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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 (PIP 2 ). 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 PIP 2 -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 PIP 2 . 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.

Graphical abstract, 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.

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.

International audience; 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 β-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.

International audience; 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.

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.

Background: Cardiac arrhythmias are a leading cause of morbidity and mortality. MicroRNAs (miRs) regulate the (electro) physiology of the heart and remodeling by canonical RNAi mechanism. Hypothesis: miRs maintain cardiac physiology also through noncanonical mechanisms. Methods: The physical binding between cardiac predominant miR--miR1 and ion channels was explored by EMSA, in situ PLA, RNA pull down and RIP assays. Electrophysiology of cardiomyocytes (CMs) and ex vivo miR1-deficient hearts were studied to reveal the functional outcome and the pathophysiological significance. Results: miR1 physically binds with an inward rectifier K + channel Kir2.1, which exists endogenously in CMs. This miR1-Kir2.1 binding is evolutionarily conserved. Functionally, miR1, at sub-pmol/L concentration, significantly suppresses IK1, depolarizes resting membrane potential, and prolongs final repolarization of action potentials in CMs. Mechanistically, miR1 binds to the pore-facing G-loop of Kir2.1 though the core sequence AAGAAG, which is outside the seed region. This biophysical modulation is involved in the dysregulation of a gain-of-function mutation Kir2.1-M301K in short-QT/AF patients. An AF-associated miR1-hSNP14A/G specifically disrupts the biophysical modulation while maintains miR1’s RNAi function. Significantly, miR1 but not hSNP14A/G eliminates the high inducibility of arrhythmia in miR1-deficient hearts. Conclusion: We reveal a novel function of miRs and develop a ground-breaking concept that endogenous miRs can physically bind with ion channels and rapidly modulate cardiac electrophysiology before its long-term effect of conventional RNAi mechanism. Our study provides more comprehensive understanding of ion-channel dysregulation associated with cardiac arrhythmias.

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.

International audience; There is a constant need for direct counting of biotic nanoparticles such as viruses to unravel river functioning. We used, for the first time in freshwater, a new method based on interferometry differentiating viruses from other particles such as membrane vesicles. In the French Marne River, viruses represented between 42 and 72% of the particles. A spring monitoring in 2014 revealed their increase (2.1 × 107 to 2.1 × 108 mL−1) linked to an increase in algal biomass and diversity of bacterial plankton. Predicted virus size distributions were in agreement with transmission electron microscopy analysis suggesting a dominance of large viruses (≥60 nm).

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 I K1 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.

International audience; > De récentes avancées technologiques révo-lutionnent le domaine des biologistes structu-ralistes. Plus précisément, des progrès spec-taculaires liés au développement de nouvelles technologies de capture d'images de microscope électronique (la détection directe d'électrons) ainsi que la mise à disposition de nouveaux logiciels d'analyse d'images ont conduit à une percée en terme de résolution en cryo-micros-copie électronique à transmission. Il est ainsi possible de calculer relativement rapidement des structures à haute résolution de molécules biologiques dont l'étude résiste aux méthodes plus classiques comme la diffraction des rayons X ou la résonance magnétique nucléaire (RMN). Ces structures ainsi obtenues peuvent aussi venir en complément des informations structurales déjà décrites par d'autres méthodes. Certaines de ces nouvelles structures résolues grâce à la cryo-microscopie électronique révèlent pour la première fois le fonctionnement précis de méca-nismes essentiels au bon déroulement physiolo-gique d'une cellule. La capacité à résoudre ces structures à la résolution du détail atomique est une condition essentielle pour le développement de nouveaux médicaments ayant comme cible thérapeutique ces protéines d'intérêt. Grâce à ces avancées techniques que nous résumons ici, des questions biologiques et médicales sont maintenant devenues accessibles, ce qui était inconcevable il y a seulement cinq ans.

International audience; The inactivated polio vaccine (IPV) contains poliovirus (PV) samples that belong to serotypes 1, 2 and 3.All three serotypes contain the D-antigen, which induces protective antibodies. The antigenic structureof PVs consists of at least four different antigenic sites and the D-antigen content represents the combinedactivity of multiple epitopes (Ferguson et al., 1993; Minor, 1990; Minor et al., 1986). The potencyof IPV vaccines is determined by measuring the D-antigen content. Several ELISA methods have beendeveloped using polyclonal or monoclonal antibodies (Mabs) in order to quantify the D-antigen content.Characterization of the epitopes recognized by the different Mabs is crucial to map the entire virus surfaceand ensure the presence of epitopes able to induce neutralizing antibodies. Using a new approachthat we developed to study the interaction between monoclonal antibodies and poliovirus type 2, whichcombines cryo-electron microscopy, image analysis and X-ray crystallography along with identificationof exposed amino acids, we have mapped in 3D the epitope sites recognized by three specific Fabs at thesurface of poliovirus type 2 (PV2) and characterized precisely the antigenic sites for these Fabs.

Here we study the intact stoichiometry and top-down fragmentation behavior of three integral membrane proteins which were natively reconstituted into detergent micelles: the mechano-sensitive ion channel of large conductance (MscL), the Kirbac potassium channel and the p7 viroporin from the hepatitis C virus. By releasing the proteins under nondenaturing conditions inside the mass spectrometer, we obtained their oligomeric sizes. Increasing the ion activation (collision energy) causes unfolding and subsequent ejection of a highly charged monomer from the membrane protein complexes. Further increase of the ion activation then causes collision-induced dissociation (CID) of the ejected monomers, with fragments observed which were predominantly found to stem from membrane-embedded regions. These experiments show how in a single experiment, we can probe the relation between higher-order structure and protein sequence, by combining the native MS data with fragmentation obtained from top-down MS.

Membrane proteins are key cellular components that perform essential functions. They are major therapeutic targets. Electron crystallography can provide structural experimental information at atomic scale for membrane proteins forming two-dimensional (2D) crystals. There are two different methods to produce 2D crystals of membrane proteins. (1) either directly in the bulk of the solution (2) or under a lipid monolayer at the air-water interface. This extra lipid monolayer helps to pre-orient the proteins in order to facilitate the growth of 2D crystals. We present here these two methods for 2D crystallization of membrane proteins implemented in a fully automated robot called CRACAM. These methods require small volume of low concentration of proteins, making it possible to explore more conditions with the same amount of protein. These automated methods outperform traditional 2D crystallization approaches in terms of accuracy, flexibility, and throughput.

The invention of the electron microscope has greatly enhanced the view scientists have of small structural details. Since its implementation, this technology has undergone considerable evolution and the resolution that can be obtained for biological objects has been extended. In addition, the latest generation of cryo-electron microscopes equipped with direct electron detectors and software for the automated collection of images, in combination with the use of advanced image-analysis methods, has dramatically improved the performance of this technique in terms of resolution. While calculating a sub-10 Å resolution structure was an accomplishment less than a decade ago, it is now common to generate structures at sub-5 Å resolution and even better. It is becoming possible to relatively quickly obtain high-resolution structures of biological molecules, in particular large ones (>500 kDa) which, in some cases, have resisted more conventional methods such as X-ray crystallography or nuclear magnetic resonance (NMR). Such newly resolved structures may, for the first time, shed light on the precise mechanisms that are essential for cellular physiological processes. The ability to attain atomic resolution may support the development of new drugs that target these proteins, allowing medicinal chemists to understand the intimacy of the relationship between their molecules and targets. In addition, recent developments in cryo-electron microscopy combined with image analysis can provide unique information on the conformational variability of macromolecular complexes. Conformational flexibility of macromolecular complexes can be investigated using cryo-electron microscopy and multiconformation reconstruction methods. However, the biochemical quality of the sample remains the major bottleneck to routine cryo-electron microscopy-based determination of structures at very high resolution.

Membrane proteins are key cellular components that perform essential functions. They are major therapeutic targets. Electron crystallography can provide structural experimental information at atomic scale for membrane proteins forming two-dimensional (2D) crystals. There are two different methods to produce 2D crystals of membrane proteins. (1) either directly in the bulk of the solution (2) or under a lipid monolayer at the air-water interface. This extra lipid monolayer helps to pre-orient the proteins in order to facilitate the growth of 2D crystals. We present here these two methods for 2D crystallization of membrane proteins implemented in a fully automated robot called CRACAM. These methods require small volume of low concentration of proteins, making it possible to explore more conditions with the same amount of protein. These automated methods outperform traditional 2D crystallization approaches in terms of accuracy, flexibility, and throughput.

International audience; The ATP-sensitive potassium (K ATP) channel is a hetero-octameric complex that links cell metabolism to membrane electrical activity in many cells, thereby controlling physiological functions such as insulin release, muscle contraction and neuronal activity. It consists of four pore-forming Kir6.2 and four regulatory sulfonylurea receptor (SUR) subunits. SUR2B serves as the regulatory subunit in smooth muscle and some neurones. An integrative approach, combining electron microscopy and homology modelling, has been used to obtain information on the structure of this large (megadalton) membrane protein complex. Single-particle electron microscopy of purified SUR2B tethered to a lipid monolayer revealed that it assembles as a tetramer of four SUR2B subunits surrounding a central hole. In the absence of an X-ray structure, a homology model for SUR2B based on the X-ray structure of the related ABC transporter Sav1866 was used to fit the experimental images. The model indicates that the central hole can readily accommodate the transmembrane domains of the Kir tetramer, suggests a location for the first transmembrane domains of SUR2B (which are absent in Sav1866) and suggests the relative orientation of the SUR and Kir6.2 subunits. Structured digital abstract SUR2B and SUR2B bind by electron microscopy (View interaction) SUR2B and SUR2B bind by molecular sieving (View interaction)

Recent technological advances have revolutionized the field of structural biologists. Specifically, dramatic progress related to the development of new electron microscopes and image capture (direct electron detection camera) and the provision of new image analysis software has led to a breakthrough in terms of resolution attained using cryo-electron transmission microscopy. It is thus possible to calculate relatively quickly high-resolution structures of biological molecules whom structural study still resists to more conventional methods such as X-ray diffraction or nuclear magnetic resonance (NMR). These structures thus obtained may also bring complementary structural information to those already described by other methods. Some of these new structures resolved through cryo-electron microscopy revealed for the first time the precise operation of essential mechanisms necessary for the good physiological process of a cell. The ability to solve these structures at atomic resolution detail is essential for the development of new drugs that target these proteins of therapeutic interest. Thanks to these advanced techniques that we summarize in this revew, biological and medical issues have now become accessible, whereas this approach was inconceivable only five yeras ago. ‡.

Negative stain electron microscopy and saturation transfer electron spin resonance spectroscopy have been used to compare the lattice ordering and in-plane membrane mobility of full-length and C-terminally cleaved squid rhodopsin. The C-terminus of squid rhodopsin contains a negatively charged region followed by 9–10 repeats of a proline-rich sequence, not found in rhodopsins other than those of cephalopod invertebrates, but similar proline repeats are found in other, unrelated membrane proteins. We find that the proline repeats cluster the rhodopsins into small groups, interfering with two-dimensional crystallization and maintaining their mobility in the membrane.

We have shown previously that normal mouse prion protein (MoPrP) binds copper ions during protein refolding and acquires antioxidant activity. In this report, we probe the structure of the copper refolded form of MoPrP to determine how copper binding alters the secondary and tertiary features of the protein. Circular dichroism showed that recombinant MoPrP prepared in the presence of copper (as Cu(++)) showed an increased signal in the 210-220 nm range of the spectrum. Changes in protein conformation were localised to the N-terminal region of MoPrP using a panel of antibodies to assess epitope accessibility. The copper refolded recombinant prion protein had reduced proteinase K (PK) sensitivity when compared to the non-copper liganded form. Reduced PK sensitivity was not due to aggregation however as high resolution electron microscopy showed a homogenous preparation with little aggregate when compared to the non-copper form. Finally, disruption of the single disulphide linkage in MoPrP significantly diminished the antioxidant activity of the copper refolded form suggesting that activity was not solely dependent on bound copper but also on a conformation enabled by the formation of the disulphide bond.

A high concentration of cadherin molecules at cell–cell adhesion sites is believed to be essential for generating strong intercellular junctions. In order to determine the interactions of cadherin domains involved in the early stages of lateral cluster formation on the cell surface, a recombinant fragment encompassing the first four domains of human VE-cadherin with a His-tag at the C terminus (VE-EC1-4-His) was produced. Two-dimensional crystals of VE-EC1-4-His were formed at the air–water interface using conventional lipids modified to contain a Ni2+-chelating group, which provides a specific site for interaction with the polyhistidine tag. The VE-EC1-4-His was monomeric at the concentration employed for crystal formation; however, the crystals exhibited a p2 symmetry and the presence of cis-dimer interactions between symmetry-related molecules. The VE-EC1-4-His molecules in the crystalline array have a remarkably compact conformation in contrast to the elongated “string of pearls” conformation seen in the hexameric assembly of VE-EC1-4-His in solution, and as seen in the crystal structure of C-cadherin. These results indicate that VE-cadherin can exist in at least two oligomeric states with different interactions between domains and can adopt highly different conformational states. We suggest that the compact cis-dimeric state may occur on isolated cells and that the compact form may serve to protect the molecule from degradation. As previously proposed we suppose that the trans-hexameric form is involved in intercellular adhesion.

International audience; 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 premRNA 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, coimmunoprecipitation 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. Key words: Fe(II)- and 2-oxoglutarate-dependent oxygenase, JmjC, lysine hydroxylation, nucleolus, polyserine domain, pre-mRNA splicing

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase composed of approximately 13 distinct subunits required for progression through meiosis, mitosis, and the G1 phase of the cell cycle. Despite its central role in these processes, information concerning its composition and structure is limited. Here, we determined the structure of yeast APC/C by cryo-electron microscopy (cryo-EM). Docking of tetratricopeptide repeat (TPR)-containing subunits indicates that they likely form a scaffold-like outer shell, mediating assembly of the complex and providing potential binding sites for regulators and substrates. Quantitative determination of subunit stoichiometry indicates multiple copies of specific subunits, consistent with a total APC/C mass of approximately 1.7 MDa. Moreover, yeast APC/C forms both monomeric and dimeric species. Dimeric APC/C is a more active E3 ligase than the monomer, with greatly enhanced processivity. Our data suggest that multimerisation and/or the presence of multiple active sites facilitates the APC/C's ability to elongate polyubiquitin chains.

A key molecular event in prion diseases is the conversion of the prion protein (PrP) from its normal cellular form (PrPC) to the disease-specific form (PrPSc). The transition from PrPC to PrPSc involves a major conformational change, resulting in amorphous protein aggregates and fibrillar amyloid deposits with increased beta-sheet structure. Using recombinant PrP refolded into a beta-sheet-rich form (beta-PrP) we have studied the fibrillization of beta-PrP both in solution and in association with raft membranes. In low ionic strength thick dense fibrils form large networks, which coexist with amorphous aggregates. High ionic strength results in less compact fibrils, that assemble in large sheets packed with globular PrP particles, resembling diffuse aggregates found in ex vivo preparations of PrPSc. Here we report on the finding of a beta-turn-rich conformation involved in prion fibrillization that is toxic to neuronal cells in culture. This is the first account of an intermediate in prion fibril formation that is toxic to neuronal cells. We propose that this unusual beta-turn-rich form of PrP may be a precursor of PrPSc and a candidate for the neurotoxic molecule in prion pathogenesis.

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 .

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 subuint 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.

Numerous cell surface receptors propagate activation signals to the interior of the cell via tyrosine phosphorylation of transmembrane proteins. This leads to the phosphotyrosine (PiY)-mediated recruitment of cytoplasmic signalling protein complexes which catalyze crucial biochemical signalling reactions. Here we describe the first in vitro reconstitution of such PiY-nucleated protein complexes on an artificial lipid membrane. A tyrosine phosphorylated recombinant variant of the transmembrane adaptor protein Linker for Activation of T cells (PiYLAT) was anchored in liposomes. These PiYLAT proteoliposomes specifically recruited cooperative high avidity signalling protein complexes from Jurkat cytosol. Nucleation of signalling protein assemblies readily occurred on PiYLAT liposomes composed of phosphatidylserine, but not on PiYLAT liposomes composed of phosphatidylcholine. Purified recombinant grb2 alone did not stably associate with tyrosine phosphorylated LAT proteoliposomes. However, when grb2 was presented to the PiYLAT proteoliposomes in the context of Jurkat cytosol it was incorporated into multiprotein signalling complexes. Together the data suggest that these reconstituted high-avidity signalling protein complexes represent a cooperative protein network. This novel in vitro approach offers a novel technology permitting biochemical, structural, and pharmacological analyses of plasma membrane receptor signalling complexes.

Phosphorylase kinase (PhK) coordinates hormonal and neuronal signals to initiate the breakdown of glycogen. The enzyme catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a. We present a 9.9 angstroms resolution structure of PhK heterotetramer (alphabetagammadelta)4 determined by cryo-electron microscopy single-particle reconstruction. The enzyme has a butterfly-like shape comprising two lobes with 222 symmetry. This three-dimensional structure has allowed us to dock the catalytic gamma subunit to the PhK holoenzyme at a location that is toward the ends of the lobes. We have also determined the structure of PhK decorated with GPb at 18 angstroms resolution, which shows the location of the substrate near the kinase subunit. The PhK preparation contained a number of smaller particles whose structure at 9.8 angstroms resolution was consistent with a proteolysed activated form of PhK that had lost the alpha subunits and possibly the gamma subunits.

Hydrogen uptake protein regulator (HupR) is a member of the nitrogen regulatory protein C (NtrC) family of response regulators. These proteins activate transcription by RNA polymerase (RNAP) in response to a change in environment. This change is detected through the phosphorylation of their receiver domain as part of a two-component signalling pathway. HupR is an unusual member of this family as it activates transcription when unphosphorylated, and transcription is inhibited by phosphorylation. Also, HupR activates transcription through the more general sigma(70) transcription initiation factor, which does not require activation by ATPase, in contrast to other NtrC family members that utilise sigma(54). Hence, its mode of action is expected to be different from those of the more conventional NtrC family members. We have determined the structures of the unphosphorylated N-terminal receiver domain of wild-type HupR, the mutant HupR(D55E)(N) (which cannot be phosphorylated and down-regulated), and HupR in the presence of the phosphorylation mimic BeF(3)(-). The structures show a typical response regulator fold organised as a dimer whose interface involves alpha4-beta5-alpha5 elements. The interactions across the interface are slightly different between apo and phospho mimics, and these reflect a rearrangement of key conserved residues around the active site aspartate that have been implicated in domain activation in other receiver domain proteins. We also show that the wild-type HupR receiver domain forms a weak dimer in solution, which is strengthened in the presence of the phosphorylation mimic BeF(3)(-). The results indicate many features similar to those that have been observed in other systems, including NtrC (where phosphorylation is activatory), and indicate that recognition properties, which allow HupR to be active in the absence of phosphorylation, lie in the transmission of phosphorylation signals through the linker region to the other domains of the protein.

Prion diseases are characterised at autopsy by neuronal loss and accumulation of amorphous protein aggregates and/or amyloid fibrils in the brains of humans and animals. These protein deposits result from the conversion of the cellular, mainly alpha-helical prion protein (PrP(C)) to the beta-sheet-rich isoform (PrP(Sc)). Although the pathogenic mechanism of prion diseases is not fully understood, it appears that protein aggregation is itself neurotoxic and not the product of cell death. The precise nature of the neurotoxic species and mechanism of cell death are yet to be determined, although recent studies with other amyloidogenic proteins suggest that ordered pre-fibrillar or oligomeric forms may be responsible for cellular dysfunction. In this study we have refolded recombinant prion protein (rPrP) to two distinct forms rich in beta-sheet structure with an intact disulphide bond. Here we report on the structural properties of globular aggregates and pre-fibrils of rPrP and show that both states are toxic to neuronal cells in culture. We show that exogenous rPrP aggregates are internalised by neuronal cells and found in the cytoplasm. We also measured the changes in electrophysiological properties of cultured neuronal cells on exposure to exogenous prion aggregates and discuss the implications of these findings.

The work reported herein deals with the synthesis and the preliminary physical-chemical analysis of new hemifluorinated surfactant made up of one fluorinated chain linked to a tricarboxylic acid polar head which is able to complex a Ni atom and should favor the two-dimensional crystallization of membrane proteins. Such a compound forms a Langmuir film which is a fluid at 20 °C and not perturbed by the presence of hydrocarbon detergent in aqueous solution. © 2008 Elsevier Ltd. All rights reserved.

KirBac3.1 belongs to a family of transmembrane potassium (K(+)) channels that permit the selective flow of K-ions across biological membranes and thereby regulate cell excitability. They are crucial for a wide range of biological processes and mutations in their genes cause multiple human diseases. Opening and closing (gating) of Kir channels may occur spontaneously but is modulated by numerous intracellular ligands that bind to the channel itself. These include lipids (such as PIP(2)), G-proteins, nucleotides (such as ATP) and ions (e.g. H(+), Mg(2+), Ca(2+)). We have used high-resolution atomic force microscopy (AFM) to examine KirBac3.1 in two different configurations. AFM imaging of the cytoplasmic surface of KirBac3.1 embedded in a lipid bilayer has allowed visualization of the tetrameric assembly of the ligand-binding domain. In the absence of Mg(2+), the four subunits appeared as four protrusions surrounding a central depression corresponding to the cytoplasmic pore. They did not display 4-fold symmetry, but formed a dimer-of-dimers with 2-fold symmetry. Upon addition of Mg(2+), a marked rearrangement of the intracellular ligand-binding domains was observed: the four protrusions condensed into a single protrusion per tetramer, and there was an accompanying increase in protrusion height. The central cavity within the four intracellular domains also disappeared on addition of Mg(2+), indicating constriction of the cytoplasmic pore. These structural changes are likely transduced to the transmembrane helices, which gate the K(+) channel. This is the first time AFM has been used as an interactive tool to study K(+) channels. It has enabled us to directly measure the conformational changes in the protein surface produced by ligand binding.

The ATP-sensitive potassium (K(ATP)) channel couples glucose metabolism to insulin secretion in pancreatic beta-cells. It comprises regulatory sulfonylurea receptor 1 and pore-forming Kir6.2 subunits. Binding and/or hydrolysis of Mg-nucleotides at the nucleotide-binding domains of sulfonylurea receptor 1 stimulates channel opening and leads to membrane hyperpolarization and inhibition of insulin secretion. We report here the first purification and functional characterization of sulfonylurea receptor 1. We also compared the ATPase activity of sulfonylurea receptor 1 with that of the isolated nucleotide-binding domains (fused to maltose-binding protein to improve solubility). Electron microscopy showed that nucleotide-binding domains purified as ring-like complexes corresponding to approximately 8 momomers. The ATPase activities expressed as maximal turnover rate [in nmol P(i).s(-1).(nmol protein)(-1)] were 0.03, 0.03, 0.13 and 0.08 for sulfonylurea receptor 1, nucleotide-binding domain 1, nucleotide-binding domain 2 and a mixture of nucleotide-binding domain 1 and nucleotide-binding domain 2, respectively. Corresponding K(m) values (in mm) were 0.1, 0.6, 0.65 and 0.56, respectively. Thus sulfonylurea receptor 1 has a lower K(m) than either of the isolated nucleotide-binding domains, and a lower maximal turnover rate than nucleotide-binding domain 2. Similar results were found with GTP, but the K(m) values were lower. Mutation of the Walker A lysine in nucleotide-binding domain 1 (K719A) or nucleotide-binding domain 2 (K1385M) inhibited the ATPase activity of sulfonylurea receptor 1 by 60% and 80%, respectively. Beryllium fluoride (K(i) 16 microm), but not MgADP, inhibited the ATPase activity of sulfonylurea receptor 1. In contrast, both MgADP and beryllium fluoride inhibited the ATPase activity of the nucleotide-binding domains. These data demonstrate that the ATPase activity of sulfonylurea receptor 1 differs from that of the isolated nucleotide-binding domains, suggesting that the transmembrane domains may influence the activity of the protein.

International audience; 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. 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 small angle X-ray scattering (SAXS) and electron microscopy data. Solution scattering data suggests structural flexibility in EF-hand domains indicative of conformational plasticity within TbbVSP1.

International audience; 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 conforma-tional changes during MscL gating as small as 3%. Our findings will allow studying native structure of many other membrane proteins.

Potassium channels allow the selective flow of K(+) ions across membranes. In response to external gating signals, the potassium channel can move reversibly through a series of structural conformations from a closed to an open state. 2D crystals of the inwardly rectifying K(+) channel KirBac3.1 from Magnetospirillum magnetotacticum have been captured in two distinct conformations, providing "snap shots" of the gating process. Analysis by electron cryomicroscopy of these KirBac3.1 crystals has resulted in reconstructed images in projection at 9 A resolution. Kir channels are tetramers of four subunits arranged as dimers of dimers. Each subunit has two transmembrane helices (inner and outer). In one crystal form, the pore is blocked; in the other crystal form, the pore appears open. Modeling based on the KirBac1.1 (closed) crystal structure shows that opening of the ion conduction pathway could be achieved by bending of the inner helices and significant movements of the outer helices.

Summary autoinhibitory domain (residues 298–386). Within the regulatorydomainaretwosequences(residues302–326 Phosphorylase kinase (PhK) integrates hormonal and and 342–366) that bind tightly to calmodulin [4, 5]. Whileneuronal signals and is a key enzyme in the control it is established that phosphorylation of the subunits of glycogen metabolism. PhK is one of the largest of (and to a lesser extent the subunits) gives rise to activa-the protein kinases and is composed of four types of tion [6], the mechanism of conformational changes pro- subunit, with stoichiometry ( ) 4 and a total MW duced by this posttranslational modification are notof 1.3 6 10 . PhK catalyzes the phosphorylation of understood.Theregulatorysubunitsappeartobeinhibi- inactive glycogen phosphorylase b (GPb), resulting in tory in that they restrain, either directly or indirectly, thethe formation of active glycogen phosphorylase a activity of the kinase domain. Partial proteolysis of the (GPa) and the stimulation of glycogenolysis. We have

The inactivated polio vaccine (IPV) contains poliovirus (PVs) samples that belong to serotypes 1, 2 and 3. All three serotypes contain the D-antigen, which induces protective antibodies. The antigenic structure of PVs consists of at least four different antigenic sites and the D-antigen content represents the combined activity of multiple epitopes (Ferguson et al., 1993; Minor, 1990; Minor et al., 1986). The potency of IPV vaccines is determined by measuring the D-antigen content. Several ELISA methods have been developed using polyclonal or monoclonal antibodies (Mabs) in order to quantify the D-antigen content. Characterization of the epitopes recognized by the different Mabs is crucial to map the entire virus surface and ensure the presence of epitopes able to induce neutralizing antibodies. In a new approach, combining cryo-electron microscopy and image analysis with X-ray crystallography data available along with identification of exposed amino acids we have mapped in 3D the epitope sites recognized by five specific Fabs and one Mab and characterized precisely the antigenic sites for these Mabs. We propose this method to be used to map the entire “epitopic” surface of virus.

The prion protein is a glycoprotein expressed by neurones and other cells. In its holo-form it binds copper and exhibits superoxide dismutase activity. Studies in mice have led to the description of two distinct alleles. Differences in these alleles are linked to long and short incubation times following infection with scrapie. We studied recombinant mouse protein corresponding to the products of either allele and two intermediates carrying single amino-acid residue substitutions. The different forms of the prion protein exhibited differences in superoxide dismutase (SOD) activity and conformation. Intermediates with single substitutions were less stable than either allelic product. The findings provide insight into the differences between the two alleles and might have consequences for understanding differences in susceptibility to prion disease.

Large, well-ordered two-dimensional crystals of the histidine-tagged-HupR protein, a transcriptional regulator from the photosynthetic bacterium Rhodobacter capsulatus, were obtained by specific interaction with a Ni(2+)-chelated lipid monolayer. HupR is a response regulator of the NtrC subfamily; it activates the transcription of the structural genes hupSLC, of [NiFe]hydrogenase. A projection map of the full-length protein at 9 A resolution was obtained by electron cryo-microscopy and image analysis of frozen-hydrated two-dimensional crystals. The crystals have a p6 plane group with unit cell dimensions of a=b=111.6(+/-1.0) A, gamma=120.4(+/-0.5) degrees. The structure of the N-terminal domain of NtrC, the family to which HupR belongs, had been determined previously by NMR. The atomic coordinates of the N-terminal domain of NtrC, were compared to the structure obtained by cryo-electron microscope techniques of the whole HupR. These results provide the first structure at medium resolution of a whole transcription factor, HupR from the NtrC family.

We present here some sensitive optical and mechanical experiments for monitoring the process of formation and growth of two-dimensional (2D) crystals of proteins on a lipid monolayer at an air-water interface. The adsorption of proteins on the lipid monolayer was monitored by ellipsometry measurements. An instrument was developed to measure the shear elastic constant (in plane rigidity) of the monolayer. These experiments have been done using cholera toxin B subunit (CTB) and annexin V as model proteins interacting with a monosialoganglioside (GM1) and dioleoylphosphatidylserine (DOPS), respectively. Electron microscopy observations of the protein-lipid layer transferred to grids were systematically used as a control. We found a good correlation between the measured in-plane rigidity of the monolayer and the presence of large crystalline domains observed by electron microscopy grids. Our interpretation of these data is that the crystallization process of proteins on a lipid monolayer passes through at least three successive stages: 1) molecular recognition between protein and lipid-ligand, i.e., adsorption of the protein on the lipid layer; 2) nucleation and growth of crystalline patches whose percolation is detected by the appearance of a non-zero in-plane rigidity; and 3) annealing of the layer producing a slower increase of the lateral or in-plane rigidity.

This method of two-dimensional crystallization of proteins on a lipid monolayer aims at producing 2D crystals of membrane proteins, which can provide structural information at high resolution by electron crystallography. A lipid monolayer is spread over the whole air-water interface of a drop, which provides a substrate for protein binding. The protein of interest is then adsorbed onto the lipid monolayer and forms a closely packed layer. The reconstitution step of the membrane protein into a lipid bilayer is realized by elimination of detergent. The combined effect of the elevated protein concentration, the alignment of the protein on the lipid monolayer, and the fluid monolayer film properties are conducive, in some cases, to the formation of 2D crystals. The use of locally fluorinated lipids in avoiding solubilization of the lipid monolayer by detergents is presented and discussed.