Characterization of the flexibility of the peripheral stalk of prokaryotic rotary A‐ATPases by atomistic simulations

Rotary ATPases are involved in numerous physiological processes, with the three distinct types (F/A/V‐ATPases) sharing functional properties and structural features. The basic mechanism involves the counter rotation of two motors, a soluble ATP hydrolyzing/synthesizing domain and a membrane‐embedded ion pump connected through a central rotor axle and a stator complex. Within the A/V‐ATPase family conformational flexibility of the EG stators has been shown to accommodate catalytic cycling and is considered to be important to function. For the A‐ATPase three EG structures have been reported, thought to represent conformational states of the stator during different stages of rotary catalysis. Here we use long, detailed atomistic simulations to show that those structures are conformers explored through thermal fluctuations, but do not represent highly populated states of the EG stator in solution. We show that the coiled coil tail domain has a high persistence length (～100 nm), but retains the ability to adapt to different conformational states through the presence of two hinge regions. Moreover, the stator network of the related V‐ATPase has been suggested to adapt to subunit interactions in the collar region in addition to the nucleotide occupancy of the catalytic domain. The MD simulations reported here, reinforce this observation showing that the EG stators have enough flexibility to adapt to significantly different structural re‐arrangements and accommodate structural changes in the catalytic domain whilst resisting the large torque generated by catalytic cycling. These results are important to understand the role the stators play in the rotary‐ATPase mechanism. Proteins 2016; 84:1203–1212. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Structural divergence of the rotary ATPases

The rotary ATPase family of membrane protein complexes may have only three members, but each one plays a fundamental role in biological energy conversion. The F?F(o)-ATPase (F-ATPase) couples ATP synthesis to the electrochemical membrane potential in bacteria, mitochondria and chloroplasts, while the vacuolar H?-ATPase (V-ATPase) operates as an ATP-driven proton pump in eukaryotic membranes. In different species of archaea and bacteria, the A?A(o)-ATPase (A-ATPase) can function as either an ATP synthase or an ion pump. All three of these multi-subunit complexes are rotary molecular motors, sharing a fundamentally similar mechanism in which rotational movement drives the energy conversion process. By analogy to macroscopic systems, individual subunits can be assigned to rotor, axle or stator functions. Recently, three-dimensional reconstructions from electron microscopy and single particle image processing have led to a significant step forward in understanding of the overall architecture of all three forms of these complexes and have allowed the organisation of subunits within the rotor and stator parts of the motors to be more clearly mapped out. This review describes the emerging consensus regarding the organisation of the rotor and stator components of V-, A- and F-ATPases, examining core similarities that point to a common evolutionary origin, and highlighting key differences. In particular, it discusses how newly revealed variation in the complexity of the inter-domain connections may impact on the mechanics and regulation of these molecular machines.     

Lipid nanotechnologies for structural studies of membrane‐associated proteins

We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane‐associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryo‐electron microscopy (cryo‐EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ～20 nm inner diameter and a few microns in length, that self‐assemble in aqueous solutions. The lipid nanodisks (NDs) are self‐assembled discoid lipid bilayers of ～10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane‐associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane‐bound coagulation factor VIII in vitro for structure determination by cryo‐EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three‐dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane‐associated proteins and complexes for structural studies by cryo‐EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane‐associated proteins, such as the coagulation factors, at a close to physiological environment. Proteins 2014; 82:2902–2909. © 2014 Wiley Periodicals, Inc.     

Molecular architecture of the N‐type ATPase rotor ring from Burkholderia pseudomallei

The genome of the highly infectious bacterium Burkholderia pseudomallei harbors an atp operon that encodes an N‐type rotary ATPase, in addition to an operon for a regular F‐type rotary ATPase. The molecular architecture of N‐type ATPases is unknown and their biochemical properties and cellular functions are largely unexplored. We studied the B. pseudomallei N₁N_o‐type ATPase and investigated the structure and ion specificity of its membrane‐embedded c‐ring rotor by single‐particle electron cryo‐microscopy. Of several amphiphilic compounds tested for solubilizing the complex, the choice of the low‐density, low‐CMC detergent LDAO was optimal in terms of map quality and resolution. The cryoEM map of the c‐ring at 6.1 Å resolution reveals a heptadecameric oligomer with a molecular mass of ~141 kDa. Biochemical measurements indicate that the c₁₇ ring is H⁺ specific, demonstrating that the ATPase is proton‐coupled. The c₁₇ ring stoichiometry results in a very high ion‐to‐ATP ratio of 5.7. We propose that this N‐ATPase is a highly efficient proton pump that helps these melioidosis‐causing bacteria to survive in the hostile, acidic environment of phagosomes.     

Cryo‐EM map interpretation and protein model‐building using iterative map segmentation

A procedure for building protein chains into maps produced by single‐particle electron cryo‐microscopy (cryo‐EM) is described. The procedure is similar to the way an experienced structural biologist might analyze a map, focusing first on secondary structure elements such as helices and sheets, then varying the contour level to identify connections between these elements. Since the high density in a map typically follows the main‐chain of the protein, the main‐chain connection between secondary structure elements can often be identified as the unbranched path between them with the highest minimum value along the path. This chain‐tracing procedure is then combined with finding side‐chain positions based on the presence of density extending away from the main path of the chain, allowing generation of a C_α model. The C_α model is converted to an all‐atom model and is refined against the map. We show that this procedure is as effective as other existing methods for interpretation of cryo‐EM maps and that it is considerably faster and produces models with fewer chain breaks than our previous methods that were based on approaches developed for crystallographic maps.     

Stairway to translocation: AAA+ motor structures reveal the mechanisms of ATP‐dependent substrate translocation

Translocases of the AAA+ (ATPases Associated with various cellular Activities) family are powerful molecular machines that use the mechano‐chemical coupling of ATP hydrolysis and conformational changes to thread DNA or protein substrates through their central channel for many important biological processes. These motors comprise hexameric rings of ATPase subunits, in which highly conserved nucleotide‐binding domains form active‐site pockets near the subunit interfaces and aromatic pore‐loop residues extend into the central channel for substrate binding and mechanical pulling. Over the past 2 years, 41 cryo‐EM structures have been solved for substrate‐bound AAA+ translocases that revealed spiral‐staircase arrangements of pore‐loop residues surrounding substrate polypeptides and indicating a conserved hand‐over‐hand mechanism for translocation. The subunits' vertical positions within the spiral arrangements appear to be correlated with their nucleotide states, progressing from ATP‐bound at the top to ADP or apo states at the bottom. Studies describing multiple conformations for a particular motor illustrate the potential coupling between ATP‐hydrolysis steps and subunit movements to propel the substrate. Experiments with double‐ring, Type II AAA+ motors revealed an offset of hydrolysis steps between the two ATPase domains of individual subunits, and the upper ATPase domains lacking aromatic pore loops frequently form planar rings. This review summarizes the critical advances provided by recent studies to our structural and functional understanding of hexameric AAA+ translocases, as well as the important outstanding questions regarding the underlying mechanisms for coordinated ATP‐hydrolysis and mechano‐chemical coupling.     

Towards correlative super‐resolution fluorescence and electron cryo‐microscopy

Correlative light and electron microscopy (CLEM) has become a powerful tool in life sciences. Particularly cryo‐CLEM, the combination of fluorescence cryo‐microscopy (cryo‐FM) permitting for non‐invasive specific multi‐colour labelling, with electron cryo‐microscopy (cryo‐EM) providing the undisturbed structural context at a resolution down to the Ångstrom range, has enabled a broad range of new biological applications. Imaging rare structures or events in crowded environments, such as inside a cell, requires specific fluorescence‐based information for guiding cryo‐EM data acquisition and/or to verify the identity of the structure of interest. Furthermore, cryo‐CLEM can provide information about the arrangement of specific proteins in the wider structural context of their native nano‐environment. However, a major obstacle of cryo‐CLEM currently hindering many biological applications is the large resolution gap between cryo‐FM (typically in the range of ～400 nm) and cryo‐EM (single nanometre to the Ångstrom range). Very recently, first proof of concept experiments demonstrated the feasibility of super‐resolution cryo‐FM imaging and the correlation with cryo‐EM. This opened the door towards super‐resolution cryo‐CLEM, and thus towards direct correlation of structural details from both imaging modalities.     

Direct imaging electron microscopy (EM) methods in modern structural biology: Overview and comparison with X‐ray crystallography and single‐particle cryo‐EM reconstruction in the studies of large macromolecules

Determining the structure of macromolecules is important for understanding their function. The fine structure of large macromolecules is currently studied primarily by X‐ray crystallography and single‐particle cryo‐electron microscopy (EM) reconstruction. Before the development of these techniques, macromolecular structure was often examined by negative‐staining, rotary‐shadowing and freeze‐etching EM, which are categorised here as ‘direct imaging EM methods’. In this review, the results are summarised by each of the above techniques and compared with respect to four macromolecules: the ryanodine receptor, cadherin, rhodopsin and the ribosome–translocon complex (RTC). The results of structural analysis of the ryanodine receptor and cadherin are consistent between each technique. The results obtained for rhodopsin vary to some extent within each technique and between the different techniques. Finally, the results for RTC are inconsistent between direct imaging EM and other analytical techniques, especially with respect to the space within RTC, the reasons for which are discussed. Then, the role of direct imaging EM methods in modern structural biology is discussed. Direct imaging methods should support and verify the results obtained by other analytical methods capable of solving three‐dimensional molecular architecture, and they should still be used as a primary tool for studying macromolecule structure in vivo.     

Developments,applications, and prospects of cryo‐electron microscopy

Cryo‐electron microscopy (cryo‐EM) is a structural biological method that is used to determine the 3D structures of biomacromolecules. After years of development, cryo‐EM has made great achievements, which has led to a revolution in structural biology. In this article, the principle, characteristics, history, current situation, workflow, and common problems of cryo‐EM are systematically reviewed. In addition, the new development direction of cryo‐EM—cryo‐electron tomography (cryo‐ET), is discussed in detail. Also, cryo‐EM is prospected from the following aspects: the structural analysis of small proteins, the improvement of resolution and efficiency, and the relationship between cryo‐EM and drug development. This review is dedicated to giving readers a comprehensive understanding of the development and application of cryo‐EM, and to bringing them new insights.     

Probing the activity of a recombinant Zn2+‐transporting P‐type ATPase

P‐type ATPase proteins maintain cellular homeostasis and uphold critical concentration gradients by ATP‐driven ion transport across biological membranes. Characterization of single‐cycle dynamics by time‐resolved X‐ray scattering techniques in solution could resolve structural intermediates not amendable to for example crystallization or cryo‐electron microscopy sample preparation. To pave way for such time‐resolved experiments, we used biochemical activity measurements, Attenuated Total Reflectance (ATR) and time‐dependent Fourier‐Transform Infra‐Red (FTIR) spectroscopy to identify optimal conditions for activating a Zn²⁺‐transporting Type‐I ATPase from Shigella sonnei (ssZntA) at high protein concentration using caged ATP. The highest total activity was observed at a protein concentration of 25 mg/mL, at 310 K, pH 7, and required the presence of 20% (v/v) glycerol as stabilizing agent. Neither the presence of caged ATP nor increasing lipid‐to‐protein ratio affected the hydrolysis activity significantly. This work also paves way for characterization of recombinant metal‐transporting (Type‐I) ATPase mutants with medical relevance.     

Breaking up and making up: The secret life of the vacuolar H+‐ATPase

The vacuolar ATPase (V‐ATPase; V₁V_o‐ATPase) is a large multisubunit proton pump found in the endomembrane system of all eukaryotic cells where it acidifies the lumen of subcellular organelles including lysosomes, endosomes, the Golgi apparatus, and clathrin‐coated vesicles. V‐ATPase function is essential for pH and ion homeostasis, protein trafficking, endocytosis, mechanistic target of rapamycin (mTOR), and Notch signaling, as well as hormone secretion and neurotransmitter release. V‐ATPase can also be found in the plasma membrane of polarized animal cells where its proton pumping function is involved in bone remodeling, urine acidification, and sperm maturation. Aberrant (hypo or hyper) activity has been associated with numerous human diseases and the V‐ATPase has therefore been recognized as a potential drug target. Recent progress with moderate to high‐resolution structure determination by cryo electron microscopy and X‐ray crystallography together with sophisticated single‐molecule and biochemical experiments have provided a detailed picture of the structure and unique mode of regulation of the V‐ATPase. This review summarizes the recent advances, focusing on the structural and biophysical aspects of the field.     

qFit 3: Protein and ligand multiconformer modeling for X‐ray crystallographic and single‐particle cryo‐EM density maps

New X‐ray crystallography and cryo‐electron microscopy (cryo‐EM) approaches yield vast amounts of structural data from dynamic proteins and their complexes. Modeling the full conformational ensemble can provide important biological insights, but identifying and modeling an internally consistent set of alternate conformations remains a formidable challenge. qFit efficiently automates this process by generating a parsimonious multiconformer model. We refactored qFit from a distributed application into software that runs efficiently on a small server, desktop, or laptop. We describe the new qFit 3 software and provide some examples. qFit 3 is open‐source under the MIT license, and is available at https://github.com/ExcitedStates/qfit-3.0 .     

Probing the structural dynamics of the SNARE recycling machine based on coarse‐grained modeling

Membrane fusion in eukaryotes is driven by the formation of a four‐helix bundle by three SNARE proteins. To recycle the SNARE proteins, they must be disassembled by the ATPase NSF and four SNAP proteins which together form a 20S supercomplex. Recently, the first high‐resolution structures of the NSF (in both ATP and ADP state) and 20S (in four distinct states termed I, II, IIIa, and IIIb) were solved by cryo‐electron microscopy (cryo‐EM), which have paved the way for structure‐driven studies of the SNARE recycling mechanism. To probe the structural dynamics of SNARE disassembly at amino‐acid level of details, a systematic coarse‐grained modeling based on an elastic network model and related analyses were performed. Our normal mode analysis of NSF, SNARE, and 20S predicted key modes of collective motions that partially account for the observed structural changes, and illuminated how the SNARE complex can be effectively destabilized by untwisting and bending motions of the SNARE complex driven by the amino‐terminal domains of NSF in state II. Our flexibility analysis identified regions with high/low flexibility that coincide with key functional sites (such as the NSF‐SNAPs‐SNARE binding sites). A subset of hotspot residues that control the above collective motions, which will make promising targets for future mutagenesis studies were also identified. Finally, the conformational changes in 20S as induced by the transition of NSF from ATP to ADP state were modeled, and a concerted untwisting motion of SNARE/SNAPs and a sideway flip of two amino‐terminal domains were observed. In sum, the findings have offered new structural and dynamic details relevant to the SNARE disassembly mechanism, and will guide future functional studies of the SNARE recycling machinery. Proteins 2016; 84:1055–1066. © 2016 Wiley Periodicals, Inc.     

ATP synthase: two motors, two fuels

FoF1 ATPase is the universal protein responsible for ATP synthesis. The enzyme comprises two reversible rotary motors: Fo is either an ion 'turbine' or an ion pump, and F1 is either a hydrolysis motor or an ATP synthesizer. Recent biophysical and biochemical studies have helped to elucidate the operating principles for both motors.     

Membrane dynamics of γ‐secretase with the anterior pharynx‐defective 1B subunit

The four‐subunit protease complex γ‐secretase cleaves many single‐pass transmembrane (TM) substrates, including Notch and β‐amyloid precursor protein to generate amyloid‐β (Aβ), central to Alzheimer's disease. Two of the subunits anterior pharynx‐defective 1 (APH‐1) and presenilin (PS) exist in two homologous forms APH1‐A and APH1‐B, and PS1 and PS2. The consequences of these variations are poorly understood and could affect Aβ production and γ‐secretase medicine. Here, we developed the first complete structural model of the APH‐1B subunit using the published cryo‐electron microscopy (cryo‐EM) structures of APH1‐A (Protein Data Bank: 5FN2, 5A63, and 6IYC). We then performed all‐atom molecular dynamics simulations at 303 K in a realistic bilayer system to understand both APH‐1B alone and in γ‐secretase without and with substrate C83‐bound. We show that APH‐1B adopts a 7TM topology with a water channel topology similar to APH‐1A. We demonstrate direct transport of water through this channel, mainly via Glu84, Arg87, His170, and His196. The apo and holo states closely resemble the experimental cryo‐EM structures with APH‐1A, however with subtle differences: The substrate‐bound APH‐1B γ‐secretase was quite stable, but some TM helices of PS1 and APH‐1B rearranged in the membrane consistent with the disorder seen in the cryo‐EM data. This produces different accessibility of water molecules for the catalytic aspartates of PS1, critical for Aβ production. In particular, we find that the typical distance between the catalytic aspartates of PS1 and the C83 cleavage sites are shorter in APH‐1B, that is, it represents a more closed state, due to interactions with the C‐terminal fragment of PS1. Our structural‐dynamic model of APH‐1B alone and in γ‐secretase suggests generally similar topology but some notable differences in water accessibility which may be relevant to the protein's existence in two forms and their specific function and location.     

The integrative role of cryo electron microscopy in molecular and cellular structural biology

After gradually moving away from preparation methods prone to artefacts such as plastic embedding and negative staining for cell sections and single particles, the field of cryo electron microscopy (cryo‐EM) is now heading off at unprecedented speed towards high‐resolution analysis of biological objects of various sizes. This ‘revolution in resolution’ is happening largely thanks to new developments of new‐generation cameras used for recording the images in the cryo electron microscope which have much increased sensitivity being based on complementary metal oxide semiconductor devices. Combined with advanced image processing and 3D reconstruction, the cryo‐EM analysis of nucleoprotein complexes can provide unprecedented insights at molecular and atomic levels and address regulatory mechanisms in the cell. These advances reinforce the integrative role of cryo‐EM in synergy with other methods such as X‐ray crystallography, fluorescence imaging or focussed‐ion beam milling as exemplified here by some recent studies from our laboratory on ribosomes, viruses, chromatin and nuclear receptors. Such multi‐scale and multi‐resolution approaches allow integrating molecular and cellular levels when applied to purified or in situ macromolecular complexes, thus illustrating the trend of the field towards cellular structural biology.     

Higher order structures of Adalimumab,Infliximab and their complexes with TNFα revealed by electron microscopy

Adalimumab and Infliximab are recombinant IgG1 monoclonal antibodies (mAbs) that bind and neutralize human tumor necrosis factor alpha (TNFα). TNFα forms a stable homotrimer with unique surface‐exposed sites for Adalimumab, Infliximab, and TNF receptor binding. Here, we report the structures of Adalimumab‐TNFα and Infliximab‐TNFα complexes modeled from negative stain EM and cryo‐EM images. EM images reveal complex structures consisting of 1:1, 1:2, 2:2, and 3:2 complexes of Adalimumab‐TNFα and Infliximab‐TNFα. The 2:2 complex structures of Adalimumab‐TNFα and Infliximab‐TNFα show diamond‐shaped profiles and the 2D class averages reveal distinct orientations of the Fab domains, indicating different binding modes by Adalimumab and Infliximab to TNFα. After separation by size exclusion chromatography and analysis by negative stain EM, the 3:2 complexes of Adalimumab‐TNFα or Infliximab‐TNFα complexes are more complicated but retain features recognized in the 2:2 complexes. Preliminary cryo‐EM analysis of 3:2 Adalimumab‐TNFα complex generated a low‐resolution density consistent with a TNFα trimer bound with three Fab domains from three individual antibody molecules, while each antibody molecule binds to two molecules of TNFα trimer. The Fc domains are not visible in the reconstruction. These results show the two mAbs form structurally distinct complexes with TNFα.     

Assembly of anthrax toxin pore: Lethal‐factor complexes into lipid nanodiscs

We have devised a procedure to incorporate the anthrax protective antigen (PA) pore complexed with the N‐terminal domain of anthrax lethal factor (LF_N) into lipid nanodiscs and analyzed the resulting complexes by negative‐stain electron microscopy. Insertion into nanodiscs was performed without relying on primary and secondary detergent screens. The preparations were relatively pure, and the percentage of PA pore inserted into nanodiscs on EM grids was high (～43%). Three‐dimensional analysis of negatively stained single particles revealed the LF_N‐PA nanodisc complex mirroring the previous unliganded PA pore nanodisc structure, but with additional protein density consistent with multiple bound LF_N molecules on the PA cap region. The assembly procedure will facilitate collection of higher resolution cryo‐EM LF_N‐PA nanodisc structures and use of advanced automated particle selection methods.