Showing papers in "Nature Structural & Molecular Biology in 2001"
TL;DR: The 2.7 Å structure of wheat HSP16.9, a member of the small heat shock proteins (sHSPs), indicates how its α-crystallin domain and flanking extensions assemble into a dodecameric double disk, and provides a model by which members of the sHSP protein family bind unfolded substrates.
Abstract: The 2.7 A structure of wheat HSP16.9, a member of the small heat shock proteins (sHSPs), indicates how its alpha-crystallin domain and flanking extensions assemble into a dodecameric double disk. The folding of the monomer and assembly of the oligomer are mutually interdependent, involving strand exchange, helix swapping, loose knots and hinged extensions. In support of the chaperone mechanism, the substrate-bound dimers, in temperature-dependent equilibrium with higher assembly forms, have unfolded N-terminal arms and exposed conserved hydrophobic binding sites on the alpha-crystallin domain. The structure also provides a model by which members of the sHSP protein family bind unfolded substrates, which are involved in a variety of neurodegenerative diseases and cataract formation.
710 citations
TL;DR: The N-terminal domain of the influenza hemagglutinin is deduced to be a kinked, predominantly helical amphipathic structure that could perturb lipid packing and facilitate lipid mixing between juxtaposed membranes.
Abstract: The N-terminal domain of the influenza hemagglutinin (HA) is the only portion of the molecule that inserts deeply into membranes of infected cells to mediate the viral and the host cell membrane fusion. This domain constitutes an autonomous folding unit in the membrane, causes hemolysis of red blood cells and catalyzes lipid exchange between juxtaposed membranes in a pH-dependent manner. Combining NMR structures determined at pHs 7.4 and 5 with EPR distance constraints, we have deduced the structures of the N-terminal domain of HA in the lipid bilayer. At both pHs, the domain is a kinked, predominantly helical amphipathic structure. At the fusogenic pH 5, however, the domain has a sharper bend, an additional 3(10)-helix and a twist, resulting in the repositioning of Glu 15 and Asp 19 relative to that at the nonfusogenic pH 7.4. Rotation of these charged residues out of the membrane plane creates a hydrophobic pocket that allows a deeper insertion of the fusion domain into the core of the lipid bilayer. Such an insertion mode could perturb lipid packing and facilitate lipid mixing between juxtaposed membranes.
448 citations
TL;DR: The discovery of RNAi has changed the understanding of how cells guard their genomes, led to the development of new strategies for blocking gene function, and may yet yield RNA-based drugs to treat human disease.
Abstract: The term RNA interference (RNAi) describes the use of double-stranded RNA to target specific mRNAs for degradation, thereby silencing their expression. RNAi is one manifestation of a broad class of RNA silencing phenomena that are found in plants, animals and fungi. The discovery of RNAi has changed our understanding of how cells guard their genomes, led to the development of new strategies for blocking gene function, and may yet yield RNA-based drugs to treat human disease.
447 citations
TL;DR: The crystal structure of H. pylori urease is determined and provides a novel example of a molecular assembly adapted for acid resistance that, together with the low Km value of the enzyme, is likely to enable the organism to inhabit the hostile niche.
Abstract: Helicobacter pylori, an etiologic agent in a variety of gastroduodenal diseases, produces a large amount of urease, which is believed to neutralize gastric acid by producing ammonia for the survival of the bacteria. Up to 30% of the enzyme associates with the surface of intact cells upon lysis of neighboring bacteria. The role of the enzyme at the extracellular location has been a subject of controversy because the purified enzyme is irreversibly inactivated below pH 5. We have determined the crystal structure of H. pylori urease, which has a 1.1 MDa spherical assembly of 12 catalytic units with an outer diameter of approximately 160 A. Under physiologically relevant conditions, the activity of the enzyme remains unaffected down to pH 3. Activity assays under different conditions indicated that the cluster of the 12 active sites on the supramolecular assembly may be critical for the survival of the enzyme at low pH. The structure provides a novel example of a molecular assembly adapted for acid resistance that, together with the low Km value of the enzyme, is likely to enable the organism to inhabit the hostile niche.
438 citations
TL;DR: The BRCA1–BARD1 structure provides a model for its ubiquitin ligase activity, illustrates how the BRCa1 RING domain can be involved in associations with multiple protein partners and provides a framework for understanding cancer-causing mutations at the molecular level.
Abstract: The RING domain of the breast and ovarian cancer tumor suppressor BRCA1 interacts with multiple cognate proteins, including the RING protein BARD1. Proper function of the BRCA1 RING domain is critical, as evidenced by the many cancer-predisposing mutations found within this domain. We present the solution structure of the heterodimer formed between the RING domains of BRCA1 and BARD1. Comparison with the RING homodimer of the V(D)J recombination-activating protein RAG1 reveals the structural diversity of complexes formed by interactions between different RING domains. The BRCA1-BARD1 structure provides a model for its ubiquitin ligase activity, illustrates how the BRCA1 RING domain can be involved in associations with multiple protein partners and provides a framework for understanding cancer-causing mutations at the molecular level.
424 citations
TL;DR: This is the first reported crystal structure of a member of this newly-described MarR protein family, which shows MarR as a dimer with each subunit containing a winged-helix DNA binding motif.
Abstract: MarR is a regulator of multiple antibiotic resistance in Escherichia coli. It is the prototypical member of the MarR family of regulatory proteins found in bacteria and archaea that play important roles in the development of antibiotic resistance, a global health problem. Here we describe the crystal structure of the MarR protein, determined at a resolution of 2.3 A. This is the first reported crystal structure of a member of this newly-described protein family. The structure shows MarR as a dimer with each subunit containing a winged-helix DNA binding motif.
401 citations
TL;DR: The three-dimensional fold of the 19 kDa (177 residues) transmembrane domain of the outer membrane protein A of Escherichia coli in dodecylphosphocholine (DPC) micelles in solution is determined using heteronuclear NMR.
Abstract: We have determined the three-dimensional fold of the 19 kDa (177 residues) transmembrane domain of the outer membrane protein A of Escherichia coli in dodecylphosphocholine (DPC) micelles in solution using heteronuclear NMR. The structure consists of an eight-stranded beta-barrel connected by tight turns on the periplasmic side and larger mobile loops on the extracellular side. The solution structure of the barrel in DPC micelles is similar to that in n-octyltetraoxyethylene (C(8)E(4)) micelles determined by X-ray diffraction. Moreover, data from NMR dynamic experiments reveal a gradient of conformational flexibility in the structure that may contribute to the membrane channel function of this protein.
392 citations
TL;DR: The crystal structures of Newcastle disease virus HN alone and in complex with either an inhibitor or with the β-anomer of sialic acid reveal a typical neuraminidase active site within a β-propeller fold.
Abstract: Paramyxoviruses are the main cause of respiratory disease in children. One of two viral surface glycoproteins, the hemagglutinin-neuraminidase (HN), has several functions in addition to being the major surface antigen that induces neutralizing antibodies. Here we present the crystal structures of Newcastle disease virus HN alone and in complex with either an inhibitor or with the β-anomer of sialic acid. The inhibitor complex reveals a typical neuraminidase active site within a β-propeller fold. Comparison of the structures of the two complexes reveal differences in the active site, suggesting that the catalytic site is activated by a conformational switch. This site may provide both sialic acid binding and hydrolysis functions since there is no evidence for a second sialic acid binding site in HN. Evidence for a single site with dual functions is examined and supported by mutagenesis studies. The structure provides the basis for the structure-based design of inhibitors for a range of paramyxovirus-induced diseases.
386 citations
TL;DR: Novel relaxation dispersion NMR techniques are used to kinetically and thermodynamically characterize a transition between a highly populated ground state conformation and an excited state that is 2.0 kcal mol−1 higher in free energy.
Abstract: Protein structure is inherently dynamic, with function often predicated on excursions from low to higher energy conformations. For example, X-ray studies of a cavity mutant of T4 lysozyme, L99A, show that the cavity is sterically inaccessible to ligand, yet the protein is able to bind substituted benzenes rapidly. We have used novel relaxation dispersion NMR techniques to kinetically and thermodynamically characterize a transition between a highly populated (97%, 25 degrees C) ground state conformation and an excited state that is 2.0 kcal mol(-1) higher in free energy. A temperature-dependent study of the rates of interconversion between ground and excited states allows the separation of the free energy change into enthalpic (Delta H = 7.1 kcal mol(-1)) and entropic (T Delta S = 5.1 kcal mol(-1), 25 degrees C) components. The residues involved cluster about the cavity, providing evidence that the excited state facilitates ligand entry.
386 citations
TL;DR: The crystal structure of human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of cysteine proteases, reveals how the protein refolds to produce very tight two-fold symmetric dimers while retaining the secondary structure of the monomeric form.
Abstract: The crystal structure of human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of cysteine proteases, reveals how the protein refolds to produce very tight two-fold symmetric dimers while retaining the secondary structure of the monomeric form. The dimerization occurs through three-dimensional domain swapping, a mechanism for forming oligomeric proteins. The reconstituted monomer-like domains are similar to chicken cystatin except for one inhibitory loop that unfolds to form the 'open interface' of the dimer. The structure explains the tendency of human cystatin C to dimerize and suggests a mechanism for its aggregation in the brain arteries of elderly people with amyloid angiopathy. A more severe 'conformational disease' is associated with the L68Q mutant of human cystatin C, which causes massive amyloidosis, cerebral hemorrhage and death in young adults. The structure of the three-dimensional domain-swapped dimers shows how the L68Q mutation destabilizes the monomers and makes the partially unfolded intermediate less unstable. Higher aggregates may arise through the three-dimensional domain-swapping mechanism occurring in an open-ended fashion in which partially unfolded molecules are linked into infinite chains.
378 citations
TL;DR: The structure of IGF1RK reveals how the enzyme recognizes peptides containing hydrophobic residues at the P+1 and P+3 positions and how autophosphorylation stabilizes the activation loop in a conformation that facilitates catalysis.
Abstract: The insulin-like growth factor 1 (IGF1) receptor is closely related to the insulin receptor. However, the unique biological functions of IGF1 receptor make it a target for therapeutic intervention in human cancer. Using its isolated tyrosine kinase domain, we show that the IGF1 receptor is regulated by intermolecular autophosphorylation at three sites within the kinase activation loop. Steady-state kinetic analyses of the isolated phosphorylated forms of the IGF1 receptor kinase (IGF1RK) reveal that each autophosphorylation event increases enzyme turnover number and decreases Km for ATP and peptide. We have determined the 2.1 A-resolution crystal structure of the tris-phosphorylated form of IGF1RK in complex with an ATP analog and a specific peptide substrate. The structure of IGF1RK reveals how the enzyme recognizes peptides containing hydrophobic residues at the P+1 and P+3 positions and how autophosphorylation stabilizes the activation loop in a conformation that facilitates catalysis. Although the nucleotide binding cleft is conserved between IGF1RK and the insulin receptor kinase, sequence differences in the nearby interlobe linker could potentially be exploited for anticancer drug design.
TL;DR: The 1.7 Å crystal structure of the 323 amino acid catalytic core of human SIRT2, a homolog of yeast Sir2, reveals an NAD-binding domain, which is a variant of the Rossmann fold, and a smaller domain composed of a helical module and a zinc-binding module.
Abstract: Sir2 is an NAD-dependent histone deacetylase that mediates transcriptional silencing at mating-type loci, telomeres and ribosomal gene clusters, and has a critical role in the determination of life span in yeast and Caenorhabditis elegans. The 1.7 A crystal structure of the 323 amino acid catalytic core of human SIRT2, a homolog of yeast Sir2, reveals an NAD-binding domain, which is a variant of the Rossmann fold, and a smaller domain composed of a helical module and a zinc-binding module. A conserved large groove at the interface of the two domains is the likely site of catalysis based on mutagenesis. Intersecting this large groove, there is a pocket formed by the helical module. The pocket is lined with hydrophobic residues conserved within each of the five Sir2 classes, suggesting that it is a class-specific protein-binding site.
TL;DR: The structure explains the unique primed phosphorylation mechanism of G SK3β and how GSK3β relies on a phosphoserine in the substrate for the alignment of the β- and α-helical domains.
Abstract: GSK3beta was identified as the kinase that phosphorylates glycogen synthase but is now known to be involved in multiple signaling pathways. GSK3beta prefers prior phosphorylation of its substrates. We present the structure of unphosphorylated GSK3beta at 2.7 A. The orientation of the two domains and positioning of the activation loop of GSK3beta are similar to those observed in activated kinases. A phosphate ion held by Arg 96, Arg 180 and Lys 205 occupies the same position as the phosphate group of the phosphothreonine in activated p38gamma, CDK2 or ERK2. A loop from a neighboring molecule in the crystal occupies a portion of the substrate binding groove. The structure explains the unique primed phosphorylation mechanism of GSK3beta and how GSK3beta relies on a phosphoserine in the substrate for the alignment of the beta- and alpha-helical domains.
TL;DR: This work evaluates different strategies for optimizing information return on effort and concludes that the strategy that maximizes structural coverage requires about seven times fewer structure determinations compared with the strategy in which targets are selected at random.
Abstract: Structural genomics has the goal of obtaining useful, three-dimensional models of all proteins by a combination of experimental structure determination and comparative model building. We evaluate different strategies for optimizing information return on effort. The strategy that maximizes structural coverage requires about seven times fewer structure determinations compared with the strategy in which targets are selected at random. With a choice of reasonable model quality and the goal of 90% coverage, we extrapolate the estimate of the total effort of structural genomics. It would take ∼16,000 carefully selected structure determinations to construct useful atomic models for the vast majority of all proteins. In practice, unless there is global coordination of target selection, the total effort will likely increase by a factor of three. The task can be accomplished within a decade provided that selection of targets is highly coordinated and significant funding is available.
TL;DR: The 2 Å crystal structures of the complex of LgtC with manganese and UDP 2-deoxy-2-fluoro-galactose in the presence and absence of the acceptor sugar analog 4′-deoxylactose give valuable insights into the unique catalytic mechanism and, as the first structure of a glycosyltransferase in complex with both the donor and acceptor sugars, provide a starting point for inhibitor design.
Abstract: Many bacterial pathogens express lipooligosaccharides that mimic human cell surface glycoconjugates, enabling them to attach to host receptors and to evade the immune response. In Neisseria meningitidis, the galactosyltransferase LgtC catalyzes a key step in the biosynthesis of lipooligosaccharide structure by transferring alpha-d-galactose from UDP-galactose to a terminal lactose. The product retains the configuration of the donor sugar glycosidic bond; LgtC is thus a retaining glycosyltranferase. We report the 2 A crystal structures of the complex of LgtC with manganese and UDP 2-deoxy-2-fluoro-galactose (a donor sugar analog) in the presence and absence of the acceptor sugar analog 4'-deoxylactose. The structures, together with results from site-directed mutagenesis and kinetic analysis, give valuable insights into the unique catalytic mechanism and, as the first structure of a glycosyltransferase in complex with both the donor and acceptor sugars, provide a starting point for inhibitor design.
TL;DR: The three-dimensional structure of the Tudor domain of human SMN is determined and it is shown that a conserved negatively charged surface that is shown to interact with the C-terminal Arg and Gly-rich tails of Sm proteins.
Abstract: Spinal muscular atrophy (SMA) is a common motor neuron disease that results from mutations in the Survival of Motor Neuron (SMN) gene. The SMN protein plays a crucial role in the assembly of spliceosomal uridine-rich small nuclear ribonucleoprotein (U snRNP) complexes via binding to the spliceosomal Sm core proteins. SMN contains a central Tudor domain that facilitates the SMN-Sm protein interaction. A SMA-causing point mutation (E134K) within the SMN Tudor domain prevents Sm binding. Here, we have determined the three-dimensional structure of the Tudor domain of human SMN. The structure exhibits a conserved negatively charged surface that is shown to interact with the C-terminal Arg and Gly-rich tails of Sm proteins. The E134K mutation does not disrupt the Tudor structure but affects the charge distribution within this binding site. An intriguing structural similarity between the Tudor domain and the Sm proteins suggests the presence of an additional binding interface that resembles that in hetero-oligomeric complexes of Sm proteins. Our data provide a structural basis for a molecular defect underlying SMA.
TL;DR: The difference between the crystal and solution structures of Ca2+–calmodulin indicates considerable backbone plasticity within the domains of calmodulin, which is key to their ability to bind a wide range of targets.
Abstract: The solution structure of Ca2+-ligated calmodulin is determined from residual dipolar couplings measured in a liquid crystalline medium and from a large number of heteronuclear J couplings for defining side chains. Although the C-terminal domain solution structure is similar to the X-ray crystal structure, the EF hands of the N-terminal domain are considerably less open. The substantial differences in interhelical angles correspond to negligible changes in short interproton distances and, therefore, cannot be identified by comparison of NOEs and X-ray data. NOE analysis, however, excludes a two-state equilibrium in which the closed apo conformation is partially populated in the Ca2+-ligated state. The difference between the crystal and solution structures of Ca2+–calmodulin indicates considerable backbone plasticity within the domains of calmodulin, which is key to their ability to bind a wide range of targets. In contrast, the vast majority of side chains making up the target binding surface are locked into the same χ1 rotameric states as in complexes with target peptide.
TL;DR: It is shown that Rad23 and Ddi1 interact directly with ubiquitin and that this interaction is dependent on their UBA domains, providing a possible mechanism for UBA-dependent cell cycle control.
Abstract: Rad23 is a highly conserved protein involved in nucleotide excision repair (NER) that associates with the proteasome via its N-terminus. Its C-terminal ubiquitin-associated (UBA) domain is evolutionarily conserved from yeast to humans. However, the cellular function of UBA domains is not completely understood. Recently, RAD23 and DDI1, both DNA damage-inducible genes encoding proteins with UBA domains, were implicated genetically in Pds1-dependent mitotic control in yeast. The UBA domains of RAD23 and DDI1 are required for these interactions. Timely degradation of Pds1 via the ubiquitin/proteasome pathway allows anaphase onset and is crucial for chromosome maintenance. Here, we show that Rad23 and Ddi1 interact directly with ubiquitin and that this interaction is dependent on their UBA domains, providing a possible mechanism for UBA-dependent cell cycle control. Moreover, we show that a hydrophobic surface on the UBA domain, which from structural work had been predicted to be a protein–protein interaction interface, is indeed required for ubiquitin binding. By demonstrating that UBA domains interact with ubiquitin, we have provided the first indication of a cellular function for the UBA domain.
TL;DR: In this article, the mitochondrial proteins cytochrome c and Smac/DIABLO are released into the cytosol, where they synergistically activate caspases by activating Apaf-1 and relieving the apoptotic inhibition by IAPs.
Abstract: Mitochondria-mediated apoptosis plays a central role in animal development and tissue homeostasis, and its alteration results in a range of malignant disorders including cancer. Upon apoptotic stimuli, the mitochondrial proteins cytochrome c and Smac/DIABLO are released into the cytosol, where they synergistically activate caspases by activating Apaf-1 and relieving the apoptotic inhibition by IAPs. Recent biochemical and structural studies reveal a molecular basis for these important events and identify an evolutionarily conserved mechanism of apoptosis from fruit flies to mammals.
TL;DR: Proteins imported into the mitochondrial matrix are synthesized in the cytosol with an N-terminal presequence and are translocated through hetero-oligomeric translocase complexes of the outer and inner mitochondrial membranes, where purified Tim23 forms a hydrophilic, ∼13–24 Å wide channel characteristic of the mitochondrial presequence translocases.
Abstract: Proteins imported into the mitochondrial matrix are synthesized in the cytosol with an N-terminal presequence and are translocated through hetero-oligomeric translocase complexes of the outer and inner mitochondrial membranes. The channel across the inner membrane is formed by the presequence translocase, which consists of roughly six distinct subunits; however, it is not known which subunits actually form the channel. Here we report that purified Tim23 forms a hydrophilic, approximately 13-24 A wide channel characteristic of the mitochondrial presequence translocase. The Tim23 channel is cation selective and activated by a membrane potential and presequences. The channel is formed by the C-terminal domain of Tim23 alone, whereas the N-terminal domain is required for selectivity and a high-affinity presequence interaction. Thus, Tim23 forms a voltage-sensitive high-conductance channel with specificity for mitochondrial presequences.
TL;DR: Current insights into the nature of protein dynamics and their potential influence on protein structure, stability and function are reviewed and particular emphasis is placed on the potential of fast side chain motion to report on the residual conformational entropy of proteins.
Abstract: Recent developments in solution NMR methods have allowed for an unprecedented view of protein dynamics. Current insights into the nature of protein dynamics and their potential influence on protein structure, stability and function are reviewed. Particular emphasis is placed on the potential of fast side chain motion to report on the residual conformational entropy of proteins and how this entropy can enter into both the thermodynamic and kinetic aspects of protein function.
TL;DR: The first crystal structure of the complex between maize leaf Fd and Fd-NADP+ oxidoreductase (FNR) is reported, suggesting that this type of molecular communication not only determines the optimal orientation of the two proteins for electron transfer, but also contributes to the modulation of the enzymatic properties of FNR.
Abstract: All oxygenic photosynthetically derived reducing equivalents are utilized by combinations of a single multifuctional electron carrier protein, ferredoxin (Fd), and several Fd-dependent oxidoreductases. We report the first crystal structure of the complex between maize leaf Fd and Fd-NADP(+) oxidoreductase (FNR). The redox centers in the complex--the 2Fe-2S cluster of Fd and flavin adenine dinucleotide (FAD) of FNR--are in close proximity; the shortest distance is 6.0 A. The intermolecular interactions in the complex are mainly electrostatic, occurring through salt bridges, and the interface near the prosthetic groups is hydrophobic. NMR experiments on the complex in solution confirmed the FNR recognition sites on Fd that are identified in the crystal structure. Interestingly, the structures of Fd and FNR in the complex and in the free state differ in several ways. For example, in the active site of FNR, Fd binding induces the formation of a new hydrogen bond between side chains of Glu 312 and Ser 96 of FNR. We propose that this type of molecular communication not only determines the optimal orientation of the two proteins for electron transfer, but also contributes to the modulation of the enzymatic properties of FNR.
TL;DR: The high resolution crystal structure of LTA4H in complex with the competitive inhibitor bestatin reveals a protein folded into three domains that together create a deep cleft harboring the catalytic Zn2+ site, which provides detailed insight into mechanisms of catalysis.
Abstract: Leukotriene (LT) A4 hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc enzyme that catalyzes the biosynthesis of LTB4, a potent lipid chemoattractant involved in inflammation, immune responses, host defense against infection, and PAF-induced shock. The high resolution crystal structure of LTA4H in complex with the competitive inhibitor bestatin reveals a protein folded into three domains that together create a deep cleft harboring the catalytic Zn2+ site. A bent and narrow pocket, shaped to accommodate the substrate LTA4, constitutes a highly confined binding region that can be targeted in the design of specific anti-inflammatory agents. Moreover, the structure of the catalytic domain is very similar to that of thermolysin and provides detailed insight into mechanisms of catalysis, in particular the chemical strategy for the unique epoxide hydrolase reaction that generates LTB4.
TL;DR: These two OMTs constitute the first plant methyltransferases to be structurally characterized and reveal a novel oligomerization domain and the molecular determinants for substrate selection and this work provides a structural basis for understanding the substrate specificity of the diverse family of plant O MTs and facilitates the engineering of novel activities in this extensive class of natural product biosynthetic enzymes.
Abstract: Chalcone O-methyltransferase (ChOMT) and isoflavone O-methyltransferase (IOMT) are S-adenosyl-l-methionine (SAM) dependent plant natural product methyltransferases involved in secondary metabolism in Medicago sativa (alfalfa). Here we report the crystal structure of ChOMT in complex with the product S-adenosyl-l-homocysteine and the substrate isoliquiritigenin (4,2′,4′-trihydroxychalcone) refined to 1.8 A as well as the crystal structure of IOMT in complex with the products S-adenosyl-l-homocysteine and isoformononetin (4′-hydroxy-7-methoxyisoflavone) refined to 1.4 A. These two OMTs constitute the first plant methyltransferases to be structurally characterized and reveal a novel oligomerization domain and the molecular determinants for substrate selection. As such, this work provides a structural basis for understanding the substrate specificity of the diverse family of plant OMTs and facilitates the engineering of novel activities in this extensive class of natural product biosynthetic enzymes.
TL;DR: It is shown on the basis of chemical shifts that dimeric p53 both containing and lacking the C-terminal domain are identical in conformation and that theC-terminus does not interact with other p53 domains, ruling out an allosteric model for the regulation of p53.
Abstract: p53 is a nuclear phosphoprotein that regulates cellular fate after genotoxic stress through its role as a transcriptional regulator of genes involved in cell cycle control and apoptosis. The C-terminal region of p53 is known to negatively regulate sequence specific DNA-binding of p53; modifications to the C-terminus relieve this inhibition. Two models have been proposed to explain this latency: (i) an allosteric model in which the C-terminal domain interacts with another domain of p53 or (ii) a competitive model in which the C-terminal and the core domains compete for DNA binding. We have characterized latent and active forms of dimeric p53 using gel mobility shift assays and NMR spectroscopy. We show on the basis of chemical shifts that dimeric p53 both containing and lacking the C-terminal domain are identical in conformation and that the C-terminus does not interact with other p53 domains. Similarly, NMR spectra of isolated core and tetramerization domains confirm a modular p53 architecture. The data presented here rule out an allosteric model for the regulation of p53.
TL;DR: Repeated stretching and relaxing of the fiber in the absence of egg extract showed that the loss of histone octamers was irreversible, comparable to forces reported for RNA- and DNA-polymerases.
Abstract: Single chromatin fibers were assembled directly in the flow cell of an optical tweezers setup. A single λ phage DNA molecule, suspended between two polystyrene beads, was exposed to a Xenopus laevis egg extract, leading to chromatin assembly with concomitant apparent shortening of the DNA molecule. Assembly was force-dependent and could not take place at forces exceeding 10 pN. The assembled single chromatin fiber was subjected to stretching by controlled movement of one of the beads with the force generated in the molecule continuously monitored with the second bead trapped in the optical trap. The force displayed discrete, sudden drops upon fiber stretching, reflecting discrete opening events in fiber structure. These opening events were quantized at increments in fiber length of ∼65 nm and are attributed to unwrapping of the DNA from around individual histone octamers. Repeated stretching and relaxing of the fiber in the absence of egg extract showed that the loss of histone octamers was irreversible. The forces measured for individual nucleosome disruptions are in the range of 20–40 pN, comparable to forces reported for RNA- and DNA-polymerases.
TL;DR: Striking conformational rearrangements are observed in both the chaperone and target enzyme upon complex formation, and the functionally essential C-terminal domain of yCCS is well positioned to play a key role in the metal ion transfer mechanism.
Abstract: The copper chaperone for superoxide dismutase (CCS) activates the eukaryotic antioxidant enzyme copper, zinc superoxide dismutase (SOD1). The 2.9 A resolution structure of yeast SOD1 complexed with yeast CCS (yCCS) reveals that SOD1 interacts with its metallochaperone to form a complex comprising one monomer of each protein. The heterodimer interface is remarkably similar to the SOD1 and yCCS homodimer interfaces. Striking conformational rearrangements are observed in both the chaperone and target enzyme upon complex formation, and the functionally essential C-terminal domain of yCCS is well positioned to play a key role in the metal ion transfer mechanism. This domain is linked to SOD1 by an intermolecular disulfide bond that may facilitate or regulate copper delivery.
TL;DR: A tripeptide 'IGF' in E. coli ClpX that is essential for ClpP recognition is identified and mapping of the IGF loop onto a homolog of known structure suggests a model forClpX–ClpP docking.
Abstract: The Clp/Hsp100 ATPases are hexameric protein machines that catalyze the unfolding, disassembly and disaggregation of specific protein substrates in bacteria, plants and animals. Many family members also interact with peptidases to form ATP-dependent proteases. In Escherichia coli, for instance, the ClpXP protease is assembled from the ClpX ATPase and the ClpP peptidase. Here, we have used multiple sequence alignments to identify a tripeptide 'IGF' in E. coli ClpX that is essential for ClpP recognition. Mutations in this IGF sequence, which appears to be part of a surface loop, disrupt ClpXP complex formation and prevent protease function but have no effect on other ClpX activities. Homologous tripeptides are found only in a subset of Clp/Hsp100 ATPases and are a good predictor of family members that have a ClpP partner. Mapping of the IGF loop onto a homolog of known structure suggests a model for ClpX-ClpP docking.
TL;DR: The structure provides a basis to predict the structural consequences of uncharacterized BRCA1 mutations and may represent a general mode of interaction between homologous domains within proteins that interact to regulate the cellular response to DNA damage.
Abstract: The C-terminal BRCT region of BRCA1 is essential for its DNA repair, transcriptional regulation and tumor suppressor functions. Here we determine the crystal structure of the BRCT domain of human BRCA1 at 2.5 A resolution. The domain contains two BRCT repeats that adopt similar structures and are packed together in a head-to-tail arrangement. Cancer-causing missense mutations occur at the interface between the two repeats and destabilize the structure. The manner by which the two BRCT repeats interact in BRCA1 may represent a general mode of interaction between homologous domains within proteins that interact to regulate the cellular response to DNA damage. The structure provides a basis to predict the structural consequences of uncharacterized BRCA1 mutations.
TL;DR: The hinge loop of the major dimer, connecting the swapped β-strand to the protein core, resembles a short segment of the polar zipper proposed by Perutz and suggests a model for aggregate formation by 3D domain swapping with a polar zipper.
Abstract: Bovine pancreatic ribonuclease (RNase A) forms two types of dimers (a major and a minor component) upon concentration in mild acid. These two dimers exhibit different biophysical and biochemical properties. Earlier we reported that the minor dimer forms by swapping its N-terminal α-helix with that of an identical molecule. Here we find that the major dimer forms by swapping its C-terminal β-strand, thus revealing the first example of three-dimensional (3D) domain swapping taking place in different parts of the same protein. This feature permits RNase A to form tightly bonded higher oligomers. The hinge loop of the major dimer, connecting the swapped β-strand to the protein core, resembles a short segment of the polar zipper proposed by Perutz and suggests a model for aggregate formation by 3D domain swapping with a polar zipper.