Showing papers on "Structural biology published in 2015"

Sampling the conformational space of the catalytic subunit of human γ-secretase

Abstract: An enzyme called gamma-secretase cuts other proteins in cells into smaller pieces. Like most enzymes, gamma-secretase is expected to move through several different three-dimensional shapes to perform its role, and identifying these structures could help us to understand how the enzyme works. One of the proteins that is targeted by gamma-secretase is called amyloid precursor protein, and cutting this protein results in the formation of so-called amyloid-beta peptides. When gamma-secretase doesn't work properly, these amyloid-beta peptides can accumulate in the brain and large accumulations of these peptides have been observed in the brains of patients with Alzheimer's disease. Earlier in 2015, a group of researchers used a technique called cryo-electron microscopy (cryo-EM) to produce a three-dimensional model of gamma-secretase. This revealed that the active site of the enzyme, that is, the region that is used to cut the other proteins, is particularly flexible. Now, Bai et al. – including many of the researchers from the earlier work – studied this flexibility in more detail. For the experiments, gamma-secretase was exposed to an inhibitor molecule that stopped it from cutting other proteins. This meant that the structure of gamma-secretase became more rigid than normal, which made it possible to collect more detailed structural information using cryo-EM. Bai et al. also developed new methods for processing images to separate the images of individual enzyme molecules based on the different shapes they had adopted at the time. These methods make it possible to view a mixture of very similar enzyme structures that differ only in a small region of the protein (in this case the active site). In the future, it would be useful to repeat these imaging experiments using a range of different molecules that alter the activity of gamma-secretase. Furthermore, the new image processing methods developed by Bai et al. could be used to study flexibility in the shapes of other proteins.

In situ structural analysis of the human nuclear pore complex

Abstract: Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block--although compositionally identical--engage in different local sets of interactions and conformations.

Structure of the immature HIV-1 capsid in intact virus particles at 8.8 Å resolution

Abstract: Human immunodeficiency virus type 1 (HIV-1) assembly proceeds in two stages. First, the 55 kilodalton viral Gag polyprotein assembles into a hexameric protein lattice at the plasma membrane of the infected cell, inducing budding and release of an immature particle. Second, Gag is cleaved by the viral protease, leading to internal rearrangement of the virus into the mature, infectious form. Immature and mature HIV-1 particles are heterogeneous in size and morphology, preventing high-resolution analysis of their protein arrangement in situ by conventional structural biology methods. Here we apply cryo-electron tomography and sub-tomogram averaging methods to resolve the structure of the capsid lattice within intact immature HIV-1 particles at subnanometre resolution, allowing unambiguous positioning of all α-helices. The resulting model reveals tertiary and quaternary structural interactions that mediate HIV-1 assembly. Strikingly, these interactions differ from those predicted by the current model based on in vitro-assembled arrays of Gag-derived proteins from Mason-Pfizer monkey virus. To validate this difference, we solve the structure of the capsid lattice within intact immature Mason-Pfizer monkey virus particles. Comparison with the immature HIV-1 structure reveals that retroviral capsid proteins, while having conserved tertiary structures, adopt different quaternary arrangements during virus assembly. The approach demonstrated here should be applicable to determine structures of other proteins at subnanometre resolution within heterogeneous environments.

Thermodynamics of protein destabilization in live cells

Abstract: Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.

2.8 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome using cryo-electron microscopy

Abstract: Proteins perform many critical tasks within cells, and to do so, they must first fold into specific shapes. Being able to visualize these shapes can help scientists to understand how proteins work, and help them create drugs that can interact with the proteins to treat diseases. The past few years have seen the rapid development of an imaging technique called single-particle cryo-electron microscopy (or cryoEM for short), and this technique is now increasingly used to investigate protein structures. First, proteins are embedded in a thin film of non-crystalline ice by rapidly cooling to around the temperature of liquid nitrogen (below −180°C). This traps the protein in the shape it has in solution. High-energy electrons are then transmitted through the protein sample and their interaction with the atoms in the protein is recorded by a direct electron camera. The analysis of a large series of images recorded in this way can be used to determine the approximate positions of the atoms in the protein. Previously, single-particle cryoEM techniques have not produced a detailed enough protein structure to be useful to scientists interested in drug development. By refining these techniques, Campbell, Veesler et al. have now obtained the most detailed cryoEM protein structure to date—a structure of an enzyme complex that helps get rid of proteins that are misfolded or that have become too abundant. The structure is so detailed that it reveals the shapes of some small groups of atoms that stick out from the sides of amino acids in the enzyme complex. (Amino acids are the building blocks of enzymes and all other proteins.) Moreover, the structure shows where individual water molecules are positioned around the protein. The level of detail in the structure produced by Campbell, Veesler et al. is high enough to be useful to drug researchers. Furthermore, because only 10% of the images Campbell, Veesler et al. collected were used to produce the structure, future work will investigate whether incorporating more of the images could reveal structures in even greater detail.

The Bcl-2 family: structures, interactions and targets for drug discovery

Abstract: Two phylogenetically and structurally distinct groups of proteins regulate stress induced intrinsic apoptosis, the programmed disassembly of cells. Together they form the B cell lymphoma-2 (Bcl-2) family. Bcl-2 proteins appeared early in metazoan evolution and are identified by the presence of up to four short conserved sequence blocks known as Bcl-2 homology (BH) motifs, or domains. The simple BH3-only proteins bear only a BH3-motif and are intrinsically disordered proteins and antagonize or activate the other group, the multi-motif Bcl-2 proteins that have up to four BH motifs, BH1-BH4. Multi-motif Bcl-2 proteins are either pro-survival or pro-apoptotic in action and have remarkably similar α-helical bundle structures that provide a binding groove formed from the BH1, BH2, and BH3-motifs for their BH3-bearing antagonists. In mammals a network of interactions between Bcl-2 members regulates mitochondrial outer membrane permeability (MOMP) and efflux of cytochrome c and other death inducing factors from mitochondria to initiate the apoptotic caspase cascade, but the molecular events leading to MOMP are uncertain. Dysregulation of the Bcl-2 family occurs in many diseases and pathogenic viruses have assimilated pro-survival Bcl-2 proteins to evade immune responses. Their role in disease has made the Bcl-2 family the focus of drug design attempts and clinical trials are showing promise for 'BH3-mimics', drugs that mimic the ability of BH3-only proteins to neutralize selected pro-survival proteins to induce cell death in tumor cells. This review focuses on the structural biology of Bcl-2 family proteins, their interactions and attempts to harness them as targets for drug design.

Structural basis for activation, assembly and membrane binding of ESCRT-III Snf7 filaments

Abstract: A cell constantly remodels its surface to adapt to its environment, as well as to replace old or damaged proteins. To achieve this, cell-surface receptors are taken inside the cell and delivered to organelles called endosomes, where a molecular machine called ESCRT governs the receptors’ fate. Distinct ESCRT complexes remodel the endosomal membrane to form vesicle packages that encapsulate the receptor proteins. These vesicles bud off into the endosome, which is then targeted to another organelle called the lysosome where the receptor proteins are degraded. If the vesicles are unable to make their deliveries, the resulting sustained receptor activity can lead to numerous developmental and neurodegenerative diseases, as well as cancer. Remarkably, the ESCRT machinery also plays critical roles during cell division and the release of the human immunodeficiency virus (HIV) from host cells. Previous studies demonstrated that a particular ESCRT complex, called ESCRT-III, forms spiraling filaments on membranes to generate vesicles. However, how the individual components of ESCRT-III assemble into such filaments was a mystery. Now, Tang et al. have determined the first X-ray crystal structures of the main component of ESCRT-III, a polymer of the protein called Snf7, and thus uncovered how these membrane-bound Snf7 spirals assemble. Using a combination of cell biology, genetics and biochemistry techniques, Tang et al. also demonstrated that the Snf7 structures are necessary for ESCRT-III to work correctly inside living cells. Despite this achievement, key questions remain. The main one is how the other subunits of ESCRT-III interact and work together to remodel the membrane to form the vesicle packages at the endosomes.

Visualizing the functional architecture of the endocytic machinery.

Abstract: Cells take up proteins and other useful material (called cargo) from their external environment through a process known as endocytosis. To start with, the cargo accumulates in a patch on the surface of the cell. On the inner side of the cell's membrane, a protein called clathrin gathers around the patch of cargo. Clathrin molecules and many other proteins bind together to make a lattice-like coat that causes the membrane to curve inwards and form a pocket that contains the cargo. This continues until the cargo is completely surrounded by membrane and eventually forms a bubble-like structure, or ‘vesicle’, that moves into the cell. More than 50 other proteins are involved in the endocytosis. These proteins arrive at the site of endocytosis in a particular order, complete their tasks and then move away to be used in further rounds of endocytosis. It is not clear how these proteins are organized to complete these steps because it is technically difficult to track the movements of many proteins at the same time. Here, Picco et al. developed a new fluorescence microscopy method that enabled them to track the positions of many of the proteins involved in endocytosis in yeast cells in real time. The experiments revealed when the proteins arrived at the site of endocytosis and how they assembled in relation to the membrane. For example, a group of proteins called N-BAR proteins formed an extended lattice covering the sides of the pocket that forms as the membrane curves inwards. To transform the flat membrane into a vesicle, a network of filaments made of a protein called actin needs to form at the site of endocytosis. The new method shows that the actin filaments grow in a small region at the base of the developing vesicle. By combining different types of microscopy data, Picco et al. were able to build a comprehensive model describing when the proteins involved in endocytosis move and assemble. The next challenge will be to understand the physics behind the molecular machine composed of these many proteins and the cell membrane.

Probing a cell-embedded megadalton protein complex by DNP-supported solid-state NMR

Abstract: Studying biomolecules at atomic resolution in their native environment is the ultimate aim of structural biology. We investigated the bacterial type IV secretion system core complex (T4SScc) by cellular dynamic nuclear polarization–based solid-state nuclear magnetic resonance spectroscopy to validate a structural model previously generated by combining in vitro and in silico data. Our results indicate that T4SScc is well folded in the cellular setting, revealing protein regions that had been elusive when studied in vitro.

Chemical cross‐linking and native mass spectrometry: A fruitful combination for structural biology

Abstract: Mass spectrometry (MS) is becoming increasingly popular in the field of structural biology for analyzing protein three-dimensional-structures and for mapping protein-protein interactions. In this review, the specific contributions of chemical crosslinking and native MS are outlined to reveal the structural features of proteins and protein assemblies. Both strategies are illustrated based on the examples of the tetrameric tumor suppressor protein p53 and multisubunit vinculin-Arp2/3 hybrid complexes. We describe the distinct advantages and limitations of each technique and highlight synergistic effects when both techniques are combined. Integrating both methods is especially useful for characterizing large protein assemblies and for capturing transient interactions. We also point out the future directions we foresee for a combination of in vivo crosslinking and native MS for structural investigation of intact protein assemblies.

Structures of the scanning and engaged states of the mammalian SRP-ribosome complex

Abstract: Proteins are long chain-like molecules built from smaller building blocks, called amino acids, by a large molecular machine known as a ribosome. Although all proteins are assembled inside cells, some of them must be delivered to the outside or inserted into cell membranes. It is important to understand how this selective delivery system works because secreted proteins (i.e., those delivered outside) and membrane-embedded proteins are essential for cells to communicate with their surroundings. Proteins destined for secretion or membrane insertion contain characteristic stretches of amino acids that act as a targeting signal for delivery to the membrane. These targeting signals are recognized by the ‘signal recognition particle’ (or SRP for short), a large complex found in all living organisms. The SRP has the task of finding ribosomes that are assembling proteins with a targeting signal, and then taking them to the membrane. The protein being assembled can then either cross the membrane for secretion by the cell, or get embedded within the membrane. So, how can the SRP scan the broad range of proteins that are made by the ribosome and engage with only those containing targeting signals? Voorhees and Hegde investigated this question by analyzing SRPs bound to ribosomes that were at different stages of building a membrane protein. The experiment was devised so that SRP would be in two different states: in the first state, the SRP was scanning for its targeting signal and, in the second, it was engaged with the targeting signal. Voorhees and Hegde took many thousands of pictures of these samples using a technique called cryo-electron microscopy, and reconstructed the three-dimensional structures of both states. This revealed fine details of how SRP positions itself immediately next to the part of the ribosome where newly formed protein chains emerge. From here, the SRP scans the protein until the targeting signal emerges and then it engages with the protein. Engaging the targeting signal just as it emerges from the ribosome is probably important because targeting signals tend to aggregate if they are exposed to the contents of a cell. The new structures show how SRP cradles the targeting signal inside a binding groove and covers it with a protective lid to minimize its risk of aggregation. The next challenges are to figure out how SRP chooses which ribosomes to scan, and how it releases the targeting signal when it has delivered it to the membrane.

Structural determinants of nuclear export signal orientation in binding to exportin CRM1

Abstract: Many organisms keep their DNA within a structure inside their cells called the nucleus. Two layers of membrane surround the nucleus and keep the DNA separate from the rest of the cell's contents. Yet, proteins and other molecules can move in and out of the nucleus by passing through small pores in this nuclear membrane. To travel through these pores, larger molecules such as proteins rely on the assistance of transport receptors, including one called CRM1. This transport receptor helps to export hundreds of different proteins from the nucleus by recognizing a part of their structure called the ‘nuclear export signal’. Earlier work has shown that three different nuclear export signals interact with CRM1 in a similar ways by binding to a groove on its outer surface. But, there are several different types of nuclear export signal, and many are predicted to have three-dimensional structures that would seem to prevent them from binding to CRM1 in this way. As yet, it remains unknown how these diverse signals interact with this important transporter receptor. Protein crystallization is a technique that is used to visualize a protein's three-dimensional structure. Fung et al. have now used this approach to investigate how a particular class of nuclear export signals (called ‘class 3’) bind to CRM1. First, a modified form of CRM1 was crystallized once it had bound to a small fragment of protein that contains a class 3 nuclear export signal. The protein's molecular structure was then revealed by performing X-ray diffraction on the crystals. The results show, unexpectedly, that two different nuclear export signals in class 3 bind to the groove of CRM1 in the opposite direction to that reported previously. Additional biochemical and structural experiments then identified a particular feature or motif in the nuclear export signals that determines which way round they bind to CRM1. This discovery advances our understanding of how these signals work, which will allow us to more accurately identify new nuclear export signals from genome sequences. As more CRM1-binding nuclear export signals are discovered in the future, the experimental data sets used to train the computational programs that are currently used to locate these signals in genomic sequences will be diversified and improved.

Dynamics and Membrane Interactions of Protein Kinase C

Abstract: Protein kinase C (PKC) is a family of Ser/Thr kinases that regulate a multitude of cellular processes through participation in the phosphoinositide signaling pathway. Significant research efforts have been directed at understanding the structure, function, and regulatory modes of the enzyme since its discovery and identification as the first receptor for tumor-promoting phorbol esters. The activation of PKC involves a transition from the cytosolic autoinhibited latent form to the membrane-associated active form. The membrane recruitment step is accompanied by the conformational rearrangement of the enzyme, which relieves autoinhibitory interactions and thereby allows PKC to phosphorylate its targets. The multidomain structure and intrinsic flexibility of PKC present remarkable challenges and opportunities for the biophysical and structural biology studies of this class of enzymes and their interactions with membranes, the major focus of this Current Topic. I will highlight the recent advances in the field, outline the current challenges, and identify areas where biophysics and structural biology approaches can provide insight into the isoenzyme-specific regulation of PKC activity.

Rapid search for tertiary fragments reveals protein sequence–structure relationships

Abstract: Finding backbone substructures from the Protein Data Bank that match an arbitrary query structural motif, composed of multiple disjoint segments, is a problem of growing relevance in structure prediction and protein design. Although numerous protein structure search approaches have been proposed, methods that address this specific task without additional restrictions and on practical time scales are generally lacking. Here, we propose a solution, dubbed MASTER, that is both rapid, enabling searches over the Protein Data Bank in a matter of seconds, and provably correct, finding all matches below a user-specified root-mean-square deviation cutoff. We show that despite the potentially exponential time complexity of the problem, running times in practice are modest even for queries with many segments. The ability to explore naturally plausible structural and sequence variations around a given motif has the potential to synthesize its design principles in an automated manner; so we go on to illustrate the utility of MASTER to protein structural biology. We demonstrate its capacity to rapidly establish structure-sequence relationships, uncover the native designability landscapes of tertiary structural motifs, identify structural signatures of binding, and automatically rewire protein topologies. Given the broad utility of protein tertiary fragment searches, we hope that providing MASTER in an open-source format will enable novel advances in understanding, predicting, and designing protein structure.

Computational Prediction of RNA-Binding Proteins and Binding Sites

Abstract: Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%-8% of all proteins are RNA-binding proteins (RBPs). Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein-RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein-RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

A Molecular Pharmacologist's Guide to G Protein-Coupled Receptor Crystallography.

Abstract: G protein-coupled receptor (GPCR) structural biology has progressed dramatically in the last decade. There are now over 120 GPCR crystal structures deposited in the Protein Data Bank of 32 different receptors from families scattered across the phylogenetic tree, including class B, C, and Frizzled GPCRs. These structures have been obtained in combination with a wide variety of ligands and captured in a range of conformational states. This surge in structural knowledge has enlightened research into the molecular recognition of biologically active molecules, the mechanisms of receptor activation, the dynamics of functional selectivity, and fueled structure-based drug design efforts for GPCRs. Here we summarize the innovations in both protein engineering/molecular biology and crystallography techniques that have led to these advances in GPCR structural biology and discuss how they may influence the resulting structural models. We also provide a brief molecular pharmacologist's guide to GPCR X-ray crystallography, outlining some key aspects in the process of structure determination, with the goal to encourage noncrystallographers to interrogate structures at the molecular level. Finally, we show how chemogenomics approaches can be used to marry the wealth of existing receptor pharmacology data with the expanding repertoire of structures, providing a deeper understanding of the mechanistic details of GPCR function.

mtsslSuite: Probing Biomolecular Conformation by Spin-Labeling Studies.

Abstract: EPR long-range distance measurements on spin-labeled macromolecules have recently become a popular tool in structural biology. The method can be used to obtain coarse-grained structures of biomolecules, to track conformational changes and dynamics, to dock macromolecular complexes, or to localize spin centers within macromolecules using trilateration. Because the conformation of the spin label is usually unknown, it is often necessary to construct conformational models of the spin label on the macromolecules for data interpretation. For this purpose, so-called in silico spin-labeling approaches have been developed. In this chapter, a comprehensive summary of the mtsslSuite is provided, one of the in silico spin-labeling software packages. The package currently contains three programs: mtsslWizard, mtsslDock, and mtsslTrilaterate. Worked examples for the usage of all three programs during the planning- and interpretation stages of the EPR experiment are given.

The Ras-Membrane Interface: Isoform-specific Differences in The Catalytic Domain.

Abstract: The small GTPase Ras is mutated in about 20% of human cancers, primarily at active site amino acid residues G12, G13, and Q61. Thus, structural biology research has focused on the active site, impairment of GTP hydrolysis by oncogenic mutants, and characterization of protein-protein interactions in the effector lobe half of the protein. The C-terminal hypervariable region has increasingly gained attention due to its importance in H-Ras, N-Ras, and K-Ras differences in membrane association. A high-resolution molecular view of the Ras-membrane interaction involving the allosteric lobe of the catalytic domain has lagged behind, although evidence suggests that it contributes to isoform specificity. The allosteric lobe has recently gained interest for harboring potential sites for more selective targeting of this elusive "undruggable" protein. The present review reveals critical insight that isoform-specific differences appear prominently at these potentially targetable sites and integrates these differences with knowledge of Ras plasma membrane localization, with the intent to better understand the structure-function relationships needed to design isoform-specific Ras inhibitors.