Asymmetric Small Protein Structure Determination by Individual Particle Electron Tomography

Commonly used methods for determining protein structure, including X-ray crystallography and single-particle reconstruction, often provide a single and unique three-dimensional (3D) structure. However, in these methods, the protein dynamics and flexibility/fluctuation remain mostly unknown. Here, we utilized advances in electron tomography (ET) to study the antibody flexibility and fluctuation through structural determination of individual antibody particles rather than averaging multiple antibody particles together. Through individual-particle electron tomography (IPET) 3D reconstruction from negatively-stained ET images, we obtained 120 ab-initio 3D density maps at an intermediate resolution (~1–3 nm) from 120 individual IgG1 antibody particles. Using these maps as a constraint, we derived 120 conformations of the antibody via structural flexible docking of the crystal structure to these maps by targeted molecular dynamics simulations. Statistical analysis of the various conformations disclosed the antibody 3D conformational flexibility through the distribution of its domain distances and orientations. This blueprint approach, if extended to other flexible proteins, may serve as a useful methodology towards understanding protein dynamics and functions.

Both single-particle analysis and electron tomography are special tools for the structural analysis of biological components by transmission electron microscopy. Single-particle analysis enables us to determine the structures of relatively large proteins and macromolecular complexes without crystallisation. Electron tomography is often used to visualise organelles and subcellular structures at molecular resolution. Recently, the scope of electron tomography has been widened due to the introduction of an automatic data collection system, cryo-electron microscopy, and improved sample preparation techniques. Application of electron tomography to single-particle analysis will be capable of analysing dynamics of large proteins. The techniques described in this review are expected to bridge the gap between the atomic resolution structures of protein molecules and cellular structures obtained by light microscopy.

Cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids, including cholesteryl esters (CEs) and triglycerides (TGs), between HDL, LDL and VLDL. Lipoprotein particles contain a neutral lipid core composed of CE and TG surrounded by a surface monolayer of phospholipids (PL), free cholesterol (FC), and apolipoproteins, most notably, apo B-100 in LDL and VLDL and apo A-I in HDL. An elevated level of LDL-cholesterol (LDL-C) and/or a low level of HDL-cholesterol (HDL-C) in human plasma are major risk factors for cardiovascular disease (CVD). Since increased CETP can reduce HDL-C concentration and CETP deficiency is associated with elevated HDL-C levels, CETP inhibitors, including torcetrapib, anacetrapib and dalcetrapib have been investigated in clinical trials for treating CVD. Despite the intense clinical interest in CETP inhibition, little is known concerning the molecular mechanisms of CETP-mediated lipid transfer among lipoproteins, or even how CETP interacts with lipoproteins. CETP is a hydrophobic glycoprotein of 476 amino acids (∼53 kDa, before posttranslational modification). Its crystal structure reveals a banana-shaped molecule with N- and C-terminal β-barrel domains, a central β-sheet, and a ∼60 Å-long hydrophobic central cavity. Three CETP neutral lipid transfer hypotheses were proposed more than two decades ago: 1) a shuttle mechanism that involves CETP collecting CEs from one lipoprotein and delivering them through the aqueous phase to another lipoprotein; 2) a tunnel mechanism in which CETP bridges two lipoproteins forming a ternary complex, with lipids flowing from the donor to acceptor lipoprotein through the CETP molecule; and 3) a modified tunnel mechanism implicating a CETP dimer. One difficulty in investigating CETP mechanisms using structural methods is that interaction with CETP can alter the size, shape, and composition of lipoproteins, especially HDL. We validated an optimized negative-staining electron microscopy (NS-EM) protocol in which flash-fixation of lipoprotein particles preserves a near native-state conformation for direct visualization of individual molecular or macromolecular particles. We applied this protocol to study the mechanisms by which CETP interacts with spherical HDL, LDL and VLDL. Three-dimensional (3D) reconstructions of CETP, free and HDL-bound, were obtained by single-particle techniques. In addition, we used inhibitory CETP antibodies to identify the regions of CETP that interact with HDL and LDL. Finally molecular dynamics (MD) simulation was used to assess the molecular mobility of CETP and predict the likely conformational changes that are associated with lipid transfer. We discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into HDL and its C-terminal domain interacting with LDL or VLDL. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.

The engineering of immunoglobulin-G molecules (IgGs) is of wide interest for improving therapeutics, for example by modulating the activity or multiplexing the specificity of IgGs to recognize more than one antigen. Optimization of engineered IgG requires knowledge of three-dimensional (3D) structure of synthetic IgG. However, due to flexible nature of the molecules, their structural characterization is challenging. Here, we use our reported individual-particle electron tomography (IPET) method with optimized negative-staining (OpNS) for direct 3D reconstruction of individual IgG hole-hole homodimer molecules. The hole-hole homodimer is an undesired variant generated during the production of a bispecific antibody using the knob-into-hole heterodimer technology. A total of 64 IPET 3D density maps at ~15 Å resolutions were reconstructed from 64 individual molecules, revealing 64 unique conformations. In addition to the known Y-shaped conformation, we also observed an unusual X-shaped conformation. The 3D structure of the X-shaped conformation contributes to our understanding of the structural details of the interaction between two heavy chains in the Fc domain. The IPET approach, as an orthogonal technique to characterize the 3D structure of therapeutic antibodies, provides insight into the 3D structural variety and dynamics of heterogeneous IgG molecules.

Cholesteryl ester transfer protein (CETP) mediates the exchange of triglycerides (TGs), cholesteryl esters (CEs) and phospholipids (PLs) between lipoproteins in the plasma. In order to better understand the lipid transfer process, we have used recombinant human CETP expressed in cultured mammalian cells, purified to homogeneity by immunoaffinity chromatography. Purified recombinant CETP had a weight-average relative molecular mass (MW) of 69561, determined by sedimentation equilibrium, and a specific absorption coefficient of 0.83 litre.g-1.cm-1. The corresponding hydrodynamic diameter (Dh) of the protein, determined by dynamic light scattering, was 14 nm, which is nearly twice the expected value for a spheroidal protein of this molecular mass. These data suggest that CETP has a non-spheroidal shape in solution. The secondary structure of CETP was estimated by CD to contain 32% alpha-helix, 35% beta-sheet, 17% turn and 16% random coil. Like the natural protein from plasma, the recombinant protein consisted of several glycoforms that could be only partially deglycosylated using N-glycosidase F. Organic extraction of CETP followed by TLC showed that CE, unesterified cholesterol (UC), PL, TG and fatty acids (FA) were associated with the pure protein. Quantitative analyses verified that each mol of CETP contained 1.0 mol of cholesterol, 0.5 mol of TG and 1.3 mol of PL. CETP mediated the transfer of CE, TG, PL, and UC between lipoproteins, or between protein-free liposomes. In dual-label transfer experiments, the transfer rates for CE or TG from HDL to LDL were found to be proportional to the initial concentrations of the respective ligands in the donor HDL particles. Kinetic analysis of CE transfer was consistent with a carrier mechanism, having a Km of 700 nM for LDL particles and of 2000 nM for HDL particles, and a kcat of 2 s-1. The Km values were thus in the low range of the normal physiological concentration for each substrate. The carrier mechanism was verified independently for CE, TG, PL and UC in 'half-reaction' experiments.

Electron tomography (ET) is an outstanding technique to access information about nanoparticles whose properties are critically dependent on their 3D shape and structure. A particularly relevant application of ET concerns plasmonic chiral nanoparticles, which exhibit strong correlation between optical activity and asymmetric morphology. Thus, it is crucial to go beyond qualitative visual inspection of ET reconstructions and extract quantitative shape or chirality descriptors. However, the time‐consuming nature of conventional ET, where a series of 2D projection images is used for one 3D reconstruction, makes it challenging to collect statistically representative data. A recently proposed semi‐automated approach for the acquisition of the required tilt series can reduce the data collection time from typically ≈ 45 minutes–1 hour to less than 10 minutes. What remains unclear is how this method influences the quality of the reconstruction and the accuracy of the samples’ quantitative descriptors. Here, two sets of chiral gold nanoparticles with wrinkled or twisted morphological chirality are used. It is demonstrated that the fast tilt series acquisitions yield results that are consistent with conventional ET, provided a standardized data processing protocol is used. Finally, a practical example of how fast ET can be used to obtain more statistically relevant measurements is presented.

High Density Lipoproteins (HDL) perform a variety of atheroprotective functions in the artery wall. These functions are driven by the protein and lipid components of the HDL particle. Almost 100 different proteins have been detected on HDL and over 20% are known regulators of protease activity. Inflammatory cells in the artery wall secrete proteases, such as elastase, that promote atherosclerosis progression by propagating inflammatory signaling and causing structural damage, which may destabilize lesions. We have previously shown that alpha-1-antitrypsin (A1AT), an endogenous inhibitor of elastase, associates with HDL in the circulation and can suppress elastase triggered inflammatory processes while bound to HDL. This interaction also prevents elastase-mediated degradation of HDL proteins and preserves HDL functions including cholesterol efflux, cholesterol esterification by LCAT, and antioxidant activity. In the present study, we report that a reciprocal protective effect occurs whereby the function of A1AT is preserved during interaction with HDL. Free or HDL-bound A1AT were treated with H 2 O 2 prior to measurement of elastase inhibitor activity. HDL-bound A1AT is resistant to oxidative inactivation (IC 50 28.6 vs 49.9 mM; p<0.0001). Using individual particle electron tomography and chemical crosslinking, we demonstrate that the reactive center loop (RCL) of A1AT is the site of contact with HDL. Oxidation of two methionine (Met) residues within the RCL of A1AT is known to impair elastase inhibitor activity. To test the hypothesis that RCL Met residues are protected from oxidative modification when bound to HDL, we used mass spectrometry to measure Met oxidation in H 2 O 2 treated free and HDL-bound A1AT. Met oxidation on HDL-bound A1AT treated with 100 mM H 2 O 2 was half that of free A1AT. This suggests that shielding of the RCL by interaction with HDL is the mechanism for functional preservation. In conclusion, our studies reveal that the RCL of A1AT is responsible for interaction with HDL and that physical contact results in a mutually beneficial arrangement whereby both HDL and A1AT function are preserved. These findings may serve as a paradigm for the interaction of other serine protease inhibitors with HDL as many share similar a functional motif.

In micrographs acquired with a transmission electron microscope, 3-dimensional (3D) objects are superimposed onto a 2D screen. This reduction in dimension necessarily leads to a degradation of image resolution. To overcome this problem, 3D microscopy techniques, such as tomography and single particle analysis, have been developed. Tomography has been used to visualize cells in 3D, and single particle analysis has been used to investigate macromolecules and viral particles. In this chapter we will describe how we have collected tilting series micrographs from plant cells and how we have reconstructed the cellular volumes using dual axis electron tomography.

Electron tomography is the leading technique to elucidate the structure of complex biological specimens. Due to the resolution needs, huge reconstructions are required. Grid computing has the potential to face the significant computational demands involved. However, there are a number of key issues, such as stability or difficult user-grid interaction, that currently preclude fully exploitation of its potential. EGEETomo is a user-friendly application that facilitates the interaction with the grid for the non-specialized user and automates job submission and supervision. In addition, EGEETomo is supplied with an automated fault recovery mechanism, which is key to make all the work transparent to the user. EGEETomo significantly accelerates tomographic reconstruction by exploiting the computational resources in the EGEE grid with minimal user intervention. http://www.ace.ual.es/~jrbcast/EGEETomo.tar.gz

Modern cryo-transmission electron microscopes (cryo-TEMs) are sophisticated instruments capable of recording data for multiple experimental modalities. At the Simons Electron Microscopy Center (SEMC), the primary cryo-TEM modalities requested from our biomedical-focused user community include single particle analysis (SPA) and electron tomography (ET). In the past 5 years, there has been growing interest in electron diffraction (ED) capabilities. SEMC houses multiple instruments capable of collecting diffraction data. Differences in the experimental setup for ED impact the allocation of time dedicated for SPA and ET that is in high demand. In addition, successful implementation of ED protocols requires knowledge transfer between microscopists and crystallographers to ensure usable data is collected. As a national nexus for cryoEM, SEMC is well poised to join the conversation around the balance of microscope flexibility and “productivity”. Here we discuss the workflow we have established for the collection of ED data, current demand from our user community, and subsequent influence on time allocation for different modes of data collection. In addition, we will discuss our work towards fostering a community of ED capable facilities more broadly through cross-training and focused workshops.

Single particle and molecular assembly analysis of polyribosomes by single- and double-tilt cryo electron tomography

Compressed sensing electron tomography

Globulins are an important group of seed storage proteins in dicotyledonous plants. They are synthesized during seed development, assembled into very compact protein complexes, and finally stored in protein storage vacuoles (PSVs). Here, we report a proteomic investigation on the native composition and structure of cruciferin, the 12 S globulin of Brassica napus. PSVs were directly purified from mature seeds by differential centrifugations. Upon analyses by blue native (BN) PAGE, two major types of cruciferin complexes of ∼ 300-390 kDa and of ∼470 kDa are resolved. Analyses by two-dimensional BN/SDS-PAGE revealed that both types of complexes are composed of several copies of the cruciferin α and β polypeptide chains, which are present in various isoforms. Protein analyses by two-dimensional isoelectric focusing (IEF)/SDS-PAGE not only revealed different α and β isoforms but also several further versions of the two polypeptide chains that most likely differ with respect to posttranslational modifications. Overall, more than 30 distinct forms of cruciferin were identified by mass spectrometry. To obtain insights into the structure of the cruciferin holocomplex, a native PSV fraction was analyzed by single particle electron microscopy. More than 20,000 images were collected, classified, and used for the calculation of detailed projection maps of the complex. In contrast to previous reports on globulin structure in other plant species, the cruciferin complex of Brassica napus has an octameric barrel-like structure, which represents a very compact building block optimized for maximal storage of amino acids within minimal space.

The soluble cyclic AMP-dependent protein kinase of bovine heart has been purified to homogeneity. It is an asymmetric protein with a molecular weight of 174,000 and is composed of two globular catalytic subunits (molecular weight, 38,000) and an asymmetric cyclic nucleotide-binding protein (molecular weight, 98,000). The cyclic AMP-binding protein consists of two polypeptide chains of equal size and is able to serve as substrate for a protein kinase-catalyzed phosphotransferase reaction. The physiological significance of this “autophosphorylation” of protein kinase has not been established. The protein kinase activity associated with the plasma membrane of the human erythrocyte has many properties in common with soluble protein kinases including the ability to be dissociated into cyclic AMP-independent kinase and cyclic AMP-binding activities. Membrane-bound protein kinase catalyzes the phosphorylation of three endogenous membrane proteins, two of them in a cyclic nucleotide-dependent fashion. Both the cyclic AMP-binding and phosphotransferase activities of the human erythrocyte membrane appear to be localized on the inner cytoplasmic surface of the membrane.

Lipoprotein metabolism in FIC1 deficiency due to ATP8B1 mutations has never been studied sufficiently. This study was performed to investigate the detailed lipoprotein metabolism in benign recurrent intrahepatic cholestasis (BRIC) caused by FIC1 deficiency. Lipoprotein profile and major lipoprotein regulators such as lecithin:cholesterol acyltransferase (LCAT), hepatic triglyceride lipase (HTGL), lipoprotein lipase, and cholesteryl ester transfer protein in a Japanese patient with BRIC were serially examined during a bout of cholestasis. Liver expression of farnesoid X receptor (FXR), which suppresses high-density lipoprotein (HDL) generation, was also examined. Hypercholesterolemia and lipoprotein X accumulation were never observed throughout this study. When the cholestasis was severe, triglyceride-rich low-density lipoprotein (LDL) accounted for most of the plasma lipoproteins whereas HDL was hardly detectable. Concurrently, activities of all regulators were decreased, together with decreases of the serum parameter for liver protein synthesis. In particular, suppressions of LCAT and HTGL activities were severe and greatly contributed to the appearance of triglyceride-rich LDL. As the cholestasis improved, this LDL gradually transformed into normal LDL with the recoveries of LCAT and HTGL activities. The activities of all regulators for the last 1 to 2 months were normal but HDL remained depleted. His liver showed low FXR expression compared with control livers. The present study showed an appearance of triglyceride-rich LDL due to suppressions of LCAT and HTGL activities and a depletion of HDL that is not able to be explained by lipoprotein regulators or FXR in our patient.