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Showing papers on "Structural biology published in 2008"


Journal ArticleDOI
TL;DR: Glycosaminoglycans (GAGs) are important complex carbohydrates that participate in many biological processes through the regulation of their various protein partners, such as growth factors, anti-thrombin, cytokines and cell adhesion molecules as mentioned in this paper.
Abstract: Glycosaminoglycans (GAGs) are important complex carbohydrates that participate in many biological processes through the regulation of their various protein partners. Biochemical, structural biology and molecular modelling approaches have assisted in understanding the molecular basis of such interactions, creating an opportunity to capitalize on the large structural diversity of GAGs in the discovery of new drugs. The complexity of GAG–protein interactions is in part due to the conformational flexibility and underlying sulphation patterns of GAGs, the role of metal ions and the effect of pH on the affinity of binding. Current understanding of the structure of GAGs and their interactions with proteins is here reviewed: the basic structures and functions of GAGs and their proteoglycans, their clinical significance, the three-dimensional features of GAGs, their interactions with proteins and the molecular modelling of heparin binding sites and GAG–protein interactions. This review focuses on some key aspects of GAG structure–function relationships using classical examples that illustrate the specificity of GAG–protein interactions, such as growth factors, anti-thrombin, cytokines and cell adhesion molecules. New approaches to the development of GAG mimetics as possible new glycotherapeutics are also briefly covered.

798 citations


Journal ArticleDOI
TL;DR: In this review, insights are provided into the structural complexity of p53, the molecular mechanisms of its inactivation in cancer, and therapeutic strategies for the pharmacological rescue of p 53 function in tumors.
Abstract: The tumor suppressor protein p53 induces or represses the expression of a variety of target genes involved in cell cycle control, senescence, and apoptosis in response to oncogenic or other cellular stress signals. It exerts its function as guardian of the genome through an intricate interplay of independently folded and intrinsically disordered functional domains. In this review, we provide insights into the structural complexity of p53, the molecular mechanisms of its inactivation in cancer, and therapeutic strategies for the pharmacological rescue of p53 function in tumors. p53 emerges as a paradigm for a more general understanding of the structural organization of modular proteins and the effects of disease-causing mutations.

593 citations


Journal ArticleDOI
04 Dec 2008-Nature
TL;DR: The route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells is defined and it is proposed that ectodomain cleavage represents a strategy to restrict receptor activation to endophilic compartments and prevent TLRs from responding to self nucleic acids.
Abstract: Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells. The ectodomains of TLR9 and TLR7 are cleaved in the endolysosome, such that no full-length protein is detectable in the compartment where ligand is recognized. Notably, although both the full-length and cleaved forms of TLR9 are capable of binding ligand, only the processed form recruits MyD88 on activation, indicating that this truncated receptor, rather than the full-length form, is functional. Furthermore, conditions that prevent receptor proteolysis, including forced TLR9 surface localization, render the receptor non-functional. We propose that ectodomain cleavage represents a strategy to restrict receptor activation to endolysosomal compartments and prevent TLRs from responding to self nucleic acids.

562 citations


Journal ArticleDOI
TL;DR: Examination of the current three-dimensional structures of the outer membrane receptors, PBPs, and ABC transporters provides an overview of the structural biology of iron uptake in bacteria.

470 citations


Journal ArticleDOI
TL;DR: Technological advances are emerging for effective expression, solubilisation, purification and crystallisation of membrane proteins, which will lead to a rapid increase in the rate at which membrane protein structures are solved in the near future.

451 citations


Journal ArticleDOI
TL;DR: The biology of the Hsp90 molecular chaperone is reviewed, emphasizing recent progress in the understanding of structure-function relationships and the identification of new client proteins.
Abstract: The molecular chaperone Hsp90 (90 kDa heat-shock protein) is a remarkably versatile protein involved in the stress response and in normal homoeostatic control mechanisms. It interacts with 'client proteins', including protein kinases, transcription factors and others, and either facilitates their stabilization and activation or directs them for proteasomal degradation. By this means, Hsp90 displays a multifaceted ability to influence signal transduction, chromatin remodelling and epigenetic regulation, development and morphological evolution. Hsp90 operates as a dimer in a conformational cycle driven by ATP binding and hydrolysis at the N-terminus. The cycle is also regulated by a group of co-chaperones and accessory proteins. Here we review the biology of the Hsp90 molecular chaperone, emphasizing recent progress in our understanding of structure-function relationships and the identification of new client proteins. In addition we describe the exciting progress that has been made in the development of Hsp90 inhibitors, which are now showing promise in the clinic for cancer treatment. We also identify the gaps in our current understanding and highlight important topics for future research.

422 citations


Journal ArticleDOI
26 Jun 2008-Nature
TL;DR: An analysis of over 5,000 unique atomic structures shows that the quaternary structure of homomers is conserved in over 70% of protein pairs sharing as little as 30% sequence identity, and allows reliable prediction of evolution and assembly of a complex solely from its crystal structure.
Abstract: A homomer is formed by self-interacting copies of a protein unit. This is functionally important, as in allostery, and structurally crucial because mis-assembly of homomers is implicated in disease. Homomers are widespread, with 50-70% of proteins with a known quaternary state assembling into such structures. Despite their prevalence, their role in the evolution of cellular machinery and the potential for their use in the design of new molecular machines, little is known about the mechanisms that drive formation of homomers at the level of evolution and assembly in the cell. Here we present an analysis of over 5,000 unique atomic structures and show that the quaternary structure of homomers is conserved in over 70% of protein pairs sharing as little as 30% sequence identity. Where quaternary structure is not conserved among the members of a protein family, a detailed investigation revealed well-defined evolutionary pathways by which proteins transit between different quaternary structure types. Furthermore, we show by perturbing subunit interfaces within complexes and by mass spectrometry analysis, that the (dis)assembly pathway mimics the evolutionary pathway. These data represent a molecular analogy to Haeckel's evolutionary paradigm of embryonic development, where an intermediate in the assembly of a complex represents a form that appeared in its own evolutionary history. Our model of self-assembly allows reliable prediction of evolution and assembly of a complex solely from its crystal structure.

396 citations


Journal ArticleDOI
12 Jun 2008-Nature
TL;DR: The molecular mechanisms underlying the regulated protease and chaperone function of DegP from Escherichia coli are described and it is shown that binding of misfolded proteins transforms hexameric DegP into large, catalytically active 12-meric and 24-meric multimers.
Abstract: All organisms have to monitor the folding state of cellular proteins precisely. The heat-shock protein DegP is a protein quality control factor in the bacterial envelope that is involved in eliminating misfolded proteins and in the biogenesis of outer-membrane proteins. Here we describe the molecular mechanisms underlying the regulated protease and chaperone function of DegP from Escherichia coli. We show that binding of misfolded proteins transforms hexameric DegP into large, catalytically active 12-meric and 24-meric multimers. A structural analysis of these particles revealed that DegP represents a protein packaging device whose central compartment is adaptable to the size and concentration of substrate. Moreover, the inner cavity serves antagonistic functions. Whereas the encapsulation of folded protomers of outer-membrane proteins is protective and might allow safe transit through the periplasm, misfolded proteins are eliminated in the molecular reaction chamber. Oligomer reassembly and concomitant activation on substrate binding may also be critical in regulating other HtrA proteases implicated in protein-folding diseases.

360 citations


Journal ArticleDOI
TL;DR: A protocol for rapidly screening and purifying eukaryotic membrane proteins in the yeast Saccharomyces cerevisiae and details the purification of targets that pass the quality criteria is developed.
Abstract: It is often difficult to produce eukaryotic membrane proteins in large quantities, which is a major obstacle for analyzing their biochemical and structural features. To date, yeast has been the most successful heterologous overexpression system in producing eukaryotic membrane proteins for high-resolution structural studies. For this reason, we have developed a protocol for rapidly screening and purifying eukaryotic membrane proteins in the yeast Saccharomyces cerevisiae. Using this protocol, in 1 week many genes can be rapidly cloned by homologous recombination into a 2 micro GFP-fusion vector and their overexpression potential determined using whole-cell and in-gel fluorescence. The quality of the overproduced eukaryotic membrane protein-GFP fusions can then be evaluated over several days using confocal microscopy and fluorescence size-exclusion chromatography (FSEC). This protocol also details the purification of targets that pass our quality criteria, and can be scaled up for a large number of eukaryotic membrane proteins in either an academic, structural genomics or commercial environment.

298 citations


Journal ArticleDOI
TL;DR: An NMR method for quantifying millisecond time scale dynamics that involve transitions between different molecular conformations is described, and it is shown that the methodology facilitates detection of intermediates and other excited states that are populated at low levels that cannot be observed directly in spectra, so long as they exchange with the observable ground state of the protein on the milliseconds time scale.
Abstract: Biological function depends on molecular dynamics that lead to excursions from highly populated ground states to much less populated excited states. The low populations and the transient formation of such excited states render them invisible to the conventional methods of structural biology. Thus, while detailed pictures of ground-state structures of biomolecules have emerged over the years, largely through X-ray diffraction and solution nuclear magnetic resonance (NMR) spectroscopy studies, much less structural data has been accumulated on the conformational properties of the invisible excited states that are necessary to fully explain function. NMR spectroscopy is a powerful tool for studying conformational dynamics because it is sensitive to dynamics over a wide range of time scales, extending from picoseconds to seconds and because information is, in principle, available at nearly every position in the molecule. Here an NMR method for quantifying millisecond time scale dynamics that involve transitions between different molecular conformations is described. The basic experimental approach, termed relaxation dispersion NMR spectroscopy, is outlined to provide the reader with an intuitive feel for the technology. A variety of different experiments that probe conformational exchange at different sites in proteins are described, including a brief summary of data-fitting procedures to extract both the kinetic and thermodynamic properties of the exchange process and the structural features of the invisible excited states along the exchange pathway. It is shown that the methodology facilitates detection of intermediates and other excited states that are populated at low levels, 0.5% or higher, that cannot be observed directly in spectra, so long as they exchange with the observable ground state of the protein on the millisecond time scale. The power of the methodology is illustrated by a detailed application to the study of protein folding of the small modular SH3 domain. The kinetics and thermodynamics that describe the folding of this domain have been characterized through the effects of temperature, pressure, side-chain deuteration, and mutation, and the structural features of a low-populated folding intermediate have been assessed. Despite the fact that many previous studies have shown that SH3 domains fold via a two-state mechanism, the NMR methods presented unequivocally establish the presence of an on-pathway folding intermediate. The unique capabilities of NMR relaxation dispersion follow from the fact that large numbers of residues can be probed individually in a single experiment. By contrast, many other forms of spectroscopy monitor properties that are averaged over all residues in the molecule or that make use of only one or two reporters. The NMR methodology is not limited to protein folding, and applications to enzymatic catalysis, binding, and molecular recognition are beginning to emerge.

250 citations


Journal ArticleDOI
26 Jun 2008-Nature
TL;DR: The results indicate that most hydrogen-bond interactions in membrane proteins are only modestly stabilizing, which should be reflected in considerations of membrane protein folding, dynamics, design, evolution and function.
Abstract: Understanding the energetics of molecular interactions is fundamental to all of the central quests of structural biology including structure prediction and design, mapping evolutionary pathways, learning how mutations cause disease, drug design, and relating structure to function. Hydrogen-bonding is widely regarded as an important force in a membrane environment because of the low dielectric constant of membranes and a lack of competition from water. Indeed, polar residue substitutions are the most common disease-causing mutations in membrane proteins. Because of limited structural information and technical challenges, however, there have been few quantitative tests of hydrogen-bond strength in the context of large membrane proteins. Here we show, by using a double-mutant cycle analysis, that the average contribution of eight interhelical side-chain hydrogen-bonding interactions throughout bacteriorhodopsin is only 0.6 kcal mol(-1). In agreement with these experiments, we find that 4% of polar atoms in the non-polar core regions of membrane proteins have no hydrogen-bond partner and the lengths of buried hydrogen bonds in soluble proteins and membrane protein transmembrane regions are statistically identical. Our results indicate that most hydrogen-bond interactions in membrane proteins are only modestly stabilizing. Weak hydrogen-bonding should be reflected in considerations of membrane protein folding, dynamics, design, evolution and function.

Journal ArticleDOI
TL;DR: A detailed analysis of the crystallization conditions from 121 α‐helical membrane protein structures deposited in the Protein Data Bank is undertaken so that the success of different parameters can be easily compared for different membrane protein families.
Abstract: X-ray crystallography is currently the most successful method for determining the three-dimensional structure of membrane proteins. Nevertheless, growing the crystals required for this technique presents one of the major bottlenecks in this area of structural biology. This is especially true for the α-helical type membrane proteins that are of particular interest due to their medical relevance. To address this problem we have undertaken a detailed analysis of the crystallization conditions from 121 α-helical membrane protein structures deposited in the Protein Data Bank. This information has been analyzed so that the success of different parameters can be easily compared for different membrane protein families. Concurrent with this analysis, we also present the new sparse matrix crystallization screen MemGold.

Book
01 Jan 2008
TL;DR: The structure, function, and biogenesis of membrane lipids and proteins are examined, bioinformatics and computational approaches to membrane components are introduced, and the high-resolution structures that are giving new insights into the vital roles membranes play are discussed.
Abstract: Dedication Preface 1. Introduction 2. The diversity of membrane lipids 3. Tools for studying membrane components 4. Proteins in or at the bilayer 5. Bundles and barrels 6. Functions and families 7. Protein folding and biogenesis 8. Diffraction and simulation 9. Membrane enzymes 10. Membrane receptors 11. Transporters 12. Channels 13. Electron transport and energy transduction 14. In pursuit of complexity Appendix I. Abbreviations Appendix II. Single-letter codes for amino acids Index.

Journal ArticleDOI
TL;DR: The structure of an apo steroid receptor is presented that reveals a ligand-accessible channel allowing soaking of preformed crystals and the structural basis of NFkappaB-selective signaling through the estrogen receptor is defined, thus revealing a unique conformation of the receptor that allows selective suppression of inflammatory gene expression.
Abstract: Our understanding of how steroid hormones regulate physiological functions has been significantly advanced by structural biology approaches However, progress has been hampered by misfolding of the ligand binding domains in heterologous expression systems and by conformational flexibility that interferes with crystallization Here, we show that protein folding problems that are common to steroid hormone receptors are circumvented by mutations that stabilize well-characterized conformations of the receptor We use this approach to present the structure of an apo steroid receptor that reveals a ligand-accessible channel allowing soaking of preformed crystals Furthermore, crystallization of different pharmacological classes of compounds allowed us to define the structural basis of NFkappaB-selective signaling through the estrogen receptor, thus revealing a unique conformation of the receptor that allows selective suppression of inflammatory gene expression The ability to crystallize many receptor-ligand complexes with distinct pharmacophores allows one to define structural features of signaling specificity that would not be apparent in a single structure

Journal ArticleDOI
07 Oct 2008
TL;DR: Advances in mass spectroscopy have allowed very accurate and detailed analyses of lipid compositions as well as detection of the interactions of lipid biosynthetic proteins with one another and with proteins outside the lipid pathway, which has resulted in use of E. coli and S. enterica for discovery of new antimicrobials targeted to lipid synthesis and in deciphering the molecular actions of known antimicroBials.
Abstract: The pathways in Escherichia coli and (largely by analogy) S. enterica remain the paradigm of bacterial lipid synthetic pathways, although recently considerable diversity among bacteria in the specific areas of lipid synthesis has been demonstrated. The structural biology of the fatty acid synthetic proteins is essentially complete. However, the membrane-bound enzymes of phospholipid synthesis remain recalcitrant to structural analyses. Recent advances in genetic technology have allowed the essentialgenes of lipid synthesis to be tested with rigor, and as expected most genes are essential under standard growth conditions. Conditionally lethal mutants are available in numerous genes, which facilitates physiological analyses. The array of genetic constructs facilitates analysis of the functions of genes from other organisms. Advances in mass spectroscopy have allowed very accurate and detailed analyses of lipid compositions as well as detection of the interactions of lipid biosynthetic proteins with one another and with proteins outside the lipid pathway. The combination of these advances has resulted in use of E. coli and S. enterica for discovery of new antimicrobials targeted to lipid synthesis and in deciphering the molecular actions of known antimicrobials. Finally,roles for bacterial fatty acids other than as membrane lipid structural components have been uncovered. For example, fatty acid synthesis plays major roles in the synthesis of the essential enzyme cofactors, biotin and lipoic acid. Although other roles for bacterial fatty acids, such as synthesis of acyl-homoserine quorum-sensing molecules, are not native to E. coli introduction of the relevant gene(s) synthesis of these foreign molecules readily proceeds and the sophisticated tools available can used to decipher the mechanisms of synthesis of these molecules.

Journal ArticleDOI
TL;DR: In this article, the application of transient transfection technology for the expression of a variety of recombinant proteins has been described, including antibodies, dimeric proteins and tagged proteins of various complexity.
Abstract: Recent advances in genomics, proteomics, and structural biology raised the general need for significant amounts of pure recombinant protein (r-protein). Because of the difficulty in obtaining in some cases proper protein folding in bacteria, several methods have been established to obtain large amounts of r-proteins by transgene expression in mammalian cells. We have developed three nonviral DNA transfer protocols for suspension-adapted HEK-293 and CHO cells: (1) a calcium phosphate based method (Ca-Pi), (2) a calcium-mediated method called Calfection, and (3) a polyethylenimine-based method (PEI). The first two methods have already been scaled up to 14 L and 100 L for HEK-293 cells in bioreactors. The third method, entirely serum-free, has been successfully applied to both suspension-adapted CHO and HEK-293 cells. We describe here the application of this technology to the transient expression in suspension cultivated HEK-293 EBNA cells of some out of more than 20 secreted r-proteins, including antibodies, dimeric proteins, and tagged proteins of various complexity. Most of the proteins were expressed from different plasmid vectors within 5-10 days after the availability of the DNA. Transfections were successfully performed from the small scale (1 mL in 12-well microtiter plates) to the 2 L scale. The results reported made it possible to establish an optimized cell culture and transfection protocol that minimizes batch-to-batch variations in protein expression. The work presented here proves the applicability and robustness of transient transfection technology for the expression of a variety of recombinant proteins.

Journal ArticleDOI
TL;DR: It is shown that Sgt1 bridges the Hsp90 molecular chaperone system to the substrate‐specific arm of SCF ubiquitin ligase complexes, suggesting a role in SCF assembly and regulation, and providing multiple complementary routes for ubiquitination of Hsp 90 client proteins.
Abstract: Sgt1 is an adaptor protein implicated in a variety of processes, including formation of the kinetochore complex in yeast, and regulation of innate immunity systems in plants and animals. Sgt1 has been found to associate with SCF E3 ubiquitin ligases, the CBF3 kinetochore complex, plant R proteins and related animal Nod-like receptors, and with the Hsp90 molecular chaperone. We have determined the crystal structure of the core Hsp90-Sgt1 complex, revealing a distinct site of interaction on the Hsp90 N-terminal domain. Using the structure, we developed mutations in Sgt1 interfacial residues, which specifically abrogate interaction with Hsp90, and disrupt Sgt1-dependent functions in vivo, in plants and yeast. We show that Sgt1 bridges the Hsp90 molecular chaperone system to the substrate-specific arm of SCF ubiquitin ligase complexes, suggesting a role in SCF assembly and regulation, and providing multiple complementary routes for ubiquitination of Hsp90 client proteins.

Journal ArticleDOI
TL;DR: The energy landscape theory is proposed which provides a consistent framework to better understand how a protein folds rapidly and efficiently to the compact, biologically active structure.
Abstract: Protein folding, misfolding and aggregation, as well as the way misfolded and aggregated proteins affects cell viability are emerging as key themes in molecular and structural biology and in molecular medicine. Recent advances in the knowledge of the biophysical basis of protein folding have led to propose the energy landscape theory which provides a consistent framework to better understand how a protein folds rapidly and efficiently to the compact, biologically active structure. The increased knowledge on protein folding has highlighted its strict relation to protein misfolding and aggregation, either process being in close competition with the other, both relying on the same physicochemical basis. The theory has also provided information to better understand the structural and environmental factors affecting protein folding resulting in protein misfolding and aggregation into ordered or disordered polymeric assemblies. Among these, particular importance is given to the effects of surfaces. The latter, in some cases make possible rapid and efficient protein folding but most often recruit proteins/peptides increasing their local concentration thus favouring misfolding and accelerating the rate of nucleation. It is also emerging that surfaces can modify the path of protein misfolding and aggregation generating oligomers and polymers structurally different from those arising in the bulk solution and endowed with different physical properties and cytotoxicities.

Journal ArticleDOI
TL;DR: In this paper, the DOCK180-ELMO1 interaction was analyzed, and direct interaction interfaces were mapped to the N-terminal 200 amino acids of DOCK 180, and to the C-terminals of ELMO1, comprising the ELMO 1 PH domain.
Abstract: The mammalian DOCK180 protein belongs to an evolutionarily conserved protein family, which together with ELMO proteins, is essential for activation of Rac GTPase-dependent biological processes. Here, we have analyzed the DOCK180-ELMO1 interaction, and map direct interaction interfaces to the N-terminal 200 amino acids of DOCK180, and to the C-terminal 200 amino acids of ELMO1, comprising the ELMO1 PH domain. Structural and biochemical analysis of this PH domain reveals that it is incapable of phospholipid binding, but instead structurally resembles FERM domains. Moreover, the structure revealed an N-terminal amphiphatic alpha-helix, and point mutants of invariant hydrophobic residues in this helix disrupt ELMO1-DOCK180 complex formation. A secondary interaction between ELMO1 and DOCK180 is conferred by the DOCK180 SH3 domain and proline-rich motifs at the ELMO1 C-terminus. Mutation of both DOCK180-interaction sites on ELMO1 is required to disrupt the DOCK180-ELMO1 complex. Significantly, although this does not affect DOCK180 GEF activity toward Rac in vivo, Rac signaling is impaired, implying additional roles for ELMO in mediating intracellular Rac signaling.

Journal ArticleDOI
TL;DR: Surprisingly, recent data indicate that the deviant Walker ATPase proteins also form polymer-like structures, suggesting that, although the par families harbour what initially appeared to be structurally and functionally divergent proteins, they actually utilize similar mechanisms of DNA segregation.
Abstract: DNA segregation or partition is an essential process that ensures stable genome transmission. In prokaryotes, partition is best understood for plasmids, which serve as tractable model systems to study the mechanistic underpinnings of DNA segregation at a detailed atomic level owing to their simplicity. Specifically, plasmid partition requires only three elements: a centromere-like DNA site and two proteins: a motor protein, generally an ATPase, and a centromere-binding protein. In the first step of the partition process, multiple centromere-binding proteins bind co-operatively to the centromere, which typically consists of several tandem repeats, to form a higher-order nucleoprotein complex called the partition complex. The partition complex recruits the ATPase to form the segrosome and somehow activates the ATPase for DNA separation. Two major families of plasmid par systems have been delineated based on whether they utilize ATPase proteins with deviant Walker-type motifs or actin-like folds. In contrast, the centromere-binding proteins show little sequence homology even within a given family. Recent structural studies, however, have revealed that these centromere-binding proteins appear to belong to one of two major structural groups: those that employ helix-turn-helix DNA-binding motifs or those with ribbon-helix-helix DNA-binding domains. The first structure of a higher-order partition complex was recently revealed by the structure of pSK41 centromere-binding protein, ParR, bound to its centromere site. This structure showed that multiple ParR ribbon-helix-helix motifs bind symmetrically to the tandem centromere repeats to form a large superhelical structure with dimensions suitable for capture of the filaments formed by the actinlike ATPases. Surprisingly, recent data indicate that the deviant Walker ATPase proteins also form polymer-like structures, suggesting that, although the par families harbour what initially appeared to be structurally and functionally divergent proteins, they actually utilize similar mechanisms of DNA segregation. Thus, in the present review, the known Par protein and Par-protein complex structures are discussed with regard to their functions in DNA segregation in an attempt to begin to define, at a detailed atomic level, the molecular mechanisms involved in plasmid segregation.

Journal ArticleDOI
TL;DR: The opportunities of the recently developed designed ankyrin repeat protein (DARPin) technology for structural biology and the structural aspects of the DARPin-protein complexes are reviewed.

Journal ArticleDOI
TL;DR: A sparse matrix crystallization screen consisting of 48 lipidic-sponge phase conditions and how the screen may be manipulated by incorporating specific lipids such as cholesterol is demonstrated; this modification led to crystals being recovered from a bacterial photosynthetic core complex.

Journal ArticleDOI
TL;DR: The structural biology of uPAR will be reviewed with special emphasis on its multidomain composition and the interaction with its natural protein ligands, i.e. the serine protease uPA and the matrix protein vitronectin.
Abstract: The urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycolipid-anchored membrane glycoprotein, which is responsible for focalizing plasminogen activation to the cell surface through its high-affinity binding to the serine protease uPA. This tight interaction (KD less than 1 nM) is accomplished by an unusually large and hydrophobic binding cavity in uPAR that is created by a unique interdomain assembly involving all three homologous domains of the receptor. These domains belong to the Ly-6/uPAR (LU) protein domain family, which is defined by a consensus sequence predominantly based on disulfide connectivities, and they adopt a characteristic three-finger fold. Interestingly, the gene for uPAR is localized in a cluster of 6 homologous genes encoding proteins with multiple LU-domains. The structural biology of uPAR will be reviewed with special emphasis on its multidomain composition and the interaction with its natural protein ligands, i.e. the serine protease uPA and the matrix protein vitronectin.

Journal ArticleDOI
TL;DR: This Account has constructed synthetic building blocks called bis-amino acids that are then couple through pairs of amide bonds to create water-soluble, spiroladder oligomers (bis-peptides) with well-defined three-dimensional structures, which could facilitate multifunctional catalysis and molecular recognition and lead to nanoscale molecular devices.
Abstract: Proteins catalyze specific chemical reactions and carry out highly selective molecular recognition because they adopt well-defined three-dimensional structures and position chemically reactive functional groups in specific constellations. Proteins attain these well-defined structures through the complex process of protein folding. We seek to emulate these protein functions by constructing macromolecules that are easier to engineer by avoiding folding altogether. Toward that goal, we have developed an approach for the synthesis of macromolecules with programmable shapes. As described in this Account, we have constructed synthetic building blocks called bis-amino acids that we then couple through pairs of amide bonds to create water-soluble, spiroladder oligomers (bis-peptides) with well-defined three-dimensional structures. Bis-peptides use the conformational preferences of fused rings, stereochemistry, and strong covalent bonds to define their shape, unlike natural proteins and synthetic foldamers, which ...

Journal ArticleDOI
TL;DR: Recent advances in solution NMR allowing the study of a select set of peripheral and integral membrane proteins, including surface-binding proteins, transmembrane proteins including bacterial outer membrane beta-barrel proteins and oligomeric alpha-helical proteins are described.
Abstract: In living cells, membrane proteins are essential to signal transduction, nutrient use, and energy exchange between the cell and environment. Due to challenges in protein expression, purification and crystallization, deposition of membrane protein structures in the Protein Data Bank lags far behind existing structures for soluble proteins. This review describes recent advances in solution NMR allowing the study of a select set of peripheral and integral membrane proteins. Surface-binding proteins discussed include amphitropic proteins, antimicrobial and anticancer peptides, the HIV-1 gp41 peptides, human alpha-synuclein and apolipoproteins. Also discussed are transmembrane proteins including bacterial outer membrane beta-barrel proteins and oligomeric alpha-helical proteins. These structural studies are possible due to solubilization of the proteins in membrane-mimetic constructs such as detergent micelles and bicelles. In addition to protein dynamics, protein-lipid interactions such as those between arginines and phosphatidylglycerols have been detected directly by NMR. These examples illustrate the unique role solution NMR spectroscopy plays in structural biology of membrane proteins.

Journal ArticleDOI
TL;DR: Solid-state NMR spectroscopy, and more specifically rotational echo double-resonance (REDOR) experiments, are a powerful tool to unambiguously determine the register of constituent b strands within an amyloid fibril.
Abstract: The conversion of peptides or proteins from their soluble forms into amyloid fibrils is frequently associated with pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. Although amyloid fibrils and non-disease-associated amyloid-like fibrils can be formed by peptides and proteins that share no sequence identity, they display several common properties. One hallmark of amyloid and amyloid-like fibrils is their highly ordered organization into a laminated cross-b structure, in which the b strands run perpendicular to the long fibril axis. Another characteristic is that the same protein or peptide can form fibrils of different morphologies. It has been suggested that the structural and morphological variability of fibrils is likely to form the molecular basis for the phenomenon of strains, and may play a role in amyloid diseases. Although the basis of amyloid fibril polymorphism is not well understood, there is spectroscopic evidence that it is accompanied by specific changes in the conformation and packing of the individual polypeptide chains. It has been shown that fibril polymorphism can partially be controlled by variation of the growth conditions and that seeds from fibrils with a particular morphology can induce the sample to polymerize into fibrils of the same morphology. Elucidation of the factors that control the polymorphism of amyloid fibrils is therefore of major importance for understanding amyloid and prion diseases at the molecular level. Herein we address the molecular basis of polymorphism using the example of the de novo designed peptide ccb-p as a model system. Previous studies have shown that ccb-p (Ac-SIRELEARIRELELRIG-NH2) adopts a three-stranded a-helical coiled-coil structure in aqueous solution at low temperatures. However, the peptide forms amyloid-like fibrils spontaneously and irreversibly upon raising the temperature. When formed from a solution buffered at pH 7.3, the b strands within the fibrils were shown to assume a laminated cross-b conformation in which the extended b strands form antiparallel b sheets. The b strands were found to be shifted by three amino acid residues from an in-register arrangement (see Figure 1b,d). We denote this arrangement as “+ 3 out-of-register” (+ 3-or). It was suggested that, in addition to the clustering of hydrophobic residues, extensive salt-bridge formation between the charged side chains of Glu and Arg is a stabilizing factor for this arrangement. Therefore, the protonation of the Glu side chains at low pH was suspected to potentially change the register. As a result of its sensitivity to the inverse third power of the internuclear distance, solid-state NMR spectroscopy, and more specifically rotational echo double-resonance (REDOR) experiments, are a powerful tool to unambiguously determine the register of constituent b strands within an amyloid fibril. The distance between the carbonyl carbon atom and the amide nitrogen atom is close to 4.2 ? if two amino acid residues are hydrogen-bonded partners, and larger than 5.5 ? otherwise. If the samples investigated are selectively labeled with a single C and a single N atom and the distance measured is about 4.2 ?, the corresponding register is unambiguously established. To investigate the structure of ccb-p amyloid-like fibrils at the atomic level, differently labeled peptides were prepared. Of particular interest in the following are the results from two compounds: for compound I the N label was located on Ala7, and for compound II on Ile2. Both samples contained, in addition, a C label on the carbonyl of Leu14. Compound I will lead to a strong REDOR effect for a + 3-or antiparallel bsheet structure, known to form at pH 7.3, and sample II for a 2-or arrangement (see Figure 1), which will be shown to form at low pH. Figure 2 shows the REDOR dephasing on fibrils of compound I prepared from solution at different pH values. The dephasing increases with increasing pH in the range from 2.0 to 7.3, indicating an increase of the abundance of the + 3or fibril polymorph, which indeed is the dominant structure at neutral pH. Figure 3 shows the REDOR data obtained from samples of compound II. For samples prepared at low pH values, a strong REDOR effect is visible, attesting the existence of a 2-or structure. The solid lines in Figures 2 and 3 indicate the best fit of the data by a model in which the dephasing is described by a superposition of the + 3-or and the 2-or register dephasing curves. This approach is justified because compound I will [*] Dr. R. Verel, I. T. Tomka, C. Bertozzi, R. Cadalbert, Prof. B. H. Meier Physical Chemistry ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093, Zurich (Switzerland) Fax: (+41)44-632-1621 E-mail: beme@nmr.phys.chem.ethz.ch Homepage: http://www.ssnmr.ethz.ch

Journal ArticleDOI
TL;DR: This data generate two functional hypotheses for the AKAP18 central domain, which may act as a phosphoesterase, although it did not identify a substrate, or as an AMP sensor with the potential to couple intracellular AMP levels to PKA signalling events.

Journal ArticleDOI
TL;DR: Tah1 acheives ligand discrimination by favourably binding the methionine residue in the conserved MEEVD motif (Hsp90) and positively discriminating against the first valine residues in the VEEVD logo motif (Ssa1).
Abstract: Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein. TPR-domain proteins are involved in protein–protein interactions and a number have been characterized that interact either with Hsp70 or Hsp90, but a few can bind both chaperones. Independent studies suggest that Tah1 interacts with Hsp90, but whether it can also interact with Hsp70/Ssa1 has not been investigated. Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70. Our alignments suggest that there are three TPR-domain motifs in Tah1, which is consistent with the architecture of the TPR2b domain. In the present study we find that Tah1 is specific for Hsp90, and is able to bind tightly the yeast Hsp90, and the human Hsp90α and Hsp90β proteins, but not the yeast Hsp70 Ssa1 isoform. Tah1 acheives ligand discrimination by favourably binding the methionine residue in the conserved MEEVD motif (Hsp90) and positively discriminating against the first valine residue in the VEEVD motif (Ssa1). In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.

Journal ArticleDOI
Hong Yi1, Su Qiu1, Zhijian Cao1, Yingliang Wu1, Wenxin Li1 
15 Feb 2008-Proteins
TL;DR: Combined computation methods were employed to study the specific binding of maurotoxin (MTX) peptide to Kv1.2 channel to provide intrinsically valuable structural biology information to interpret binding affinities, specificities, and diversity of K+ channel‐nature toxin interactions.
Abstract: Inhibitory peptide-channel interactions have been utilized to characterize both channels and peptides; however, the fundamental basis for these interactions remains elusive. Here, combined computation methods were employed to study the specific binding of maurotoxin (MTX) peptide to Kv1.2 channel. In the first stage, numerous predicted complexes were generated by docking an ensemble of all 35 NMR conformations of MTX to Kv1.2 channel with ZDOCK program. Then the resulted complexes were clustered and classified into four main binding modes, based on experimental information and interaction energy analysis after the energy minimization and molecular dynamics (MD) simulations. By examining the stability of the plausible candidates through unrestrained MD simulations and calculation of the binding free energies, a final reasonable MTX-Kv1.2 complex was identified, with an overall high degree of correlation between the calculation and experiment on mutational effects. In the obtained complex structure model, MTX mainly used its beta-sheet domains to associate the channel mouth instead of the well-recognized functionally important S5P linkers of Kv1.2 channel. Structure analysis characterized that the most essential Tyr(32) residue of MTX was surrounded by a "pocket" formed by many nonpolar and polar residues of Kv1.2 channel, and revealed a pore-blocking Lys(23) and an important Lys(7) stabilized by strong electrostatic interactions with Asp(379) of Kv1.2. Furthermore, a stepwise structural arrangement for both ligand and receptor was found to accompany the tighter interaction of MTX into the target channel. The starting conformation of MTX, the side-chain conformation of the most important residue Tyr(32), and proper introduction of flexibility for candidate complexes were demonstrated to be considerably important factors for obtaining the final reasonable complex structure model. All these findings should not only be helpful for identifying more plausible K(+) channel-inhibitory peptide complex structures, but also provide intrinsically valuable structural biology information to interpret binding affinities, specificities, and diversity of K(+) channel-nature toxin interactions.

Journal ArticleDOI
TL;DR: This paper provides an overview of the recent progress in the structural biology of ncRNA-modification enzymes and identifies new genes encoding enzymes responsible for ncRNAs.