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Epitranscriptomic mRNA modifications affect gene expression, with their altered balance detected in various cancers. YTHDF proteins contain the YTH reader domain recognizing the m 6 A mark on mRNA and represent valuable drug targets. Crystallographic structures have been determined for all three family members; however, discrepancies are present in the organization of the m 6 A-binding pocket. Here, we present new crystallographic structures of the YTH domain of YTHDF1, accompanied by computational studies, showing that this domain can exist in different stable conformations separated by a significant energetic barrier. During the transition, additional conformations are explored, with peculiar druggable pockets appearing and offering new opportunities for the design of YTH-interfering small molecules.

Argonaute (Ago) proteins mediate target recognition guiding miRNA to bind complementary mRNA primarily in the seed region. However, additional pairing can occur beyond the seed, forming a supplementary duplex that can contribute to the guide-target affinity. In order to shed light on the connection, between protein-RNA interactions and miRNA-mRNA seed and supplementary duplex mobility, we carried out molecular dynamics simulations at the microsecond time-scale using a different approach compared to the ones normally used. Until now, theoretical investigations with classical MD on Ago-RNA complexes have been focused primarily on pure water solvent, which mimics the natural environment of biological molecules. Here, we explored the conformational space of a human Ago2 (hAgo2) bound to the seed + supplementary miRNA-mRNA duplex, using the solvent environment as a molecular probe. MD simulations have been performed in a mixture of water/MeOH at a molar ratio of 70 : 30 as well as in pure water for comparison. Our findings revealed that the mixed solvent promotes protein RNA association, principally enhancing salt-linkages between basic amino acid side-chains and acidic phosphates of the sugar-phosphate backbone. The primary effect registered was the restriction of supplementary duplex flexibility and the stabilization of the miRNA 3' terminus. Interestingly, we observed that the influence of the solvent appears to have almost no impact on the conformation of the seed duplex.

Thanks to the considerable research which has been undertaken in the last few years to improve our understanding of the biology and mechanism of action of SARS-CoV-2, we know how the virus uses its surface spike protein to infect host cells. The transmembrane prosthesis, serine 2 (TMPRSS2) protein, located on the surface of human cells, recognizes the cleavage site in the spike protein, leading to the release of the fusion peptide and entry of the virus into the host cells. Because of its role, TMPRSS2 has been proposed as a drug target to prevent infection by the virus. In this study, we aim to increase our understanding of TMPRSS2 using long scale microsecond atomistic molecular dynamics simulations, focusing on the conformational changes over time. The comparison between simulations conducted on the protein in the native (apo) and inhibited form (holo), has shown that in the holo form the inhibitor stabilizes the catalytic site and induces rearrangements in the extracellular domain of the protein. In turn, it leads to the formation of a new cavity in the vicinity of the ligand binding pocket that is stable in the microsecond time scale. Given the low specificity of known protease inhibitors, these findings suggest a new potential drug target site that can be used to improve TMPRSS2 specific recognition by newly designed inhibitors., (© 2023 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

Peptidomimetic antimicrobials exhibit a selective interaction with bacterial cells over mammalian cells once they have achieved an optimum amphiphilic balance (hydrophobicity/hydrophilicity) in the molecular architecture. To date, hydrophobicity and cationic charge have been considered the crucial parameters to attain such amphiphilic balance. However, optimization of these properties is not enough to circumvent unwanted toxicity towards mammalian cells. Hence, herein, we report new isoamphipathic antibacterial molecules (IAMs: 1-3 ) where positional isomerism was introduced as one of the guiding factors for molecular design. This class of molecules displayed good (MIC = 1-8 μg mL -1 or μM) to moderate [MIC = 32-64 μg mL -1 (32.2-64.4 μM)] antibacterial activity against multiple Gram-positive and Gram-negative bacteria. Positional isomerism showed a strong influence on regulating antibacterial activity and toxicity for ortho [IAM-1: MIC = 1-32 μg mL -1 (1-32.2 μM), HC 50 = 650 μg mL -1 (654.6 μM)], meta [IAM-2: MIC = 1-16 μg mL -1 (1-16.1 μM), HC 50 = 98 μg mL -1 (98.7 μM)] and para [IAM-3: MIC = 1-16 μg mL -1 (1-16.1 μM), HC 50 = 160 μg mL -1 (161.1 μM)] isomers. Co-culture studies and investigation of membrane dynamics indicated that ortho isomer, IAM-1 exerted more selective activity towards bacterial over mammalian membranes, compared to meta and para isomers. Furthermore, the mechanism of action of the lead molecule (IAM-1) has been characterized through detailed molecular dynamics simulations. In addition, the lead molecule displayed substantial efficacy against dormant bacteria and mature biofilms, unlike conventional antibiotics. Importantly, IAM-1 exhibited moderate in vivo activity against MRSA wound infection in a murine model with no detectable dermal toxicity. Altogether, the report explored the design and development of isoamphipathic antibacterial molecules to establish the role of positional isomerism in achieving selective and potential antibacterial agents., Competing Interests: The authors declare no conflicts of interest., (This journal is © The Royal Society of Chemistry.)

Theoretical work suggests that collective spatiotemporal behavior of integral membrane proteins should be modulated by boundary lipids sheathing their membrane anchors. Here, we show evidence for this prediction while investigating the mechanism for maintaining a steady amount of the active form of integral membrane protein Lck kinase (Lck A ) by Lck trans-autophosphorylation regulated by the phosphatase CD45. We used super-resolution microscopy, flow cytometry, and pharmacological and genetic perturbation to gain insight into the spatiotemporal context of this process. We found that Lck A is generated exclusively at the plasma membrane, where CD45 maintains it in a ceaseless dynamic equilibrium with its unphosphorylated precursor. Steady Lck A shows linear dependence, after an initial threshold, over a considerable range of Lck expression levels. This behavior fits a phenomenological model of trans-autophosphorylation that becomes more efficient with increasing Lck A . We then challenged steady Lck A formation by genetically swapping the Lck membrane anchor with structurally divergent ones, such as that of Src or the transmembrane domains of LAT, CD4, palmitoylation-defective CD4 and CD45 that were expected to drastically modify Lck boundary lipids. We observed small but significant changes in Lck A generation, except for the CD45 transmembrane domain that drastically reduced Lck A due to its excessive lateral proximity to CD45. Comprehensively, Lck A formation and maintenance can be best explained by lipid bilayer critical density fluctuations rather than liquid-ordered phase-separated nanodomains, as previously thought, with "like/unlike" boundary lipids driving dynamical proximity and remoteness of Lck with itself and with CD45., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the pandemic that broke out in 2020 and continues to be the cause of massive global upheaval. Coronaviruses are positive-strand RNA viruses with a genome of ~30 kb. The genome is replicated and transcribed by RNA-dependent RNA polymerase together with accessory factors. One of the latter is the protein helicase (NSP13), which is essential for viral replication. The recently solved helicase structure revealed a tertiary structure composed of five domains. Here, we investigated NSP13 from a structural point of view, comparing its RNA-free form with the RNA-engaged form by using atomistic molecular dynamics (MD) simulations at the microsecond timescale. Structural analyses revealed conformational changes that provide insights into the contribution of the different domains, identifying the residues responsible for domain-domain interactions in both observed forms. The RNA-free system appears to be more flexible than the RNA-engaged form. This result underlies the stabilizing role of the nucleic acid and the functional core role of these domains.

Diverticula of the choledochus, better known as Todani type II cysts, are very rare and represent a predominantly pediatric pathology. Their identification by radiological methods, even if occasional, requires clinical doctors to request a surgical consultation, even for asymptomatic subjects, to proceed with their removal, given the risk of associated neoplasms. The laparoscopic approach for surgical treatment of these cysts has been recently introduced with excellent results. Due to the poor clinical records, currently there are neither shared protocols about their management nor long-term follow-up of operated patients. We report a case of an adult female suffering for years from biliary colic due to the presence of a duodenal diverticulum associated with microlithiasis' cholecystitis, who was laparoscopically treated, with excellent results in terms of symptomatic regression, reduced hospitalization, and no surgery-related complications.

Background and Aims: The aim of this study was to apply machine learning models and a nomogram to differentiate critically ill from non-critically ill COVID-19 pneumonia patients., Methods: Clinical symptoms and signs, laboratory parameters, cytokine profile, and immune cellular data of 63 COVID-19 pneumonia patients were retrospectively reviewed. Outcomes were followed up until Mar 12, 2020. A logistic regression function (LR model), Random Forest, and XGBoost models were developed. The performance of these models was measured by area under receiver operating characteristic curve (AUC) analysis., Results: Univariate analysis revealed that there was a difference between critically and non-critically ill patients with respect to levels of interleukin-6, interleukin-10, T cells, CD4 +  T, and CD8 + T cells. Interleukin-10 with an AUC of 0.86 was most useful predictor of critically ill patients with COVID-19 pneumonia. Ten variables (respiratory rate, neutrophil counts, aspartate transaminase, albumin, serum procalcitonin, D-dimer and B-type natriuretic peptide, CD4 + T cells, interleukin-6 and interleukin-10) were used as candidate predictors for LR model, Random Forest (RF) and XGBoost model application. The coefficients from LR model were utilized to build a nomogram. RF and XGBoost methods suggested that Interleukin-10 and interleukin-6 were the most important variables for severity of illness prediction. The mean AUC for LR, RF, and XGBoost model were 0.91, 0.89, and 0.93 respectively (in two-fold cross-validation). Individualized prediction by XGBoost model was explained by local interpretable model-agnostic explanations (LIME) plot., Conclusions: XGBoost exhibited the highest discriminatory performance for prediction of critically ill patients with COVID-19 pneumonia. It is inferred that the nomogram and visualized interpretation with LIME plot could be useful in the clinical setting. Additionally, interleukin-10 could serve as a useful predictor of critically ill patients with COVID-19 pneumonia., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hong, Zhou, Jin, Lu, Pan, Lin, Yang, Xu, Basharat, Zippi, Fiorino, Tsukanov, Stock, Grottesi, Chen and Pan.)

The COVID-19 pandemic is caused by SARS-CoV-2. Currently, most of the research efforts towards the development of vaccines and antibodies against SARS-CoV-2 were mainly focused on the spike (S) protein, which mediates virus entry into the host cell by binding to ACE2. As the virus SARS-CoV-2 continues to spread globally, variants have emerged, characterized by multiple mutations of the S glycoprotein. Herein, we employed microsecond-long molecular dynamics simulations to study the impact of the mutations of the S glycoprotein in SARS-CoV-2 Variant of Concern 202012/01 (B.1.1.7), termed the "UK variant", in comparison with the wild type, with the aim to decipher the structural basis of the reported increased infectivity and virulence. The simulations provided insights on the different dynamics of UK and wild-type S glycoprotein, regarding in particular the Receptor Binding Domain (RBD). In addition, we investigated the role of glycans in modulating the conformational transitions of the RBD. The overall results showed that the UK mutant experiences higher flexibility in the RBD with respect to wild type; this behavior might be correlated with the increased transmission reported for this variant. Our work also adds useful structural information on antigenic "hotspots" and epitopes targeted by neutralizing antibodies.

Cocaine abuse is rising in the young population, triggering uncommon and potentially life-threatening causes of acute abdomen in this age group. The authors present the case of a 30-year-old man with emergency admission due to abdominal pain, with no history of drug abuse. Several signs and symptoms elicited toxicologic blood screening, which disclosed high serum levels of cocaine and its metabolites. Twelve hours after admission, the onset of acute abdomen with signs of diffuse peritonitis prompted surgical exploration through a minimally invasive approach. Two segmental small bowel ischemic loops and diffuse peritonitis, but no bowel perforation, were identified and treated by laparoscopic peritoneal lavage with 5 l of heated saline and intravenous administration of sodium heparin, 10 000 IU. Postoperative course was uneventful with home discharge on postoperative day 5. High index of suspicion is required to establish a prompt diagnosis and treatment of this uncommon cocaine abuse-related disease., (Published by Oxford University Press and JSCR Publishing Ltd. © The Author(s) 2021.)

The conformational state of the activation loop (A-loop) is pivotal for the activity of most protein kinases. Hence, the characterization of the conformational dynamics of the A-loop is important to increase our understanding of the molecular processes related to diseases and to support the discovery of small molecule kinase inhibitors. Here, we carry out a combination of molecular dynamics (MD) and essential dynamics (ED) analyses to fully map the effects of phosphorylation, ADP, and conformation disrupting (CD) inhibitors (i.e., CD532 and MLN8054) on the dynamics of the A-loop of Aurora-A. MD revealed that the stability of the A-loop in an open conformation is enhanced by single phospho-Thr-288, while paradoxically, the presence of a second phosphorylation at Thr-287 decreases such stability and renders the A-loop more fluctuant in time and space. Moreover, we found that this post-translational modification has a significant effect on the direction of the A-loop motions. ED analysis suggests that the presence of the phosphate moiety induces the dynamics of Aurora-A to sample two distinct energy minima, instead of a single large minimum, as in unphosphorylated Aurora-A states. This observation indicates that the conformational distributions of Aurora-A with both single and double phospho-threonine modifications are remarkably different from the unphosphorylated state. In the closed states, binding of CD532 and MLN8054 inhibitors has the effect of increasing the distance of the N- and C-lobes of the kinase domain of Aurora-A, and the angle analysis between those two lobes during MD simulations showed that the N- and C-lobes are kept more open in presence of CD532, compared to MLN8054. As the A-loop is a common feature of Aurora protein kinases, our studies provide a general description of the conformational dynamics of this structure upon phosphorylation and different ligands binding.

Given the enormous social and health impact of the pandemic triggered by severe acute respiratory syndrome 2 (SARS-CoV-2), the scientific community made a huge effort to provide an immediate response to the challenges posed by Coronavirus disease 2019 (COVID-19). One of the most important proteins of the virus is an enzyme, called 3CLpro or main protease, already identified as an important pharmacological target also in SARS and Middle East respiratory syndrome virus (MERS) viruses. This protein triggers the production of a whole series of enzymes necessary for the virus to carry out its replicating and infectious activities. Therefore, it is crucial to gain a deeper understanding of 3CLpro structure and function in order to effectively target this enzyme. All-atoms molecular dynamics (MD) simulations were performed to examine the different conformational behaviors of the monomeric and dimeric form of SARS-CoV-2 3CLpro apo structure, as revealed by microsecond time scale MD simulations. Our results also shed light on the conformational dynamics of the loop regions at the entry of the catalytic site. Studying, at atomic level, the characteristics of the active site and obtaining information on how the protein can interact with its substrates will allow the design of molecules able to block the enzymatic function crucial for the virus.

PPARγ represents a key target for the treatment of type 2 diabetes and metabolic syndrome. Synthetic antidiabetic drugs activating PPARγ are accompanied by serious undesirable side effects related to their agonism. In the search for new PPARγ regulators, inhibitors of PPARγ phosphorylation on S245 mediated by CDK5 represent an opportunity for the development of an improved generation of antidiabetic drugs acting through this nuclear receptor. We have employed a multidisciplinary approach, including protein-protein docking, X-ray crystallography, NMR, HDX, MD simulations, and site-directed mutagenesis to investigate conformational changes in PPARγ that impair the ability of CDK5 to interact with PPARγ and hence inhibit PPARγ phosphorylation. Finally, we describe an alternative inhibition mechanism adopted by a ligand bound far from the phosphorylation site.

The thyroid-stimulating hormone receptor (TSHR) has been indicated as a putative domestication gene in chicken. Comparison of WGS identified a variant in residue 558 of the transmembrane domain (TM) of TSHR, where the domestic chicken (GGD) presents an arginine, whereas the red jungle fowl (RJF) shares a conserved glycine with other vertebrates. This variant has been demonstrated to be associated with phenotypes that are important for domestication and related to thyroid regulation, such as less fearful behavior, reduced aggressive behavior and reduced dependence on seasonal reproduction in GGD as compared with RJF. By means of molecular dynamics simulations, we highlighted the structural and dynamic differences of variant Gly558Arg in the TSHR TM domain. Alterations in TM helix flexibility, structure and protein overall motion are described. The so-called 'arginine snorkeling' of residue 568 in GGD is observed and we hypothesize it as the originating force that produces the observed whole-protein perturbation in the helix bundle dynamics, capable of altering the TSHR signal transduction. The results are discussed in the context of their implications for a better understanding of biological mechanisms in chicken under control of the thyroid, such as body metabolism, as well as for their usefulness in biomedical research., (© 2019 Stichting International Foundation for Animal Genetics.)

The most frequently used approach for protein structure prediction is currently homology modeling. The 3D model building phase of this methodology is critical for obtaining an accurate and biologically useful prediction. The most widely employed tool to perform this task is MODELLER. This program implements the "modeling by satisfaction of spatial restraints" strategy and its core algorithm has not been altered significantly since the early 1990s. In this work, we have explored the idea of modifying MODELLER with two effective, yet computationally light strategies to improve its 3D modeling performance. Firstly, we have investigated how the level of accuracy in the estimation of structural variability between a target protein and its templates in the form of σ values profoundly influences 3D modeling. We show that the σ values produced by MODELLER are on average weakly correlated to the true level of structural divergence between target-template pairs and that increasing this correlation greatly improves the program's predictions, especially in multiple-template modeling. Secondly, we have inquired into how the incorporation of statistical potential terms (such as the DOPE potential) in the MODELLER's objective function impacts positively 3D modeling quality by providing a small but consistent improvement in metrics such as GDT-HA and lDDT and a large increase in stereochemical quality. Python modules to harness this second strategy are freely available at https://github.com/pymodproject/altmod. In summary, we show that there is a large room for improving MODELLER in terms of 3D modeling quality and we propose strategies that could be pursued in order to further increase its performance., Competing Interests: The authors have declared that no competing interests exist.

Histidine decarboxylase is a pyridoxal 5'-phosphate enzyme catalyzing the conversion of histidine to histamine, a bioactive molecule exerting its role in many modulatory processes. The human enzyme is involved in many physiological functions, such as neurotransmission, gastrointestinal track function, cell growth, and differentiation. Here, we studied the functional properties of the human enzyme and, in particular, the effects exerted at the protein level by two cysteine residues: Cys-180 and Cys-418. Surprisingly, the enzyme exists in an equilibrium between a reduced and an oxidized form whose extent depends on the redox state of Cys-180. Moreover, we determined that (i) the two enzymatic redox species exhibit modest structural changes in the coenzyme microenvironment and (ii) the oxidized form is slightly more active and stable than the reduced one. These data are consistent with the model proposed by bioinformatics analyses and molecular dynamics simulations in which the Cys-180 redox state could be responsible for a structural transition affecting the C-terminal domain reorientation leading to active site alterations. Furthermore, the biochemical properties of the purified C180S and C418S variants reveal that C180S behaves like the reduced form of the wild-type enzyme, while C418S is sensitive to reductants like the wild-type enzyme, thus allowing the identification of Cys-180 as the redox sensitive switch. On the other hand, Cys-418 appears to be a residue involved in aggregation propensity. A possible role for Cys-180 as a regulatory switch in response to different cellular redox conditions could be suggested.

Molecular dynamics (MD) simulation allows one to predict the time evolution of a system of interacting particles. It is widely used in physics, chemistry and biology to address specific questions about the structural properties and dynamical mechanisms of model systems. MD earned a great success in genome research, as it proved to be beneficial in sorting pathogenic from neutral genomic mutations. Considering their computational requirements, simulations are commonly performed on HPC computing devices, which are generally expensive and hard to administer. However, variables like the software tool used for modeling and simulation or the size of the molecule under investigation might make one hardware type or configuration more advantageous than another or even make the commodity hardware definitely suitable for MD studies. This work aims to shed lights on this aspect.

A 2-yr-old boy presented profound developmental delay, failure to thrive, ataxia, hypotonia, and tonic-clonic seizures that caused the death of the patient. Targeted and whole exome sequencing revealed two heterozygous missense variants: a novel mutation in the KCNJ10 gene that encodes for the inward-rectifying K + channel Kir4.1 and another previously characterized mutation in KCNT1 that encodes for the Na + -activated K + channel known as Slo2.2 or SLACK. The objectives of this study were to perform the clinical and genetic characterization of the proband and his family and to examine the functional consequence of the Kir4.1 mutation. The mutant and wild-type KCNJ10 constructs were generated and heterologously expressed in Xenopus laevis oocytes, and whole cell K + currents were measured using the two-electrode voltage-clamp technique. The KCNJ10 mutation c.652C>T resulted in a p.L218F substitution at a highly conserved residue site. Wild-type KCNJ10 expression yielded robust Kir current, whereas currents from oocytes expressing the mutation were reduced, remarkably. Western Blot analysis revealed reduced protein expression by the mutation. Kir5.1 subunits display selective heteromultimerization with Kir4.1 constituting channels with unique kinetics. The effect of the mutation on Kir4.1/5.1 channel activity was twofold: a reduction in current amplitudes and an increase in the pH-dependent inhibition. We thus report a novel loss-of-function mutation in Kir4.1 found in a patient with a coexisting mutation in SLACK channels that results in a fatal disease. NEW & NOTEWORTHY We present and characterize a novel mutation in KCNJ10 Unlike previously reported EAST/SeSAME patients, our patient was heterozygous, and contrary to previous studies, mimicking the heterozygous state by coexpression resulted in loss of channel function. We report in the same patient co-occurrence of a KCNT1 mutation resulting in a more severe phenotype. This study provides new insights into the phenotypic spectrum and to the genotype-phenotype correlations associated with EAST/SeSAME and MMFSI., (Copyright © 2017 the American Physiological Society.)

Nucleophosmin (NPM1) is a multifunctional nucleolar protein implicated in ribogenesis, centrosome duplication, cell cycle control, regulation of DNA repair and apoptotic response to stress stimuli. The majority of these functions are played through the interactions with a variety of protein partners. NPM1 is frequently overexpressed in solid tumors of different histological origin. Furthermore NPM1 is the most frequently mutated protein in acute myeloid leukemia (AML) patients. Mutations map to the C-terminal domain and lead to the aberrant and stable localization of the protein in the cytoplasm of leukemic blasts. Among NPM1 protein partners, a pivotal role is played by the tumor suppressor Fbw7γ, an E3-ubiquitin ligase that degrades oncoproteins like c-MYC, cyclin E, Notch and c-jun. In AML with NPM1 mutations, Fbw7γ is degraded following its abnormal cytosolic delocalization by mutated NPM1. This mechanism also applies to other tumor suppressors and it has been suggested that it may play a key role in leukemogenesis. Here we analyse the interaction between NPM1 and Fbw7γ, by identifying the protein surfaces implicated in recognition and key aminoacids involved. Based on the results of computational methods, we propose a structural model for the interaction, which is substantiated by experimental findings on several site-directed mutants. We also extend the analysis to two other NPM1 partners (HIV Tat and CENP-W) and conclude that NPM1 uses the same molecular surface as a platform for recognizing different protein partners. We suggest that this region of NPM1 may be targeted for cancer treatment.

Globular denatured proteins have structural properties similar to those of random coils. Experiments on denatured proteins have shown that when the temperature is increased thermal compaction may take place, resulting in a reduction of their radius of gyration R g to range between 5% and 35% of its initial value. This phenomenon has been attributed to various causes, namely entropic, hydrophobic, and structural factors. The intrinsically disordered protein tau, which helps in nucleating and stabilizing microtubules in the axons of the neurons, also undergoes a relevant compaction process: when its temperature is increased from 293 K to 333 K its gyration radius decreases by 18%. We have performed an atomistic simulation of this molecule, at the lowest and highest temperatures of the mentioned interval, using both standard molecular dynamics and metadynamics, in parallel with small-angle X-ray scattering experiments. Using the fit of the experimental data and a genetic algorithm to select the most probable configurations among those produced in both atomistic simulations (standard MD and metadynamics), we were able to compute relevant changes, related to the temperature increase, in the average angles between residues, in the transient secondary structures, in the solvent accessible surface area, and in the number of intramolecular H-bonds. The analysis of the data showed how to decompose the compaction phenomenon into three contributions. An estimate of the entropic contribution to the compaction was obtained using the changes in the mean values of the angles between contiguous residues. The computation of the solvent accessible surface at the two temperatures allowed an estimation of the second factor contributing to the compaction, namely the increase in the hydrophobic interaction. We also measured the change in the average number of residues temporarily being in α-helices, 3-helices, PP II helices, β-sheets and β-turns. Those changes in the secondary structure population produce a reduction in the contour length of the protein, yielding a structural contribution to the reduction of R g . This analysis shows that in tau the entropic factor accounts for about 60% of the compaction, the hydrophobic factor for about 25%, and the change in the secondary structure for about 15%.

The conformational behavior of the wild-type endonucleases I-DmoI and two of its mutants has been studied in the presence and in the absence of DNA target sequences by means of extended molecular dynamics simulations. Our results show that in the absence of DNA, the three protein forms explore a similar essential conformational space, whereas when bound to the same DNA target sequence of 25 base pairs, they diversify and restrain the subspace explored. In addition, the differences in the essential subspaces explored by the residues near the catalytic site for both the bound and unbound forms are discussed in background of the experimental protein activity., (© 2016 Wiley Periodicals, Inc.)

Dysfunction of the inwardly-rectifying potassium channels Kir4.1 (KCNJ10) represents a pathogenic mechanism contributing to Autism-Epilepsy comorbidity. To define the role of Kir4.1 variants in the disorder, we sequenced KCNJ10 in a sample of affected individuals, and performed genotype-phenotype correlations. The effects of mutations on channel activity, protein trafficking, and astrocyte function were investigated in Xenopus laevis oocytes, and in human astrocytoma cell lines. An in vivo model of the disorder was also explored through generation of kcnj10a morphant zebrafish overexpressing the mutated human KCNJ10. We detected germline heterozygous KCNJ10 variants in 19/175 affected children. Epileptic spasms with dysregulated sensory processing represented the main disease phenotype. When investigated on astrocyte-like cells, the p.R18Q mutation exerted a gain-of-function effect by enhancing Kir4.1 membrane expression and current density. Similarly, the p.R348H variant led to gain of channel function through hindrance of pH-dependent current inhibition. The frequent polymorphism p.R271C seemed, instead, to have no obvious functional effects. Our results confirm that variants in KCNJ10 deserve attention in autism-epilepsy, and provide insight into the molecular mechanisms of autism and seizures. Similar to neurons, astrocyte dysfunction may result in abnormal synaptic transmission and electrical discharge, and should be regarded as a possible pharmacological target in autism-epilepsy.

Marine ladder-shaped polyether toxins are implicated in neurological symptoms of fish-borne food poisonings. The toxin gambierol, produced by the marine dinoflagellate Gambierdiscus toxicus, belongs to the group of ladder-shaped polyether toxins and inhibits Kv3.1 channels with nanomolar affinity through a mechanism of gating modification. Binding determinants for gambierol localize at the lipid-exposed interface of the pore forming S5 and S6 segments, suggesting that gambierol binds outside of the permeation pathway. To explore a possible involvement of the voltage-sensing domain (VSD), we made different chimeric channels between Kv3.1 and Kv2.1, exchanging distinct parts of the gating machinery. Our results showed that neither the electro-mechanical coupling nor the S1-S3a region of the VSD affect gambierol sensitivity. In contrast, the S3b-S4 part of the VSD (paddle motif) decreased gambierol sensitivity in Kv3.1 more than 100-fold. Structure determination by homology modeling indicated that the position of the S3b-S4 paddle and its primary structure defines the shape and∖or the accessibility of the binding site for gambierol, explaining the observed differences in gambierol affinity between the channel chimeras. Furthermore, these findings explain the observed difference in gambierol affinity for the closed and open channel configurations of Kv3.1, opening new possibilities for exploring the VSDs as selectivity determinants in drug design., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

An autosomal dominant protein aggregate myopathy, characterized by high plasma creatine kinase and calsequestrin-1 (CASQ1) accumulation in skeletal muscle, has been recently associated with a missense mutation in CASQ1 gene. The mutation replaces an evolutionarily-conserved aspartic acid with glycine at position 244 (p.D244G) of CASQ1, the main sarcoplasmic reticulum (SR) Ca2+ binding and storage protein localized at the terminal cisternae of skeletal muscle cells. Here, immunocytochemical analysis of myotubes, differentiated from muscle-derived primary myoblasts, shows that sarcoplasmic vacuolar aggregations positive for CASQ1 are significantly larger in CASQ1-mutated cells than control cells. A strong co-immuno staining of both RyR1 and CASQ1 was also noted in the vacuoles of myotubes and muscle biopsies derived from patients. Electrophysiological recordings and sarcoplasmic Ca2+ measurements provide evidence for less Ca2+ release from the SR of mutated myotubes when compared to that of controls. These findings further clarify the pathogenic nature of the p.D244G variant and point out defects in sarcoplasmic Ca2+ homeostasis as a mechanism underlying this human disease, which could be distinctly classified as "CASQ1-couplonopathy".

Episodic ataxia type 1 (EA1) is an autosomal dominant K(+) channelopathy which manifests with short attacks of cerebellar ataxia and dysarthria, and may also show interictal myokymia. Episodes can be triggered by emotional or physical stress, startle response, sudden postural change or fever. Here we describe a 31-year-old man displaying markedly atypical symptoms, including long-lasting attacks of jerking muscle contractions associated with hyperthermia, severe migraine, and a relatively short-sleep phenotype. A single nucleotide change in KCNA1 (c.555C>G) was identified that changes a highly conserved residue (p.C185W) in the first transmembrane segment of the voltage-gated K(+) channel Kv1.1. The patient is heterozygous and the mutation was inherited from his asymptomatic mother. Next generation sequencing revealed no variations in the CACNA1A, CACNB4, KCNC3, KCNJ10, PRRT2 or SCN8A genes of either the patient or mother, except for a benign variant in SLC1A3. Functional analysis of the p.C185W mutation in KCNA1 demonstrated a deleterious dominant-negative phenotype where the remaining current displayed slower activation kinetics, subtle changes in voltage-dependence and faster recovery from slow inactivation. Structural modeling also predicts the C185W mutation to be functionally deleterious. This description of novel clinical features, associated with a Kv1.1 mutation highlights a possibly unrecognized relationship between K(+) channel dysfunction, hyperthermia and migraine in EA1, and suggests that thorough assessments for these symptoms should be carefully considered for all patients affected by EA1.

Short QT3 syndrome (SQT3S) is a cardiac disorder characterized by a high risk of mortality and associated with mutations in Kir2.1 (KCNJ2) channels. The molecular mechanisms leading to channel dysfunction, cardiac rhythm disturbances and neurodevelopmental disorders, potentially associated with SQT3S, remain incompletely understood. Here, we report on monozygotic twins displaying a short QT interval on electrocardiogram recordings and autism-epilepsy phenotype. Genetic screening identified a novel KCNJ2 variant in Kir2.1 that (i) enhanced the channel's surface expression and stability at the plasma membrane, (ii) reduced protein ubiquitylation and degradation, (iii) altered protein compartmentalization in lipid rafts by targeting more channels to cholesterol-poor domains and (iv) reduced interactions with caveolin 2. Importantly, our study reveals novel physiological mechanisms concerning wild-type Kir2.1 channel processing by the cell, such as binding to both caveolin 1 and 2, protein degradation through the ubiquitin-proteasome pathway; in addition, it uncovers a potential multifunctional site that controls Kir2.1 surface expression, protein half-life and partitioning to lipid rafts. The reported mechanisms emerge as crucial also for proper astrocyte function, suggesting the need for a neuropsychiatric evaluation in patients with SQT3S and offering new opportunities for disease management., (© The Author 2014. Published by Oxford University Press.)

Protein kinases work because their flexibility allows to continuously switch from inactive to active form. Despite the large number of structures experimentally determined in such states, the mechanism of their conformational transitions as well as the transition pathways are not easily to capture. In this regard, computational methods can help to shed light on such an issue. However, due to the intrinsic sampling limitations, much efforts have been done to model in a realistic way the conformational changes occurring in protein kinases. In this review we will address the principal biological achievements and structural aspects in studying kinases conformational transitions and will focus on the main challenges related to computational approaches such as molecular modeling and MD simulations.

ATP-sensitive K(+) (K(ATP)) channels, comprised of pore-forming Kir6.x and regulatory SURx subunits, play important roles in many cellular functions; because of their sensitivity to inhibition by intracellular ATP, K(ATP) channels provide a link between cell metabolism and membrane electrical activity. We constructed structural homology models of Kir6.2 and a series of Kir6.2 channels carrying mutations within the putative ATP-binding site. Computational docking was carried out to determine the conformation of ATP in its binding site. The Linear Interaction Energy (LIE) method was used to estimate the free-energy of ATP binding to wild-type and mutant Kir6.2 channels. Comparisons of the theoretical binding free energies for ATP with those determined from mutational experiments enabled the identification of the most probable conformation of ATP bound to the Kir6.2 channel. A set of LIE parameters was defined that may enable prediction of the effects of additional Kir6.2 mutations within the ATP binding site on the affinity for ATP., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

The 37/67-kDa human laminin receptor(LamR) is a cell surface protein that interacts with molecules located in the extra-cellular matrix. In particular, interactions between LamR and laminins play a major role in mediating changes in the cellular environment that affect cell adhesion, neurite outgrowth, tumor growth and metastasis. The exact interaction mode of laminin-1 and LamR is not fully understood. Laminin-1 is thought to bind to LamR through interaction with the so-called peptide G (residues 161–180) and the C-terminal helix (residues 205–229). Here we performed 100-ns atomistic force field based molecular dynamics simulations to explore the structure and dynamics of LamR related to laminin-1 interactions. Our main finding is that loop 188–197 in the C-terminal region is highly flexible. It undergoes a major change resulting in a conformational switch that partially solvent exposes the R180 residue in the final part of the G peptide. So, R180 could contribute to laminin-1 binding. Projection of the simulations along the first two principal components also confirms the importance of this conformational switch in the LamR. This may be a basic prerequisite to clarify the key structural determinants of the interaction of LamR with laminin-1.

The inwardly-rectifying potassium channel Kir4.1 is a major player in the astrocyte-mediated regulation of [K(+)](o) in the brain, which is essential for normal neuronal activity and synaptic functioning. KCNJ10, encoding Kir4.1, has been recently linked to seizure susceptibility in humans and mice, and is a possible candidate gene for Autism Spectrum Disorders (ASD). In this study, we performed a mutational screening of KCNJ10 in 52 patients with epilepsy of "unknown cause" associated with impairment of either cognitive or communicative abilities, or both. Among them, 14 patients fitted the diagnostic criteria for ASD. We identified two heterozygous KCNJ10 mutations (p.R18Q and p.V84M) in three children (two unrelated families) with seizures, ASD, and intellectual disability. The mutations replaced amino acid residues that are highly conserved throughout evolution and were undetected in about 500 healthy chromosomes. The effects of mutations on channel activity were functionally assayed using a heterologous expression system. These studies indicated that the molecular mechanism contributing to the disorder relates to an increase in either surface-expression or conductance of the Kir4.1 channel. Unlike previous syndromic associations of genetic variants in KCNJ10, the pure neuropsychiatric phenotype in our patients suggests that the new mutations affect K(+) homeostasis mainly in the brain, by acting through gain-of-function defects. Dysfunction in astrocytic-dependent K(+) buffering may contribute to autism/epilepsy phenotype, by altering neuronal excitability and synaptic function, and may represent a new target for novel therapeutic approaches., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Episodic ataxia type 1 (EA1) is an autosomal dominant disorder characterized by continuous myokymia and episodic attacks of ataxia. Mutations in the gene KCNA1 that encodes the voltage-gated potassium channel Kv1.1 are responsible for EA1. In several brain areas, Kv1.1 coassembles with Kv1.4, which confers N-type inactivating properties to heteromeric channels. It is therefore likely that the rate of inactivation will be determined by the number of Kv1.4 inactivation particles, as set by the precise subunit stoichiometry. We propose that EA1 mutations affect the rate of N-type inactivation either by reduced subunit surface expression, giving rise to a reduced number of Kv1.1 subunits in heterotetramer Kv1.1-Kv1.4 channels, or by reduced affinity for the Kv1.4 inactivation domain. To test this hypothesis, quantified amounts of mRNA for Kv1.4 or Kv1.1 containing selected EA1 mutations either in the inner vestibule of Kv1.1 on S6 or in the transmembrane regions were injected into Xenopus laevis oocytes and the relative rates of inactivation and stoichiometry were determined. The S6 mutations, V404I and V408A, which had normal surface expression, reduced the rate of inactivation by a decreased affinity for the inactivation domain while the mutations I177N in S1 and E325D in S5, which had reduced subunit surface expression, increased the rate of N-type inactivation due to a stoichiometric increase in the number of Kv1.4 subunits.

The energetic profile of an ion translated along the axis of an ion channel should reveal whether the structure corresponds to a functionally open or closed state of the channel. In this study, we explore the combined use of Poisson-Boltzmann electrostatic calculations and evaluation of van der Waals interactions between ion and pore to provide an initial appraisal of the gating state of a channel. This approach is exemplified by its application to the bacterial inward rectifier potassium channel KirBac3.1, where it reveals the closed gate to be formed by a ring of leucine (L124) side chains. We have extended this analysis to a comparative survey of gating profiles, including model hydrophobic nanopores, the nicotinic acetylcholine receptor, and a number of potassium channel structures and models. This enables us to identify three gating regimes, and to show the limitation of this computationally inexpensive method. For a (closed) gate radius of 0.4 nm < R < 0.8 nm, a hydrophobic gate may be present. For a gate radius of 0.2 nm < R < 0.4 nm, both electrostatic and van der Waals interactions will contribute to the barrier height. Below R = 0.2 nm, repulsive van der Waals interactions are likely to dominate, resulting in a sterically occluded gate. In general, the method is more useful when the channel is wider; for narrower channels, the flexibility of the protein may allow otherwise-unsurmountable energetic barriers to be overcome.

Shaker-like (KV1.1) channels contain a highly conserved Pro-Val-Pro (PVP) motif at the base of S6 that produces a kink in the S6 helices and provides a flexible element thought to be essential for channel gating. The role of proline-induced kinks in transmembrane helices is well known, but the contribution of the small hydrophobic valine between these two prolines is not known, and interestingly, Shab-like (KV2.1) channels possess an isoleucine at this position (PIP). Here we show that the exact nature of this central hydrophobic residue within the PXP motif confers unique functional properties to KV1 channels, including changes in activation and deactivation kinetics, voltage-dependent properties and open probabilities, but single-channel conductance and cell expression levels are not affected. In support of these functional changes, molecular dynamic simulations demonstrate that valine and isoleucine contribute differently to S6 flexibility within this motif. These results therefore indicate that the nature of the central hydrophobic residue in the PXP motif is an important functional determinant of KV channel gating by contributing, at least in part, to the relative flexibility of this motif.

Voltage-dependent K(+) channels transfer the voltage sensor movement into gate opening or closure through an electromechanical coupling. To test functionally whether an interaction between the S4-S5 linker (L45) and the cytoplasmic end of S6 (S6(T)) constitutes this coupling, the L45 in hKv1.5 was replaced by corresponding hKv2.1 sequence. This exchange was not tolerated but could be rescued by also swapping S6(T). Exchanging both L45 and S6(T) transferred hKv2.1 kinetics to an hKv1.5 background while preserving the voltage dependence. A one-by-one residue substitution scan of L45 and S6(T) in hKv1.5 further shows that S6(T) needs to be alpha-helical and forms a "crevice" in which residues I422 and T426 of L45 reside. These residues transfer the mechanical energy onto the S6(T) crevice, whereas other residues in S6(T) and L45 that are not involved in the interaction maintain the correct structure of the coupling.

Potassium (K (+)) channels can regulate ionic conduction through their pore by a mechanism, involving the selectivity filter, known as C-type inactivation. This process is rapid in the hERG K (+) channel and is fundamental to its physiological role. Although mutations within hERG are known to remove this process, a structural basis for the inactivation mechanism has yet to be characterized. Using MD simulations based on homology modeling, we observe that the carbonyl of the filter aromatic, Phe627, forming the S 0 K (+) binding site, swiftly rotates away from the conduction axis in the wild-type channel. In contrast, in well-characterized non-inactivating mutant channels, this conformational change occurs less frequently. In the non-inactivating channels, interactions with a water molecule located behind the selectivity filter are critical to the enhanced stability of the conducting state. We observe comparable conformational changes in the acid sensitive TASK-1 channel and propose a common mechanism in these channels for regulating efflux of K (+) ions through the selectivity filter.

Missense PTPN11 mutations cause Noonan and LEOPARD syndromes (NS and LS), two developmental disorders with pleiomorphic phenotypes. PTPN11 encodes SHP2, an SH2 domain-containing protein tyrosine phosphatase functioning as a signal transducer. Generally, different substitutions of a particular amino acid residue are observed in these diseases, indicating that the crucial factor is the residue being replaced. For a few codons, only one substitution is observed, suggesting the possibility of specific roles for the residue introduced. We analyzed the biochemical behavior and ligand-binding properties of all possible substitutions arising from single-base changes affecting codons 42, 139, 279, 282 and 468 to investigate the mechanisms underlying the invariant occurrence of the T42A, E139D and I282V substitutions in NS and the Y279C and T468M changes in LS. Our data demonstrate that the isoleucine-to-valine change at codon 282 is the only substitution at that position perturbing the stability of SHP2's closed conformation without impairing catalysis, while the threonine-to-alanine change at codon 42, but not other substitutions of that residue, promotes increased phosphopeptide-binding affinity. The recognition specificity of the C-SH2 domain bearing the E139D substitution differed substantially from its wild-type counterpart acquiring binding properties similar to those observed for the N-SH2 domain, revealing a novel mechanism of SHP2's functional dysregulation. Finally, while functional selection does not seem to occur for the substitutions at codons 279 and 468, we point to deamination of the methylated cytosine at nucleotide 1403 as the driving factor leading to the high prevalence of the T468M change in LS.

The Kv1-4 families of K+ channels contain a tandem proline motif (PXP) in the S6 helix that is crucial for channel gating. In human Kv1.5, replacing the first proline by an alanine resulted in a nonfunctional channel. This mutant was rescued by introducing another proline at a nearby position, changing the sequence into AVPP. This resulted in a channel that activated quickly (ms range) upon the first depolarization. However, thereafter, the channel became trapped in another gating mode that was characterized by slow activation kinetics (s range) with a shallow voltage dependence. The switch in gating mode was observed even with very short depolarization steps, but recovery to the initial "fast" mode was extremely slow. Computational modeling suggested that switching occurred during channel deactivation. To test the effect of the altered PXP sequence on the mobility of the S6 helix, we used molecular dynamics simulations of the isolated S6 domain of wild type (WT) and mutants starting from either a closed or open conformation. The WT S6 helix displayed movements around the PXP region with simulations starting from either state. However, the S6 with a AVPP sequence displayed flexibility only when started from the closed conformation and was rigid when started from the open state. These results indicate that the region around the PXP motif may serve as a "hinge" and that changing the sequence to AVPP results in channels that deactivate to a state with an alternate configuration that renders them "reluctant" to open subsequently.

The structures of three bacterial outer membrane proteins (OmpA, OmpX and PagP) have been determined by both X-ray diffraction and NMR. We have used multiple (7 x 15 ns) MD simulations to compare the conformational dynamics resulting from the X-ray versus the NMR structures, each protein being simulated in a lipid (DMPC) bilayer. Conformational drift was assessed via calculation of the root mean square deviation as a function of time. On this basis the 'quality' of the starting structure seems mainly to influence the simulation stability of the transmembrane beta-barrel domain. Root mean square fluctuations were used to compare simulation mobility as a function of residue number. The resultant residue mobility profiles were qualitatively similar for the corresponding X-ray and NMR structure-based simulations. However, all three proteins were generally more mobile in the NMR-based than in the X-ray simulations. Principal components analysis was used to identify the dominant motions within each simulation. The first two eigenvectors (which account for >50% of the protein motion) reveal that such motions are concentrated in the extracellular loops and, in the case of PagP, in the N-terminal alpha-helix. Residue profiles of the magnitude of motions corresponding to the first two eigenvectors are similar for the corresponding X-ray and NMR simulations, but the directions of these motions correlate poorly reflecting incomplete sampling on a approximately 10 ns timescale.

VSTx1 is a tarantula venom toxin which binds to the archaebacterial voltage-gated potassium channel KvAP. VSTx1 is thought to access the voltage sensor domain of the channel via the lipid bilayer phase. In order to understand its mode of action and implications for the mechanism of channel activation, it is important to characterize the interactions of VSTx1 with lipid bilayers. Molecular dynamics (MD) simulations (for a total simulation time in excess of 0.2 micros) have been used to explore VSTx1 localization and interactions with zwitterionic (POPC) and with anionic (POPE/POPG) lipid bilayers. In particular, three series of MD simulations have been used to explore the net drift of VSTx1 relative to the center of a bilayer, starting from different locations of the toxin. The preferred location of the toxin is at the membrane/water interface. Although there are differences between POPC and POPE/POPG bilayers, in both cases the toxin forms favorable interactions at the interface, maximizing H-bonding to lipid headgroups and to water molecules while retaining interactions with the hydrophobic core of the bilayer. A 30 ns unrestrained simulation reveals dynamic partitioning of VSTx1 into the interface of a POPC bilayer. The preferential location of VSTx1 at the interface is discussed in the context of Kv channel gating models and provides support for a mode of action in which the toxin interacts with the Kv voltage sensor "paddle" formed by the S3 and S4 helices.

Analysis of the crystal structures of the intact voltage-sensitive potassium channel KvAP (from Aeropyrum pernix) and Kv1.2 (from rat brain), along with the isolated voltage sensor (VS) domain from KvAP, raises the question of the exact nature of the voltage-sensing conformational change that triggers activation of Kv and related voltage-gated channels. Molecular dynamics simulations of the isolated VS of KvAP in a detergent micelle environment at two different temperatures (300 K and 368 K) have been used to probe the intrinsic flexibility of this domain on a tens-of-nanoseconds timescale. The VS contains a positively charged (S4) helix which is packed against a more hydrophobic S3 helix. The simulations at elevated temperature reveal an intrinsic flexibility/conformational instability of the S3a region (i.e., the C-terminus of the S3 helix). It is also evident that the S4 helix undergoes hinge bending and swiveling about its central I130 residue. The conformational instability of the S3a region facilitates the motion of the N-terminal segment of S4 (i.e., S4a). These simulations thus support a gating model in which, in response to depolarization, an S3b-S4a "paddle" may move relative to the rest of the VS domain. The flexible S3a region may in turn act to help restore the paddle to its initial conformation upon repolarization.

KirBac1.1 and 3.1 are bacterial homologues of mammalian inward rectifier K channels. We have performed extended molecular dynamics simulations (five simulations, each of >20 ns duration) of the transmembrane domain of KirBac in two membrane environments, a palmitoyl oleoyl phosphatidylcholine bilayer and an octane slab. Analysis of these simulations has focused on the conformational dynamics of the pore-lining M2 helices, which form the cytoplasmic hydrophobic gate of the channel. Principal components analysis reveals bending of M2, with a molecular hinge at the conserved glycine (Gly134 in KirBac1.1, Gly120 in KirBac3.1). More detailed analysis reveals a dimer-of-dimers type motion. The first two eigenvectors describing the motions of M2 correspond to helix kink and swivel motions. The conformational flexibility of M2 seen in these simulations correlates with differences in M2 conformation between that seen in the X-ray structures of closed channels (KcsA and KirBac) in which the helix is undistorted, and in open channels (e.g. MthK) in which the M2 helix is kinked. Thus, the simulations, albeit on a time scale substantially shorter than that required for channel gating, suggest a gating model in which the intrinsic flexibility of M2 about a molecular hinge is coupled to conformational transitions of an intracellular 'gatekeeper' domain, the latter changing conformation in response to ligand binding.

Molecular dynamics simulations may be used to probe the interactions of membrane proteins with lipids and with detergents at atomic resolution. Examples of such simulations for ion channels and for bacterial outer membrane proteins are described. Comparison of simulations of KcsA (an alpha-helical bundle) and OmpA (a beta-barrel) reveals the importance of two classes of side chains in stabilizing interactions with the head groups of lipid molecules: (i) tryptophan and tyrosine; and (ii) arginine and lysine. Arginine residues interacting with lipid phosphate groups play an important role in stabilizing the voltage-sensor domain of the KvAP channel within a bilayer. Simulations of the bacterial potassium channel KcsA reveal specific interactions of phosphatidylglycerol with an acidic lipid-binding site at the interface between adjacent protein monomers. A combination of molecular modelling and simulation reveals a potential phosphatidylinositol 4,5-bisphosphate-binding site on the surface of Kir6.2.

The recently determined structure of a mammalian voltage-gated potassium channel has important implications for our understanding of voltage-sensing and gating mechanisms in channels. It is also the first crystal structure of an overexpressed eukaryotic membrane protein.

Inward rectifier (Kir) potassium channels are characterized by two transmembrane helices per subunit, plus an intracellular C-terminal domain that controls channel gating in response to changes in concentration of various ligands. Based on the crystal structure of the tetrameric C-terminal domain of Kir3.1, it is possible to build a homology model of the ATP-binding C-terminal domain of Kir6.2. Molecular dynamics simulations have been used to probe the dynamics of Kir C-terminal domains and to explore the relationship between their dynamics and possible mechanisms of channel gating. Multiple simulations, each of 10 ns duration, have been performed for Kir3.1 (crystal structure) and Kir6.2 (homology model), in both their monomeric and tetrameric forms. The Kir6.2 simulations were performed with and without bound ATP. The results of the simulations reveal comparable conformational stability for the crystal structure and the homology model. There is some decrease in conformational flexibility when comparing the monomers with the tetramers, corresponding mainly to the subunit interfaces in the tetramer. The beta-phosphate of ATP interacts with the side chain of K185 in the Kir6.2 model and simulations. The flexibility of the Kir6.2 tetramer is not changed greatly by the presence of bound ATP, other than in two loop regions. Principal components analysis of the simulated dynamics suggests loss of symmetry in both the Kir3.1 and Kir6.2 tetramers, consistent with "dimer-of-dimers" motion of subunits in C-terminal domains of the corresponding Kir channels. This is suggestive of a gating model in which a transition between exact tetrameric symmetry and dimer-of-dimers symmetry is associated with a change in transmembrane helix packing coupled to gating of the channel. Dimer-of-dimers motion of the C-terminal domain tetramer is also supported by coarse-grained (anisotropic network model) calculations. It is of interest that loss of exact rotational symmetry has also been suggested to play a role in gating in the bacterial Kir homolog, KirBac1.1, and in the nicotinic acetylcholine receptor channel.

Molecular modeling and simulations enable extrapolation for the structure of bacterial potassium channels to the function of their mammalian homologues. Molecular dynamics simulations have revealed the concerted single-file motion of potassium ions and water molecules through the selectivity filter of K channels and the role of filter flexibility in ion permeation and in "fast gating." Principal components analysis of extended K channel simulations suggests that hinge-bending of pore-lining M2 (or S6) helices plays a key role in K channel gating. Based on these and other simulations, a molecular model for gating of inward rectifier K channel gating is presented.

In a previous article (Zbilut et al., Biophys J 2003;85:3544-3557), we demonstrated how an aggregation versus folding choice could be approached considering hydrophobicity distribution and charge. In this work, our aim is highlighting the mutual interaction of charge and hydrophobicity distribution in the aggregation process. Use was made of two different peptides, both derived from a transmembrane protein (amyloid precursor protein; APP), namely, Abeta(1-28) and Abeta(1-40). Abeta(1-28) has a much lower aggregation propensity than Abeta(1-40). The results obtained by means of molecular dynamics simulations show that, when submitted to the most "aggregation-prone" environment, corresponding to the isoelectric point and consequently to zero net charge, both peptides acquire their maximum flexibility, but Abeta(1-40) has a definitely higher conformational mobility than Abeta(1-28). The absence of a hydrophobic "tail," which is the most mobile part of the molecule in Abeta(1-40), is the element lacking in Abeta(1-28) for obtaining a "fully aggregating" phenotype. Our results suggest that conformational flexibility, determined by both hydrophobicity and charge effect, is the main mechanistic determinant of aggregation propensity., ((c) 2004 Wiley-Liss, Inc.)

The bacterial channel KirBac1.1 provides a structural homolog of mammalian inward rectifier potassium (Kir) channels. The conformational dynamics of the selectivity filter of Kir channels are of some interest in the context of possible permeation and gating mechanisms for this channel. Molecular dynamics simulations of KirBac have been performed on a 10-ns timescale, i.e., comparable to that of ion permeation. The results of five simulations (total simulation time 50 ns) based on three different initial ion configurations and two different model membranes are reported. These simulation data provide evidence for limited (<0.1 nm) filter flexibility during the concerted motion of ions and water molecules within the filter, such local changes in conformation occurring on an approximately 1-ns timescale. In the absence of K(+) ions, the KirBac selectivity filter undergoes more substantial distortions. These resemble those seen in comparable simulations of other channels (e.g., KcsA and KcsA-based homology models) and are likely to lead to functional closure of the channel. This suggests filter distortions may provide a mechanism of K-channel gating in addition to changes in the hydrophobic gate formed at the intracellular crossing point of the M2 helices. The simulation data also provide evidence for interactions of the "slide" (pre-M1) helix of KirBac with phospholipid headgroups.

Ion channels are gated, i.e. they can switch conformation between a closed and an open state. Molecular dynamics simulations may be used to study the conformational dynamics of ion channels and of simple channel models. Simulations on model nanopores reveal that a narrow (<4 A) hydrophobic region can form a functionally closed gate in the channel and can be opened by either a small (approximately 1 A) increase in pore radius or an increase in polarity. Modelling and simulation studies confirm the importance of hydrophobic gating in K channels, and support a model in which hinge-bending of the pore-lining M2 (or S6 in Kv channels) helices underlies channel gating. Simulations of a simple outer membrane protein, OmpA, indicate that a gate may also be formed by interactions of charged side chains within a pore, as is also the case in ClC channels.

Although the use of surgical staples is a well established practice in intestinal tract surgery on adults, their role in biliodigestive anastomoses in adults and children has been more limited. The Authors describe a 12-year-old girl with a type-IV choledochal cyst, who was successfully treated with cyst excision and Roux-en-Y hepaticojejunostomy created with a surgical stapler.