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Journal of Molecular Biology
H-Bonding and Positive Charge at the N(5)/O(4) Locus Are Critical for Covalent Flavin Attachment in Trametes Pyranose 2-Oxidase.
9 hours 46 min agoPublication Date: 2010 Aug 12 PMID: 20708626
Authors: Tan, T. C. - Pitsawong, W. - Wongnate, T. - Spadiut, O. - Haltrich, D. - Chaiyen, P. - Divne, C.
Journal: J Mol Biol
Flavoenzymes perform a wide range of redox reactions in nature, and a subclass of flavoenzymes carry covalently bound cofactor. The enzyme-flavin bond helps to increase the flavin's redox potential to facilitate substrate oxidation in several oxidases. The formation of the enzyme-flavin covalent bond-the flavinylation reaction-has been studied for the past 40 years. For the most advocated mechanism of autocatalytic flavinylation, the quinone methide mechanism, appropriate stabilization of developing negative charges at the flavin N(1) and N(5) loci is crucial. Whereas the structural basis for stabilization at N(1) is relatively well studied, the structural requisites for charge stabilization at N(5) remain less clear. Here, we show that flavinylation of histidine 167 of pyranose 2-oxidase from Trametes multicolor requires hydrogen bonding at the flavin N(5)/O(4) locus, which is offered by the side chain of Thr169 when the enzyme is in its closed, but not open, state. Moreover, our data show that additional stabilization at N(5) by histidine 548 is required to ensure high occupancy of the histidyl-flavin bond. The combination of structural and spectral data on pyranose 2-oxidase mutants supports the quinone methide mechanism. Our results demonstrate an elaborate structural fine-tuning of the active site to complete its own formation that couples efficient holoenzyme synthesis to conformational substates of the substrate-recognition loop and concerted movements of side chains near the flavinylation ligand.
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Crystal Structure of the Cyanobacterial Signal Transduction Protein P(II) in Complex with PipX.
9 hours 46 min agoPublication Date: 2010 Aug 12 PMID: 20708625
Authors: Zhao, M. X. - Jiang, Y. L. - Xu, B. Y. - Chen, Y. - Zhang, C. C. - Zhou, C. Z.
Journal: J Mol Biol
P(II) proteins are highly conserved signal transducers in bacteria, archaea, and plants. They have a large flexible loop (T-loop) that adopts different conformations after covalent modification or binding to different effectors to regulate the functions of diverse protein partners. The P(II) partner PipX (P(II)interaction protein X), first identified from Synechococcus sp. PCC 7942, exists uniquely in cyanobacteria. PipX also interacts with the cyanobacterial global nitrogen regulator NtcA. The mutually exclusive binding of P(II) and NtcA by PipX in a 2-oxoglutarate (2-OG)-dependent manner enables P(II) to indirectly regulate the transcriptional activity of NtcA. However, the structural basis for these exclusive interactions remains unknown. We solved the crystal structure of the P(II)-PipX complex from the filamentous cyanobacterium Anabaena sp. PCC 7120 at 1.90 A resolution. A homotrimeric P(II) captures three subunits of PipX through the T-loops. Similar to P(II) from Synechococcus, the core structure consists of an antiparallel beta-sheet with four beta-strands and two alpha-helices at the lateral surface. PipX adopts a novel structure composed of five twisted antiparallel beta-strands and two alpha-helices, which is reminiscent of the P(II) structure. The T-loop of each P(II) subunit extends from the core structure as an antenna that is stabilized at the cleft between two PipX monomers via hydrogen bonds. In addition, the interfaces between the beta-sheets of PipX and P(II) core structures partially contribute to complex formation. Comparative structural analysis indicated that PipX and 2-OG share a common binding site that overlaps with the 14 signature residues of cyanobacterial P(II) proteins. Our structure of PipX and the recently solved NtcA structure enabled us to propose a putative model for the NtcA-PipX complex. Taken together, these findings provide structural insights into how P(II) regulates the transcriptional activity of NtcA via PipX upon accumulation of the metabolite 2-OG.
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Structure of the AML1-ETO NHR3-PKA(RIIalpha) Complex and Its Contribution to AML1-ETO Activity.
9 hours 46 min agoPublication Date: 2010 Aug 11 PMID: 20708017
Authors: Corpora, T. - Roudaia, L. - Oo, Z. M. - Chen, W. - Manuylova, E. - Cai, X. - Chen, M. J. - Cierpecki, T. - Speck, N. A. - Bushweller, J. H.
Journal: J Mol Biol
AML1-ETO is the chimeric protein product of t(8;21) in acute myeloid leukemia. The ETO portion of the fusion protein includes the nervy homology region (NHR) 3 domain, which shares homology with A-kinase anchoring proteins and interacts with the regulatory subunit of type II cAMP-dependent protein kinase A (PKA(RIIalpha)). We determined the solution structure of a complex between the AML1-ETO NHR3 domain and PKA(RIIalpha). Based on this structure, a key residue in AML1-ETO for PKA(RIIalpha) association was mutated. This mutation did not disrupt AML1-ETO's ability to enhance the clonogenic capacity of primary mouse bone marrow cells or its ability to repress proliferation or granulocyte differentiation. Introduction of the mutation into AML1-ETO had minimal impact on in vivo leukemogenesis. Therefore, the NHR3-PKA(RIIalpha) protein interaction does not appear to significantly contribute to AML1-ETO's ability to induce leukemia.
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Conformation Changes in E. coli Rho Monitored by Hydrogen/Deuterium Exchange and Mass Spectrometry: Response to Ligand Binding.
9 hours 46 min agoPublication Date: 2010 Aug 11 PMID: 20708016
Authors: Stitt, B. L. - Xiao, H.
Journal: J Mol Biol
Escherichia coli Rho is a doughnut-shaped homohexameric ATP-dependent RNA-DNA helicase that releases newly synthesized RNA molecules from transcription complexes. Rho binds 60-80 bases of RNA among six primary RNA binding sites around the inside of its N-terminal crown; the RNA then passes through the central hole of the hexamer. Here it triggers ATP hydrolysis and is moved with respect to the protein. We study protein conformation changes upon ligand binding using amide proton hydrogen/deuterium exchange and mass spectrometry. Global-exchange studies indicate net mass differences of about 15 Da after 1 h of exchange in the presence-versus in the absence-of the ligand MgATP or the RNA poly(C). Sites of ligand-dependent exchange differences were localized by mass determination of the peptic peptides of Rho. A peptide of the N-terminal domain near the known primary RNA sites (aa 56-63) was protected from amide proton exchange in the presence of poly(C), as was a novel N-terminal domain peptide that is not near RNA in the crystal structures or in NMR structures with RNA oligomers (aa 37-46). This result may further define the primary interaction site of RNA with Rho. The Q-loop-containing peptide in the central hole of the protein that interacts with RNA was also protected by RNA (aa 271-286). The exchange rate of one peptide near the ATPase active site (aa 206-218) slowed in the presence of MgATP and increased in the presence of RNA. Overall, the results show changes in a few protein segments rather than a different overall conformation.
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Crystal structure of the 2-oxoglutarate- and Fe(II)-dependent lysyl hydroxylase JMJD6.
9 hours 46 min agoPublication Date: 2010 Aug 13 PMID: 20684070
Authors: Mantri, M. - Krojer, T. - Bagg, E. A. - Webby, C. J. - Butler, D. S. - Kochan, G. - Kavanagh, K. L. - Oppermann, U. - McDonough, M. A. - Schofield, C. J.
Journal: J Mol Biol
Lysyl and prolyl hydroxylations are well-known post-translational modifications to animal and plant proteins with extracellular roles. More recent work has indicated that the hydroxylation of intracellular animal proteins may be common. JMJD6 catalyses the iron- and 2-oxoglutarate-dependent hydroxylation of lysyl residues in arginine-serine-rich domains of RNA splicing-related proteins. We report crystallographic studies on the catalytic domain of JMJD6 in complex with Ni(II) substituting for Fe(II). Together with mutational studies, the structural data suggest how JMJD6 binds its lysyl residues such that it can catalyse C-5 hydroxylation rather than Nepsilon-demethylation, as for analogous enzymes.
MeSH Categories: Amino Acid Sequence, Amino Acid Substitution, Base Sequence, Catalytic Domain, Crystallography, X-Ray, DNA Primers/genetics, Humans, Iron/metabolism, Jumonji Domain-Containing Histone, Demethylases/*chemistry/genetics/metabolism, Ketoglutaric Acids/metabolism, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutant Proteins/chemistry/genetics/metabolism, Nickel/metabolism, Procollagen-Lysine, 2-Oxoglutarate, 5-Dioxygenase/*chemistry/genetics/metabolism, Protein Folding, Recombinant Proteins/chemistry/genetics/metabolism, Sequence Homology, Amino Acid, Static Electricity
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Mutually-induced conformational switching of RNA and coat protein underpins efficient assembly of a viral capsid.
9 hours 46 min agoPublication Date: 2010 Aug 13 PMID: 20684044
Authors: Rolfsson, O. - Toropova, K. - Ranson, N. A. - Stockley, P. G.
Journal: J Mol Biol
Single-stranded RNA viruses package their genomes into capsids enclosing fixed volumes. We assayed the ability of bacteriophage MS2 coat protein to package large, defined fragments of its genomic, single-stranded RNA. We show that the efficiency of packaging into a T=3 capsid in vitro is inversely proportional to RNA length, implying that there is a free-energy barrier to be overcome during assembly. All the RNAs examined have greater solution persistence lengths than the internal diameter of the capsid into which they become packaged, suggesting that protein-mediated RNA compaction must occur during assembly. Binding ethidium bromide to one of these RNA fragments, which would be expected to reduce its flexibility, severely inhibited packaging, consistent with this idea. Cryo-EM structures of the capsids assembled in these experiments with the sub-genomic RNAs show a layer of RNA density beneath the coat protein shell but lack density for the inner RNA shell seen in the wild-type virion. The inner layer is restored when full-length virion RNA is used in the assembly reaction, implying that it becomes ordered only when the capsid is filled, presumably because of the effects of steric and/or electrostatic repulsions. The cryo-EM results explain the length dependence of packaging. In addition, they show that for the sub-genomic fragments the strongest ordered RNA density occurs below the coat protein dimers forming the icosahedral 5-fold axes of the capsid. There is little such density beneath the proteins at the 2-fold axes, consistent with our model in which coat protein dimers binding to RNA stem-loops located at sites throughout the genome leads to switching of their preferred conformations, thus regulating the placement of the quasi-conformers needed to build the T=3 capsid. The data are consistent with mutual chaperoning of both RNA and coat protein conformations, partially explaining the ability of such viruses to assemble so rapidly and accurately.
MeSH Categories: Base Sequence, Capsid/*chemistry/ultrastructure, Capsid Proteins/*chemistry/genetics, Cryoelectron Microscopy, DNA Primers/genetics, Imaging, Three-Dimensional, Levivirus/chemistry/enzymology/physiology/ultrastructure, Models, Molecular, Nucleic Acid Conformation, Protein Conformation, RNA, Viral/*chemistry/genetics, Recombinant Proteins/chemistry/genetics, Virus Assembly/*physiology
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Structure and Engineering of l-Arabinitol 4-Dehydrogenase from Neurospora crassa.
9 hours 46 min agoPublication Date: 2010 Sep 10 PMID: 20655316
Authors: Bae, B. - Sullivan, R. P. - Zhao, H. - Nair, S. K.
Journal: J Mol Biol
l-Arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of l-arabinitol into l-xylulose with concomitant NAD(+) reduction. It is an essential enzyme in the development of recombinant organisms that convert l-arabinose into fuels and chemicals using the fungal l-arabinose catabolic pathway. Here we report the crystal structure of LAD from the filamentous fungus Neurospora crassa at 2.6 A resolution. In addition, we created a number of site-directed variants of N. crassa LAD that are capable of utilizing NADP(+) as cofactor, yielding the first example of LAD with an almost completely switched cofactor specificity. This work represents the first structural data on any LAD and provides a molecular basis for understanding the existing literature on the substrate specificity and cofactor specificity of this enzyme. The engineered LAD mutants with altered cofactor specificity should be useful for applications in industrial biotechnology.
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HIV Fusion Peptide Penetrates, Disorders, and Softens T-Cell Membrane Mimics.
9 hours 46 min agoPublication Date: 2010 Sep 10 PMID: 20655315
Authors: Tristram-Nagle, S. - Chan, R. - Kooijman, E. - Uppamoochikkal, P. - Qiang, W. - Weliky, D. P. - Nagle, J. F.
Journal: J Mol Biol
This work investigates the interaction of N-terminal gp41 fusion peptide (FP) of human immunodeficiency virus type 1 (HIV-1) with model membranes in order to elucidate how FP leads to fusion of HIV and T-cell membranes. FP constructs were (i) wild-type FP23 (23 N-terminal amino acids of gp41), (ii) water-soluble monomeric FP that adds six lysines on the C-terminus of FP23 (FPwsm), and (iii) the C-terminus covalently linked trimeric version (FPtri) of FPwsm. Model membranes were (i) LM3 (a T-cell mimic), (ii) 1,2-dioleoyl-sn-glycero-3-phosphocholine, (iii) 1,2-dioleoyl-sn-glycero-3-phosphocholine/30 mol% cholesterol, (iv) 1,2-dierucoyl-sn-glycero-3-phosphocholine, and (v) 1,2-dierucoyl-sn-glycero-3-phosphocholine/30 mol% cholesterol. Diffuse synchrotron low-angle x-ray scattering from fully hydrated samples, supplemented by volumetric data, showed that FP23 and FPtri penetrate into the hydrocarbon region and cause membranes to thin. Depth of penetration appears to depend upon a complex combination of factors including bilayer thickness, presence of cholesterol, and electrostatics. X-ray data showed an increase in curvature in hexagonal phase 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, which further indicates that FP23 penetrates into the hydrocarbon region rather than residing in the interfacial headgroup region. Low-angle x-ray scattering data also yielded the bending modulus K(C), a measure of membrane stiffness, and wide-angle x-ray scattering yielded the S(xray) orientational order parameter. Both FP23 and FPtri decreased K(C) and S(xray) considerably, while the weak effect of FPwsm suggests that it did not partition strongly into LM3 model membranes. Our results are consistent with the HIV FP disordering and softening the T-cell membrane, thereby lowering the activation energy for viral membrane fusion.
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Crystal Structure of the GerBC Component of a Bacillus subtilis Spore Germinant Receptor.
9 hours 46 min agoPublication Date: 2010 Sep 10 PMID: 20654628
Authors: Li, Y. - Setlow, B. - Setlow, P. - Hao, B.
Journal: J Mol Biol
The nutrient germinant receptors (nGRs) of spores of Bacillus species are clusters of three proteins that play a critical role in triggering the germination of dormant spores in response to specific nutrient molecules. Here, we report the crystal structure of the C protein of the GerB germinant receptor, so-called GerBC, of Bacillus subtilis spores at 2.3 A resolution. The GerBC protein adopts a previously uncharacterized type of protein fold consisting of three distinct domains, each of which is centered by a beta sheet surrounded by multiple alpha helices. Secondary-structure prediction and structure-based sequence alignment suggest that the GerBC structure represents the prototype for C subunits of nGRs from spores of all Bacillales and Clostridiales species and defines two highly conserved structural regions in this family of proteins. GerBC forms an interlocked dimer in the crystalline state but is predominantly monomeric in solution, pointing to the possibility that GerBC oligomerizes as a result of either high local protein concentrations or interaction with other nGR proteins in spores. Our findings provide the first structural view of the nGR subunits and a molecular framework for understanding the architecture, conservation, and function of nGRs.
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Conformational Switching of the Diphtheria Toxin T Domain.
9 hours 46 min agoPublication Date: 2010 Sep 10 PMID: 20654627
Authors: Rodnin, M. V. - Kyrychenko, A. - Kienker, P. - Sharma, O. - Posokhov, Y. O. - Collier, R. J. - Finkelstein, A. - Ladokhin, A. S.
Journal: J Mol Biol
The diphtheria toxin T domain translocates the catalytic C domain across the endosomal membrane in response to acidification. To elucidate the role of histidine protonation in modulating pH-dependent membrane action of the T domain, we have used site-directed mutagenesis coupled with spectroscopic and physiological assays. Replacement of H257 with an arginine (but not with a glutamine) resulted in dramatic unfolding of the protein at neutral pH, accompanied by a substantial loss of helical structure and greatly increased exposure of the buried residues W206 and W281. This unfolding and spectral shift could be reversed by the interaction of the H257R mutant with model lipid membranes. Remarkably, this greatly unfolded mutant exhibited wild-type-like activity in channel formation, N-terminus translocation, and cytotoxicity assays. Moreover, membrane permeabilization caused by the H257R mutant occurs already at pH 6, where wild type protein is inactive. We conclude that protonation of H257 acts as a major component of the pH-dependent conformational switch, resulting in destabilization of the folded structure in solution and thereby promoting the initial membrane interactions necessary for translocation.
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Structure of the Complex between HER2 and an Antibody Paratope Formed by Side Chains from Tryptophan and Serine.
9 hours 46 min agoPublication Date: 2010 Sep 10 PMID: 20654626
Authors: Fisher, R. D. - Ultsch, M. - Lingel, A. - Schaefer, G. - Shao, L. - Birtalan, S. - Sidhu, S. S. - Eigenbrot, C.
Journal: J Mol Biol
Engineered antibody paratopes with limited sequence diversity permit assessment of the roles played by different amino acid side chains in creating the high-affinity, high-specificity interactions characteristic of antibodies. We describe a paratope raised against the human ErbB family member HER2, using a binary diversity tryptophan/serine library displayed on phage. Fab37 binds to the extracellular domain of HER2 with sub-nanomolar affinity. An X-ray structure at 3.2 A resolution reveals a contact paratope composed almost entirely of tryptophan and serine residues. Mutagenesis experiments reveal which of these side chains are more important for direct antigen interactions and which are more important for conformational flexibility. The crystal lattice contains an unprecedented trimeric arrangement of HER2 closely related to previously observed homodimers of the related epidermal growth factor receptor.
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Molecular Basis for Complement Recognition and Inhibition Determined by Crystallographic Studies of the Staphylococcal Complement Inhibitor (SCIN) Bound to C3c and C3b.
9 hours 46 min agoPublication Date: 2010 Sep 10 PMID: 20654625
Authors: Garcia, B. L. - Ramyar, K. X. - Tzekou, A. - Ricklin, D. - McWhorter, W. J. - Lambris, J. D. - Geisbrecht, B. V.
Journal: J Mol Biol
The human complement system plays an essential role in innate and adaptive immunity by marking and eliminating microbial intruders. Activation of complement on foreign surfaces results in proteolytic cleavage of complement component 3 (C3) into the potent opsonin C3b, which triggers a variety of immune responses and participates in a self-amplification loop mediated by a multi-protein assembly known as the C3 convertase. The human pathogen Staphylococcus aureus has evolved a sophisticated and potent complement evasion strategy, which is predicated upon an arsenal of potent inhibitory proteins. One of these, the staphylococcal complement inhibitor (SCIN), acts at the level of the C3 convertase (C3bBb) and impairs downstream complement function by trapping the convertase in a stable but inactive state. Previously, we have shown that SCIN binds C3b directly and competitively inhibits binding of human factor H and, to a lesser degree, that of factor B to C3b. Here, we report the co-crystal structures of SCIN bound to C3b and C3c at 7.5 and 3.5 A limiting resolution, respectively, and show that SCIN binds a critical functional area on C3b. Most significantly, the SCIN binding site sterically occludes the binding sites of both factor H and factor B. Our results give insight into SCIN binding to activated derivatives of C3, explain how SCIN can recognize C3b in the absence of other complement components, and provide a structural basis for the competitive C3b-binding properties of SCIN. In the future, this may suggest templates for the design of novel complement inhibitors based upon the SCIN structure.
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The M-Domain Controls Hsp104 Protein Remodeling Activity in an Hsp70/Hsp40-Dependent Manner.
9 hours 46 min agoPublication Date: 2010 Sep 10 PMID: 20654624
Authors: Sielaff, B. - Tsai, F. T.
Journal: J Mol Biol
Yeast Hsp104 is a ring-forming ATP-dependent protein disaggregase that, together with the cognate Hsp70 chaperone system, has the remarkable ability to rescue stress-damaged proteins from a previously aggregated state. Both upstream and downstream functions for the Hsp70 system have been reported, but it remains unclear how Hsp70/Hsp40 is coupled to Hsp104 protein remodeling activity. Hsp104 is a multidomain protein that possesses an N-terminal domain, an M-domain, and two tandem AAA(+) domains. The M-domain forms an 85-A long coiled coil and is a hallmark of the Hsp104 chaperone family. While the three-dimensional structure of Hsp104 has been determined, the function of the M-domain is unclear. Here, we demonstrate that the M-domain is essential for protein disaggregation, but dispensable for Hsp104 ATPase- and substrate-translocating activities. Remarkably, replacing the Hsp104 M-domain with that of bacterial ClpB, and vice versa, switches species specificity so that our chimeras now cooperate with the noncognate Hsp70/DnaK chaperone system. Our results demonstrate that the M-domain controls Hsp104 protein remodeling activities in an Hsp70/Hsp40-dependent manner, which is required to unleash Hsp104 protein disaggregating activity.
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Residue-Resolved Stability of Full-Consensus Ankyrin Repeat Proteins Probed by NMR.
9 hours 46 min agoPublication Date: 2010 Sep 10 PMID: 20654623
Authors: Wetzel, S. K. - Ewald, C. - Settanni, G. - Jurt, S. - Pluckthun, A. - Zerbe, O.
Journal: J Mol Biol
We investigated the stability determinants and the unfolding characteristics of full-consensus designed ankyrin repeat proteins (DARPins) by NMR. Despite the repeating sequence motifs, the resonances could be fully assigned using (2)H,(15)N,(13)C triple-labeled proteins. To remove further ambiguities, we attached paramagnetic spin labels to either end of these elongated proteins, which attenuate the resonances of the spatially closest residues. Deuterium exchange experiments of DARPins with two and three internal repeats between N- and C-terminal capping repeats (NI(2)C, NI(3)C) and NI(3)C_Mut5, where the C-cap had been reengineered, indicate that the stability of the full-consensus ankyrin repeat proteins is strongly dependent on the coupling between repeats, as the stabilized cap decreases the exchange rate throughout the whole protein. Some amide protons require more than a year to exchange at 37 degrees C, highlighting the extraordinary stability of the proteins. Denaturant-induced unfolding, followed by deuterium exchange, chemical shift change, and heteronuclear nuclear Overhauser effects, is consistent with an Ising-type description of equilibrium folding for NI(3)C_Mut5, while for native-state deuterium exchange, we postulate local fluctuations to dominate exchange as unfolding events are too slow in these very stable proteins. The location of extraordinarily slowly exchanging protons indicates a very stable core structure in the DARPins that combines hydrophobic shielding with favorable electrostatic interactions. These investigations help the understanding of repeat protein architecture and the further design of DARPins for biomedical applications where high stability is required.
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The impact of viral RNA on assembly pathway selection.
9 hours 46 min agoPublication Date: 2010 Aug 13 PMID: 20621589
Authors: Morton, V. L. - Dykeman, E. C. - Stonehouse, N. J. - Ashcroft, A. E. - Twarock, R. - Stockley, P. G.
Journal: J Mol Biol
Many single-stranded RNA viruses self-assemble their protein containers around their genomes. The roles that the RNA plays in this assembly process have mostly been ignored, resulting in a protein-centric view of assembly that is unable to explain adequately the fidelity and speed of assembly in such viruses. Using bacteriophage MS2, we demonstrate here via a combination of mass spectrometry and kinetic modelling how viral RNA can bias assembly towards only a small number of the many possible assembly pathways, thus increasing assembly efficiency. Assembly reactions have been studied in vitro using phage coat protein dimers, the known building block of the T=3 shell, and short RNA stem-loops based on the translational operator of the replicase cistron, a 19 nt fragment (TR). Mass spectrometry has unambiguously identified two on-pathway intermediates in such reactions that have stoichiometry consistent with formation of either a particle 3-fold or 5-fold axis. These imply that there are at least two sub-pathways to the final capsid. The flux through each pathway is controlled by the length of the RNA stem-loop triggering the assembly reaction and this effect can be understood in structural terms. The kinetics of intermediate formation have been studied and show steady-state concentrations for intermediates between starting materials and the T=3 shell, consistent with an assembly process in which all the steps are in equilibrium. These data have been used to derive a kinetic model of the assembly reaction that in turn allows us to determine the dominant assembly pathways explicitly, and to estimate the effect of the RNA on the free energy of association between the assembling protein subunits. The results reveal that there are only a small number of dominant assembly pathways, which vary depending on the relative ratios of RNA and protein. These results suggest that the genomic RNA plays significant roles in defining the precise assembly sub-pathway followed to create the final capsid.
MeSH Categories: Base Sequence, Capsid/chemistry, Capsid Proteins/chemistry, Kinetics, Levivirus/chemistry/genetics/physiology, Macromolecular Substances/chemistry, Mass Spectrometry, Models, Molecular, Nucleic Acid Conformation, Protein Subunits, RNA, Viral/*chemistry/genetics, Thermodynamics, Virus Assembly/genetics/*physiology
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The Role of the T7 Gp2 Inhibitor of Host RNA Polymerase in Phage Development.
9 hours 46 min agoPublication Date: 2010 Sep 10 PMID: 20650282
Authors: Savalia, D. - Robins, W. - Nechaev, S. - Molineux, I. - Severinov, K.
Journal: J Mol Biol
Bacteriophage T7 relies on its own RNA polymerase (RNAp) to transcribe its middle and late genes. Early genes, which include the viral RNAp gene, are transcribed by the host RNAp from three closely spaced strong promoters-A1, A2, and A3. One middle T7 gene product, gp2, is a strong inhibitor of the host RNAp. Gp2 is essential and is required late in infection, during phage DNA packaging. Here, we explore the role of gp2 in controlling host RNAp transcription during T7 infection. We demonstrate that in the absence of gp2, early viral transcripts continue to accumulate throughout the infection. Decreasing transcription from early promoter A3 is sufficient to make gp2 dispensable for phage infection. Gp2 also becomes dispensable when an antiterminating element boxA, located downstream of early promoters, is deleted. The results thus suggest that antiterminated transcription by host RNAp from the A3 promoter is interfering with phage development and that the only essential role for gp2 is to prevent this transcription.
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Multiplexing RMCE: Versatile Extensions of the Flp-Recombinase-Mediated Cassette-Exchange Technology.
9 hours 46 min agoPublication Date: 2010 Sep 10 PMID: 20650281
Authors: Turan, S. - Kuehle, J. - Schambach, A. - Baum, C. - Bode, J.
Journal: J Mol Biol
There are strong indications, but as yet no proof, that extended 48-bp Flp recombinase targets (FRTs) represent unique targets in all eukaryotic genomes investigated, and that recombinase-mediated cassette exchange is not hampered by the occurrence of genomic pseudo sites. This encouraged the present study in which we explore the feasibility of exchanging, in a given cell, two distinct genomically anchored cassettes, each flanked by a unique set of two heterospecific FRT sites. Mutant FRTs have to meet two major prerequisites for successful recombinase-mediated cassette exchange: (i) a self-recognition capacity comparable to a pair of FRT wild-type sites (FRTxFRT), and (ii) a negligible cross-interaction if part of a set of heterospecific sites (F'xF). We apply a two-step strategy to explore various newly created FRT spacer mutants for these properties. As a result of our screening steps, we identify combinations of sites that are successfully applied to parallel Flp-mediated genomic targeting ("multiplexing") reactions (i.e., the simultaneous exchange of two separate target cassettes in a given cell).
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Cooperative Binding of MgATP and MgADP in the Trimeric P(II) Protein GlnK2 from Archaeoglobus fulgidus.
9 hours 46 min agoPublication Date: 2010 Sep 10 PMID: 20643148
Authors: Helfmann, S. - Lu, W. - Litz, C. - Andrade, S. L.
Journal: J Mol Biol
P(II)-like proteins, such as GlnK, found in a wide variety of organisms from prokaryotes to plants constitute a family of cytoplasmic signaling proteins that play a central regulatory role in the assimilation of nitrogen for biosyntheses. They specifically bind and are modulated by effector molecules such as adenosine triphosphate, adenosine diphosphate and 2-oxoglutarate. Their highly conserved, trimeric structure suggests that cooperativity in effector binding might be the basis for the ability to integrate and respond to a wide range of concentrations, but to date no direct quantification of this cooperative behavior has been presented. The hyperthermophilic archaeon Archaeoglobus fulgidus contains three GlnK proteins, functionally associated with ammonium transport proteins (Amt). We have characterized GlnK2 and its interaction with effectors by high-resolution X-ray crystallography and isothermal titration calorimetry. Binding of adenosine nucleotides resulted in distinct, cooperative behavior for ATP and ADP. While 2-oxoglutarate has been shown to interact with other GlnK proteins, GlnK2 was completely insensitive to this key indicator of a low level of intracellular nitrogen. These findings point to different regulation and modulation patterns and add to our understanding of the flexibility and versatility of the GlnK family of signaling proteins.
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Computational Mapping of Anchoring Spots on Protein Surfaces.
9 hours 46 min agoPublication Date: 2010 Sep 10 PMID: 20643147
Authors: Ben-Shimon, A. - Eisenstein, M.
Journal: J Mol Biol
Protein-protein and protein-peptide interactions are often controlled by few strong contacts that involve hot spot residues. Computational detection of such contacts, termed here anchoring spots, is important for understanding recognition processes and for predicting interactions; it is an essential step in designing interaction interfaces and therapeutic agents. We describe ANCHORSMAP, an algorithm for computational mapping of amino acid side chains on protein surfaces. The algorithm consists of two stages: A geometry based stage (LSMdet), in which sub-pockets adequate for binding single side chains are detected and amino acid probes are scattered near them, and an energy based stage in which optimal positions of the probes are determined through repeated energy minimization and clustering of nearby poses and their DeltaG are calculated. ANCHORSMAP employs a new function for DeltaG calculations, which is specifically designed for the context of protein-protein recognition by introducing a correction in the electrostatic energy term that compensates for the dielectric shielding exerted by a hypothetical protein bound to the probe. The algorithm successfully detects known anchoring sites and accurately positions the probes. The calculated DeltaG rank high the correct anchoring spots in maps produced for unbound proteins. We find that Arg, Trp, Glu and Tyr, which are favorite hot spot residues, are also more selective of their binding environment. The usefulness of anchoring spots mapping is demonstrated by detecting the binding surfaces in the protein-protein complex barnase/barstar and the protein-peptide complex kinase/PKI, and by identifying phenylalanine anchoring sites on the surface of the nuclear transporter NTF2, C-terminus anchors on PDZ domains and phenol anchors on thermolysin. Finally, we discuss the role of anchoring spots in molecular recognition processes.
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The PAX3 Paired Domain and Homeodomain Function as a Single Binding Module In Vivo to Regulate Subnuclear Localization and Mobility by a Mechanism That Requires Base-Specific Recognition.
9 hours 46 min agoPublication Date: 2010 Sep 10 PMID: 20643146
Authors: Corry, G. N. - Raghuram, N. - Missiaen, K. K. - Hu, N. - Hendzel, M. J. - Underhill, D. A.
Journal: J Mol Biol
The transcription factor PAX3 is essential for myogenesis and neural crest development, and is one of several genes mutated in human Waardenburg syndrome. Analysis of disease-causing missense mutations in PAX3 has established the interdependence of its two DNA-binding domains, the paired domain (PD) and the homeodomain (HD), as well as defects in localization and mobility. Paradoxically, mutants that retained DNA binding activity exhibited the greatest defects in localization and mobility, regardless of the domain in which they reside. In the present study, structure-function analyses were used to determine the mechanistic basis of this effect. In the context of the isolated DNA-binding domains, HD mutants adopted an increase in mobility proportional to their loss in DNA binding, while PD mutants continued to display the inverse relationship observed in the full-length protein. At the structural level, this reflected an unexpected dependence on base-specific contacts in the PD, whereas HD mobility was more severely affected by loss of backbone contacts, as has been observed with other DNA-binding proteins. This requires that the HD switch to a base-specific mode in the full-length protein. Moreover, both domains underwent substantial reduction in mobility and altered localization when in a contiguous polypeptide with the endogenous linker segment. Notably, although the HD conferred localization to heterochromatin, this activity was masked when linked to the PD, despite the absence of determinants for subnuclear compartmentalization in the PD or linker. Last, the propensity for PAX3 heterochromatin localization was modulated by sequences at the amino and carboxy termini, supporting a model in which alternate conformations lead to unmasking of the HD. These data indicate that the PD and the HD functionally interact in vivo and behave as a single binding module whose mobility and localization are dependent on sequence-specific contacts.
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