Molecular interactions of FG nucleoporin repeats at high resolution.

Proteins that contain repeat phenylalanine-glycine (FG) residues phase separate into oncogenic transcription factor condensates in malignant leukaemias, form the permeability barrier of the nuclear pore complex and mislocalize in neurodegenerative diseases. Insights into the molecular interactions of FG-repeat nucleoporins have, however, remained largely elusive. Using a combination of NMR spectroscopy and cryoelectron microscopy, we have identified uniformly spaced segments of transient ß-structure and a stable preformed α-helix recognized by messenger RNA export factors in the FG-repeat domain of human nucleoporin 98 (Nup98). In addition, we have determined at high resolution the molecular organization of reversible FG-FG interactions in amyloid fibrils formed by a highly aggregation-prone segment in Nup98. We have further demonstrated that amyloid-like aggregates of the FG-repeat domain of Nup98 have low stability and are reversible. Our results provide critical insights into the molecular interactions underlying the self-association and phase separation of FG-repeat nucleoporins in physiological and pathological cell activities.

Endo-lysosomal Aß concentration and pH trigger formation of Aß oligomers that potently induce Tau missorting.

Amyloid-ß peptide (Aß) forms metastable oligomers >50 kDa, termed AßOs, that are more effective than Aß amyloid fibrils at triggering Alzheimer's disease-related processes such as synaptic dysfunction and Tau pathology, including Tau mislocalization. In neurons, Aß accumulates in endo-lysosomal vesicles at low pH. Here, we show that the rate of AßO assembly is accelerated 8,000-fold upon pH reduction from extracellular to endo-lysosomal pH, at the expense of amyloid fibril formation. The pH-induced promotion of AßO formation and the high endo-lysosomal Aß concentration together enable extensive AßO formation of Aß42 under physiological conditions. Exploiting the enhanced AßO formation of the dimeric Aß variant dimAß we furthermore demonstrate targeting of AßOs to dendritic spines, potent induction of Tau missorting, a key factor in tauopathies, and impaired neuronal activity. The results suggest that the endosomal/lysosomal system is a major site for the assembly of pathomechanistically relevant AßOs.

Clustering polymorphs of tau and IAPP fibrils with the CHEP algorithm.

Recent steps towards automation have improved the quality and efficiency of the entire cryo-electron microscopy workflow, from sample preparation to image processing. Most of the image processing steps are now quite automated, but there are still a few steps which need the specific intervention of researchers. One such step is the identification and separation of helical protein polymorphs at early stages of image processing. Here, we tested and evaluated our recent clustering approach on three datasets containing amyloid fibrils, demonstrating that the proposed unsupervised clustering method automatically and effectively identifies the polymorphs from cryo-EM images. As an automated polymorph separation method, it has the potential to complement automated helical picking, which typically cannot easily distinguish between polymorphs with subtle differences in morphology, and is therefore a useful tool for the image processing and structure determination of helical proteins.

High-Resolution Cryo-EM Structure of the Cardiac Actomyosin Complex.

Heart contraction depends on a complicated array of interactions between sarcomeric proteins required to convert chemical energy into mechanical force. Cyclic interactions between actin and myosin molecules, controlled by troponin and tropomyosin, generate the sliding force between the actin-based thin and myosin-based thick filaments. Alterations in this sophisticated system due to missense mutations can lead to cardiovascular diseases. Numerous structural studies proposed pathological mechanisms of missense mutations at the myosin-myosin, actin-tropomyosin, and tropomyosin-troponin interfaces. However, despite the central role of actomyosin interactions a detailed structural description of the cardiac actomyosin interface remained unknown. Here, we report a cryo-EM structure of a cardiac actomyosin complex at 3.8 Å resolution. The structure reveals the molecular basis of cardiac diseases caused by missense mutations in myosin and actin proteins.

Cryo-EM structure of islet amyloid polypeptide fibrils reveals similarities with amyloid-ß fibrils.

Amyloid deposits consisting of fibrillar islet amyloid polypeptide (IAPP) in pancreatic islets are associated with beta-cell loss and have been implicated in type 2 diabetes (T2D). Here, we applied cryo-EM to reconstruct densities of three dominant IAPP fibril polymorphs, formed in vitro from synthetic human IAPP. An atomic model of the main polymorph, built from a density map of 4.2-Å resolution, reveals two S-shaped, intertwined protofilaments. The segment 21-NNFGAIL-27, essential for IAPP amyloidogenicity, forms the protofilament interface together with Tyr37 and the amidated C terminus. The S-fold resembles polymorphs of Alzheimer's disease (AD)-associated amyloid-ß (Aß) fibrils, which might account for the epidemiological link between T2D and AD and reports on IAPP-Aß cross-seeding in vivo. The results structurally link the early-onset T2D IAPP genetic polymorphism (encoding Ser20Gly) with the AD Arctic mutation (Glu22Gly) of Aß and support the design of inhibitors and imaging probes for IAPP fibrils.

ß-Hairpin of Islet Amyloid Polypeptide Bound to an Aggregation Inhibitor.

In type 2 diabetes, the formation of islet amyloid consisting of islet amyloid polypeptide (IAPP) is associated with reduction in ß-cell mass and contributes to the failure of islet cell transplantation. Rational design of inhibitors of IAPP amyloid formation has therapeutic potential, but is hampered by the lack of structural information on inhibitor complexes of the conformationally flexible, aggregation-prone IAPP. Here we characterize a ß-hairpin conformation of IAPP in complex with the engineered binding protein ß-wrapin HI18. The ß-strands correspond to two amyloidogenic motifs, 12-LANFLVH-18 and 22-NFGAILS-28, which are connected by a turn established around Ser-20. Besides backbone hydrogen bonding, the IAPP:HI18 interaction surface is dominated by non-polar contacts involving hydrophobic side chains of the IAPP ß-strands. Apart from monomers, HI18 binds oligomers and fibrils and inhibits IAPP aggregation and toxicity at low substoichiometric concentrations. The IAPP ß-hairpin can serve as a molecular recognition motif enabling control of IAPP aggregation.

Ligand-induced structural changes in the cyclic nucleotide-modulated potassium channel MloK1.

Cyclic nucleotide-modulated ion channels are important for signal transduction and pacemaking in eukaryotes. The molecular determinants of ligand gating in these channels are still unknown, mainly because of a lack of direct structural information. Here we report ligand-induced conformational changes in full-length MloK1, a cyclic nucleotide-modulated potassium channel from the bacterium Mesorhizobium loti, analysed by electron crystallography and atomic force microscopy. Upon cAMP binding, the cyclic nucleotide-binding domains move vertically towards the membrane, and directly contact the S1-S4 voltage sensor domains. This is accompanied by a significant shift and tilt of the voltage sensor domain helices. In both states, the inner pore-lining helices are in an 'open' conformation. We propose a mechanism in which ligand binding can favour pore opening via a direct interaction between the cyclic nucleotide-binding domains and voltage sensors. This offers a simple mechanistic hypothesis for the coupling between ligand gating and voltage sensing in eukaryotic HCN channels.

Visualizing GroEL/ES in the act of encapsulating a folding protein.

The GroEL/ES chaperonin system is required for the assisted folding of many proteins. How these substrate proteins are encapsulated within the GroEL-GroES cavity is poorly understood. Using symmetry-free, single-particle cryo-electron microscopy, we have characterized a chemically modified mutant of GroEL (EL43Py) that is trapped at a normally transient stage of substrate protein encapsulation. We show that the symmetric pattern of the GroEL subunits is broken as the GroEL cis-ring apical domains reorient to accommodate the simultaneous binding of GroES and an incompletely folded substrate protein (RuBisCO). The collapsed RuBisCO folding intermediate binds to the lower segment of two apical domains, as well as to the normally unstructured GroEL C-terminal tails. A comparative structural analysis suggests that the allosteric transitions leading to substrate protein release and folding involve concerted shifts of GroES and the GroEL apical domains and C-terminal tails.

The crystal structures of the eukaryotic chaperonin CCT reveal its functional partitioning.

In eukaryotes, CCT is essential for the correct and efficient folding of many cytosolic proteins, most notably actin and tubulin. Structural studies of CCT have been hindered by the failure of standard crystallographic analysis to resolve its eight different subunit types at low resolutions. Here, we exhaustively assess the R value fit of all possible CCT models to available crystallographic data of the closed and open forms with resolutions of 3.8 Å and 5.5 Å, respectively. This unbiased analysis finds the native subunit arrangements with overwhelming significance. The resulting structures provide independent crystallographic proof of the subunit arrangement of CCT and map major asymmetrical features of the particle onto specific subunits. The actin and tubulin substrates both bind around subunit CCT6, which shows other structural anomalies. CCT is thus clearly partitioned, both functionally and evolutionary, into a substrate-binding side that is opposite to the ATP-hydrolyzing side.

Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycle.

The eukaryotic group II chaperonin TRiC/CCT is a 16-subunit complex with eight distinct but similar subunits arranged in two stacked rings. Substrate folding inside the central chamber is triggered by ATP hydrolysis. We present five cryo-EM structures of TRiC in apo and nucleotide-induced states without imposing symmetry during the 3D reconstruction. These structures reveal the intra- and inter-ring subunit interaction pattern changes during the ATPase cycle. In the apo state, the subunit arrangement in each ring is highly asymmetric, whereas all nucleotide-containing states tend to be more symmetrical. We identify and structurally characterize an one-ring closed intermediate induced by ATP hydrolysis wherein the closed TRiC ring exhibits an observable chamber expansion. This likely represents the physiological substrate folding state. Our structural results suggest mechanisms for inter-ring-negative cooperativity, intra-ring-positive cooperativity, and protein-folding chamber closure of TRiC. Intriguingly, these mechanisms are different from other group I and II chaperonins despite their similar architecture.

The refined structure of functional unit h of keyhole limpet hemocyanin (KLH1-h) reveals disulfide bridges.

Hemocyanins are multimeric oxygen-transport proteins in the hemolymph of many arthropods and mollusks. The overall molecular architecture of arthropod and molluscan hemocyanin is very different, although they possess a similar binuclear type 3 copper center to bind oxygen in a side-on conformation. Gastropod hemocyanin is a 35 nm cylindrical didecamer (2 × 10-mer) based on a 400 kDa subunit. The latter is subdivided into eight paralogous "functional units" (FU-a to FU-h), each with an active site. FU-a to FU-f contribute to the cylinder wall, whereas FU-g and FU-h form the internal collar complex. Atomic structures of FU-e and FU-g, and a 9 Å cryoEM structure of the 8 MDa didecamer are available. Recently, the structure of keyhole limpet hemocyanin FU-h (KLH1-h) was presented as a C(α) -trace at 4 Å resolution. Unlike the other seven FU types, FU-h contains an additional C-terminal domain with a cupredoxin-like fold. Because of the resolution limit of 4 Å, in some loops, the course of the protein backbone could not be established with high certainty yet. Here, we present a refined atomic structure of FU-h (KLH1-h) obtained from low-resolution refinement, which unambiguously establishes the course of the polypeptide backbone and reveals the disulfide bridges as well as the orientation of bulky amino acids.

A spring-loaded release mechanism regulates domain movement and catalysis in phosphoglycerate kinase.

Phosphoglycerate kinase (PGK) is the enzyme responsible for the first ATP-generating step of glycolysis and has been implicated extensively in oncogenesis and its development. Solution small angle x-ray scattering (SAXS) data, in combination with crystal structures of the enzyme in complex with substrate and product analogues, reveal a new conformation for the resting state of the enzyme and demonstrate the role of substrate binding in the preparation of the enzyme for domain closure. Comparison of the x-ray scattering curves of the enzyme in different states with crystal structures has allowed the complete reaction cycle to be resolved both structurally and temporally. The enzyme appears to spend most of its time in a fully open conformation with short periods of closure and catalysis, thereby allowing the rapid diffusion of substrates and products in and out of the binding sites. Analysis of the open apoenzyme structure, defined through deformable elastic network refinement against the SAXS data, suggests that interactions in a mostly buried hydrophobic region may favor the open conformation. This patch is exposed on domain closure, making the open conformation more thermodynamically stable. Ionic interactions act to maintain the closed conformation to allow catalysis. The short time PGK spends in the closed conformation and its strong tendency to rest in an open conformation imply a spring-loaded release mechanism to regulate domain movement, catalysis, and efficient product release.

Mechanism of folding chamber closure in a group II chaperonin.

Group II chaperonins are essential mediators of cellular protein folding in eukaryotes and archaea. These oligomeric protein machines, approximately 1 megadalton, consist of two back-to-back rings encompassing a central cavity that accommodates polypeptide substrates. Chaperonin-mediated protein folding is critically dependent on the closure of a built-in lid, which is triggered by ATP hydrolysis. The structural rearrangements and molecular events leading to lid closure are still unknown. Here we report four single particle cryo-electron microscopy (cryo-EM) structures of Mm-cpn, an archaeal group II chaperonin, in the nucleotide-free (open) and nucleotide-induced (closed) states. The 4.3 A resolution of the closed conformation allowed building of the first ever atomic model directly from the single particle cryo-EM density map, in which we were able to visualize the nucleotide and more than 70% of the side chains. The model of the open conformation was obtained by using the deformable elastic network modelling with the 8 A resolution open-state cryo-EM density restraints. Together, the open and closed structures show how local conformational changes triggered by ATP hydrolysis lead to an alteration of intersubunit contacts within and across the rings, ultimately causing a rocking motion that closes the ring. Our analyses show that there is an intricate and unforeseen set of interactions controlling allosteric communication and inter-ring signalling, driving the conformational cycle of group II chaperonins. Beyond this, we anticipate that our methodology of combining single particle cryo-EM and computational modelling will become a powerful tool in the determination of atomic details involved in the dynamic processes of macromolecular machines in solution.

Single-molecule FRET measures bends and kinks in DNA.

We present advances in the use of single-molecule FRET measurements with flexibly linked dyes to derive full 3D structures of DNA constructs based on absolute distances. The resolution obtained by this single-molecule approach harbours the potential to study in detail also protein- or damage-induced DNA bending. If one is to generate a geometric structural model, distances between fixed positions are needed. These are usually not experimentally accessible because of unknown fluorophore-linker mobility effects that lead to a distribution of FRET efficiencies and distances. To solve this problem, we performed studies on DNA double-helices by systematically varying donor acceptor distances from 2 to 10 nm. Analysis of dye-dye quenching and fluorescence anisotropy measurements reveal slow positional and fast orientational fluorophore dynamics, that results in an isotropic average of the FRET efficiency. We use a nonlinear conversion function based on MD simulations that allows us to include this effect in the calculation of absolute FRET distances. To obtain unique structures, we performed a quantitative statistical analysis for the conformational search in full space based on triangulation, which uses the known helical nucleic acid features. Our higher accuracy allowed the detection of sequence-dependent DNA bending by 16 degrees . For DNA with bulged adenosines, we also quantified the kink angles introduced by the insertion of 1, 3 and 5 bases to be 32 degrees +/- 6 degrees , 56 degrees +/- 4 degrees and 73 +/- 2 degrees , respectively. Moreover, the rotation angles and shifts of the helices were calculated to describe the relative orientation of the two arms in detail.

Simulation of fluorescence anisotropy experiments: probing protein dynamics.

Time-resolved fluorescence anisotropy decay experiments on a protein-attached dye can probe local protein dynamics and steric restrictions, but are difficult to interpret at the structural level. Aiming at an atomistic description, we have carried out molecular dynamics simulations of such experiments. Our simulations describe an Alexa488 fluorescent dye maleimide derivative covalently attached via a single cysteine to the AB-loop of bacteriorhodopsin. Fluorescence anisotropy decay curves obtained from the simulations agree well with the measured ones. Three anisotropy decay components were resolved and assigned to: 1), the fast dynamics of the attached dye on the picosecond timescale; 2), the slower dynamics of the loop at the one nanosecond timescale; and 3), the overall tumbling of the molecule. For the biologically relevant 1-ns component we identified two processes from simulations, the motion of the flexible loop as well as slow conformational dynamics of the dye. These two processes are not separable by experiment alone. Furthermore, analysis of the correlation between the dye and the protein motion revealed which part and which motion of the protein is actually probed by the experiment. Finally, our simulations allowed us to test the usual and inevitable assumption underlying these types of spectroscopic measurements that the attached dye probe does not severely perturb the protein dynamics. For the case at hand, by comparison with a simulation of the dye-free protein, the perturbation was quantified and found to be small.