ErbB2's autoregulation processes. In addition, the transient formation of polyproline II helices is compliant with previously suggested interactions with SH3 domains. All in all, our in-depth structural study opens perspectives in the mechanistic understanding of ErbB2.

Neural recruitment by ephaptic coupling in epilepsy.

OBJECTIVE: One of the challenges in treating patients with drug-resistant epilepsy is that the mechanisms of seizures are unknown. Most current interventions are based on the assumption that epileptic activity recruits neurons and progresses by synaptic transmission. However, several experimental studies have shown that neural activity in rodent hippocampi can propagate independently of synaptic transmission. Recent studies suggest these waves are self-propagating by electric field (ephaptic) coupling. In this study, we tested the hypothesis that neural recruitment during seizures can occur by electric field coupling. METHODS: 4-Aminopyridine was used in both in vivo and in vitro preparation to trigger seizures or epileptiform activity. A transection was made in the in vivo hippocampus and in vitro hippocampal and cortical slices to study whether the induced seizure activity can recruit neurons across the gap. A computational model was built to test whether ephaptic coupling alone can account for neural recruitment across the transection. The model prediction was further validated by in vitro experiments. RESULTS: Experimental results show that electric fields generated by seizure-like activity in the hippocampus both in vitro and in vivo can recruit neurons locally and through a transection of the tissue. The computational model suggests that the neural recruitment across the transection is mediated by electric field coupling. With in vitro experiments, we show that a dielectric material can block the recruitment of epileptiform activity across a transection, and that the electric fields measured within the gap are similar to those predicted by model simulations. Furthermore, this nonsynaptic neural recruitment is also observed in cortical slices, suggesting that this effect is robust in brain tissue. SIGNIFICANCE: These results indicate that ephaptic coupling, a nonsynaptic mechanism, can underlie neural recruitment by a small electric field generated by seizure activity and could explain the low success rate of surgical transections in epilepsy patients.

YB-1, an abundant core mRNA-binding protein, has the capacity to form an RNA nucleoprotein filament: a structural analysis.

The structural rearrangements accompanying mRNA during translation in mammalian cells remain poorly understood. Here, we discovered that YB-1 (YBX1), a major partner of mRNAs in the cytoplasm, forms a linear nucleoprotein filament with mRNA, when part of the YB-1 unstructured C-terminus has been truncated. YB-1 possesses a cold-shock domain (CSD), a remnant of bacterial cold shock proteins that have the ability to stimulate translation under the low temperatures through an RNA chaperone activity. The structure of the nucleoprotein filament indicates that the CSD of YB-1 preserved its chaperone activity also in eukaryotes and shows that mRNA is channeled between consecutive CSDs. The energy benefit needed for the formation of stable nucleoprotein filament relies on an electrostatic zipper mediated by positively charged amino acid residues in the YB-1 C-terminus. Thus, YB-1 displays a structural plasticity to unfold structured mRNAs into extended linear filaments. We anticipate that our findings will shed the light on the scanning of mRNAs by ribosomes during the initiation and elongation steps of mRNA translation.

Structural ensemble and biological activity of DciA intrinsically disordered region.

DciA is a newly discovered bacterial protein involved in loading the replicative helicase DnaB onto DNA at the initiation step of chromosome replication. Its three-dimensional structure is composed of a folded N-terminal domain (residues 1-111) resembling K Homology domains and a long disordered C-terminal tail (residues 112-157) which structure-activity relationship remains to be elucidated. In the present study on Vibrio cholerae DciA, we emphasize the importance of its disordered region to load DnaB onto DNA using surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC). Then we characterize the conformational ensemble of the full-length protein using a combination of circular dichroism (CD), small angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations. The atomic-level structural ensemble generated by MD simulations is in very good agreement with SAXS data. From initial conformations of the C-terminal tail without any secondary structure, our simulations bring to light several transient helical structures in this segment, which might be molecular recognition features (MoRFs) for the binding to DnaB and its recruitment and loading onto DNA.

Quantitative live-cell imaging and 3D modeling reveal critical functional features in the cytosolic complex of phagocyte NADPH oxidase.

Phagocyte NADPH oxidase produces superoxide anions, a precursor of reactive oxygen species (ROS) critical for host responses to microbial infections. However, uncontrolled ROS production contributes to inflammation, making NADPH oxidase a major drug target. It consists of two membranous (Nox2 and p22phox) and three cytosolic subunits (p40phox, p47phox, and p67phox) that undergo structural changes during enzyme activation. Unraveling the interactions between these subunits and the resulting conformation of the complex could shed light on NADPH oxidase regulation and help identify inhibition sites. However, the structures and the interactions of flexible proteins comprising several well-structured domains connected by intrinsically disordered protein segments are difficult to investigate by conventional techniques such as X-ray crystallography, NMR, or cryo-EM. Here, we developed an analytical strategy based on FRET-fluorescence lifetime imaging (FLIM) and fluorescence cross-correlation spectroscopy (FCCS) to structurally and quantitatively characterize NADPH oxidase in live cells. We characterized the inter- and intramolecular interactions of its cytosolic subunits by elucidating their conformation, stoichiometry, interacting fraction, and affinities in live cells. Our results revealed that the three subunits have a 1:1:1 stoichiometry and that nearly 100% of them are present in complexes in living cells. Furthermore, combining FRET data with small-angle X-ray scattering (SAXS) models and published crystal structures of isolated domains and subunits, we built a 3D model of the entire cytosolic complex. The model disclosed an elongated complex containing a flexible hinge separating two domains ideally positioned at one end of the complex and critical for oxidase activation and interactions with membrane components.

Calmodulin fishing with a structurally disordered bait triggers CyaA catalysis.

Once translocated into the cytosol of target cells, the catalytic domain (AC) of the adenylate cyclase toxin (CyaA), a major virulence factor of Bordetella pertussis, is potently activated by binding calmodulin (CaM) to produce supraphysiological levels of cAMP, inducing cell death. Using a combination of small-angle X-ray scattering (SAXS), hydrogen/deuterium exchange mass spectrometry (HDX-MS), and synchrotron radiation circular dichroism (SR-CD), we show that, in the absence of CaM, AC exhibits significant structural disorder, and a 75-residue-long stretch within AC undergoes a disorder-to-order transition upon CaM binding. Beyond this local folding, CaM binding induces long-range allosteric effects that stabilize the distant catalytic site, whilst preserving catalytic loop flexibility. We propose that the high enzymatic activity of AC is due to a tight balance between the CaM-induced decrease of structural flexibility around the catalytic site and the preservation of catalytic loop flexibility, allowing for fast substrate binding and product release. The CaM-induced dampening of AC conformational disorder is likely relevant to other CaM-activated enzymes.

The structure of the TsaB/TsaD/TsaE complex reveals an unexpected mechanism for the bacterial t6A tRNA-modification.

The universal N6-threonylcarbamoyladenosine (t6A) modification at position A37 of ANN-decoding tRNAs is essential for translational fidelity. In bacteria the TsaC enzyme first synthesizes an l-threonylcarbamoyladenylate (TC-AMP) intermediate. In cooperation with TsaB and TsaE, TsaD then transfers the l-threonylcarbamoyl-moiety from TC-AMP onto tRNA. We determined the crystal structure of the TsaB-TsaE-TsaD (TsaBDE) complex of Thermotoga maritima in presence of a non-hydrolysable AMPCPP. TsaE is positioned at the entrance of the active site pocket of TsaD, contacting both the TsaB and TsaD subunits and prohibiting simultaneous tRNA binding. AMPCPP occupies the ATP binding site of TsaE and is sandwiched between TsaE and TsaD. Unexpectedly, the binding of TsaE partially denatures the active site of TsaD causing loss of its essential metal binding sites. TsaE interferes in a pre- or post-catalytic step and its binding to TsaBD is regulated by ATP hydrolysis. This novel binding mode and activation mechanism of TsaE offers good opportunities for antimicrobial drug development.

Structural Characterization of N-WASP Domain V Using MD Simulations with NMR and SAXS Data.

Because of their large conformational heterogeneity, structural characterization of intrinsically disordered proteins (IDPs) is very challenging using classical experimental methods alone. In this study, we use NMR and small-angle x-ray scattering (SAXS) data with multiple molecular dynamics (MD) simulations to describe the conformational ensemble of the fully disordered verprolin homology domain of the neural Aldrich syndrome protein involved in the regulation of actin polymerization. First, we studied several back-calculation software of SAXS scattering intensity and optimized the adjustable parameters to accurately calculate the SAXS intensity from an atomic structure. We also identified the most appropriate force fields for MD simulations of this IDP. Then, we analyzed four conformational ensembles of neural Aldrich syndrome protein verprolin homology domain, two generated with the program flexible-meccano with or without NMR-derived information as input and two others generated by MD simulations with two different force fields. These four conformational ensembles were compared to available NMR and SAXS data for validation. We found that MD simulations with the AMBER-03w force field and the TIP4P/2005s water model are able to correctly describe the conformational ensemble of this 67-residue IDP at both local and global level.

Slow periodic activity in the longitudinal hippocampal slice can self-propagate non-synaptically by a mechanism consistent with ephaptic coupling.

KEY POINTS: Slow periodic activity can propagate with speeds around 0.1 m s-1 and be modulated by weak electric fields. Slow periodic activity in the longitudinal hippocampal slice can propagate without chemical synaptic transmission or gap junctions, but can generate electric fields which in turn activate neighbouring cells. Applying local extracellular electric fields with amplitude in the range of endogenous fields is sufficient to modulate or block the propagation of this activity both in the in silico and in the in vitro models. Results support the hypothesis that endogenous electric fields, previously thought to be too small to trigger neural activity, play a significant role in the self-propagation of slow periodic activity in the hippocampus. Experiments indicate that a neural network can give rise to sustained self-propagating waves by ephaptic coupling, suggesting a novel propagation mechanism for neural activity under normal physiological conditions. ABSTRACT: Slow oscillations are a standard feature observed in the cortex and the hippocampus during slow wave sleep. Slow oscillations are characterized by low-frequency periodic activity (<1 Hz) and are thought to be related to memory consolidation. These waves are assumed to be a reflection of the underlying neural activity, but it is not known if they can, by themselves, be self-sustained and propagate. Previous studies have shown that slow periodic activity can be reproduced in the in vitro preparation to mimic in vivo slow oscillations. Slow periodic activity can propagate with speeds around 0.1 m s-1 and be modulated by weak electric fields. In the present study, we show that slow periodic activity in the longitudinal hippocampal slice is a self-regenerating wave which can propagate with and without chemical or electrical synaptic transmission at the same speeds. We also show that applying local extracellular electric fields can modulate or even block the propagation of this wave in both in silico and in vitro models. Our results support the notion that ephaptic coupling plays a significant role in the propagation of the slow hippocampal periodic activity. Moreover, these results indicate that a neural network can give rise to sustained self-propagating waves by ephaptic coupling, suggesting a novel propagation mechanism for neural activity under normal physiological conditions.

Molecular Dynamics Simulations Combined with Nuclear Magnetic Resonance and/or Small-Angle X-ray Scattering Data for Characterizing Intrinsically Disordered Protein Conformational Ensembles.

The concept of intrinsically disordered proteins (IDPs) has emerged relatively slowly, but over the past 20 years, it has become an intense research area in structural biology. Indeed, because of their considerable flexibility and structural heterogeneity, the determination of IDP conformational ensemble is particularly challenging and often requires a combination of experimental measurements and computational approaches. With the improved accuracy of all-atom force fields and the increasing computing performances, molecular dynamics (MD) simulations have become more and more reliable to generate realistic conformational ensembles. And the combination of MD simulations with experimental approaches, such as nuclear magnetic resonance (NMR) and/or small-angle X-ray scattering (SAXS) allows one to converge toward a more accurate and exhaustive description of IDP structures. In this Review, we discuss the state of the art of MD simulations of IDP conformational ensembles, with a special focus on studies that back-calculated and directly compared theoretical and experimental NMR or SAXS observables, such as chemical shifts (CS), 3J-couplings (3Jc), residual dipolar couplings (RDC), or SAXS intensities. We organize the review in three parts. In the first section, we discuss the studies which used NMR and/or SAXS data to test and validate the development of force fields or enhanced sampling techniques. In the second part, we explore different methods for the refinement of MD-derived structural ensembles, such as NMR or SAXS data-restrained MD simulations or ensemble reweighting to better fit experiments. Finally, we survey some recent studies combining MD simulations with NMR and/or SAXS measurements to investigate the relationship between IDP conformational ensemble and biological activity, as well as their implication in human diseases. From this review, we noticed that quite a few studies compared MD-generated conformational ensembles with both NMR and SAXS measurements to validate IDP structures at both local and global levels. Yet, beside the IDP propensity to form local secondary structures, their dynamic extension or compactness also appears important for their activity. Thus, we believe that a close synergy between MD simulations, NMR, and SAXS experiments would be greatly appropriate to address the challenges of characterizing the disordered structures of proteins and their complexes, relative to their biological functions.

The crystal structure of Trz1, the long form RNase Z from yeast.

tRNAs are synthesized as precursor RNAs that have to undergo processing steps to become functional. Yeast Trz1 is a key endoribonuclease involved in the 3΄ maturation of tRNAs in all domains of life. It is a member of the ß-lactamase family of RNases, characterized by an HxHxDH sequence motif involved in coordination of catalytic Zn-ions. The RNase Z family consists of two subfamilies: the short (250-400 residues) and the long forms (about double in size). Short form RNase Z enzymes act as homodimers: one subunit embraces tRNA with a protruding arm, while the other provides the catalytic site. The long form is thought to contain two fused ß-lactamase domains within a single polypeptide. Only structures of short form RNase Z enzymes are known. Here we present the 3.1 Å crystal structure of the long-form Trz1 from Saccharomyces cerevisiae. Trz1 is organized into two ß-lactamase domains connected by a long linker. The N-terminal domain has lost its catalytic residues, but retains the long flexible arm that is important for tRNA binding, while it is the other way around in the C-terminal domain. Trz1 likely evolved from a duplication and fusion of the gene encoding the monomeric short form RNase Z.

Concomitant contact-allergic reactions to iodopropynyl butylcarbamate and iodine.

BACKGROUND: Iodopropynyl butylcarbamate (IPBC) is a broad-spectrum preservative for use in several product types, including cosmetics, in which its concentrations have been limited by EU legislation because of concerns related to its iodine content and release, and the risk of subsequent iodine overdose. OBJECTIVES: To report on concomitant patch test reactions observed with iodine and IPBC in patients sensitized to iodine-containing antiseptics. PATIENTS: Between 2012 and 2018, seven patients, six from Belgium and one from France, two suffering from acute dermatitis during surgical interventions, four from dermatitis caused by wound treatment, and one from occupational dermatitis, were shown to be sensitized to iodine and/or povidone-iodine (PVP-I), which was considered to be relevant for their dermatitis. All patients were coincidentally also patch tested with IPBC. RESULTS: All patients showed positive patch test reactions to several other allergens, including IPBC. No relevance could be detected for IPBC. CONCLUSIONS: We suspect that, notwithstanding the absence of firm evidence for IPBC being dehalogenated to produce free iodine in animals or in humans, the patch test reactions to IPBC in iodine-allergic subjects were possibly caused by free iodine released from this preservative agent, thus underlining the EU restrictions regarding the use of this preservative in cosmetics.

Investigation into Early Steps of Actin Recognition by the Intrinsically Disordered N-WASP Domain V.

Cellular regulation or signaling processes are mediated by many proteins which often have one or several intrinsically disordered regions (IDRs). These IDRs generally serve as binders to different proteins with high specificity. In many cases, IDRs undergo a disorder-to-order transition upon binding, following a mechanism between two possible pathways, the induced fit or the conformational selection. Since these mechanisms contribute differently to the kinetics of IDR associations, it is important to investigate them in order to gain insight into the physical factors that determine the biomolecular recognition process. The verprolin homology domain (V) of the Neural Wiskott-Aldrich Syndrome Protein (N-WASP), involved in the regulation of actin polymerization, is a typical example of IDR. It is composed of two WH2 motifs, each being able to bind one actin molecule. In this study, we investigated the early steps of the recognition process of actin by the WH2 motifs of N-WASP domain V. Using docking calculations and molecular dynamics simulations, our study shows that actin is first recognized by the N-WASP domain V regions which have the highest propensity to form transient α -helices. The WH2 motif consensus sequences "LKKV" subsequently bind to actin through large conformational changes of the disordered domain V.

Dissociation of the Dimer of the Intrinsically Disordered Domain of RNase Y upon Antibody Binding.

Although RNase Y acts as the key enzyme initiating messenger RNA decay in Bacillus subtilis and likely in many other Gram-positive bacteria, its three-dimensional structure remains unknown. An antibody belonging to the rare immunoglobulin G (IgG) 2b λx isotype was raised against a 12-residue conserved peptide from the N-terminal noncatalytic domain of B. subtilis RNase Y (BsRNaseY) that is predicted to be intrinsically disordered. Here, we show that this domain can be produced as a stand-alone protein called Nter-BsRNaseY that undergoes conformational changes between monomeric and dimeric forms. Circular dichroism and size exclusion chromatography coupled with multiangle light scattering or with small angle x-ray scattering indicate that the Nter-BsRNaseY dimer displays an elongated form and a high content of α-helices, in agreement with the existence of a central coiled-coil structure appended with flexible ends, and that the monomeric state of Nter-BsRNaseY is favored upon binding the fragment antigen binding (Fab) of the antibody. The dissociation constants of the IgG/BsRNaseY, IgG/Nter-BsRNaseY, and IgG/peptide complexes indicate that the affinity of the IgG for Nter-BsRNaseY is in the nM range and suggest that the peptide is less accessible in BsRNaseY than in Nter-BsRNaseY. The crystal structure of the Fab in complex with the peptide antigen shows that the peptide adopts an elongated U-shaped conformation in which the unique hydrophobic residue of the peptide, Leu6, is completely buried. The peptide/Fab complex may mimic the interaction of a microdomain of the N-terminal domain of BsRNaseY with one of its cellular partners within the degradosome complex. Altogether, our results suggest that BsRNaseY may become accessible for protein interaction upon dissociation of its N-terminal domain into the monomeric form.

Corpus callosum low-frequency stimulation suppresses seizures in an acute rat model of focal cortical seizures.

OBJECTIVE: Low-frequency fiber-tract stimulation has been shown to be effective in treating mesial temporal lobe epilepsies through activation of the hippocampal commissure in rodents and human patients. The corpus callosum is a major pathway connecting the two hemispheres of the brain; however, few experiments have documented corpus callosum stimulation. The objective is to determine the efficacy of corpus callosum stimulation at low frequencies to suppress cortical seizures. METHODS: 4-Aminopyridine was injected in the primary motor cortex of 24 rats under anesthesia. Recording electrodes were placed in the contralateral motor cortex and hippocampus. Three pairs of stimulating electrodes were inserted into the corpus callosum along its longitudinal axis. Local field potentials were recorded 1 hour before, during, and after stimulation to determine the effect of stimulation on seizure duration. Stimulation was delivered from each pair of electrodes independently in separate experiments. Furthermore, electrical stimulation was applied to the region of the corpus callosum with the highest degree of innervation of the seizure focus to compare the efficacy of different stimulation frequencies (1-30 Hz) on seizure suppression. RESULTS: Corpus callosum stimulation was effective at suppressing seizures at 10 Hz by 76% (P < 0.05, n = 5) and at 20 Hz by 95% (P < 0.0001, n = 14). Stimulation at frequencies of 1 and 30 Hz did not have a significant effect on reducing the total time spent seizing (P > 0.9999, n = 5). Furthermore, stimulation was only effective at suppressing seizures when the pair of electrodes was placed within the section of corpus callosum containing fibers innervating the seizure focus. Secondarily generalized seizures in the hippocampus were eliminated when seizures in the cortical focus were suppressed. SIGNIFICANCE: Low-frequency fiber-tract stimulation of the corpus callosum suppresses both cortical and cortically induced hippocampal seizures in an acute model of focal cortical seizures. The stimulation paradigm is selective, as it is only effective when targeted to specific regions of the corpus callosum that project maximally to cortical regions generating the seizure activity. Selective placement of stimulation electrodes along the corpus callosum could be used as a patient-specific treatment for cortical epilepsies.