Cryo-electron microscopy reveals hydrogen positions and water networks in photosystem II.

Photosystem II starts the photosynthetic electron transport chain that converts solar energy into chemical energy and thus sustains life on Earth. It catalyzes two chemical reactions: water oxidation to molecular oxygen and plastoquinone reduction. Coupling of electron and proton transfer is crucial for efficiency; however, the molecular basis of these processes remains speculative owing to uncertain water binding sites and the lack of experimentally determined hydrogen positions. We thus collected high-resolution cryo-electron microscopy data of fully hydrated photosystem II from the thermophilic cyanobacterium Thermosynechococcus vestitus to a final resolution of 1.71 angstroms. The structure reveals several previously undetected partially occupied water binding sites and more than half of the hydrogen and proton positions. This clarifies the pathways of substrate water binding and plastoquinone B protonation.

Exploiting the full potential of cryo-EM maps.

The Coulomb potential maps generated by electron microscopy (EM) experiments contain not only information about the position but also about the charge state of the atom. This feature of EM maps allows the identification of specific ions and the protonation state of amino acid side chains in the sample. Here, we summarize qualitative observations of charges in EM maps, discuss the difficulties in interpreting the charge in Coulomb potential maps with respect to distinguishing it from radiation damage, and outline considerations to implement the correct charge in fitting algorithms.

Stabilization of membrane topologies by proteinaceous remorin scaffolds.

In plants, the topological organization of membranes has mainly been attributed to the cell wall and the cytoskeleton. Additionally, few proteins, such as plant-specific remorins have been shown to function as protein and lipid organizers. Root nodule symbiosis requires continuous membrane re-arrangements, with bacteria being finally released from infection threads into membrane-confined symbiosomes. We found that mutations in the symbiosis-specific SYMREM1 gene result in highly disorganized perimicrobial membranes. AlphaFold modelling and biochemical analyses reveal that SYMREM1 oligomerizes into antiparallel dimers and may form a higher-order membrane scaffolding structure. This was experimentally confirmed when expressing this and other remorins in wall-less protoplasts is sufficient where they significantly alter and stabilize de novo membrane topologies ranging from membrane blebs to long membrane tubes with a central actin filament. Reciprocally, mechanically induced membrane indentations were equally stabilized by SYMREM1. Taken together we describe a plant-specific mechanism that allows the stabilization of large-scale membrane conformations independent of the cell wall.

Antimicrobial Polymers of Linear and Bottlebrush Architecture: Probing the Membrane Interaction and Physicochemical Properties.

Polymeric antimicrobial peptide mimics are a promising alternative for the future management of the daunting problems associated with antimicrobial resistance. However, the development of successful antimicrobial polymers (APs) requires careful control of factors such as amphiphilic balance, molecular weight, dispersity, sequence, and architecture. While most of the earlier developed APs focus on random linear copolymers, the development of APs with advanced architectures proves to be more potent. It is recently developed multivalent bottlebrush APs with improved antibacterial and hemocompatibility profiles, outperforming their linear counterparts. Understanding the rationale behind the outstanding biological activity of these newly developed antimicrobials is vital to further improving their performance. This work investigates the physicochemical properties governing the differences in activity between linear and bottlebrush architectures using various spectroscopic and microscopic techniques. Linear copolymers are more solvated, thermo-responsive, and possess facial amphiphilicity resulting in random aggregations when interacting with liposomes mimicking Escheria coli membranes. The bottlebrush copolymers adopt a more stable secondary conformation in aqueous solution in comparison to linear copolymers, conferring rapid and more specific binding mechanism to membranes. The advantageous physicochemical properties of the bottlebrush topology seem to be a determinant factor in the activity of these promising APs.

Protein Nanopore Membranes Prepared by a Simple Langmuir-Schaefer Approach.

Filtration through membranes with nanopores is typically associated with high transmembrane pressures and high energy consumption. This problem can be addressed by reducing the respective membrane thickness. Here, a simple procedure is described to prepare ultrathin membranes based on protein nanopores, which exhibit excellent water permeance, two orders of magnitude superior to comparable, industrially applied membranes. Furthermore, incorporation of either closed or open protein nanopores allows tailoring the membrane's ion permeability. To form such membranes, the transmembrane protein ferric hydroxamate uptake protein component A (FhuA) or its open-pore variant are assembled at the air-water interface of a Langmuir trough, compressed to a dense film, crosslinked by glutaraldehyde, and transferred to various support materials. This approach allows to prepare monolayer or multilayer membranes with a very high density of protein nanopores. Freestanding membranes covering holes up to 5 µm in diameter are visualized by atomic force microscopy (AFM), helium ion microscopy, and transmission electron microscopy. AFM PeakForce quantitative nanomechanical property mapping (PeakForce QNM)  demonstrates remarkable mechanical stability and elastic properties of freestanding monolayer membranes with a thickness of only 5 nm. The new protein membrane can pave the way to energy-efficient nanofiltration.

Construction of Highly Ordered Glyco-Inside Nano-Assemblies through RAFT Dispersion Polymerization of Galactose-Decorated Monomer.

Glyco-assemblies derived from amphiphilic sugar-decorated block copolymers (ASBCs) have emerged prominently due to their wide application, for example, in biomedicine and as drug carriers. However, to efficiently construct these glyco-assemblies is still a challenge. Herein, we report an efficient technology for the synthesis of glyco-inside nano-assemblies by utilizing RAFT polymerization of a galactose-decorated methacrylate for polymerization-induced self-assembly (PISA). Using this approach, a series of highly ordered glyco-inside nano-assemblies containing intermediate morphologies were fabricated by adjusting the length of the hydrophobic glycoblock and the polymerization solids content. A specific morphology of complex vesicles was captured during the PISA process and the formation mechanism is explained by the morphology of its precursor and intermediate. Thus, this method establishes a powerful route to fabricate glyco-assemblies with tunable morphologies and variable sizes, which is significant to enable the large-scale fabrication and wide application of glyco-assemblies.

Cryo-EM structures reveal intricate Fe-S cluster arrangement and charging in Rhodobacter capsulatus formate dehydrogenase.

Metal-containing formate dehydrogenases (FDH) catalyse the reversible oxidation of formate to carbon dioxide at their molybdenum or tungsten active site. They display a diverse subunit and cofactor composition, but structural information on these enzymes is limited. Here we report the cryo-electron microscopic structures of the soluble Rhodobacter capsulatus FDH (RcFDH) as isolated and in the presence of reduced nicotinamide adenine dinucleotide (NADH). RcFDH assembles into a 360 kDa dimer of heterotetramers revealing a putative interconnection of electron pathway chains. In the presence of NADH, the RcFDH structure shows charging of cofactors, indicative of an increased electron load.

Structural Mapping of Missense Mutations in the Pex1/Pex6 Complex.

Peroxisome biogenesis disorders (PBDs) are nontreatable hereditary diseases with a broad range of severity. Approximately 65% of patients are affected by mutations in the peroxins Pex1 and Pex6. The proteins form the heteromeric Pex1/Pex6 complex, which is important for protein import into peroxisomes. To date, no structural data are available for this AAA+ ATPase complex. However, a wealth of information can be transferred from low-resolution structures of the yeast scPex1/scPex6 complex and homologous, well-characterized AAA+ ATPases. We review the abundant records of missense mutations described in PBD patients with the aim to classify and rationalize them by mapping them onto a homology model of the human Pex1/Pex6 complex. Several mutations concern functionally conserved residues that are implied in ATP hydrolysis and substrate processing. Contrary to fold destabilizing mutations, patients suffering from function-impairing mutations may not benefit from stabilizing agents, which have been reported as potential therapeutics for PBD patients.

Nuclear Transport of Yeast Proteasomes.

Proteasomes are key proteases in regulating protein homeostasis. Their holo-enzymes are composed of 40 different subunits which are arranged in a proteolytic core (CP) flanked by one to two regulatory particles (RP). Proteasomal proteolysis is essential for the degradation of proteins which control time-sensitive processes like cell cycle progression and stress response. In dividing yeast and human cells, proteasomes are primarily nuclear suggesting that proteasomal proteolysis is mainly required in the nucleus during cell proliferation. In yeast, which have a closed mitosis, proteasomes are imported into the nucleus as immature precursors via the classical import pathway. During quiescence, the reversible absence of proliferation induced by nutrient depletion or growth factor deprivation, proteasomes move from the nucleus into the cytoplasm. In the cytoplasm of quiescent yeast, proteasomes are dissociated into CP and RP and stored in membrane-less cytoplasmic foci, named proteasome storage granules (PSGs). With the resumption of growth, PSGs clear and mature proteasomes are transported into the nucleus by Blm10, a conserved 240 kDa protein and proteasome-intrinsic import receptor. How proteasomes are exported from the nucleus into the cytoplasm is unknown.

Mechanism of Enzyme Repair by the AAA+ Chaperone Rubisco Activase.

How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) in metabolic repair of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits containing eight catalytic sites. Rubisco is prone to inhibition by tight-binding sugar phosphates, whose removal is catalyzed by Rca. We engineered a stable Rca hexamer ring and analyzed its functional interaction with Rubisco. Hydrogen/deuterium exchange and chemical crosslinking showed that Rca structurally destabilizes elements of the Rubisco active site with remarkable selectivity. Cryo-electron microscopy revealed that Rca docks onto Rubisco over one active site at a time, positioning the C-terminal strand of RbcL, which stabilizes the catalytic center, for access to the Rca hexamer pore. The pulling force of Rca is fine-tuned to avoid global destabilization and allow for precise enzyme repair.

AAA-ATPases in Protein Degradation.

Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.

Structure and Function of p97 and Pex1/6 Type II AAA+ Complexes.

Protein complexes of the Type II AAA+ (ATPases associated with diverse cellular activities) family are typically hexamers of 80-150 kDa protomers that harbor two AAA+ ATPase domains. They form double ring assemblies flanked by associated domains, which can be N-terminal, intercalated or C-terminal to the ATPase domains. Most prominent members of this family include NSF (N-ethyl-maleimide sensitive factor), p97/VCP (valosin-containing protein), the Pex1/Pex6 complex and Hsp104 in eukaryotes and ClpB in bacteria. Tremendous efforts have been undertaken to understand the conformational dynamics of protein remodeling type II AAA+ complexes. A uniform mode of action has not been derived from these works. This review focuses on p97/VCP and the Pex1/6 complex, which both structurally remodel ubiquitinated substrate proteins. P97/VCP plays a role in many processes, including ER- associated protein degradation, and the Pex1/Pex6 complex dislocates and recycles the transport receptor Pex5 from the peroxisomal membrane during peroxisomal protein import. We give an introduction into existing knowledge about the biochemical and cellular activities of the complexes before discussing structural information. We particularly emphasize recent electron microscopy structures of the two AAA+ complexes and summarize their structural differences.

Structure and mechanism of the Rubisco-assembly chaperone Raf1.

Biogenesis of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits, requires assembly chaperones. Here we analyzed the role of Rubisco accumulation factor1 (Raf1), a dimer of ∼40-kDa subunits. We find that Raf1 from Synechococcus elongatus acts downstream of chaperonin-assisted RbcL folding by stabilizing RbcL antiparallel dimers for assembly into RbcL8 complexes with four Raf1 dimers bound. Raf1 displacement by RbcS results in holoenzyme formation. Crystal structures show that Raf1 from Arabidopsis thaliana consists of a ß-sheet dimerization domain and a flexibly linked α-helical domain. Chemical cross-linking and EM reconstruction indicate that the ß-domains bind along the equator of each RbcL2 unit, and the α-helical domains embrace the top and bottom edges of RbcL2. Raf1 fulfills a role similar to that of the assembly chaperone RbcX, thus suggesting that functionally redundant factors ensure efficient Rubisco biogenesis.

Molecular snapshots of the Pex1/6 AAA+ complex in action.

The peroxisomal proteins Pex1 and Pex6 form a heterohexameric type II AAA+ ATPase complex, which fuels essential protein transport across peroxisomal membranes. Mutations in either ATPase in humans can lead to severe peroxisomal disorders and early death. We present an extensive structural and biochemical analysis of the yeast Pex1/6 complex. The heterohexamer forms a trimer of Pex1/6 dimers with a triangular geometry that is atypical for AAA+ complexes. While the C-terminal nucleotide-binding domains (D2) of Pex6 constitute the main ATPase activity of the complex, both D2 harbour essential substrate-binding motifs. ATP hydrolysis results in a pumping motion of the complex, suggesting that Pex1/6 function involves substrate translocation through its central channel. Mutation of the Walker B motif in one D2 domain leads to ATP hydrolysis in the neighbouring domain, giving structural insights into inter-domain communication of these unique heterohexameric AAA+ assemblies.

Proteasome assembly from 15S precursors involves major conformational changes and recycling of the Pba1-Pba2 chaperone.

The chaperones Ump1 and Pba1-Pba2 promote efficient biogenesis of 20S proteasome core particles from its subunits via 15S intermediates containing alpha and beta subunits, except beta7. Here we elucidate the structural role of these chaperones in late steps of core particle biogenesis using biochemical, electron microscopy, cross-linking and mass spectrometry analyses. In 15S precursor complexes, Ump1 is largely unstructured, lining the inner cavity of the complex along the interface between alpha and beta subunits. The alpha and beta subunits form loosely packed rings with a wider alpha ring opening than in the 20S core particle, allowing for the Pba1-Pba2 heterodimer to be partially embedded in the central alpha ring cavity. During biogenesis, the heterodimer is expelled from the alpha ring by a restructuring event that organizes the beta ring and leads to tightening of the alpha ring opening. In this way, the Pba1-Pba2 chaperone is recycled for a new round of proteasome assembly.

Structure of the VipA/B type VI secretion complex suggests a contraction-state-specific recycling mechanism.

The bacterial type VI secretion system is a multicomponent molecular machine directed against eukaryotic host cells and competing bacteria. An intracellular contractile tubular structure that bears functional homology with bacteriophage tails is pivotal for ejection of pathogenic effectors. Here, we present the 6 Å cryoelectron microscopy structure of the contracted Vibrio cholerae tubule consisting of the proteins VipA and VipB. We localized VipA and VipB in the protomer and identified structural homology between the C-terminal segment of VipB and the tail-sheath protein of T4 phages. We propose that homologous segments in VipB and T4 phages mediate tubule contraction. We show that in type VI secretion, contraction leads to exposure of the ClpV recognition motif, which is embedded in the type VI-specific four-helix-bundle N-domain of VipB. Disaggregation of the tubules by the AAA+ protein ClpV and recycling of the VipA/B subunits are thereby limited to the contracted state.

Head-to-tail interactions of the coiled-coil domains regulate ClpB activity and cooperation with Hsp70 in protein disaggregation.

The hexameric AAA+ chaperone ClpB reactivates aggregated proteins in cooperation with the Hsp70 system. Essential for disaggregation, the ClpB middle domain (MD) is a coiled-coil propeller that binds Hsp70. Although the ClpB subunit structure is known, positioning of the MD in the hexamer and its mechanism of action are unclear. We obtained electron microscopy (EM) structures of the BAP variant of ClpB that binds the protease ClpP, clearly revealing MD density on the surface of the ClpB ring. Mutant analysis and asymmetric reconstructions show that MDs adopt diverse positions in a single ClpB hexamer. Adjacent, horizontally oriented MDs form head-to-tail contacts and repress ClpB activity by preventing Hsp70 interaction. Tilting of the MD breaks this contact, allowing Hsp70 binding, and releasing the contact in adjacent subunits. Our data suggest a wavelike activation of ClpB subunits around the ring.DOI: http://dx.doi.org/10.7554/eLife.02481.001.

Conserved distal loop residues in the Hsp104 and ClpB middle domain contact nucleotide-binding domain 2 and enable Hsp70-dependent protein disaggregation.

The homologous hexameric AAA(+) proteins, Hsp104 from yeast and ClpB from bacteria, collaborate with Hsp70 to dissolve disordered protein aggregates but employ distinct mechanisms of intersubunit collaboration. How Hsp104 and ClpB coordinate polypeptide handover with Hsp70 is not understood. Here, we define conserved distal loop residues between middle domain (MD) helix 1 and 2 that are unexpectedly critical for Hsp104 and ClpB collaboration with Hsp70. Surprisingly, the Hsp104 and ClpB MD distal loop does not contact Hsp70 but makes intrasubunit contacts with nucleotide-binding domain 2 (NBD2). Thus, the MD does not invariably project out into solution as in one structural model of Hsp104 and ClpB hexamers. These intrasubunit contacts as well as those between MD helix 2 and NBD1 are different in Hsp104 and ClpB. NBD2-MD contacts dampen disaggregase activity and must separate for protein disaggregation. We demonstrate that ClpB requires DnaK more stringently than Hsp104 requires Hsp70 for protein disaggregation. Thus, we reveal key differences in how Hsp104 and ClpB coordinate polypeptide handover with Hsp70, which likely reflects differential tuning for yeast and bacterial proteostasis.

Structure and function of the AAA+ nucleotide binding pocket.

Members of the diverse superfamily of AAA+ proteins are molecular machines responsible for a wide range of essential cellular processes. In this review we summarise structural and functional data surrounding the nucleotide binding pocket of these versatile complexes. Protein Data Bank (PDB) structures of closely related AAA+ ATPase are overlaid and biologically relevant motifs are displayed. Interactions between protomers are illustrated on the basis of oligomeric structures of each AAA+ subgroup. The possible role of conserved motifs in the nucleotide binding pocket is assessed with regard to ATP binding and hydrolysis, oligomerisation and inter-subunit communication. Our comparison indicates that in particular the roles of the arginine finger and sensor 2 residues differ subtly between AAA+ subgroups, potentially providing a means for functional diversification.