MDFF_NM: Improved Molecular Dynamics Flexible Fitting into Cryo-EM Density Maps with a Multireplica Normal Mode-Based Search.

Molecular Dynamics Flexible Fitting (MDFF) is a widely used tool to refine high-resolution structures into cryo-EM density maps. Despite many successful applications, MDFF is still limited by its high computational cost, overfitting, accuracy, and performance issues due to entrapment within wrong local minima. Modern ensemble-based MDFF tools have generated promising results in the past decade. In line with these studies, we present MDFF_NM, a stochastic hybrid flexible fitting algorithm combining Normal Mode Analysis (NMA) and simulation-based flexible fitting. Initial tests reveal that, besides accelerating the fitting process, MDFF_NM increases the diversity of fitting routes leading to the target, uncovering ensembles of conformations in closer agreement with experimental data. The potential integration of MDFF_NM with other existing methods and integrative modeling pipelines is also discussed.

Overview of Membrane Protein Sample Preparation for Single-Particle Cryo-Electron Microscopy Analysis.

Single-particle cryo-electron microscopy (cryo-EM SPA) has recently emerged as an exceptionally well-suited technique for determining the structure of membrane proteins (MPs). Indeed, in recent years, huge increase in the number of MPs solved via cryo-EM SPA at a resolution better than 3.0 Å in the Protein Data Bank (PDB) has been observed. However, sample preparation remains a significant challenge in the field. Here, we evaluated the MPs solved using cryo-EM SPA deposited in the PDB in the last two years at a resolution below 3.0 Å. The most critical parameters for sample preparation are as follows: (i) the surfactant used for protein extraction from the membrane, (ii) the surfactant, amphiphiles, nanodiscs or other molecules present in the vitrification step, (iii) the vitrification method employed, and (iv) the type of grids used. The aim is not to provide a definitive answer on the optimal sample conditions for cryo-EM SPA of MPs but rather assess the current trends in the MP structural biology community towards obtaining high-resolution cryo-EM structures.

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.

Global structural changes of an ion channel during its gating are followed by ion mobility mass spectrometry.

Mechanosensitive ion channels are sensors probing membrane tension in all species; despite their importance and vital role in many cell functions, their gating mechanism remains to be elucidated. Here, we determined the conditions for releasing intact mechanosensitive channel of large conductance (MscL) proteins from their detergents in the gas phase using native ion mobility-mass spectrometry (IM-MS). By using IM-MS, we could detect the native mass of MscL from Escherichia coli, determine various global structural changes during its gating by measuring the rotationally averaged collision cross-sections, and show that it can function in the absence of a lipid bilayer. We could detect global conformational changes during MscL gating as small as 3%. Our findings will allow studying native structure of many other membrane proteins.

Structural and Functional Highlights of Vacuolar Soluble Protein 1 from Pathogen Trypanosoma brucei brucei.

Trypanosoma brucei (T. brucei) is responsible for the fatal human disease called African trypanosomiasis, or sleeping sickness. The causative parasite, Trypanosoma, encodes soluble versions of inorganic pyrophosphatases (PPase), also called vacuolar soluble proteins (VSPs), which are localized to its acidocalcisomes. The latter are acidic membrane-enclosed organelles rich in polyphosphate chains and divalent cations whose significance in these parasites remains unclear. We here report the crystal structure of T. brucei brucei acidocalcisomal PPases in a ternary complex with Mg(2+) and imidodiphosphate. The crystal structure reveals a novel structural architecture distinct from known class I PPases in its tetrameric oligomeric state in which a fused EF hand domain arranges around the catalytic PPase domain. This unprecedented assembly evident from TbbVSP1 crystal structure is further confirmed by SAXS and TEM data. SAXS data suggest structural flexibility in EF hand domains indicative of conformational plasticity within TbbVSP1.

Control of KirBac3.1 potassium channel gating at the interface between cytoplasmic domains.

KirBac channels are prokaryotic homologs of mammalian inwardly rectifying potassium (Kir) channels, and recent structures of KirBac3.1 have provided important insights into the structural basis of gating in Kir channels. In this study, we demonstrate that KirBac3.1 channel activity is strongly pH-dependent, and we used x-ray crystallography to determine the structural changes that arise from an activatory mutation (S205L) located in the cytoplasmic domain (CTD). This mutation stabilizes a novel energetically favorable open conformation in which changes at the intersubunit interface in the CTD also alter the electrostatic potential of the inner cytoplasmic cavity. These results provide a structural explanation for the activatory effect of this mutation and provide a greater insight into the role of the CTD in Kir channel gating.

Separating speed and ability to displace roadblocks during DNA translocation by FtsK.

FtsK translocates dsDNA directionally at >5 kb/s, even under strong forces. In vivo, the action of FtsK at the bacterial division septum is required to complete the final stages of chromosome unlinking and segregation. Despite the availability of translocase structures, the mechanism by which ATP hydrolysis is coupled to DNA translocation is not understood. Here, we use covalently linked translocase subunits to gain insight into the DNA translocation mechanism. Covalent trimers of wild-type subunits dimerized efficiently to form hexamers with high translocation activity and an ability to activate XerCD-dif chromosome unlinking. Covalent trimers with a catalytic mutation in the central subunit formed hexamers with two mutated subunits that had robust ATPase activity. They showed wild-type translocation velocity in single-molecule experiments, activated translocation-dependent chromosome unlinking, but had an impaired ability to displace either a triplex oligonucleotide, or streptavidin linked to biotin-DNA, during translocation along DNA. This separation of translocation velocity and ability to displace roadblocks is more consistent with a sequential escort mechanism than stochastic, hand-off, or concerted mechanisms.

The polyserine domain of the lysyl-5 hydroxylase Jmjd6 mediates subnuclear localization.

Jmjd6 (jumonji-domain-containing protein 6) is an Fe(II)- and 2OG (2-oxoglutarate)-dependent oxygenase that catalyses hydroxylation of lysine residues in proteins involved in pre-mRNA splicing. Jmjd6 plays an essential role in vertebrate embryonic development and has been shown to modulate alternative splicing in response to hypoxic stress. In the present study we show that an alternatively spliced version of Jmjd6 lacking the polyS (polyserine) domain localizes to the nucleolus, predominantly in the fibrillar centre. Jmjd6 with the polyS domain deleted also interacts with nucleolar proteins. Furthermore, co-immunoprecipitation experiments and F2H (fluorescent 2-hybrid) assays demonstrate that Jmjd6 homo-oligomerization occurs in cells. In correlation with the observed variations in the subnuclear distribution of Jmjd6, the structure of Jmjd6 oligomers in vitro changes in the absence of the polyS domain, possibly reflecting the role of the polyS domain in nuclear/nucleolar shuttling of Jmjd6.

Human Kir2.1 Potassium Channel: Protocols for Cryo-EM Data Processing and Molecular Dynamics Simulations.

Kir channels are potassium (K+) channels responsible for the mechanism of inward rectification, which plays a fundamental role in maintaining the resting membrane potential. There are seven Kir subfamilies, and their opening and closing mechanism is regulated by different regulatory factors. Genetically inherited defects in Kir channels are responsible for several rare human diseases, and for most of them, there are currently no effective therapeutic treatments. High-resolution structural information is not available for several members within the Kir subfamilies. Recently, our group achieved a significant breakthrough by utilizing cryo-EM single-particle analysis to elucidate the first structure of the human Kir2.1 channel. We present here the data processing protocol of the cryo-EM data of the human Kir2.1 channel, which is applicable to the structural determination of other ion channels by cryo-EM single-particle analysis. We also introduce a protocol designed to assess the structural heterogeneity within the cryo-EM data, allowing for the identification of other possible protein structure conformations present in the collected data. Moreover, we present a protocol for conducting all-atom molecular dynamics (MD) simulations for K+ channels, which can be incorporated into various membrane models to simulate different environments. We also propose some methods for analyzing the MD simulations, with a particular emphasis on assessing the local mobility of protein residues.

Growth of large and highly ordered 2D crystals of a K⁺ channel, structural role of lipidic environment.

2D crystallography has proven to be an excellent technique to determine the 3D structure of membrane proteins. Compared to 3D crystallography, it has the advantage of visualizing the protein in an environment closer to the native one. However, producing good 2D crystals is still a challenge and little statistical knowledge can be gained from literature. Here, we present a thorough screening of 2D crystallization conditions for a prokaryotic inwardly rectifying potassium channel (>130 different conditions). Key parameters leading to very large and well-organized 2D crystals are discussed. In addition, the problem of formation of multilayers during the growth of 2D crystals is also addressed. An intermediate resolution projection map of KirBac3.1 at 6 Å is presented, which sheds (to our knowledge) new light on the structure of this channel in a lipid environment.

Mouse TSPO in a lipid environment interacting with a functionalized monolayer.

Translocator protein TSPO is a membrane protein highly conserved in evolution which does not belong to any structural known family. TSPO is involved in physiological functions among which transport of molecules such as cholesterol to form steroids and bile salts in mammalian cells. Membrane protein structure determination remains a difficult task and needs concomitant approaches (for instance X-ray- or Electron-crystallography and NMR). Electron microscopy and two-dimensional crystallization under functionalized monolayers have been successfully developed for recombinant tagged proteins. The difficulty comes from the detergent carried by membrane proteins that disrupt the lipid monolayer. We identified the best conditions for injecting the histidine tagged recombinant TSPO in detergent in the subphase and to keep the protein stable. Reconstituted recombinant protein into a lipid bilayer favors its adsorption to functionalized monolayers and limits the disruption of the monolayer by reducing the amount of detergent. Finally, we obtained the first transmission electron microscopy images of recombinant mouse TSPO negatively stained bound to the lipid monolayer after injection into the subphase of pre-reconstituted TSPO in lipids. Image analysis reveals that circular objects could correspond to an association of at least four monomers of mouse TSPO. The different amino acid compositions and the location of the polyhistidine tag between bacterial and mouse TSPO could account for the formation of dimer versus tetramer, respectively. The difference in the loop between the first and second putative transmembrane domain may contribute to distinct monomer interaction, this is supported by differences in ligand binding parameters and biological functions of both proteins.

Evidence for the assembly of a bacterial tripartite multidrug pump with a stoichiometry of 3:6:3.

The multiple transferable resistance (mTR) pump from Neisseria gonorrhoeae MtrCDE multidrug pump is assembled from the inner and outer membrane proteins MtrD and MtrE and the periplasmic membrane fusion protein MtrC. Previously we established that while there is a weak interaction of MtrD and MtrE, MtrC binds with relatively high affinity to both MtrD and MtrE. MtrD conferred antibiotic resistance only when it was expressed with MtrE and MtrC, suggesting that these proteins form a functional tripartite complex in which MtrC bridges MtrD and MtrE. Furthermore, we demonstrated that MtrC interacts with an intraprotomer groove on the surface of MtrE, inducing channel opening. However, a second groove is apparent at the interface of the MtrE subunits, which might also be capable of engaging MtrC. We have now established that MtrC can be cross-linked to cysteines placed in this interprotomer groove and that mutation of residues in the groove impair the ability of the pump to confer antibiotic resistance by locking MtrE in the closed channel conformation. Moreover, MtrE K390C forms an intermolecular disulfide bond with MtrC E149C locking MtrE in the open channel conformation, suggesting that a functional salt bridge forms between these residues during the transition from closed to open channel conformations. MtrC forms dimers that assemble into hexamers, and electron microscopy studies of single particles revealed that these hexamers are arranged into ring-like structures with an internal aperture sufficiently large to accommodate the MtrE trimer. Cross-linking of single cysteine mutants of MtrC to stabilize the dimer interface in the presence of MtrE, trapped an MtrC-MtrE complex with a molecular mass consistent with a stoichiometry of 3:6 (MtrE(3)MtrC(6)), suggesting that dimers of MtrC interact with MtrE, presumably by binding to the two grooves. As both MtrE and MtrD are trimeric, our studies suggest that the functional pump is assembled with a stoichiometry of 3:6:3.

Structure of human aspartyl aminopeptidase complexed with substrate analogue: insight into catalytic mechanism, substrate specificity and M18 peptidase family.

BACKGROUND: Aspartyl aminopeptidase (DNPEP), with specificity towards an acidic amino acid at the N-terminus, is the only mammalian member among the poorly understood M18 peptidases. DNPEP has implicated roles in protein and peptide metabolism, as well as the renin-angiotensin system in blood pressure regulation. Despite previous enzyme and substrate characterization, structural details of DNPEP regarding ligand recognition and catalytic mechanism remain to be delineated. RESULTS: The crystal structure of human DNPEP complexed with zinc and a substrate analogue aspartate-ß-hydroxamate reveals a dodecameric machinery built by domain-swapped dimers, in agreement with electron microscopy data. A structural comparison with bacterial homologues identifies unifying catalytic features among the poorly understood M18 enzymes. The bound ligands in the active site also reveal the coordination mode of the binuclear zinc centre and a substrate specificity pocket for acidic amino acids. CONCLUSIONS: The DNPEP structure provides a molecular framework to understand its catalysis that is mediated by active site loop swapping, a mechanism likely adopted in other M18 and M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy. Small differences in the substrate binding pocket such as shape and positive charges, the latter conferred by a basic lysine residue, further provide the key to distinguishing substrate preference. Together, the structural knowledge will aid in the development of enzyme-/family-specific aminopeptidase inhibitors.

The 3-dimensional structure of a hepatitis C virus p7 ion channel by electron microscopy.

Infection with the hepatitis C virus (HCV) has a huge impact on global health putting more than 170 million people at risk of developing severe liver disease. The HCV encoded p7 ion channel is essential for the production of infectious viruses. Despite a growing body of functional data, little is known about the 3-dimensional (3D) structure of the channel. Here, we present the 3D structure of a full-length viroporin, the detergent-solubilized hexameric 42 kDa form of the HCV p7 ion channel, as determined by single-particle electron microscopy using the random conical tilting approach. The reconstruction of such a small protein complex was made possible by a combination of high-contrast staining, the symmetry, and the distinct structural features of the channel. The orientation of the p7 monomers within the density was established using immunolabeling with N and C termini specific F(ab) fragments. The density map at a resolution of approximately 16 A reveals a flower-shaped protein architecture with protruding petals oriented toward the ER lumen. This broadest part of the channel presents a comparatively large surface area providing potential interaction sites for cellular and virally encoded ER resident proteins.

Tactical maneuvering and calculated risks: independent child migrants and the complex terrain of flight.

Similar to refugees in general, independent child migrants are frequently constructed in academic and popular discourse as passive and powerless or as untrustworthy and potentially threatening. Such portrayals fail to capture how these youth actively navigate the complex experiences of forced migration. Drawing on interviews with independent child migrants who arrived in Canada and on the conceptual framework of social navigation, we argue that contrary to being powerless, and despite significant structural barriers, these youth deliberately and thoughtfully navigate flight by making strategic decisions and taking calculated risks thereby ensuring their survival and well-being.

Re-centering labour in local food: local washing and the growing reliance on permanently temporary migrant farmworkers in Nova Scotia.

This article explores the labour behind local food in the Canadian Atlantic province of Nova Scotia. Based on surveys and interviews with farmers, migrant farmworkers, and farmers' market consumers in the province, we suggest that the celebration of local food by government and industry is a form of "local washing." Local washing hides key aspects of the social relations of production: in this case, it hides insufficient financial and policy supports for Nova Scotian farms and the increased reliance on migrant farmworkers via the Seasonal Agricultural Worker Program and the Temporary Foreign Worker Program. Our research found that a growing reliance on migrant farmworkers was not just the case for larger, industrial farms, but also for smaller farms participating in local and alternative food initiatives, like farmers' markets and fresh produce subscription boxes. Additionally, our surveys show that while farmers' market shoppers expressed an interest in supporting local foods, they reported knowing little about farm workers or working conditions. Our paper contributes to the literature on local and alternative food initiatives by connecting the relations of production to consumption. Rather than focusing solely on the nature of the relationships between farmers and consumers and the values embodied in direct agricultural markets, this research explores the central role of permanently temporary migrant workers in local agriculture.

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.

Regulation of flagellum number by FliA and FlgM and role in biofilm formation by Rhodobacter sphaeroides.

The FlgM secretion checkpoint plays a crucial role in coordinating bacterial flagellar assembly. Here we identify a new role for FlgM and FliA as part of a complex regulatory network which controls flagellum number and is essential for efficient swimming and biofilm formation in the monotrichous bacterium Rhodobacter sphaeroides.