Structural insights into actin filament recognition by commonly used cellular actin markers.

Cellular studies of filamentous actin (F-actin) processes commonly utilize fluorescent versions of toxins, peptides, and proteins that bind actin. While the choice of these markers has been largely based on availability and ease, there is a severe dearth of structural data for an informed judgment in employing suitable F-actin markers for a particular requirement. Here, we describe the electron cryomicroscopy structures of phalloidin, lifeAct, and utrophin bound to F-actin, providing a comprehensive high-resolution structural comparison of widely used actin markers and their influence towards F-actin. Our results show that phalloidin binding does not induce specific conformational change and lifeAct specifically recognizes closed D-loop conformation, i.e., ADP-Pi or ADP states of F-actin. The structural models aided designing of minimal utrophin and a shorter lifeAct, which can be utilized as F-actin marker. Together, our study provides a structural perspective, where the binding sites of utrophin and lifeAct overlap with majority of actin-binding proteins and thus offering an invaluable resource for researchers in choosing appropriate actin markers and generating new marker variants.

Structure of mammalian respiratory complex I.

Complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in the cell, powers ATP synthesis in mammalian mitochondria by using the reducing potential of NADH to drive protons across the inner mitochondrial membrane. Mammalian complex I (ref. 1) contains 45 subunits, comprising 14 core subunits that house the catalytic machinery (and are conserved from bacteria to humans) and a mammalian-specific cohort of 31 supernumerary subunits. Knowledge of the structures and functions of the supernumerary subunits is fragmentary. Here we describe a 4.2-Å resolution single-particle electron cryomicroscopy structure of complex I from Bos taurus. We have located and modelled all 45 subunits, including the 31 supernumerary subunits, to provide the entire structure of the mammalian complex. Computational sorting of the particles identified different structural classes, related by subtle domain movements, which reveal conformationally dynamic regions and match biochemical descriptions of the 'active-to-de-active' enzyme transition that occurs during hypoxia. Our structures therefore provide a foundation for understanding complex I assembly and the effects of mutations that cause clinically relevant complex I dysfunctions, give insights into the structural and functional roles of the supernumerary subunits and reveal new information on the mechanism and regulation of catalysis.

Hexameric assembly of the AAA+ protein McrB is necessary for GTPase activity.

McrBC is one of the three modification-dependent restriction enzymes encoded by the Escherichia coli K12 chromosome. Amongst restriction enzymes, McrBC and its close homologues are unique in employing the AAA+ domain for GTP hydrolysis-dependent activation of DNA cleavage. The GTPase activity of McrB is stimulated by the endonuclease subunit McrC. It had been reported previously that McrB and McrC subunits oligomerise together into a high molecular weight species. Here we conclusively demonstrate using size exclusion chromatography coupled multi-angle light scattering (SEC-MALS) and images obtained by electron cryomicroscopy that McrB exists as a hexamer in solution. Furthermore, based on SEC-MALS and SAXS analyses of McrBC and the structure of McrB, we propose that McrBC is a complex of two McrB hexamers bridged by two subunits of McrC, and that the complete assembly of this complex is integral to its enzymatic activity. We show that the nucleotide-dependent oligomerisation of McrB precedes GTP hydrolysis. Mutational studies show that, unlike other AAA+ proteins, the catalytic Walker B aspartate is required for oligomerisation.

Architecture of mammalian respiratory complex I.

Complex I (NADH:ubiquinone oxidoreductase) is essential for oxidative phosphorylation in mammalian mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across the energy-transducing inner membrane, providing electrons for respiration and driving ATP synthesis. Mammalian complex I contains 44 different nuclear- and mitochondrial-encoded subunits, with a combined mass of 1 MDa. The 14 conserved 'core' subunits have been structurally defined in the minimal, bacterial complex, but the structures and arrangement of the 30 'supernumerary' subunits are unknown. Here we describe a 5 Å resolution structure of complex I from Bos taurus heart mitochondria, a close relative of the human enzyme, determined by single-particle electron cryo-microscopy. We present the structures of the mammalian core subunits that contain eight iron-sulphur clusters and 60 transmembrane helices, identify 18 supernumerary transmembrane helices, and assign and model 14 supernumerary subunits. Thus, we considerably advance knowledge of the structure of mammalian complex I and the architecture of its supernumerary ensemble around the core domains. Our structure provides insights into the roles of the supernumerary subunits in regulation, assembly and homeostasis, and a basis for understanding the effects of mutations that cause a diverse range of human diseases.

A 2-Tyr-1-carboxylate Mononuclear Iron Center Forms the Active Site of a Paracoccus Dimethylformamidase.

N,N-dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α2 ß2 or (α2 ß2 )2 type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side-chain phenolates and one carboxylate from Glu. The Fe3+ ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism.

Structure of the mitochondrial ATP synthase from Pichia angusta determined by electron cryo-microscopy.

The structure of the intact monomeric ATP synthase from the fungus, Pichia angusta, has been solved by electron cryo-microscopy. The structure provides insights into the mechanical coupling of the transmembrane proton motive force across mitochondrial membranes in the synthesis of ATP. This mechanism requires a strong and integral stator, consisting of the catalytic α3ß3-domain, peripheral stalk, and, in the membrane domain, subunit a and associated supernumerary subunits, kept in contact with the rotor turning at speeds up to 350 Hz. The stator's integrity is ensured by robust attachment of both the oligomycin sensitivity conferral protein (OSCP) to the catalytic domain and the membrane domain of subunit b to subunit a. The ATP8 subunit provides an additional brace between the peripheral stalk and subunit a. At the junction between the OSCP and the apparently stiff, elongated α-helical b-subunit and associated d- and h-subunits, an elbow or joint allows the stator to bend to accommodate lateral movements during the activity of the catalytic domain. The stator may also apply lateral force to help keep the static a-subunit and rotating c10-ring together. The interface between the c10-ring and the a-subunit contains the transmembrane pathway for protons, and their passage across the membrane generates the turning of the rotor. The pathway has two half-channels containing conserved polar residues provided by a bundle of four α-helices inclined at ∼30° to the plane of the membrane, similar to those described in other species. The structure provides more insights into the workings of this amazing machine.

Single particle electron cryomicroscopy: trends, issues and future perspective.

There has been enormous progress during the last few years in the determination of three-dimensional biological structures by single particle electron cryomicroscopy (cryoEM), allowing maps to be obtained with higher resolution and from fewer images than required previously. This is due principally to the introduction of a new type of direct electron detector that has 2- to 3-fold higher detective quantum efficiency than available previously, and to the improvement of the computational algorithms for image processing. In spite of the great strides that have been made, quantitative analysis shows that there are still significant gains to be made provided that the problems associated with image degradation can be solved, possibly by minimising beam-induced specimen movement and charge build up during imaging. If this can be achieved, it should be possible to obtain near atomic resolution structures of smaller single particles, using fewer images and resolving more conformational states than at present, thus realising the full potential of the method. The recent popularity of cryoEM for molecular structure determination also highlights the need for lower cost microscopes, so we encourage development of an inexpensive, 100 keV electron cryomicroscope with a high-brightness field emission gun to make the method accessible to individual groups or institutions that cannot afford the investment and running costs of a state-of-the-art 300 keV installation. A key requisite for successful high-resolution structure determination by cryoEM includes interpretation of images and optimising the biochemistry and grid preparation to obtain nicely distributed macromolecules of interest. We thus include in this review a gallery of cryoEM micrographs that shows illustrative examples of single particle images of large and small macromolecular complexes.

Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay.

Rhomboid proteases are evolutionary conserved intramembrane serine proteases. Because of their emerging role in many important biological pathways, rhomboids are potential drug targets. Unfortunately, few chemical tools are available for their study. Here, we describe a mass spectrometry-based assay to measure rhomboid substrate cleavage and inhibition. We have identified isocoumarin inhibitors and developed activity-based probes for rhomboid proteases. The probes can distinguish between active and inactive rhomboids due to covalent, reversible binding of the active-site serine and stable modification of a histidine residue. Finally, the structure of an isocoumarin-based inhibitor with Escherichia coli rhomboid GlpG uncovers an unusual mode of binding at the active site and suggests that the interactions between the 3-substituent on the isocoumarin inhibitor and hydrophobic residues on the protease reflect S' subsite binding. Overall, these probes represent valuable tools for rhomboid study, and the structural insights may facilitate future inhibitor design.

The structural basis for catalysis and substrate specificity of a rhomboid protease.

Rhomboids are intramembrane proteases that use a catalytic dyad of serine and histidine for proteolysis. They are conserved in both prokaryotes and eukaryotes and regulate cellular processes as diverse as intercellular signalling, parasitic invasion of host cells, and mitochondrial morphology. Their widespread biological significance and consequent medical potential provides a strong incentive to understand the mechanism of these unusual enzymes for identification of specific inhibitors. In this study, we describe the structure of Escherichia coli rhomboid GlpG covalently bound to a mechanism-based isocoumarin inhibitor. We identify the position of the oxyanion hole, and the S1- and S2'-binding subsites of GlpG, which are the key determinants of substrate specificity. The inhibitor-bound structure suggests that subtle structural change is sufficient for catalysis, as opposed to large changes proposed from previous structures of unliganded GlpG. Using bound inhibitor as a template, we present a model for substrate binding at the active site and biochemically test its validity. This study provides a foundation for a structural explanation of rhomboid specificity and mechanism, and for inhibitor design.

Factors affecting macromolecule orientations in thin films formed in cryo-EM.

The formation of a vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the plunge-freeze method first described nearly 40 years ago. Although this is a robust method, the behaviour of different macromolecules shows great variation upon freezing and often needs to be optimized to obtain an isotropic, high-resolution reconstruction. For a macromolecule in such a film, the probability of encountering the air-water interface in the time between blotting and freezing and adopting preferred orientations is very high. 3D reconstruction using preferentially oriented particles often leads to anisotropic and uninterpretable maps. Currently, there are no general solutions to this prevalent issue, but several approaches largely focusing on sample preparation with the use of additives and novel grid modifications have been attempted. In this study, the effect of physical and chemical factors on the orientations of macromolecules was investigated through an analysis of selected well studied macromolecules, and important parameters that determine the behaviour of proteins on cryo-EM grids were revealed. These insights highlight the nature of the interactions that cause preferred orientations and can be utilized to systematically address orientation bias for any given macromolecule and to provide a framework to design small-molecule additives to enhance sample stability and behaviour.

Modeling Ligands into Maps Derived from Electron Cryomicroscopy.

Deciphering the protein-ligand interactions in a macromolecular complex is crucial for understanding the molecular mechanism, underlying biological processes, and drug development. In recent years, cryogenic sample electron microscopy (cryoEM) has emerged as a powerful technique to determine the structures of macromolecules and to investigate the mode of ligand binding at near-atomic resolution. Identifying and modeling non-protein molecules in cryoEM maps is often challenging due to anisotropic resolution across the molecule of interest and inherent noise in the data. In this article, the readers are introduced to various software and methods currently used for ligand identification, model building, and refinement of atomic coordinates using selected macromolecules. One of the simplest ways to identify the presence of a ligand, as illustrated with the enolase enzyme, is to subtract the two maps obtained with and without the ligand. The extra density of the ligand is likely to stand out in the difference map even at a higher threshold. There are instances, as shown in the case of metabotropic Glutamate receptor mGlu5, when such simple difference maps cannot be generated. The recently introduced method of deriving the Fo-Fc omit map can serve as a tool for validating and demonstrating the presence of the ligand. Finally, using the well-studied ß-galactosidase as an example, the effect of resolution on modeling the ligands and solvent molecules in cryoEM maps is analyzed, and an outlook on how cryoEM can be used in drug discovery is presented.

Exploring advances in single particle CryoEM with apoferritin: From blobs to true atomic resolution.

Deciphering the three-dimensional structures of macromolecules is of paramount importance for gaining insights into their functions and roles in human health and disease. Single particle cryoEM has emerged as a powerful technique that enables direct visualization of macromolecules and their complexes, and through subsequent averaging, achieve near atomic-level resolution. A major breakthrough was recently achieved with the determination of the apoferritin structure at true atomic resolution. In this review, we discuss the latest technological innovations across the entire single-particle workflow, which have been instrumental in driving the resolution revolution and in transforming cryoEM as a mainstream technique in structural biology. We illustrate these advancements using apoferritin as an example that has served as an excellent benchmark sample for assessing emerging technologies. We further explore whether the existing technology can routinely generate atomic structures of dynamic macromolecules that more accurately represent real-world samples, the limitations in the workflow, and the current approaches employed to overcome them.

Cryo-EM structures of pannexin 1 and 3 reveal differences among pannexin isoforms.

Pannexins are single-membrane large-pore channels that release ions and ATP upon activation. Three isoforms of pannexins 1, 2, and 3, perform diverse cellular roles and differ in their pore lining residues. In this study, we report the cryo-EM structure of pannexin 3 at 3.9 Å and analyze its structural differences with pannexin isoforms 1 and 2. The pannexin 3 vestibule has two distinct chambers and a wider pore radius in comparison to pannexins 1 and 2. We further report two cryo-EM structures of pannexin 1, with pore substitutions W74R/R75D that mimic the pore lining residues of pannexin 2 and a germline mutant of pannexin 1, R217H at resolutions of 3.2 Å and 3.9 Å, respectively. Substitution of cationic residues in the vestibule of pannexin 1 results in reduced ATP interaction propensities to the channel. The germline mutant R217H in transmembrane helix 3 (TM3), leads to a partially constricted pore, reduced ATP interaction and weakened voltage sensitivity. The study compares the three pannexin isoform structures, the effects of substitutions of pore and vestibule-lining residues and allosteric effects of a pathological substitution on channel structure and function thereby enhancing our understanding of this vital group of ATP-release channels.

Structural snapshots of Mycobacterium tuberculosis enolase reveal dual mode of 2PG binding and its implication in enzyme catalysis.

Enolase, a ubiquitous enzyme, catalyzes the reversible conversion of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP) in the glycolytic pathway of organisms of all three domains of life. The underlying mechanism of the 2PG to PEP conversion has been studied in great detail in previous work, however that of the reverse reaction remains to be explored. Here we present structural snapshots of Mycobacterium tuberculosis (Mtb) enolase in apo, PEP-bound and two 2PG-bound forms as it catalyzes the conversion of PEP to 2PG. The two 2PG-bound complex structures differed in the conformation of the bound product (2PG) viz the widely reported canonical conformation and a novel binding pose, which we refer to here as the alternate conformation. Notably, we observed two major differences compared with the forward reaction: the presence of MgB is non-obligatory for the reaction and 2PG assumes an alternate conformation that is likely to facilitate its dissociation from the active site. Molecular dynamics studies and binding free energy calculations further substantiate that the alternate conformation of 2PG causes distortions in both metal ion coordination and hydrogen-bonding interactions, resulting in an increased flexibility of the active-site loops and aiding product release. Taken together, this study presents a probable mechanism involved in PEP to 2PG catalysis that is likely to be mediated by the conformational change of 2PG at the active site.

Structures of membrane proteins.

In reviewing the structures of membrane proteins determined up to the end of 2009, we present in words and pictures the most informative examples from each family. We group the structures together according to their function and architecture to provide an overview of the major principles and variations on the most common themes. The first structures, determined 20 years ago, were those of naturally abundant proteins with limited conformational variability, and each membrane protein structure determined was a major landmark. With the advent of complete genome sequences and efficient expression systems, there has been an explosion in the rate of membrane protein structure determination, with many classes represented. New structures are published every month and more than 150 unique membrane protein structures have been determined. This review analyses the reasons for this success, discusses the challenges that still lie ahead, and presents a concise summary of the key achievements with illustrated examples selected from each class.

Decoding the Mechanism of Specific RNA Targeting by Ribosomal Methyltransferases.

Methylation of specific nucleotides is integral for ribosomal biogenesis and also serves as a common mechanism to confer antibiotic resistance by pathogenic bacteria. Here, by determining the high-resolution structure of the 30S-KsgA complex by cryo-electron microscopy, a state was captured, where KsgA juxtaposes between helices h44 and h45 of the 30S ribosome, separating them, thereby enabling remodeling of the surrounded rRNA and allowing the cognate site to enter the methylation pocket. With the structure as a guide, several mutant versions of the ribosomes, where interacting bases in the catalytic helix h45 and surrounding helices h44, h24, and h27, were mutated and evaluated for their methylation efficiency revealing factors that direct the enzyme to its cognate site with high fidelity. The biochemical studies show that the three-dimensional environment of the ribosome enables the interaction of select loop regions in KsgA with the ribosome helices paramount to maintain selectivity.

Interaction of human erythrocyte catalase with air-water interface in cryoEM.

One of the key goals in single-particle cryo-microscopy is to obtain a uniform distribution of particle orientations, so that the three-dimensional structure has isotropic resolution in Fourier space. A common problem arises from the interaction of protein molecules with the air-water interface that exists on both surfaces of the thin film of liquid that is formed prior to plunge-freezing into liquid ethane. Some proteins and other macromolecular complexes are disrupted by interaction with the air-water interface. Other proteins or macromolecules either become concentrated through their interaction with the interface or are excluded because they bind strongly to some other part of the grid or the filter paper used in blotting. In this paper, the interaction of human erythrocyte catalase with the air-water interface is investigated and minimized by the addition of certain detergents. Detergents can form an amphipathic monolayer at the air-water interface that creates a barrier and leaves the molecules free to adopt a variety of orientations, thus facilitating the 3D structure determination. These results suggest that further characterization and development of detergents for cryo-microscopy plunge-freezing would be useful.

Comparison of CryoEM and X-ray structures of dimethylformamidase.

Dimethylformamidase (DMFase) catalyzes the hydrolysis of dimethylformamide, an industrial solvent, introduced into the environment by humans. Recently, we determined the structures of dimethylformamidase by electron cryo microscopy and X-ray crystallography revealing a tetrameric enzyme with a mononuclear iron at the active site. DMFase from Paracoccus sp. isolated from a waste water treatment plant around the city of Kanpur in India shows maximal activity at 54 °C and is halotolerant. The structures determined by both techniques are mostly identical and the largest difference is in a loop near the active site. This loop could play a role in co-operativity between the monomers. A number of non-protein densities are observed in the EM map, which are modelled as water molecules. Comparison of the structures determined by the two methods reveals conserved water molecules that could play a structural role. The higher stability, unusual active site and negligible activity at low temperature makes this a very good model to study enzyme mechanism by cryoEM.

Duox-generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts.

Progenitors of the thoracic tracheal system of adult Drosophila (tracheoblasts) arrest in G2 during larval life and rekindle a mitotic program subsequently. G2 arrest is dependent on ataxia telangiectasia mutated and rad3-related kinase (ATR)-dependent phosphorylation of checkpoint kinase 1 (Chk1) that is actuated in the absence of detectable DNA damage. We are interested in the mechanisms that activate ATR/Chk1 (Kizhedathu et al., 2018; Kizhedathu et al., 2020). Here we report that levels of reactive oxygen species (ROS) are high in arrested tracheoblasts and decrease upon mitotic re-entry. High ROS is dependent on expression of Duox, an H2O2 generating dual oxidase. ROS quenching by overexpression of superoxide dismutase 1, or by knockdown of Duox, abolishes Chk1 phosphorylation and results in precocious proliferation. Tracheae deficient in Duox, or deficient in both Duox and regulators of DNA damage-dependent ATR/Chk1 activation (ATRIP/TOPBP1/claspin), can induce phosphorylation of Chk1 in response to micromolar concentrations of H2O2 in minutes. The findings presented reveal that H2O2 activates ATR/Chk1 in tracheoblasts by a non-canonical, potentially direct, mechanism.