Molecular dynamics recipes for genome research

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

Structural investigation of nucleophosmin interaction with the tumor suppressor Fbw7γ

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

Computational Study of Helicase from SARS-CoV-2 in RNA-Free and Engaged Form

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

Kv Channel Gating Requires a Compatible S4-S5 Linker and Bottom Part of S6, Constrained by Non-interacting Residues

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 (S6T) 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 S6T. Exchanging both L45 and S6T transferred hKv2.1 kinetics to an hKv1.5 background while preserving the voltage dependence. A one-by-one residue substitution scan of L45 and S6T in hKv1.5 further shows that S6T needs to be α-helical and forms a “crevice” in which residues I422 and T426 of L45 reside. These residues transfer the mechanical energy onto the S6T crevice, whereas other residues in S6T and L45 that are not involved in the interaction maintain the correct structure of the coupling

Salt Contamination of Wooden Materials: the Case of Trondheim (Norway) Warehouses

Warehouses are big architectonical structures mostly made of spruce wood and utilized as storage buildings principally by food traders in Northern Countries. Trondheim’s warehouses currently observable along the river Nidelva, date back between the 17th and half of the 19th century were mostly used to stock and process fish. Therefore, where the food goods were stored, residuals are expected to be still present and/or to be responsible for the formation of alteration products on the wooden surfaces as well as inside the wooden structure. Here we propose a characterization of residual and neo-formed compounds inside and on the surface of wooden logs by means of vacuum microbalance that allowed both to individuate the type of salts, as well as, to estimate the maximum water film thickness adsorbed on the wooden samples at 93% of RH. These data have been related to variations in the acoustic emission (AE) intensity detected at the log surface and to the wood moisture content measured with capacitive and resistance operating moisture meters. The application of three independent techniques have allowed obtaining interestingly information indicating their potentiality as decay assessment techniques in the field of historical materials and specifically in the study of salts weathering on wood. The methodology allowed identifying a clear relationship between the amount of water in logs as a function of their distance from the ground and variations in the amplitude of the acoustic emission signals

Novel phenotype associated with a mutation in the KCNA1(Kv1.1) gene

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.peer-reviewe

Gain-of-function defects of astrocytic Kir4.1 channels in children with autism spectrum disorders and epilepsy

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. Supplementary information accompanies this paper in the files section.peer-reviewe

KirBac1.1: It's an Inward Rectifying Potassium Channel

KirBac1.1 is a prokaryotic homologue of eukaryotic inward rectifier potassium (Kir) channels. The crystal structure of KirBac1.1 and related KirBac3.1 have now been used extensively to generate in silico models of eukaryotic Kir channels, but functional analysis has been limited to 86Rb+ flux experiments and bacteria or yeast complementation screens, and no voltage clamp analysis has been available. We have expressed pure full-length His-tagged KirBac1.1 protein in Escherichia coli and obtained voltage clamp recordings of recombinant channel activity in excised membrane patches from giant liposomes. Macroscopic currents of wild-type KirBac1.1 are K+ selective and spermine insensitive, but blocked by Ba2+, similar to “weakly rectifying” eukaryotic Kir1.1 and Kir6.2 channels. The introduction of a negative charge at a pore-lining residue, I138D, generates high spermine sensitivity, similar to that resulting from the introduction of a negative charge at the equivalent position in Kir1.1 or Kir6.2. KirBac1.1 currents are also inhibited by PIP2, consistent with 86Rb+ flux experiments, and reversibly inhibited by short-chain di-c8-PIP2. At the single-channel level, KirBac1.1 channels show numerous conductance states with two predominant conductances (15 pS and 32 pS at −100 mV) and marked variability in gating kinetics, similar to the behavior of KcsA in recombinant liposomes. The successful patch clamping of KirBac1.1 confirms that this prokaryotic channel behaves as a bona fide Kir channel and opens the way for combined biochemical, structural, and electrophysiological analysis of a tractable model Kir channel, as has been successfully achieved for the archetypal K+ channel KcsA