MicroRNA Biophysically Modulates Cardiac Action Potential by Direct Binding to Ion Channel.

BACKGROUND: MicroRNAs (miRs) play critical roles in regulation of numerous biological events, including cardiac electrophysiology and arrhythmia, through a canonical RNA interference mechanism. It remains unknown whether endogenous miRs modulate physiologic homeostasis of the heart through noncanonical mechanisms. METHODS: We focused on the predominant miR of the heart (miR1) and investigated whether miR1 could physically bind with ion channels in cardiomyocytes by electrophoretic mobility shift assay, in situ proximity ligation assay, RNA pull down, and RNA immunoprecipitation assays. The functional modulations of cellular electrophysiology were evaluated by inside-out and whole-cell patch clamp. Mutagenesis of miR1 and the ion channel was used to understand the underlying mechanism. The effect on the heart ex vivo was demonstrated through investigating arrhythmia-associated human single nucleotide polymorphisms with miR1-deficient mice. RESULTS: We found that endogenous miR1 could physically bind with cardiac membrane proteins, including an inward-rectifier potassium channel Kir2.1. The miR1-Kir2.1 physical interaction was observed in mouse, guinea pig, canine, and human cardiomyocytes. miR1 quickly and significantly suppressed IK1 at sub-pmol/L concentration, which is close to endogenous miR expression level. Acute presence of miR1 depolarized resting membrane potential and prolonged final repolarization of the action potential in cardiomyocytes. We identified 3 miR1-binding residues on the C-terminus of Kir2.1. Mechanistically, miR1 binds to the pore-facing G-loop of Kir2.1 through the core sequence AAGAAG, which is outside its RNA interference seed region. This biophysical modulation is involved in the dysregulation of gain-of-function Kir2.1-M301K mutation in short QT or atrial fibrillation. We found that an arrhythmia-associated human single nucleotide polymorphism of miR1 (hSNP14A/G) specifically disrupts the biophysical modulation while retaining the RNA interference function. It is remarkable that miR1 but not hSNP14A/G relieved the hyperpolarized resting membrane potential in miR1-deficient cardiomyocytes, improved the conduction velocity, and eliminated the high inducibility of arrhythmia in miR1-deficient hearts ex vivo. CONCLUSIONS: Our study reveals a novel evolutionarily conserved biophysical action of endogenous miRs in modulating cardiac electrophysiology. Our discovery of miRs' biophysical modulation provides a more comprehensive understanding of ion channel dysregulation and may provide new insights into the pathogenesis of cardiac arrhythmias.

Integrative Study of the Structural and Dynamical Properties of a KirBac3.1 Mutant: Functional Implication of a Highly Conserved Tryptophan in the Transmembrane Domain.

ATP-sensitive potassium (K-ATP) channels are ubiquitously expressed on the plasma membrane of cells in several organs, including the heart, pancreas, and brain, and they govern a wide range of physiological processes. In pancreatic ß-cells, K-ATP channels composed of Kir6.2 and SUR1 play a key role in coupling blood glucose and insulin secretion. A tryptophan residue located at the cytosolic end of the transmembrane helix is highly conserved in eukaryote and prokaryote Kir channels. Any mutation on this amino acid causes a gain of function and neonatal diabetes mellitus. In this study, we have investigated the effect of mutation on this highly conserved residue on a KirBac channel (prokaryotic homolog of mammalian Kir6.2). We provide the crystal structure of the mutant KirBac3.1 W46R (equivalent to W68R in Kir6.2) and its conformational flexibility properties using HDX-MS. In addition, the detailed dynamical view of the mutant during the gating was investigated using the in silico method. Finally, functional assays have been performed. A comparison of important structural determinants for the gating mechanism between the wild type KirBac and the mutant W46R suggests interesting structural and dynamical clues and a mechanism of action of the mutation that leads to the gain of function.

A New Strategy for Atomic Flexible Fitting in Cryo-EM Maps by Molecular Dynamics with Excited Normal Modes (MDeNM-EMfit).

Previous studies demonstrated the efficiency of the Molecular Dynamics with excited Normal Modes (MDeNM) method on the characterization of large structural changes at a low computational cost. We present here MDeNM-EMfit, an extension of the original method designed to the flexible fit of structures into cryo-EM maps. Here, instead of a uniform exploration of the collective motions described by normal modes, sampling is directed toward conformations with increased correlations with the experimental map. Future perspectives to improve the accuracy of fitting and speed of calculations are discussed in light of the results.

Molecular dynamics simulations reveal membrane lipid interactions of the full-length lymphocyte specific kinase (Lck).

The membrane-bound lymphocyte-specific protein-tyrosine kinase (Lck) triggers T cell antigen receptor signalling to initiate adaptive immune responses. Despite many structure-function studies, the mode of action of Lck and the potential role of plasma membrane lipids in regulating Lck's activity remains elusive. Advances in molecular dynamics simulations of membrane proteins in complex lipid bilayers have opened a new perspective in gathering such information. Here, we have modelled the full-length Lck open and closed conformations  using data available from different crystalographic studies and simulated its interaction with the inner leaflet of the T cell plasma membrane. In both conformations, we found that the unstructured unique domain and the structured domains including the kinase interacted with the membrane with a preference for PIP lipids. Interestingly, our simulations suggest that the Lck-SH2 domain interacts with lipids differently in the open and closed Lck conformations, demonstrating that lipid interaction can potentially regulate Lck's conformation and in turn modulate T cell signalling. Additionally, the Lck-SH2 and kinase domain residues that significantly contacted PIP lipids are found to be conserved among the Src family of kinases, thereby potentially representing similar PIP interactions within the family.

On the Tracks of the Aggregation Mechanism of the PHF6 Peptide from Tau Protein: Molecular Dynamics, Energy, and Interaction Network Investigations.

The formation of neurofibrillary tangles (NFTs), composed of tau protein aggregates, is a hallmark of some neurodegenerative diseases called tauopathies. NFTs are composed of paired helical filaments (PHFs) of tau protein with a dominant ß-sheet secondary structuration. The NFT formation mechanism is not known yet. This study focuses on PHF6, a crucial hexapeptide responsible for tau aggregation. A 2 µs molecular dynamics simulation was launched to determine the keys of the PHF6 aggregation mechanism. Hydrogen bonding, van der Waals, and other non-covalent interactions as π-stacking were investigated. Parallel aggregation was slightly preferred due to its adaptability, but antiparallel aggregation remained widely present during the PHF6 aggregation. The analysis highlighted the leading role of hydrogen bonds identified at the atomic level for each aggregation process. The aggregation study emphasized the importance of Tyr310 during the ß-sheets' complexation through π-stacking.

Cryo-electron microscopy unveils unique structural features of the human Kir2.1 channel.

We present the first structure of the human Kir2.1 channel containing both transmembrane domain (TMD) and cytoplasmic domain (CTD). Kir2.1 channels are strongly inward-rectifying potassium channels that play a key role in maintaining resting membrane potential. Their gating is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2). Genetically inherited defects in Kir2.1 channels are responsible for several rare human diseases, including Andersen's syndrome. The structural analysis (cryo-electron microscopy), surface plasmon resonance, and electrophysiological experiments revealed a well-connected network of interactions between the PIP2-binding site and the G-loop through residues R312 and H221. In addition, molecular dynamics simulations and normal mode analysis showed the intrinsic tendency of the CTD to tether to the TMD and a movement of the secondary anionic binding site to the membrane even without PIP2. Our results revealed structural features unique to human Kir2.1 and provided insights into the connection between G-loop and gating and the pathological mechanisms associated with this channel.

Unexpected Gating Behaviour of an Engineered Potassium Channel Kir.

In this study, we investigated the dynamics and functional characteristics of the KirBac3.1 S129R, a mutated bacterial potassium channel for which the inner pore-lining helix (TM2) was engineered so that the bundle crossing is trapped in an open conformation. The structure of this channel has been previously determined at high atomic resolution. We explored the dynamical characteristics of this open state channel using an in silico method MDeNM that combines molecular dynamics simulations and normal modes. We captured the global and local motions at the mutation level and compared these data with HDX-MS experiments. MDeNM provided also an estimation of the probability of the different opening states that are in agreement with our electrophysiological experiments. In the S129R mutant, the Arg129 mutation releases the two constriction points in the channel that existed in the wild type but interestingly creates another restriction point.

Biochemistry, structure, and cellular internalization of a four nanobody-bearing Fc dimer.

VHH stands for the variable regions of heavy chain only of camelid IgGs. The VHH family forms a set of interesting proteins derived from antibodies that maintain their capacity to recognize the antigen, despite their relatively small molecular weight (in the 12,000 Da range). Continuing our exploration of the possibilities of those molecules, we chose to design alternative molecules with maintained antigen recognition, but enhanced capacity, by fusing four VHH with one Fc, the fragment crystallizable region of antibodies. In doing so, we aimed at having a molecule with superior quantitative antigen recognition (×4) while maintaining its size below the 110 kDa. In the present paper, we described the building of those molecules that we coined VHH2 -Fc-VHH2 . The structure of VHH2 -Fc-VHH2 in complex with HER2 antigen was determined using electronic microscopy and modeling. The molecule is shown to bind four HER2 proteins at the end of its flexible arms. VHH2 -Fc-VHH2 also shows an internalization capacity via HER2 receptor superior to the reference anti-HER2 monoclonal antibody, Herceptin®, and to a simple fusion of two VHH with one Fc (VHH2 -Fc). This new type of molecules, VHH2 -Fc-VHH2 , could be an interesting addition to the therapeutic arsenal with multiple applications, from diagnostic to therapy.

Structural modeling of a novel membrane-bound globin-coupled sensor in Geobacter sulfurreducens.

Globin-coupled sensors (GCS) usually consist of three domains: a sensor/globin, a linker, and a transmitter domain. The globin domain (GD), activated by ligand binding and/or redox change, induces an intramolecular signal transduction resulting in a response of the transmitter domain. Depending on the nature of the transmitter domain, GCSs can have different activities and functions, including adenylate and di-guanylate cyclase, histidine kinase activity, aerotaxis and/or oxygen sensing function. The gram-negative delta-proteobacterium Geobacter sulfurreducens expresses a protein with a GD covalently linked to a four transmembrane domain, classified, by sequence similarity, as GCS (GsGCS). While its GD is fully characterized, not so its transmembrane domain, which is rarely found in the globin superfamily. In the present work, GsGCS was characterized spectroscopically and by native ion mobility-mass spectrometry in combination with cryo-electron microscopy. Although lacking high resolution, the oligomeric state and the electron density map were valuable for further rational modeling of the full-length GsGCS structure. This model demonstrates that GsGCS forms a transmembrane domain-driven tetramer with minimal contact between the GDs and with the heme groups oriented outward. This organization makes an intramolecular signal transduction less likely. Our results, including the auto-oxidation rate and redox potential, suggest a potential role for GsGCS as redox sensor or in a membrane-bound e-/H+ transfer. As such, GsGCS might act as a player in connecting energy production to the oxidation of organic compounds and metal reduction. Database searches indicate that GDs linked to a four or seven helices transmembrane domain occur more frequently than expected.

New Structural insights into Kir channel gating from molecular simulations, HDX-MS and functional studies.

Inward rectifier potassium (Kir) channels play diverse and important roles in shaping action potentials in biological membranes. An increasing number of diseases are now known to be directly associated with abnormal Kir function. However, the gating of Kir still remains unknown. To increase our understanding of its gating mechanism, a dynamical view of the entire channel is essential. Here the gating activation was studied using a recent developped in silico method, MDeNM, which combines normal mode analysis and molecular dynamics simulations that showed for the very first time the importance of interrelated collective and localized conformational movements. In particular, we highlighted the role played by concerted movements of the different regions throughout the entire protein, such as the cytoplasmic and transmembrane domains and the slide helices. In addition, the HDX-MS analysis achieved in these studies provided a comprehensive and detailed view of the dynamics associated with open/closed transition of the Kir channel in coherence with the theoretical results. MDeNM gives access to the probability of the different opening states that are in agreement with our electrophysiological experiments. The investigations presented in this article are important to remedy dysfunctional channels and are of interest for designing new pharmacological compounds.

Insights into molecular mechanisms of drug metabolism dysfunction of human CYP2C9*30.

Cytochrome P450 2C9 (CYP2C9) metabolizes about 15% of clinically administrated drugs. The allelic variant CYP2C9*30 (A477T) is associated to diminished response to the antihypertensive effects of the prodrug losartan and affected metabolism of other drugs. Here, we investigated molecular mechanisms involved in the functional consequences of this amino-acid substitution. Molecular dynamics (MD) simulations performed for the active species of the enzyme (heme in the Compound I state), in the apo or substrate-bound state, and binding energy analyses gave insights into altered protein structure and dynamics involved in the defective drug metabolism of human CYP2C9.30. Our data revealed an increased rigidity of the key Substrate Recognition Sites SRS1 and SRS5 and shifting of the ß turn 4 of SRS6 toward the helix F in CYP2C9.30. Channel and binding substrate dynamics analyses showed altered substrate channel access and active site accommodation. These conformational and dynamic changes are believed to be involved in the governing mechanism of the reduced catalytic activity. An ensemble of representative conformations of the WT and A477T mutant properly accommodating drug substrates were identified, those structures can be used for prediction of new CYP2C9 and CYP2C9.30 substrates and drug-drug interactions.