You are hereFeed aggregator / Journal of Molecular Biology
Journal of Molecular Biology
Structural Properties of EGCG-Induced, Nontoxic Alzheimer's Disease Abeta Oligomers.
17 hours 21 min agoPublication Date: 2012 Jan 28 PMID: 22300765
Authors: Lopez Del Amo, J. M. - Fink, U. - Dasari, M. - Grelle, G. - Wanker, E. E. - Bieschke, J. - Reif, B.
Journal: J Mol Biol
The green tea compound epigallocatechin-3-gallate (EGCG) inhibits Alzheimer's disease beta-amyloid peptide (Abeta) neurotoxicity. Solution-state NMR allows probing initial EGCG-Abeta interactions. We show that EGCG-induced Abeta oligomers adopt a well-defined structure and are amenable for magic angle spinning solid-state NMR investigations. We find that EGCG interferes with the aromatic hydrophobic core of Abeta. The C-terminal part of the Abeta peptide (residues 22-39) adopts a beta-sheet conformation, whereas the N-terminus (residues 1-20) is unstructured. The characteristic salt bridge involving residues D23 and K28 is present in the structure of these oligomeric Abeta aggregates as well. The structural analysis of small-molecule-induced amyloid aggregates will open new perspectives for Alzheimer's disease drug development.
post to: CiteULike
Categories: Journals
CNP-1 (ARRD-17), a Novel Substrate of Calcineurin, Is Critical for Modulation of Egg-Laying and Locomotion in Response to Food and Lysine Sensation in Caenorhabditis elegans.
17 hours 21 min agoPublication Date: 2012 Jan 28 PMID: 22300764
Authors: Jee, C. - Choi, T. W. - Kalichamy, K. - Yee, J. Z. - Song, H. O. - Ji, Y. J. - Lee, J. - Lee, J. - L'etoile, N. - Ahnn, J. - Lee, S. K.
Journal: J Mol Biol
Calcineurin is a Ca(2+)/calmodulin-dependent protein phosphatase involved in calcium signaling pathways. In Caenorhabditis elegans, the loss of calcineurin activity causes pleiotropic defects including hyperadaptation of sensory neurons, hypersensation to thermal difference and hyper-egg-laying when worms are refed after starvation. In this study, we report on arrd-17 as calcineurin-interacting protein-1 (cnp-1), which is a novel molecular target of calcineurin. CNP-1 interacts with the catalytic domain of the C. elegans calcineurin A subunit, TAX-6, in a yeast two-hybrid assay and is dephosphorylated by TAX-6 in vitro. cnp-1 is expressed in ASK, ADL, ASH and ASJ sensory neurons as TAX-6. It acts downstream of tax-6 in regulation of locomotion and egg-laying after starvation, ASH sensory neuron adaptation and lysine chemotaxis, that is known to be mediated by ASK neurons. Altogether, our biochemical and genetic evidence indicates that CNP-1 is a direct target of calcineurin and required in stimulated egg-laying and locomotion after starvation, adaptation to hyperosmolarity and attraction to lysine, which is modulated by calcineurin. We suggest that the phosphorylation status of CNP-1 plays an important role in regulation of refed stimulating behaviors after starvation and attraction to amino acid, which provides valuable nutritious information.
post to: CiteULike
Categories: Journals
Structural Basis of Substrate Binding Specificity Revealed by the Crystal Structures of Polyamine Receptors SpuD and SpuE from Pseudomonas aeruginosa.
17 hours 21 min agoPublication Date: 2012 Jan 28 PMID: 22300763
Authors: Wu, D. - Lim, S. C. - Dong, Y. - Wu, J. - Tao, F. - Zhou, L. - Zhang, L. H. - Song, H.
Journal: J Mol Biol
The type III secretion system (T3SS) of Pseudomonas aeruginosa is a key virulence determinant whose expression is induced by polyamine signals from mammalian host. SpuD and SpuE were postulated to be spermidine-preferential binding proteins, which regulate the polyamine content in this bacterial pathogen. In this study, we found that SpuD is a putrescine-preferential binding protein, while SpuE binds to spermidine exclusively. We have determined the crystal structures of SpuD in free form and in complex with putrescine and SpuE in free form and in complex with spermidine. Upon ligand binding, SpuD and SpuE undergo an "open-to-closed" conformational switch with the resultant closed ligand-bound forms, SpuD-putrescine and SpuE-spermidine, similar to their Escherichia coli counterparts PotF-putrescine and PotD-spermidine, respectively. Structural comparison suggested that two aromatic residues, Trp271 of SpuE and Phe273 of SpuD in segment II region, are the key structural determinants for putrescine/spermidine recognition specificity. Mutagenesis combined with isothermal titration calorimetry showed that substitution of Trp271 by Phe enabled SpuE gain substantial binding affinity for putrescine, while replacement of Phe273 by Trp reduced the binding affinity of SpuD toward putrescine by 250-fold. Altogether, these results revealed the molecular mechanism governing polyamine recognition specificity by SpuD and SpuE and provide the basis for further structural and functional studies of polyamine signal importation system in P. aeruginosa.
post to: CiteULike
Categories: Journals
Crystal Structure of the Urokinase Receptor in a Ligand-Free Form.
17 hours 21 min agoPublication Date: 2012 Jan 21 PMID: 22285761
Authors: Xu, X. - Gardsvoll, H. - Yuan, C. - Lin, L. - Ploug, M. - Huang, M.
Journal: J Mol Biol
The urokinase receptor urokinase-type plasminogen activator receptor (uPAR) is a surface receptor capable of not only focalizing urokinase-type plasminogen activator (uPA)-mediated fibrinolysis to the pericellular micro-environment but also promoting cell migration and chemotaxis. Consistent with this multifunctional role, uPAR binds several extracellular ligands, including uPA and vitronectin. Structural studies suggest that uPAR possesses structural flexibility. It is, however, not clear whether this flexibility is an inherent property of the uPAR structure per se or whether it is induced upon ligand binding. The crystal structure of human uPAR in its ligand-free state would clarify this issue, but such information remains unfortunately elusive. We now report the crystal structures of a stabilized, human uPAR (H47C/N259C) in its ligand-free form to 2.4 A and in complex with amino-terminal fragment (ATF) to 3.2 A. The structure of uPAR(H47C/N259C) in complex with ATF resembles the wild-type uPAR.ATF complex, demonstrating that these mutations do not perturb the uPA binding properties of uPAR. The present structure of uPAR(H47C/N259C) provides the first structural definition of uPAR in its ligand-free form, which represents one of the biologically active conformations of uPAR as defined by extensive biochemical studies. The domain boundary between uPAR DI-DII domains is more flexible than the DII-DIII domain boundary. Two important structural features are highlighted by the present uPAR structure. First, the DI-DIII domain boundary may face the cell membrane. Second, loop 130-142 of uPAR plays a dynamic role during ligand loading/unloading. Together, these studies provide new insights into uPAR structure-function relationships, emphasizing the importance of the inter-domain dynamics of this modular receptor.
post to: CiteULike
Categories: Journals
Protein Structure Determination from Pseudocontact Shifts Using ROSETTA.
17 hours 21 min agoPublication Date: 2012 Jan 18 PMID: 22285518
Authors: Schmitz, C. - Vernon, R. - Otting, G. - Baker, D. - Huber, T.
Journal: J Mol Biol
Paramagnetic metal ions generate pseudocontact shifts (PCSs) in nuclear magnetic resonance spectra that are manifested as easily measurable changes in chemical shifts. Metals can be incorporated into proteins through metal binding tags, and PCS data constitute powerful long-range restraints on the positions of nuclear spins relative to the coordinate system of the magnetic susceptibility anisotropy tensor (Deltachi-tensor) of the metal ion. We show that three-dimensional structures of proteins can reliably be determined using PCS data from a single metal binding site combined with backbone chemical shifts. The program PCS-ROSETTA automatically determines the Deltachi-tensor and metal position from the PCS data during the structure calculations, without any prior knowledge on the protein structure. The program can determine structures accurately for proteins of up to 150 residues, offering a powerful new approach to protein structure determination that relies exclusively on readily measurable backbone chemical shifts and easily discriminates between correctly and incorrectly folded conformations.
post to: CiteULike
Categories: Journals
Evidence for the Existence of a Secondary Pathway for Fibril Growth during the Aggregation of Tau.
17 hours 21 min agoPublication Date: 2012 Jan 17 PMID: 22281439
Authors: Ramachandran, G. - Udgaonkar, J. B.
Journal: J Mol Biol
The mechanism of amyloid fibril formation by proteins has been classically described by the nucleation-dependent polymerization (NDP) model, which makes certain predictions regarding the kinetics of fibrillation. All proteins whose aggregation conforms to the NDP model display a t(2) time dependence for their initial reaction profile. However, there are proteins whose aggregation reactions have kinetic signatures of a flat lag phase followed by an exponential rise in fibril mass, which does not conform to the NDP model. Amyloid fibril formation by tau, a microtubule-associated protein whose aggregation to form neurofibrillary tangles is implicated in Alzheimer's disease and other tauopathies, in the presence of inducers such as heparin and fatty acid micelles, has always been traditionally described by a ligand-induced NDP model. In this study, the existence of a secondary pathway for fibril growth during the aggregation of the functional, repeat domain of tau in the presence of heparin has been established. Both kinetic and accessory evidence are provided for the existence of this pathway, which is shown to augment the primary homogeneous nucleation pathway. From the kinetic data, the main secondary pathway that is operative appears to be fibril fragmentation but other pathways such as branching or secondary nucleation may also be operative.
post to: CiteULike
Categories: Journals
Molecular Interactions of Alzheimer's Abeta Protofilaments with Lipid Membranes.
17 hours 21 min agoPublication Date: 2012 Jan 17 PMID: 22281438
Authors: Tofoleanu, F. - Buchete, N. V.
Journal: J Mol Biol
Amyloid fibrils and peptide oligomers play central roles in the pathology of Alzheimer's disease, type 2 diabetes, Parkinson's disease, Huntington's disease, and prion-related disease. Here, we investigate the molecular interactions between preformed amyloid beta (Abeta) molecular protofilaments and lipid bilayer membranes, in the presence of explicit water molecules, using computational models and all-atom molecular dynamics. These interactions play an important role in the stability and function of both Abeta fibrils and their adjacent cellular membrane. Taking advantage of the symmetry-related and directional properties of the protofilaments, we build models that cover several relative protofilament-membrane orientations. Our molecular dynamics simulations reveal the relative contributions of different structural elements to the dynamics and stability of Abeta protofilament segments near membranes, and the first steps in the mechanism of fibril-membrane interactions. During this process, we observe a significant alteration of the side-chain contact pattern in protofilaments, although a fraction of the characteristic beta-sheet content is preserved. As a major driving force, we identify the electrostatic interactions between Abeta charged side chains, including E22, D23, and K28, and lipid headgroups. Together with hydrogen bonding with atoms from lipid headgroups, these interactions can facilitate the penetration of hydrophobic C-terminal amino acids through the lipid headgroup region, which can finally lead both to further loss of the initial fibril structure and to local membrane-thinning effects. Our results may guide new experiments that could test the extent to which the structural features of water-formed amyloid fibrils are preserved, lost, or reshaped by membrane-mediated interactions.
post to: CiteULike
Categories: Journals
Catalytic Activity and Acyl-Chain Selectivity of Diacylglycerol Kinase varepsilon Are Modulated by Residues in and near the Lipoxygenase-Like Motif.
17 hours 21 min agoPublication Date: 2012 Jan 12 PMID: 22266092
Authors: D'Souza, K. - Epand, R. M.
Journal: J Mol Biol
Diacylglycerol kinase (DGK) varepsilon plays an important role in the resynthesis of phosphatidylinositol by mediating the phosphorylation of diacylglycerol to phosphatidic acid. DGKvarepsilon is unique among mammalian DGK isoforms in that it is the only one that shows acyl-chain selectivity, preferring diacylglycerols with an sn-2 arachidonoyl group. The region responsible for this arachidonoyl specificity is the lipoxygenase (LOX)-like motif found in the accessory domain, adjacent to DGKvarepsilon's catalytic site. Many mutations within the LOX-like motif result in a loss of enzyme activity. However, the few mutants that retain significant activity exhibit some decrease in selectivity for the arachidonoyl chain. In the present work, we have explored mutations in a region adjacent to the LOX-like motif, which is also contained within the same hydrophobic segment of the protein. This adjacent region also contains a cholesterol recognition/interaction amino acid consensus motif. Being outside of the LOX-like motif, this region likely has less direct contact with the substrate, and more activity is retained with mutations. This has allowed us to probe in more detail the relationship between this region of the protein and substrate specificity. We demonstrate that this cholesterol recognition/interaction amino acid consensus domain also plays a role in acyl-chain selectivity. Despite the high degree of conservation of the amino acid sequence in this region of the protein, certain mutations result in proteins with higher activity than the wild-type protein. These mutations also result in a selective gain of acyl-chain preferences for diacylglycerols with different acyl-chain profiles. In addition to the LOX-like motif, adjacent residues also contribute to selectivity for diacylglycerols with specific acyl-chain compositions, such as those found in the phosphatidylinositol cycle.
post to: CiteULike
Categories: Journals
Temporal Regulation of Gene Expression of the Escherichia coli Bacteriophage phiEco32.
17 hours 21 min agoPublication Date: 2012 Jan 10 PMID: 22261232
Authors: Pavlova, O. - Lavysh, D. - Klimuk, E. - Djordjevic, M. - Ravcheev, D. A. - Gelfand, M. S. - Severinov, K. - Akulenko, N.
Journal: J Mol Biol
Escherichia coli phage phiEco32 encodes two proteins that bind to host RNA polymerase (RNAP): gp79, a novel protein, and gp36, a distant homolog of sigma(70) family proteins. Here, we investigated the temporal pattern of phiEco32 and host gene expression during infection. Host transcription shutoff and three distinct bacteriophage temporal gene classes (early, middle, and late) were revealed. A combination of bioinformatic and biochemical approaches allowed identification of phage promoters recognized by a host RNAP holoenzyme containing the sigma(70) factor. These promoters are located upstream of early phage genes. A combination of macroarray data, primer extension, and in vitro transcription analyses allowed identification of six promoters recognized by an RNAP holoenzyme containing gp36. These promoters are characterized by a single-consensus element tAATGTAtA and are located upstream of the middle and late phage genes. Curiously, gp79, an inhibitor of host and early phage transcription by sigma(70) holoenzyme, activated transcription by the gp36 holoenzyme in vitro.
post to: CiteULike
Categories: Journals
Roles of Extracellular Chaperones in Amyloidosis.
17 hours 21 min agoPublication Date: 2012 Jan 10 PMID: 22248589
Authors: Wyatt, A. R. - Yerbury, J. J. - Dabbs, R. A. - Wilson, M. R.
Journal: J Mol Biol
Extracellular protein misfolding and aggregation underlie many of the most serious amyloidoses including Alzheimer's disease, spongiform encephalopathies and type II diabetes. Despite this, protein homeostasis (proteostasis) research has largely focussed on characterising systems that function to monitor protein conformation and concentration within cells. We are now starting to identify elements of corresponding systems, including an expanding family of secreted chaperones, which exist in the extracellular space. Like their intracellular counterparts, extracellular chaperones are likely to play a central role in systems that maintain proteostasis; however, the precise details of how they participate are only just emerging. It is proposed that extracellular chaperones patrol biological fluids for misfolded proteins and facilitate their clearance via endocytic receptors. Importantly, many amyloidoses are associated with dysfunction in rates of protein clearance. This is consistent with a model in which disruption to, or overwhelming of, the systems responsible for extracellular proteostasis results in the accumulation of pathological protein aggregates and disease. Further characterisation of mechanisms that maintain extracellular proteostasis will shed light on why many serious diseases occur and provide us with much needed strategies to combat them.
post to: CiteULike
Categories: Journals
Unperturbing a Non-Helically Perturbed Bacterial Flagellar Filament: Salmonella typhimurium SJW23.
17 hours 21 min agoPublication Date: 2012 Jan 10 PMID: 22248588
Authors: Nisani-Bizer, K. - Trachtenberg, S.
Journal: J Mol Biol
Salmonella typhimurium SJW23 has a right-handed, non-helically perturbed filament of serotype gt with a unique surface pattern. Non-helical perturbations involve symmetry reduction along the five-start helical lines resulting in layer lines of fractional Bessel orders and a consequent seam. The flagellin gene, fliC(23), which we sequenced, differs from the sequence of the canonic, plain SJW1655 flagellin, fliC(1655). We modified discrete components of fliC(23) in order to localize, in the expressed filament, the submolecular site responsible for the non-helical perturbation. These modifications include (i) deleting the outermost domain D3(23), (ii) replacing D3(23) with D3(1655), (iii) substituting a hydrophilic alpha-helix at the interface between the neighboring domains D1 and D2 with a hydrophobic one from fliC(1655), and (iv) substituting a serine/glycine pair in the loop connecting the modified alpha-helix to its neighbor; these modifications were made in the presence and absence of D3(23). We used S. typhimurium SJW1655 both as a reference and as a source for 'spare parts'. The symmetry of the constructs was assessed from the power spectra through changes in the layer lines at a height of 1/105 and 1/35 A(-1), unique to the non-helical perturbation. Deleting D3(23), either alone or in combination with various substitutions, or replacing it with D3(1655) transforms the non-helically perturbed filament into a plain one as judged by the disappearance of the typical layer lines from the power spectra. We conclude that the non-helical perturbation is a product of unique interactions in the D3(23) density shell. Whereas other minor structural changes may occur at the filaments interior, they are all helically symmetric.
post to: CiteULike
Categories: Journals
Oligomeric Intermediates in Amyloid Formation: Structure Determination and Mechanisms of Toxicity.
17 hours 21 min agoPublication Date: 2012 Jan 12 PMID: 22248587
Authors: Fandrich, M.
Journal: J Mol Biol
Oligomeric intermediates are non-fibrillar polypeptide assemblies that occur during amyloid fibril formation and that are thought to underlie the aetiology of amyloid diseases, such as Alzheimer's disease, Parkinson's disease and Huntington's disease. Focusing primarily on the oligomeric states formed from Alzheimer's disease beta-amyloid (Abeta) peptide, this review will make references to other polypeptide systems, highlighting common principles or sequence-specific differences. The covered topics include the structural properties and polymorphism of oligomers, the biophysical mechanism of peptide self-assembly and its role for pathogenicity in amyloid disease. Oligomer-dependent toxicity mechanisms will be explained along with recently emerging possibilities of interference.
post to: CiteULike
Categories: Journals
From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression.
17 hours 21 min agoPublication Date: 2012 Jan 8 PMID: 22245580
Authors: Ohlendorf, R. - Vidavski, R. R. - Eldar, A. - Moffat, K. - Moglich, A.
Journal: J Mol Biol
Signaling photoreceptors mediate diverse organismal adaptations in response to light. As light-gated protein switches, signaling photoreceptors provide the basis for optogenetics, a term that refers to the control of organismal physiology and behavior by light. We establish as novel optogenetic tools the plasmids pDusk and pDawn, which employ blue-light photoreceptors to confer light-repressed or light-induced gene expression in Escherichia coli with up to 460-fold induction upon illumination. Key features of these systems are low background activity, high dynamic range, spatial control on the 20-mum scale, independence from exogenous factors, and ease of use. In optogenetic experiments, pDusk and pDawn can be used to specifically perturb individual nodes of signaling networks and interrogate their role. On the preparative scale, pDawn can induce by light the production of recombinant proteins and thus represents a cost-effective and readily automated alternative to conventional induction systems.
post to: CiteULike
Categories: Journals
Malleability of the Folding Mechanism of the Outer Membrane Protein PagP: Parallel Pathways and the Effect of Membrane Elasticity.
17 hours 21 min agoPublication Date: 2012 Jan 8 PMID: 22245579
Authors: Huysmans, G. H. - Radford, S. E. - Baldwin, S. A. - Brockwell, D. J.
Journal: J Mol Biol
Understanding the interactions between membrane proteins and the lipid bilayer is key to increasing our ability to predict and tailor the folding mechanism, structure and stability of membrane proteins. Here, we have investigated the effects of changing the membrane composition and the relative concentrations of protein and lipid on the folding mechanism of the bacterial outer membrane protein PagP. The folding pathway, monitored by tryptophan fluorescence, was found to be characterized by a burst phase, representing PagP adsorption to the liposome surface, followed by a time course that reflects the folding and insertion of the protein into the membrane. In 1,2-dilauroyl-sn-glycero-3-phosphocholine (diC(12:0)PC) liposomes, the post-adsorption time course fits well to a single exponential at high lipid-to-protein ratios (LPRs), but at low LPRs, a second exponential phase with a slower folding rate constant is observed. Interrupted refolding assays demonstrated that the two exponential phases reflect the presence of parallel folding pathways. Partitioning between these pathways was found to be modulated by the elastic properties of the membrane. Folding into mixed 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine:diC(12:0)PC liposomes resulted in a decrease in PagP adsorption to the liposomes and a switch to the slower folding pathway. By contrast, inclusion of 1,2-dilauroyl-sn-glycero-3-phosphoserine into diC(12:0)PC liposomes resulted in a decrease in the folding rate of the fast pathway. The results highlight the effect of lipid composition in tailoring the folding mechanism of a membrane protein, revealing that membrane proteins have access to multiple, competing folding routes to a unique native structure.
post to: CiteULike
Categories: Journals
Complex Spatial Organization and Flagellin Composition of Flagellar Propeller from Marine Magnetotactic Ovoid Strain MO-1.
17 hours 21 min agoPublication Date: 2012 Jan 5 PMID: 22245577
Authors: Zhang, W. J. - Santini, C. L. - Bernadac, A. - Ruan, J. - Zhang, S. D. - Kato, T. - Li, Y. - Namba, K. - Wu, L. F.
Journal: J Mol Biol
Marine magnetotactic ovoid bacterium MO-1 is capable of swimming along the geomagnetic field lines by means of its two sheathed flagellar bundles at a speed up to 300 mum/s. In this study, by using electron microscopy, we showed that, in each bundle, six individual flagella were organized in hexagon with a seventh in the middle. We identified 12 flagellin paralogs and 2 putative flagellins in the genome of MO-1. Among them, 13 were tandemly located on an approximately 17-kb segment while the 14th was on a separated locus. Using reverse transcription PCR and quantitative PCR, we found that all the 14 flagellin or putative flagellin genes were transcribed and that 2 of them were more abundantly expressed than others. A nLC (nanoliquid chromatography)-ESI (electrospray ionization)-MS/MS (mass spectrometry/mass spectrometry) mass spectrometry analysis identified all the 12 flagellin proteins in three glycosylated polypeptide bands resolved by one-dimensional denaturing polyacrylamide gel electrophoresis and 10 of them in 21 spots obtained by means of two-dimensional electrophoresis of flagellar extracts. Most spots contained more than one flagellin, and eight of the ten identified flagellins existed in multiple isoforms. Taken together, these results show unprecedented complexity in the spatial organization and flagellin composition of the flagellar propeller. Such architecture is observed only for ovoid-coccoid, bilophotrichously flagellated magnetotactic bacteria living in marine sediments, suggesting a species and environmental specificity.
post to: CiteULike
Categories: Journals
Non-Stressful Death of 23S rRNA Mutant G2061C Defective in Puromycin Reaction.
17 hours 21 min agoPublication Date: 2012 Jan 10 PMID: 22245576
Authors: Sergiev, P. V. - Lesnyak, D. V. - Burakovsky, D. E. - Svetlov, M. - Kolb, V. A. - Serebryakova, M. V. - Demina, I. A. - Govorun, V. M. - Dontsova, O. A. - Bogdanov, A. A.
Journal: J Mol Biol
Catalysis of peptide bond formation in the peptidyl transferase center is a major enzymatic activity of the ribosome. Mutations limiting peptidyl transferase activity are mostly lethal. However, cellular processes triggered by peptidyl transferase deficiency in the bacterial cell are largely unknown. Here we report a study of the lethal G2061C mutant of Escherichia coli 23S ribosomal RNA (rRNA). The G2061C mutation completely impaired the puromycin reaction and abolished formation of the active firefly luciferase in an in vitro translation system, while poly(U)- and short synthetic mRNA-directed peptidyl transferase reaction with aminoacylated tRNAs in vitro was seemingly unaffected. Study of the cellular proteome upon expression of the 23S rRNA gene carrying the G2061C mutation compared to cells expressing wild-type 23S rRNA gene revealed substantial differences. Most of the observed effects in the mutant were associated with reduced expression of stress response proteins and particularly proteins associated with the ppGpp-mediated stringent response.
post to: CiteULike
Categories: Journals
Engineering Domain-Swapped Binding Interfaces by Mutually Exclusive Folding.
17 hours 21 min agoPublication Date: 2012 Jan 8 PMID: 22245575
Authors: Ha, J. H. - Karchin, J. M. - Walker-Kopp, N. - Huang, L. S. - Berry, E. A. - Loh, S. N.
Journal: J Mol Biol
Domain swapping is a mechanism for forming protein dimers and oligomers with high specificity. It is distinct from other forms of oligomerization in that the binding interface is formed by reciprocal exchange of polypeptide segments. Swapping plays a physiological role in protein-protein recognition, and it can also potentially be exploited as a mechanism for controlled self-assembly. Here, we demonstrate that domain-swapped interfaces can be engineered by inserting one protein into a surface loop of another protein. The key to facilitating a domain swap is to destabilize the protein when it is monomeric but not when it is oligomeric. We achieve this condition by employing the "mutually exclusive folding" design to apply conformational stress to the monomeric state. Ubiquitin (Ub) is inserted into one of six surface loops of barnase (Bn). The 38-A amino-to-carboxy-terminal distance of Ub stresses the Bn monomer, causing it to split at the point of insertion. The 2.2-A X-ray structure of one insertion variant reveals that strain is relieved by intermolecular folding with an identically unfolded Bn domain, resulting in a domain-swapped polymer. All six constructs oligomerize, suggesting that inserting Ub into each surface loop of Bn results in a similar domain-swapping event. Binding affinity can be tuned by varying the length of the peptide linkers used to join the two proteins, which modulates the extent of stress. Engineered, swapped proteins have the potential to be used to fabricate "smart" biomaterials, or as binding modules from which to assemble heterologous, multi-subunit protein complexes.
post to: CiteULike
Categories: Journals
Propagation of the Prion Phenomenon: Beyond the Seeding Principle.
17 hours 21 min agoPublication Date: 2012 Jan 5 PMID: 22245492
Authors: Munch, C. - Bertolotti, A.
Journal: J Mol Biol
The deposition of misfolded proteins is the hallmark of the late-onset, rapidly progressive and devastating neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis. These diseases are caused by a gain of toxic properties associated with the propensity of otherwise soluble proteins to misfold. What governs the deposition of the disease-causing proteins in aged neurons is unclear, but recent evidence suggests that once misfolded, the diverse proteins associated with the neurodegenerative diseases can induce aggregation of their soluble counterpart, thereby sharing one of the defining properties of prions. In addition to the seeded polymerization, prions have the ability to replicate their aberrant conformation indefinitely and are transmissible. Are these properties also shared by diverse misfolded proteins?
post to: CiteULike
Categories: Journals
Inhibition of Amyloid Formation.
17 hours 21 min agoPublication Date: 2012 Jan 5 PMID: 22244855
Authors: Hard, T. - Lendel, C.
Journal: J Mol Biol
Amyloid is aggregated protein in the form of insoluble fibrils. Amyloid deposition in human tissue-amyloidosis-is associated with a number of diseases including all common dementias and type II diabetes. Considerable progress has been made to understand the mechanisms leading to amyloid formation. It is, however, not yet clear by which mechanisms amyloid and protein aggregates formed on the path to amyloid are cytotoxic. Strategies to prevent protein aggregation and amyloid formation are nevertheless, in many cases, promising and even successful. This review covers research on intervention of amyloidosis and highlights several examples of how inhibition of protein aggregation and amyloid formation has been achieved in practice. For instance, rational design can provide drugs that stabilize a native folded state of a protein, protein engineering can provide new binding proteins that sequester monomeric peptides from aggregation, small molecules and peptides can be designed to block aggregation or direct it into non-cytotoxic paths, and monoclonal antibodies have been developed for therapies towards neurodegenerative diseases based on amyloid inhibition and clearance.
post to: CiteULike
Categories: Journals
The Transthyretin Amyloidoses: From Delineating the Molecular Mechanism of Aggregation Linked to Pathology to a Regulatory-Agency-Approved Drug.
17 hours 21 min agoPublication Date: 2012 Jan 5 PMID: 22244854
Authors: Johnson, S. M. - Connelly, S. - Fearns, C. - Powers, E. T. - Kelly, J. W.
Journal: J Mol Biol
Transthyretin (TTR) is one of the many proteins that are known to misfold and aggregate (i.e., undergo amyloidogenesis) in vivo. The process of TTR amyloidogenesis causes nervous system and/or heart pathology. While several of these maladies are associated with mutations that destabilize the native TTR quaternary and/or tertiary structure, wild-type TTR amyloidogenesis also leads to the degeneration of postmitotic tissue. Over the past 20 years, much has been learned about the factors that influence the propensity of TTR to aggregate. This biophysical information led to the development of a therapeutic strategy, termed "kinetic stabilization," to prevent TTR amyloidogenesis. This strategy afforded the drug tafamidis which was recently approved by the European Medicines Agency for the treatment of TTR familial amyloid polyneuropathy, the most common familial TTR amyloid disease. Tafamidis is the first and currently the only medication approved to treat TTR familial amyloid polyneuropathy. Here we review the biophysical basis for the kinetic stabilization strategy and the structure-based drug design effort that led to this first-in-class pharmacologic agent.
post to: CiteULike
Categories: Journals