Showing papers on "Structural biology published in 2013"

Molecular signatures of G-protein-coupled receptors.

Abstract: G-protein-coupled receptors (GPCRs) are physiologically important membrane proteins that sense signalling molecules such as hormones and neurotransmitters, and are the targets of several prescribed drugs. Recent exciting developments are providing unprecedented insights into the structure and function of several medically important GPCRs. Here, through a systematic analysis of high-resolution GPCR structures, we uncover a conserved network of non-covalent contacts that defines the GPCR fold. Furthermore, our comparative analysis reveals characteristic features of ligand binding and conformational changes during receptor activation. A holistic understanding that integrates molecular and systems biology of GPCRs holds promise for new therapeutics and personalized medicine.

Chaperone machines for protein folding, unfolding and disaggregation

Abstract: Molecular chaperones are diverse families of multidomain proteins that have evolved to assist nascent proteins to reach their native fold, protect subunits from heat shock during the assembly of complexes, prevent protein aggregation or mediate targeted unfolding and disassembly. Their increased expression in response to stress is a key factor in the health of the cell and longevity of an organism. Unlike enzymes with their precise and finely tuned active sites, chaperones are heavy-duty molecular machines that operate on a wide range of substrates. The structural basis of their mechanism of action is being unravelled (in particular for the heat shock proteins HSP60, HSP70, HSP90 and HSP100) and typically involves massive displacements of 20–30 kDa domains over distances of 20–50 A and rotations of up to 100°.

Linkers in the structural biology of protein–protein interactions

Abstract: Linkers or spacers are short amino acid sequences created in nature to separate multiple domains in a single protein. Most of them are rigid and function to prohibit unwanted interactions between the discrete domains. However, Gly-rich linkers are flexible, connecting various domains in a single protein without interfering with the function of each domain. The advent of recombinant DNA technology made it possible to fuse two interacting partners with the introduction of artificial linkers. Often, independent proteins may not exist as stable or structured proteins until they interact with their binding partner, following which they gain stability and the essential structural elements. Gly-rich linkers have been proven useful for these types of unstable interactions, particularly where the interaction is weak and transient, by creating a covalent link between the proteins to form a stable protein-protein complex. Gly-rich linkers are also employed to form stable covalently linked dimers, and to connect two independent domains that create a ligand-binding site or recognition sequence. The lengths of linkers vary from 2 to 31 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked partners. Various structures of covalently linked protein complexes have been described using X-ray crystallography, nuclear magnetic resonance and cryo-electron microscopy techniques. In this review, we evaluate several structural studies where linkers have been used to improve protein quality, to produce stable protein-protein complexes, and to obtain protein dimers.

Protein rescue from aggregates by powerful molecular chaperone machines

Abstract: Protein quality control within the cell requires the interplay of many molecular chaperones and proteases. When this quality control system is disrupted, polypeptides follow pathways leading to misfolding, inactivity and aggregation. Among the repertoire of molecular chaperones are remarkable proteins that forcibly untangle protein aggregates, called disaggregases. Structural and biochemical studies have led to new insights into how these proteins collaborate with co-chaperones and utilize ATP to power protein disaggregation. Understanding how energy-dependent protein disaggregating machines function is universally important and clinically relevant, as protein aggregation is linked to medical conditions such as Alzheimer's disease, Parkinson's disease, amyloidosis and prion diseases.

Structural basis for protein phosphatase 1 regulation and specificity.

Abstract: The ubiquitous serine/threonine protein phosphatase 1 (PP1) regulates diverse, essential cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling. However, the free catalytic subunit of PP1, while an effective enzyme, lacks substrate specificity. Instead, it depends on a diverse set of regulatory proteins (≥ 200) to confer specificity towards distinct substrates. Here, we discuss recent advances in structural studies of PP1 holoenzyme complexes and summarize the new insights these studies have provided into the molecular basis of PP1 regulation and specificity.

Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein.

Abstract: Determination of structure of integral membrane proteins, especially in their native environment, is a formidable challenge in structural biology. Here we demonstrate that magic angle spinning solid-state NMR spectroscopy can be used to determine structures of membrane proteins reconstituted in synthetic lipids, an environment similar to the natural membrane. We combined a large number of experimentally determined interatomic distances and local torsional restraints to solve the structure of an oligomeric membrane protein of common seven-helical fold, Anabaena sensory rhodopsin (ASR). We determined the atomic resolution detail of the oligomerization interface of the ASR trimer, and the arrangement of helices, side chains and the retinal cofactor in the monomer.

Structural Biology of the Proteasome

Abstract: The proteasome refers to a collection of complexes centered on the 20S proteasome core particle (20S CP), a complex of 28 subunits that houses proteolytic sites in its hollow interior. Proteasomes are found in eukaryotes, archaea, and some eubacteria, and their activity is critical for many cellular pathways. Important recent advances include inhibitor binding studies and the structure of the immunoproteasome, whose specificity is altered by the incorporation of inducible catalytic subunits. The inherent repression of the 20S CP is relieved by the ATP-independent activators 11S and Blm10/PA200, whose structures reveal principles of proteasome mechanism. The structure of the ATP-dependent 19S regulatory particle, which mediates degradation of polyubiquitylated proteins, is being revealed by a combination of crystal or NMR structures of individual subunits and electron microscopy reconstruction of the intact complex. Other recent structural advances inform us about mechanisms of assembly and the role of conformational changes in the functional cycle.

Understanding nuclear receptor form and function using structural biology

Abstract: Nuclear receptors (NRs) are a major transcription factor family whose members selectively bind small-molecule lipophilic ligands and transduce those signals into specific changes in gene programs. For over two decades, structural biology efforts were focused exclusively on the individual ligand-binding domains (LBDs) or DNA-binding domains of NRs. These analyses revealed the basis for both ligand and DNA binding and also revealed receptor conformations representing both the activated and repressed states. Additionally, crystallographic studies explained how NR LBD surfaces recognize discrete portions of transcriptional coregulators. The many structural snapshots of LBDs have also guided the development of synthetic ligands with therapeutic potential. Yet, the exclusive structural focus on isolated NR domains has made it difficult to conceptualize how all the NR polypeptide segments are coordinated physically and functionally in the context of receptor quaternary architectures. Newly emerged crystal structures of the peroxisome proliferator-activated receptor-γ-retinoid X receptor α (PPARγ-RXRα) heterodimer and hepatocyte nuclear factor (HNF)-4α homodimer have recently revealed the higher order organizations of these receptor complexes on DNA, as well as the complexity and uniqueness of their domain-domain interfaces. These emerging structural advances promise to better explain how signals in one domain can be allosterically transmitted to distal receptor domains, also providing much better frameworks for guiding future drug discovery efforts.

Structural biology of the Bcl-2 family and its mimicry by viral proteins

Abstract: Intrinsic apoptosis in mammals is regulated by protein–protein interactions among the B-cell lymphoma-2 (Bcl-2) family. The sequences, structures and binding specificity between pro-survival Bcl-2 proteins and their pro-apoptotic Bcl-2 homology 3 motif only (BH3-only) protein antagonists are now well understood. In contrast, our understanding of the mode of action of Bax and Bak, the two necessary proteins for apoptosis is incomplete. Bax and Bak are isostructural with pro-survival Bcl-2 proteins and also interact with BH3-only proteins, albeit weakly. Two sites have been identified; the in-groove interaction analogous to the pro-survival BH3-only interaction and a site on the opposite molecular face. Interaction of Bax or Bak with activator BH3-only proteins and mitochondrial membranes triggers a series of ill-defined conformational changes initiating their oligomerization and mitochondrial outer membrane permeabilization. Many actions of the mammalian pro-survival Bcl-2 family are mimicked by viruses. By expressing proteins mimicking mammalian pro-survival Bcl-2 family proteins, viruses neutralize death-inducing members of the Bcl-2 family and evade host cell apoptosis during replication. Remarkably, structural elements are preserved in viral Bcl-2 proteins even though there is in many cases little discernible sequence conservation with their mammalian counterparts. Some viral Bcl-2 proteins are dimeric, but they have distinct structures to those observed for mammalian Bcl-2 proteins. Furthermore, viral Bcl-2 proteins modulate innate immune responses regulated by NF-κB through an interface separate from the canonical BH3-binding groove. Our increasing structural understanding of the viral Bcl-2 proteins is leading to new insights in the cellular Bcl-2 network by exploring potential alternate functional modes in the cellular context. We compare the cellular and viral Bcl-2 proteins and discuss how alterations in their structure, sequence and binding specificity lead to differences in behavior, and together with the intrinsic structural plasticity in the Bcl-2 fold enable exquisite control over critical cellular signaling pathways.

Overexpression of membrane proteins in mammalian cells for structural studies

Abstract: The number of structures of integral membrane proteins from higher eukaryotes is steadily increasing due to a number of innovative protein engineering and crystallization strategies devised over the last few years. However, it is sobering to reflect that these structures represent only a tiny proportion of the total number of membrane proteins encoded by a mammalian genome. In addition, the structures determined to date are of the most tractable membrane proteins, i.e., those that are expressed functionally and to high levels in yeast or in insect cells using the baculovirus expression system. However, some membrane proteins that are expressed inefficiently in these systems can be produced at sufficiently high levels in mammalian cells to allow structure determination. Mammalian expression systems are an under-used resource in structural biology and represent an effective way to produce fully functional membrane proteins for structural studies. This review will discuss examples of vertebrate membrane protein overexpression in mammalian cells using a variety of viral, constitutive or inducible expression systems.

Architecture and regulation of HtrA-family proteins involved in protein quality control and stress response.

Abstract: Protein quality control is vital for all living cells and sophisticated molecular mechanisms have evolved to prevent the excessive accumulation of unfolded proteins. High-temperature requirement A (HtrA) proteases have been identified as important ATP-independent quality-control factors in most species. HtrA proteins harbor a serine-protease domain and at least one peptide-binding PDZ domain to ensure efficient removal of misfolded or damaged proteins. One distinctive property of HtrAs is their ability to assemble into complex oligomers. Whereas all examined HtrAs are capable of forming pyramidal 3-mers, higher-order complexes consisting of up to 24 molecules have been reported. Tight control of chaperone and protease function is of pivotal importance in preventing deleterious HtrA-protease activity. In recent years, structural biology provided detailed insights into the molecular basis of the regulatory mechanisms, which include unique intramolecular allosteric signaling cascades and the dynamic switching of oligomeric states of HtrA proteins. Based on these results, functional models for many family members have been developed. The HtrA protein family represents a remarkable example of how structural and functional diversity is attained from the assembly of simple molecular building blocks.

Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation.

Abstract: Complement component C1, the complex that initiates the classical pathway of complement activation, is a 790-kDa assembly formed from the target-recognition subcomponent C1q and the modular proteases C1r and C1s. The proteases are elongated tetramers that become more compact when they bind to the collagen-like domains of C1q. Here, we describe a series of structures that reveal how the subcomponents associate to form C1. A complex between C1s and a collagen-like peptide containing the C1r/C1s-binding motif of C1q shows that the collagen binds to a shallow groove via a critical lysine side chain that contacts Ca2+-coordinating residues. The data explain the Ca2+-dependent binding mechanism, which is conserved in C1r and also in mannan-binding lectin-associated serine proteases, the serine proteases of the lectin pathway activation complexes. In an accompanying structure, C1s forms a compact ring-shaped tetramer featuring a unique head-to-tail interaction at its center that replicates the likely arrangement of C1r/C1s polypeptides in the C1 complex. Additional structures reveal how C1s polypeptides are positioned to enable activation by C1r and interaction with the substrate C4 inside the cage-like assembly formed by the collagenous stems of C1q. Together with previously determined structures of C1r fragments, the results reported here provide a structural basis for understanding the early steps of complement activation via the classical pathway.

High-resolution network biology: connecting sequence with function

Abstract: Proteins are not monolithic entities; rather, they can contain multiple domains that mediate distinct interactions, and their functionality can be regulated through post-translational modifications at multiple distinct sites. Traditionally, network biology has ignored such properties of proteins and has instead examined either the physical interactions of whole proteins or the consequences of removing entire genes. In this Review, we discuss experimental and computational methods to increase the resolution of protein-protein, genetic and drug-gene interaction studies to the domain and residue levels. Such work will be crucial for using interaction networks to connect sequence and structural information, and to understand the biological consequences of disease-associated mutations, which will hopefully lead to more effective therapeutic strategies.

A Gel-Encapsulated Bioreactor System for NMR Studies of Protein–Protein Interactions in Living Mammalian Cells†

Abstract: In-cell NMR spectroscopy is a method used to observe isotopically labeled molecules within living cells. The first in-cell NMR experiment was performed with an E. coli overexpressing a N-labeled protein. For the first application of the in-cell NMR method with eukaryotic cells, isotopically labeled target proteins were introduced, by microinjection, into Xenopus laevis oocytes. Recently, Inomata et al. reported a novel in-cell NMR method utilizing a cell-penetrating tag, which opens the way for the application of in-cell NMR spectroscopy to mammalian cells. Our group also reported an in-cell NMR method for mammalian cells; we used a pore-forming toxin, streptolysin O (SLO), to introduce target proteins by diffusion. By using these methods, protein–drug interactions and intracellular post-translational modifications, such as phosphorylation and acetylation, were successfully detected in vivo. However, the major limitation of the in-cell NMR experiments is the occurrence of cell death during the NMR measurement. As the suspension contains a high density of cells (> 1 10 mL), nutrient depletion occurs rapidly in the anaerobic environment within the NMR tube, thus causing the deterioration of conditions and resulting in cell death during NMR measurements. 9] Therefore, the observation of the NMR signals from proteins in living cells is hampered by the sharp NMR signals derived from the isotopically labeled proteins leaked from the cells. Therefore, in-cell NMR experiments for eukaryotic cells currently have limited applications, such as for obtaining a single NMR spectrum measured within a very short time. Although sparse sampling methods have been utilized to shorten the time required to acquire multidimensional NMR spectra, many existing in vitro NMR experiments that are used to provide information regarding dynamics and protein interactions take several hours to perform. To suppress the cell death during NMR measurements over a longer period of time, we utilized a bioreactor to perfuse the cells in the NMR sample tube. In the early in vivo NMR studies, the perfused cells were entrapped in an agarose gel in a 10 mm NMR tube to sequester them in the signal detection coil, and the P signals of intracellular ATP metabolites in yeast and mammalian cells were observed for a period of several days. 13] In this study, we developed a bioreactor for in-cell NMR spectroscopy (Figure 1); in this

Structure-Based DNA-Targeting Strategies with Small Molecule Ligands for Drug Discovery

Abstract: Nucleic acids are the molecular targets of many clinical anticancer drugs. However, compared with proteins, nucleic acids have traditionally attracted much less attention as drug targets in structure-based drug design, partially because limited structural information of nucleic acids complexed with potential drugs is available. Over the past several years, enormous progresses in nucleic acid crystallization, heavy-atom derivatization, phasing, and structural biology have been made. Many complicated nucleic acid structures have been determined, providing new insights into the molecular functions and interactions of nucleic acids, especially DNAs complexed with small molecule ligands. Thus, opportunities have been created to further discover nucleic acid-targeting drugs for disease treatments. This review focuses on the structure studies of DNAs complexed with small molecule ligands for discovering lead compounds, drug candidates, and/or therapeutics.

Structure determination of membrane proteins in their native phospholipid bilayer environment by rotationally aligned solid-state NMR spectroscopy.

Abstract: One of the most important topics in experimental structural biology is determining the structures of membrane proteins. These structures represent one-third of all of the information expressed from a genome, distinguished by their locations within the phospholipid bilayer of cells, organelles, or enveloped viruses. Their highly hydrophobic nature and insolubility in aqueous media means that they require an amphipathic environment. They have unique functions in transport, catalysis, channel formation, and signaling. Researchers are particularly interested in G-protein coupled receptors (GPCRs) because they modulate many biological processes, and about half of the approximately 800 of these proteins within the human genome are or can be turned into drug receptors that affect a wide range of diseases. Because of experimental difficulties, researchers have studied membrane proteins using a wide variety of artificial media that mimic membranes, such as mixed organic solvents or detergents. More sophisticated mimics include bilayer discs (bicelles) and the lipid cubic phase (LCP), but both of these contain a very large detergent component, which can disrupt the stability and function of membrane proteins. To have confidence in the resulting structures and their biological functions and to avoid disrupting these delicate proteins, the structures of membrane proteins should be determined in their native environment of liquid crystalline phospholipid bilayers under physiological conditions. This Account describes a recently developed general method for determining the structures of unmodified membrane proteins in phospholipid bilayers by solid-state NMR spectroscopy. Because it relies on the natural, rapid rotational diffusion of these proteins about the bilayer normal, this method is referred to as rotationally aligned (RA) solid-state NMR. This technique elaborates on oriented sample (OS) solid-state NMR, its complementary predecessor. These methods exploit the power of solid-state NMR, which enables researchers to obtain well-resolved spectra from "immobile" membrane proteins in phospholipid bilayers, to separate and measure frequencies that reflect orientations with respect to the bilayer normal, and to make complementary distance measurements. The determination of the structures of several membrane proteins, most prominently the chemokine receptor CXCR1, a 350-residue GPCR, has demonstrated this approach.

Solution structure of the two RNA recognition motifs of hnRNP A1 using segmental isotope labeling: how the relative orientation between RRMs influences the nucleic acid binding topology.

Abstract: Human hnRNP A1 is a multi-functional protein involved in many aspects of nucleic-acid processing such as alternative splicing, micro-RNA biogenesis, nucleo-cytoplasmic mRNA transport and telomere biogenesis and maintenance. The N-terminal region of hnRNP A1, also named unwinding protein 1 (UP1), is composed of two closely related RNA recognition motifs (RRM), and is followed by a C-terminal glycine rich region. Although crystal structures of UP1 revealed inter-domain interactions between RRM1 and RRM2 in both the free and bound form of UP1, these interactions have never been established in solution. Moreover, the relative orientation of hnRNP A1 RRMs is different in the free and bound crystal structures of UP1, raising the question of the biological significance of this domain movement. In the present study, we have used NMR spectroscopy in combination with segmental isotope labeling techniques to carefully analyze the inter-RRM contacts present in solution and subsequently determine the structure of UP1 in solution. Our data unambiguously demonstrate that hnRNP A1 RRMs interact in solution, and surprisingly, the relative orientation of the two RRMs observed in solution is different from the one found in the crystal structure of free UP1 and rather resembles the one observed in the nucleic-acid bound form of the protein. This strongly supports the idea that the two RRMs of hnRNP A1 have a single defined relative orientation which is the conformation previously observed in the bound form and now observed in solution using NMR. It is likely that the conformation in the crystal structure of the free form is a less stable form induced by crystal contacts. Importantly, the relative orientation of the RRMs in proteins containing multiple-RRMs strongly influences the RNA binding topologies that are practically accessible to these proteins. Indeed, RRM domains are asymmetric binding platforms contacting single-stranded nucleic acids in a single defined orientation. Therefore, the path of the nucleic acid molecule on the multiple RRM domains is strongly dependent on whether the RRMs are interacting with each other. The different nucleic acid recognition modes by multiple-RRM domains are briefly reviewed and analyzed on the basis of the current structural information.

DNA Nanotubes for NMR Structure Determination of Membrane Proteins

Abstract: Finding a way to determine the structures of integral membrane proteins using solution nuclear magnetic resonance (NMR) spectroscopy has proved to be challenging. A residual-dipolar-coupling-based refinement approach can be used to resolve the structure of membrane proteins up to 40 kDa in size, but to do this you need a weak-alignment medium that is detergent-resistant and it has thus far been difficult to obtain such a medium suitable for weak alignment of membrane proteins. We describe here a protocol for robust, large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into dilute liquid crystals for application as weak-alignment media in solution NMR structure determination of membrane proteins in detergent micelles. The DNA nanotubes are heterodimers of 400-nm-long six-helix bundles, each self-assembled from a M13-based p7308 scaffold strand and >170 short oligonucleotide staple strands. Compatibility with proteins bearing considerable positive charge as well as modulation of molecular alignment, toward collection of linearly independent restraints, can be introduced by reducing the negative charge of DNA nanotubes using counter ions and small DNA-binding molecules. This detergent-resistant liquid-crystal medium offers a number of properties conducive for membrane protein alignment, including high-yield production, thermal stability, buffer compatibility and structural programmability. Production of sufficient nanotubes for four or five NMR experiments can be completed in 1 week by a single individual.