Showing papers on "Structural biology published in 2004"
TL;DR: The recently determined structure of the anti-apoptotic Bcl-w protein, the protein was also found to have a hydrophobic groove on its surface capable of binding BH3-containing proteins and peptides, which may explain why it is found predominately in the cytoplasm prior to activation.
724 citations
TL;DR: One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels that map protein structures in NMR spectra of both weakly and completely aligned samples, and represent a convergence of solid-state and solution NMR approaches to structure determination.
Abstract: Much of postgenomic biochemistry and all of structural biology are based on the premise that the starting point for both understanding specific biochemical processes, such as affinity, reactivity, or transport, and surveying proteomes is determining the three-dimensional structures of proteins. The two well-established methods for structure determination are highly effective when applied to samples of soluble globular proteins and their complexes: witness the enormous growth of the Protein Data Bank.1 However, the vast majority of biological functions are carried out by proteins associated with supramolecular assemblies, whose samples are problematic for both X-ray crystallography and solution NMR spectroscopy, since they are generally difficult to crystallize and do not reorient rapidly even when soluble. The examples of proteins in supramolecular assemblies whose structures have been determined with atomic resolution are exceptional and highlight the importance of developing new methods of experimental protein structure determination. The essential goals of modern structural biology are to have the capability to select proteins for study based on their biological functions and to perform genuinely unbiased surveys of proteomes unfettered by considerations of the solubility, aggregation state, or other physical properties of the polypeptides. NMR spectroscopy has the potential to accomplish these goals, since it can be applied to molecules in all physical states, including the liquid crystalline environments provided by the lipids associated with membrane proteins.
Determining the atomic resolution structures of membrane proteins is of particular interest in contemporary structural biology.2 Helical membrane proteins constitute one-third of the expressed proteins encoded in a genome.3,4 Furthermore, many drugs have membrane-bound proteins as their receptors, and mutations in membrane proteins result in human diseases. They also provide daunting technical challenges for all methods of protein structure determination, including NMR spectroscopy.5
410 citations
TL;DR: The complexity of molecular recognition required to impose selectivity on substrate selection and topology of poly-ubiquitin chains is discussed, including the mechanisms of E1, E2 and E3.
Abstract: The role of protein ubiquitylation in the control of diverse cellular pathways has recently gained widespread attention. Ubiquitylation not only directs the targeted destruction of tagged proteins by the 26 S proteasome, but it also modulates protein activities, protein-protein interactions and subcellular localization. An understanding of the components involved in protein ubiquitylation (E1s, E2s and E3s) is essential to understand how specificity and regulation are conferred upon these pathways. Much of what we know about the catalytic mechanisms of protein ubiquitylation comes from structural studies of the proteins involved in this process. Indeed, structures of ubiquitin-activating enzymes (E1s) and ubiquitin-conjugating enzymes (E2s) have provided insight into their mechanistic details. E3s (ubiquitin ligases) contain most of the substrate specificity and regulatory elements required for protein ubiquitylation. Although several E3 structures are available, the specific mechanistic role of E3s is still unclear. This review will discuss the different types of ubiquitin signals and how they are generated. Recent advances in the field of protein ubiquitylation will be examined, including the mechanisms of E1, E2 and E3. In particular, we discuss the complexity of molecular recognition required to impose selectivity on substrate selection and topology of poly-ubiquitin chains.
323 citations
TL;DR: Structural analysis and mutagenesis show that binding of N‐Aha1 promotes a conformational switch in the middle‐segment catalytic loop of Hsp90 that releases the catalytic Arg 380 and enables its interaction with ATP in the N‐terminal nucleotide‐binding domain of the chaperone.
Abstract: Hsp90 is a molecular chaperone essential for the activation and assembly of many key eukaryotic signalling and regulatory proteins. Hsp90 is assisted and regulated by co-chaperones that participate in an ordered series of dynamic multiprotein complexes, linked to Hsp90 conformationally coupled ATPase cycle. The co-chaperones Aha1 and Hch1 bind to Hsp90 and stimulate its ATPase activity. Biochemical analysis shows that this activity is dependent on the N-terminal domain of Aha1, which interacts with the central segment of Hsp90. The structural basis for this interaction is revealed by the crystal structure of the N-terminal domain (1-153) of Aha1 (equivalent to the whole of Hch1) in complex with the middle segment of Hsp90 (273-530). Structural analysis and mutagenesis show that binding of N-Aha1 promotes a conformational switch in the middle-segment catalytic loop (370-390) of Hsp90 that releases the catalytic Arg 380 and enables its interaction with ATP in the N-terminal nucleotide-binding domain of the chaperone.
295 citations
TL;DR: Kinetic studies indicate that native-like secondary structure forms in one of the protein's loops in the folding transition state, but the backbone is less ordered elsewhere in the sequence, which provides an unusually detailed picture of the folding of a β-sheet protein.
Abstract: Backbone hydrogen bonds (H-bonds) are prominent features of protein structures; however, their role in protein folding remains controversial because they cannot be selectively perturbed by traditional methods of protein mutagenesis1,2,3. Here we have assessed the contribution of backbone H-bonds to the folding kinetics and thermodynamics of the PIN WW domain, a small β-sheet protein4, by individually replacing its backbone amides with esters. Amide-to-ester mutations site-specifically perturb backbone H-bonds in two ways: a H-bond donor is eliminated by replacing an amide NH with an ester oxygen, and a H-bond acceptor is weakened by replacing an amide carbonyl with an ester carbonyl5,6,7,8,9,10,11,12,13. We perturbed the 11 backbone H-bonds of the PIN WW domain by synthesizing 19 amide-to-ester mutants. Thermodynamic studies on these variants show that the protein is most destabilized when H-bonds that are enveloped by a hydrophobic cluster are perturbed. Kinetic studies indicate that native-like secondary structure forms in one of the protein's loops in the folding transition state, but the backbone is less ordered elsewhere in the sequence. Collectively, our results provide an unusually detailed picture of the folding of a β-sheet protein.
262 citations
TL;DR: A comprehensive, three-dimensional model of C1 is presented and insights into the mechanisms underlying its activation and the extraordinarily versatile recognition properties of its C1q subunit are provided.
230 citations
TL;DR: The purpose of this brief practical review was to take a snapshot of a field at the onset of its likely exponential growth phase, and to lay out the methods that have worked to date for obtaining membrane protein crystals suitable for structure determination by X-ray crystallography.
200 citations
TL;DR: High-resolution crystallographic and NMR structures of several proteins with bound fatty acids reveal the complete tertiary structure of the protein and molecular details of fatty acid-protein interactions.
141 citations
TL;DR: Mapping sequence conservation onto the molecular surface, combined with mutagenesis analysis, identified the eRF1 binding region, and revealed an essential function for the C terminus of eRF3, suggesting a novel mechanism for nucleotide exchange on e RF3 from that of other GTPases.
119 citations
TL;DR: The combination of these variables detect 78% of proteins with an HTH motif, which is a substantial improvement over previous work based purely on structural templates and is comparable to more complex methods of identifying DNA-binding proteins.
Abstract: Robust methods to detect DNA-binding proteins from structures of unknown function are important for structural biology. This paper describes a method for identifying such proteins that (i) have a solvent accessible structural motif necessary for DNAbinding and (ii) a positive electrostatic potential in the region of the binding region. We focus on three structural motifs: helix–turn-helix (HTH), helix– hairpin–helix (HhH) and helix–loop–helix (HLH). We find that the combination of these variables detect 78% of proteins with an HTH motif, which is a substantial improvement over previous work based purely on structural templates and is comparable to more complex methods of identifying DNA-binding proteins. Similar true positive fractions are achieved for the HhH and HLH motifs. We see evidence of wide evolutionary diversity for DNA-binding proteins with an HTH motif, and much smaller diversity for those with an HhH or HLH motif.
113 citations
TL;DR: Evidence from a number of laboratories suggests that AI‐2 may be used as a signal by diverse groups of bacteria, and might permit intergeneric signalling, and these two families of signalling systems are reviewed.
Abstract: Cell-cell communication via the production and detection of chemical signal molecules has been the focus of a great deal of research over the past decade. One class of chemical signals widely used by proteobacteria consists of N-acyl-homoserine lactones, which are synthesized by proteins related to LuxI of Vibrio fischeri and are detected by proteins related to the V. fischeri LuxR protein. A related marine bacterium, Vibrio harveyi, communicates using two chemical signals, one of which, autoinducer-2 (AI-2), is a furanone borate diester that is synthesized by the LuxS protein and detected by a periplasmic protein called LuxP. Evidence from a number of laboratories suggests that AI-2 may be used as a signal by diverse groups of bacteria, and might permit intergeneric signalling. These two families of signalling systems have been studied from the perspectives of physiology, ecology, biochemistry, and more recently, structural biology. Here, we review the biochemistry and structural biology of both acyl-homoserine-lactone-dependent and AI-2-dependent signalling systems.
TL;DR: Structural molecular biology and structural cell biology must merge into a single discipline, and structural biology must establish a lively intellectual complementarity with the nascent 'systems biology' of the cell.
Abstract: We know the basic principles of protein, RNA and DNA structure, and we have atomic coordinates of many proteins and RNAs. Structural biology must now expand the range of length and timescales on which we can represent the molecular reality of a cell. Structural molecular biology and structural cell biology must merge into a single discipline, and we must establish a lively intellectual complementarity with the nascent 'systems biology' of the cell.
TL;DR: The three-dimensional solution structure of the core globular domain of the C-terminal region of Ku86 (Ku86CTR(592-709)) has been determined using heteronuclear NMR spectroscopy and dynamical simulated annealing using structural restraints from nuclear Overhauser effectSpectroscopy, and scalar and residual dipolar couplings.
TL;DR: A more detailed structural understanding of the various Gram-negative secretion systems has emerged with the determination of the structures of type I and type IV secretion system components, and with the elucidation of the mechanism of fibre formation in the chaperone-usher pathway of pilus biogenesis as discussed by the authors.
TL;DR: The Consortium has established a pipeline for structural biology studies, automated modeling of ORF sequences using solved (template) structures, and a novel high-throughput approach (metallomics) to examining the metal binding to purified protein targets, and some of the experimental and bioinformatics efforts leading to structural annotation of proteins.
Abstract: Structural genomics has as its goal the provision of structural information for all possible ORF sequences through a combination of experimental and computational approaches. The access to genome sequences and cloning resources from an ever-widening array of organisms is driving high-throughput structural studies by the New York Structural Genomics Research Consortium. In this report, we outline the progress of the Consortium in establishing its pipeline for structural genomics, and some of the experimental and bioinformatics efforts leading to structural annotation of proteins. The Consortium has established a pipeline for structural biology studies, automated modeling of ORF sequences using solved (template) structures, and a novel high-throughput approach (metallomics) to examining the metal binding to purified protein targets. The Consortium has so far produced 493 purified proteins from >1077 expression vectors. A total of 95 have resulted in crystal structures, and 81 are deposited in the Protein Data Bank (PDB). Comparative modeling of these structures has generated >40,000 structural models. We also initiated a high-throughput metal analysis of the purified proteins; this has determined that 10%-15% of the targets contain a stoichiometric structural or catalytic transition metal atom. The progress of the structural genomics centers in the U.S. and around the world suggests that the goal of providing useful structural information on most all ORF domains will be realized. This projected resource will provide structural biology information important to understanding the function of most proteins of the cell.
TL;DR: The findings indicate the establishment of a biosynthetic route for producing large amounts of sumoylated recombinant proteins that will open up new avenues for studying the biochemical and structural aspects of the SUMO‐1 modification pathway.
TL;DR: The results of a double‐blind study are presented in which a new ab initio method was successfully used to predict the 3D structure of a protein designed through an experimental approach using binary patterned combinatorial libraries of de novo sequences.
Abstract: Ab initio structure prediction and de novo protein design are two problems at the forefront of research in the fields of structural biology and chemistry. The goal of ab initio structure prediction of proteins is to correctly characterize the 3D structure of a protein using only the amino acid sequence as input. De novo protein design involves the production of novel protein sequences that adopt a desired fold. In this work, the results of a double-blind study are presented in which a new ab initio method was successfully used to predict the 3D structure of a protein designed through an experimental approach using binary patterned combinatorial libraries of de novo sequences. The predicted structure, which was produced before the experimental structure was known and without consideration of the design goals, and the final NMR analysis both characterize this protein as a 4-helix bundle. The similarity of these structures is evidenced by both small RMSD values between the coordinates of the two structures and a detailed analysis of the helical packing.
TL;DR: An approach utilizing orientational restraints from residual dipolar couplings collected on solution NMR samples is presented, which can be useful in the design of new anti-bacterial agents that inhibit the biosynthesis of lipid A.
TL;DR: In this paper, yeast transformants were constructed that express the Gal4p deoxyribonucleic acid-binding domain (BD) fused to the 2 Hsp90 isoforms and the various Hsp 90 system cochaperones of yeast.
Abstract: The Hsp90 chaperone cycle involves sequential assembly of different Hsp90-containing multiprotein complexes, the accessory proteins ("cochaperones") that are associated with these complexes being exchanged as the cycle proceeds from its early to its late stages. To gain insight as to whether the 2-hybrid system could be used to probe the interactions of this Hsp90 system, yeast transformants were constructed that express the Gal4p deoxyribonucleic acid-binding domain (BD) fused to the 2 Hsp90 isoforms and the various Hsp90 system cochaperones of yeast. These "bait" fusions were then introduced by mating into other transformants expressing nearly all the 6000 proteins of yeast expressed as fusions to the Gal4p activation domain (AD). High throughput 2-hybrid screening revealed the ability of Hsp90 and Hsp90 system cochaperones to engage in stable interactions in vivo, both with each other and with the various other proteins of the yeast proteome. Consistent with the transience of most chaperone associations, interactions to Hsp90 itself were invariably weak and generally influenced by stress. Mutations within a Hsp90-BD bait fusion and an AD-Cdc37 "prey" fusion were used to provide in vivo confirmation of the in vitro data that shows that Cdc37p is interacting with the "relaxed" conformation of Hsp90 and also to provide indications that Cdc37p needs to be phosphorylated at its N-terminus for any appreciable interaction with Hsp90. A number of potentially novel cochaperone interactions were also identified, providing a framework for these to be analyzed further using other techniques.
TL;DR: The key to improved success with membrane protein structural elucidation is technology development, and the most efficient approach constitutes parallel studies on a large number of targets and evaluation of various systems for expression.
Abstract: Structural genomics, structure-based analysis of gene products, has so far mainly concentrated on soluble proteins because of their less demanding requirements for overexpression, purification and crystallisation compared to membrane proteins. This so-called "low-hanging fruit" approach has generated more than 25,000 structures deposited in databases. In contrast, the substantially more complex membrane proteins, in relation to all steps from overexpression to high-resolution structure determination, represent less than 1% of available crystal structures. This is in sharp contrast to the importance of this type of proteins, particularly G protein-coupled receptors (GPCRs), as today 60-70% of the current drug targets are based on membrane proteins. The key to improved success with membrane protein structural elucidation is technology development. The most efficient approach constitutes parallel studies on a large number of targets and evaluation of various systems for expression. Next, high throughput format solubilisation and refolding screening methods for a wide range of detergents and additives in numerous concentrations should be established. Today, several networks dealing with structural genomics approaches of membrane proteins have been initiated, among them the Membrane Protein Network (MePNet) programme that deals with the pharmaceutically important mammalian GPCRs. In MePNet, three overexpression systems have been employed for the evaluation of 101 GPCRs, which has generated large quantities of numerous recombinant GPCRs, compatible for structural biology applications.
TL;DR: Four distinct allosteric states of a mutant enzyme of Escherichia coli aspartate transcarbamoylase are trapped and their structures are determined by X-ray crystallography, shedding light on the nature and the order of internal structural rearrangements during the transition from the T to the R state.
TL;DR: Three-dimensional reconstruction of the infection device—one of the most complex multiprotein components— has been developed on the basis of cryo-electron microscopy images and will allow a better understanding of the regulation of protein folding, assembly of biological structures, and also mechanisms of functioning of the complex biological molecular machines.
Abstract: In studying bacteriophage T4--one of the basic models of molecular biology for several decades--there has come a Renaissance, and this virus is now actively used as object of structural biology. The structures of six proteins of the phage particle have recently been determined at atomic resolution by X-ray crystallography. Three-dimensional reconstruction of the infection device--one of the most complex multiprotein components--has been developed on the basis of cryo-electron microscopy images. The further study of bacteriophage T4 structure will allow a better understanding of the regulation of protein folding, assembly of biological structures, and also mechanisms of functioning of the complex biological molecular machines.
TL;DR: Two major complementary experimental techniques, X-ray crystallography and NMR spectroscopy, combined with recently developed high throughput methods have played a central role in structural proteomics research; however, an integration of these methodologies together with comparative modeling and electron microscopy would speed up the goal for completing a full dictionary of protein folding space in the near future.
Abstract: The discovery of biochemical and cellular functions of unannotated gene products begins with a database search of proteins with structure/sequence homologues based on known genes. Very recently, a number of frontier groups in structural biology proposed a new paradigm to predict biological functions of an unknown protein on the basis of its three-dimensional structure on a genomic scale. Structural proteomics (genomics), a research area for structure-based functional discovery, aims to complete the protein-folding universe of all gene products in a cell. It would lead us to a complete understanding of a living organism from protein structure. Two major complementary experimental techniques, X-ray crystallography and NMR spectroscopy, combined with recently developed high throughput methods have played a central role in structural proteomics research; however, an integration of these methodologies together with comparative modeling and electron microscopy would speed up the goal for completing a full dictionary of protein folding space in the near future.
Journal Article•
TL;DR: Of the 25,000 crystal structure entries deposited in public databases less than 1% are for membrane proteins; this gives a clear indication of the difficulties relating to structural biology and determination of this type of protein.
Abstract: Of the 25,000 crystal structure entries deposited in public databases less than 1% are for membrane proteins; this gives a clear indication of the difficulties relating to structural biology and determination of this type of protein. To date, structural genomics networks have mainly focused on soluble proteins, despite the fact that membrane proteins constitute 60 to 70% of current drug targets. Technological improvement on a broad basis is the key to higher success rates, but this is generally not possible unless large functional networks are established. Several structural genomics initiatives, concentrating partly or fully on membrane proteins, have been established during the last few years and the Membrane Protein Network (MePNet; BioXtal, Switzerland) focuses specifically on the pharmaceutically attractive G protein-coupled receptors (GPCRs). Within MePNet, over 100 full-length GPCRs have been subjected to overexpression in bacterial, yeast and mammalian cells, followed by purification and crystallization studies.
TL;DR: Comparison with the known crystal structures suggests that this 'shoe' shape requires a conformational change of the C-terminus of SCP2 to appropriately locate its peroxisomal targeting signal type-1 recognition motif into the binding pocket of the Pex5p receptor.
Abstract: Circular dichroism spectropolarimetry and X-ray scattering data, obtained using synchrotron radiation, can yield information about the secondary and tertiary structure of proteins in solution. These techniques have been used to analyse the architecture and shape of a complex of two proteins in solution. The crystal structures of two separate proteins, the C-terminal domain of Pex5p and SCP2, are available but their complex has not previously been structurally characterized. Circular dichroism spectropolarimetry indicated that complex formation requires little secondary structure rearrangement. X-ray scattering data fit an elongated irregular 'shoe'-shaped particle of the complex of the two proteins, with dimensions of the order of 30 A x 40 A x 90 A. Comparison with the known crystal structures suggests that this 'shoe' shape requires a conformational change of the C-terminus of SCP2 to appropriately locate its peroxisomal targeting signal type-1 recognition motif into the binding pocket of the Pex5p receptor. Implications of the combined use of synchrotron-based circular dichroism spectropolarimetry and X-ray scattering in structural biology and proteomics are discussed.
Journal Article•
TL;DR: The 2.85 Å crystal structure of CSL with a target DNA was reported in this article, where the Notch intracellular domain translocates to the nucleus after proteolytic release upon Notch extracellular engagement and there it displaces corepressors from DNA-bound CSL and recruits activators of Notch target genes.
Abstract: Notch signaling is a conserved pathway of communication between neighboring cells that results in cell fate specification, and CSL is the universal transcriptional effector of Notch signaling. The Notch intracellular domain translocates to the nucleus after proteolytic release upon Notch extracellular engagement, and there it displaces corepressors from DNA‐bound CSL and recruits activators of Notch target genes. Here we report the 2.85 Å crystal structure of CSL with a target DNA. CSL comprises three structurally integrated domains: its amino (NTD)‐ and carboxy (CTD)‐terminal domains are strikingly similar to those of Rel transcription factors, but a surprising beta‐trefoil domain (BTD) is inserted between them. CSL‐bound DNA is recognized specifically by conserved residues from NTD and BTD. A hydrophobic pocket on BTD is identified as the likely site of Notch interaction with CSL, which has functional implications for the mechanism of Notch signaling.
TL;DR: The gene-centric, reductionistical side of molecular biology is reviewed and seven problems are formulated, each indicating the insufficiency of the "central dogma".
TL;DR: A formal definition of a folding degree is introduced, capable of quantitative characterization, that enables ordering among protein chains based on their degree of folding, and it is demonstrated that the variation in the folding degree seen is not due to crystallization artifacts or experimental conditions.
Abstract: Comparing two or more protein structures with respect to their degree of folding is common practice in structural biology despite the fact that there is no scale for a folding degree. Here we intro...
TL;DR: It is shown that both extended duplex structures are stable, on the contrary, the crystallographic structures of the Lai and Mal kissing complexes are rapidly destabilized in aqueous environment.
Abstract: SL1 is a stem-loop RNA sequence from the genome of HIV-1 thought to be the initiation site for the dimerization of the retroviral genomic RNA. The aim of this study is to check the stability in solution of different experimental dimeric structures available in the literature. Two kinds of dimer have been evidenced: an extended duplex looking like a double helix with two internal bulges and a kissing complex in which the monomers with a stem/loop conformation are linked by intermolecular loop-loop interactions. Two divergent experimental structures of the kissing complex from the Lai isolate are reported in the literature, one obtained from NMR (Mujeeb et al., Nature Structural Biology, 1998, Vol. 5, pp. 432-436) and the other one from x-ray crystallography (Ennifar et al., Nature Structural Biology, 2001, Vol. 8, pp. 1064-1068). A crystallographic structure of the Mal isolate was also reported (Ennifar et al., Nature Structure Biology, 2001, Vol. 8, pp. 1064-1068). Concerning the extended duplex, a NMR structure is available for Lai (Girard et al., Journal of Biomolecular Structure and Dynamics, 1999, Vol. 16, pp. 1145-1157) and a crystallographic structure for Mal (Ennifar et al., Structure, 1999, Vol. 7, pp. 1439-1449). Using a molecular dynamics technique, all these experimental structures have been simulated in solution with explicit water and counterions. We show that both extended duplex structures are stable. On the contrary, the crystallographic structures of the Lai and Mal kissing complexes are rapidly destabilized in aqueous environment. Finally, the NMR structure of the Lai loop-loop kissing complex remains globally stable over a 20 ns MD simulation, although large rearrangements occur at the level of the stem/loop junctions that are flexible, as shown from free energy calculations. These results are compared to electrophoresis experiments on dimer formation.