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Structural Properties of EGCG-Induced, Nontoxic Alzheimer's Disease Abeta Oligomers.
Journal of Molecular Biology - 10 hours 45 min ago
Publication 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.
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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.
Journal of Molecular Biology - 10 hours 45 min ago
Publication 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.
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Structural Basis of Substrate Binding Specificity Revealed by the Crystal Structures of Polyamine Receptors SpuD and SpuE from Pseudomonas aeruginosa.
Journal of Molecular Biology - 10 hours 45 min ago
Publication 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 to 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.
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Crystal Structure of the Urokinase Receptor in a Ligand-Free Form.
Journal of Molecular Biology - 10 hours 45 min ago
Publication 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.
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Protein Structure Determination from Pseudocontact Shifts Using ROSETTA.
Journal of Molecular Biology - 10 hours 45 min ago
Publication 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 of 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.
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Evidence for the Existence of a Secondary Pathway for Fibril Growth during the Aggregation of Tau.
Journal of Molecular Biology - 10 hours 45 min ago
Publication 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.
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Molecular Interactions of Alzheimer's Abeta Protofilaments with Lipid Membranes.
Journal of Molecular Biology - 10 hours 45 min ago
Publication 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.
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Catalytic Activity and Acyl-Chain Selectivity of Diacylglycerol Kinase varepsilon Are Modulated by Residues in and near the Lipoxygenase-Like Motif.
Journal of Molecular Biology - 10 hours 45 min ago
Publication 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.
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Temporal Regulation of Gene Expression of the Escherichia coli Bacteriophage phiEco32.
Journal of Molecular Biology - 10 hours 45 min ago
Publication Date: 2012 Feb 24 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.
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Roles of Extracellular Chaperones in Amyloidosis.
Journal of Molecular Biology - 10 hours 45 min ago
Publication 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.
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Unperturbing a Non-Helically Perturbed Bacterial Flagellar Filament: Salmonella typhimurium SJW23.
Journal of Molecular Biology - 10 hours 45 min ago
Publication Date: 2012 Feb 24 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.
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Oligomeric Intermediates in Amyloid Formation: Structure Determination and Mechanisms of Toxicity.
Journal of Molecular Biology - 10 hours 45 min ago
Publication 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.
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From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression.
Journal of Molecular Biology - 10 hours 45 min ago
Publication 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.
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Malleability of the Folding Mechanism of the Outer Membrane Protein PagP: Parallel Pathways and the Effect of Membrane Elasticity.
Journal of Molecular Biology - 10 hours 45 min ago
Publication Date: 2012 Feb 24 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.
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Non-Stressful Death of 23S rRNA Mutant G2061C Defective in Puromycin Reaction.
Journal of Molecular Biology - 10 hours 45 min ago
Publication 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.
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Engineering Domain-Swapped Binding Interfaces by Mutually Exclusive Folding.
Journal of Molecular Biology - 10 hours 45 min ago
Publication 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.
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Evolutionary Walk between (beta/alpha)(8) Barrels: Catalytic Migration from Triosephosphate Isomerase to Thiamin Phosphate Synthase.
Journal of Molecular Biology - 10 hours 45 min ago
Publication Date: 2012 Feb 17 PMID: 22226942
Authors: Saab-Rincon, G. - Olvera, L. - Olvera, M. - Rudino-Pinera, E. - Benites, E. - Soberon, X. - Morett, E.
Journal: J Mol Biol
The functionally versatile (beta/alpha)(8) barrel scaffold was used to migrate triosephosphate isomerase (TPI) to thiamin phosphate synthase (TPS) activity, two enzymes that share the same fold but catalyze unrelated reactions through different mechanisms. The high sensitivity of the selection methodology was determinant to succeed in finding proteins with the desired activity. A combination of rational design and random mutagenesis was used to achieve the desired catalytic migration. One of the parallel directed evolution strategies followed resulted in TPI derivatives able to complement the thiamin phosphate auxotrophy phenotype of an Escherichia coli strain deleted of thiE, the gene that codes for TPS. Successive rounds of directed evolution resulted in better complementing TPI variants. Biochemical characterization of some of the evolved TPI clones demonstrated that the K(m) for the TPS substrates was similar to that of the native TPS; however and in agreement with the very slow complementation phenotype, the k(cat) was 4 orders of magnitude lower, indicating that substrate binding played a major role on selection. Interestingly, the crystal structure of the most proficient variant showed a slightly modified TPI active site occupied by a thiamin-phosphate-like molecule. Substitution of key residues in this region reduced TPS activity, strongly suggesting that this is also the catalytic site for the evolved TPS activity. The presence of the TPS reaction product at the active site explains the fast inactivation of the enzyme observed. In conclusion, by combining rational design, random mutagenesis and a very sensitive selection, it is possible to achieve enzymatic activity migration.
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Crystal Structure and Regulation Mechanisms of the CyaB Adenylyl Cyclase from the Human Pathogen Pseudomonas aeruginosa.
Journal of Molecular Biology - 10 hours 45 min ago
Publication Date: 2012 Feb 17 PMID: 22226839
Authors: Topal, H. - Fulcher, N. B. - Bitterman, J. - Salazar, E. - Buck, J. - Levin, L. R. - Cann, M. J. - Wolfgang, M. C. - Steegborn, C.
Journal: J Mol Biol
Pseudomonas aeruginosa is an opportunistic bacterial pathogen and a major cause of healthcare-associated infections. While the organism's intrinsic and acquired resistance to most antibiotics hinders treatment of P. aeruginosa infections, the regulatory networks controlling its virulence provide novel targets for drug development. CyaB, a key regulator of P. aeruginosa virulence, belongs to the Class III adenylyl cyclase (AC) family of enzymes that synthesize the second messenger cyclic adenosine 3',5'-monophosphate. These enzymes consist of a conserved catalytic domain fused to one or more regulatory domains. We describe here the biochemical and structural characterization of CyaB and its inhibition by small molecules. We show that CyaB belongs to the Class IIIb subfamily, and like other subfamily members, its activity is stimulated by inorganic carbon. CyaB is also regulated by its N-terminal MASE2 (membrane-associated sensor 2) domain, which acts as a membrane anchor. Using a genetic screen, we identified activating mutations in CyaB. By solving the crystal structure of the CyaB catalytic domain, we rationalized the effects of these mutations and propose that CyaB employs regulatory mechanisms similar to other Class III ACs. The CyaB structure further indicates subtle differences compared to other Class III ACs in both the active site and the inhibitor binding pocket. Consistent with these differences, we observed a unique inhibition profile, including identification of a CyaB selective compound. Overall, our results reveal mechanistic details of the physiological and pharmacological regulation of CyaB and provide the basis for its exploitation as a therapeutic drug target.
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Conformational properties of the unfolded state of im7 in nondenaturing conditions.
Journal of Molecular Biology - 10 hours 45 min ago
Publication Date: 2012 Feb 17 PMID: 22226836
Authors: Pashley, C. L. - Morgan, G. J. - Kalverda, A. P. - Thompson, G. S. - Kleanthous, C. - Radford, S. E.
Journal: J Mol Biol
The unfolded ensemble in aqueous solution represents the starting point of protein folding. Characterisation of this species is often difficult since the native state is usually predominantly populated at equilibrium. Previous work has shown that the four-helix protein, Im7 (immunity protein 7), folds via an on-pathway intermediate. While the transition states and folding intermediate have been characterised in atomistic detail, knowledge of the unfolded ensemble under the same ambient conditions remained sparse. Here, we introduce destabilising amino acid substitutions into the sequence of Im7, such that the unfolded state becomes predominantly populated at equilibrium in the absence of denaturant. Using far- and near-UV CD, fluorescence, urea titration and heteronuclear NMR experiments, we show that three amino acid substitutions (L18A-L19A-L37A) are sufficient to prevent Im7 folding, such that the unfolded state is predominantly populated at equilibrium. Using measurement of chemical shifts, (15)N transverse relaxation rates and sedimentation coefficients, we show that the unfolded species of L18A-L19A-L37A deviates significantly from random-coil behaviour. Specifically, we demonstrate that this unfolded species is compact (R(h)=25 A) relative to the urea-denatured state (R(h)>/=30 A) and contains local clusters of hydrophobic residues in regions that correspond to the four helices in the native state. Despite these interactions, there is no evidence for long-range stabilising tertiary interactions or persistent helical structure. The results reveal an unfolded ensemble that is conformationally restricted in regions of the polypeptide chain that ultimately form helices I, II and IV in the native state.
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N-Terminal Segments Modulate the alpha-Helical Propensities of the Intrinsically Disordered Basic Regions of bZIP Proteins.
Journal of Molecular Biology - 10 hours 45 min ago
Publication Date: 2012 Feb 17 PMID: 22226835
Authors: Das, R. K. - Crick, S. L. - Pappu, R. V.
Journal: J Mol Biol
Basic region leucine zippers (bZIPs) are modular transcription factors that play key roles in eukaryotic gene regulation. The basic regions of bZIPs (bZIP-bRs) are necessary and sufficient for DNA binding and specificity. Bioinformatic predictions and spectroscopic studies suggest that unbound monomeric bZIP-bRs are uniformly disordered as isolated domains. Here, we test this assumption through a comparative characterization of conformational ensembles for 15 different bZIP-bRs using a combination of atomistic simulations and circular dichroism measurements. We find that bZIP-bRs have quantifiable preferences for alpha-helical conformations in their unbound monomeric forms. This helicity varies from one bZIP-bR to another despite a significant sequence similarity of the DNA binding motifs (DBMs). Our analysis reveals that intramolecular interactions between DBMs and eight-residue segments directly N-terminal to DBMs are the primary modulators of bZIP-bR helicities. We test the accuracy of this inference by designing chimeras of bZIP-bRs to have either increased or decreased overall helicities. Our results yield quantitative insights regarding the relationship between sequence and the degree of intrinsic disorder within bZIP-bRs, and might have general implications for other intrinsically disordered proteins. Understanding how natural sequence variations lead to modulation of disorder is likely to be important for understanding the evolution of specificity in molecular recognition through intrinsically disordered regions (IDRs).
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