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Membrane Docking Geometry and Target Lipid Stoichiometry of Membrane-Bound PKCalpha C2 Domain: A Combined Molecular Dynamics and Experimental Study.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 23 PMID: 20659476
Authors: Lai, C. L. - Landgraf, K. E. - Voth, G. A. - Falke, J. J.
Journal: J Mol Biol
Protein kinase Calpha (PKCalpha) possesses a conserved C2 domain (PKCalpha C2) that acts as a Ca(2+)-regulated membrane targeting element. Upon activation by Ca(2+), PKCalpha C2 directs the kinase protein to the plasma membrane, thereby stimulating an array of cellular pathways. At sufficiently high Ca(2+)concentrations the binding of the C2 domain to the target lipid phosphatidylserine (PS) is sufficient to drive membrane association, but at typical physiological Ca(2+)concentrations binding both to PS and to phosphoinositidyl-4,5-bisphosphate (PIP(2)) is required for specific plasma membrane targeting. Recent EPR studies have revealed the membrane docking geometries of PKCalpha C2 docked to (i) PS alone, and to (ii) both PS and PIP(2) simultaneously. These two EPR docking geometries exhibit significantly different tilt angles relative to the plane of the membrane, presumably induced by the large size of the PIP(2) headgroup. The present study utilizes the two EPR docking geometries as starting points for molecular dynamics simulations that investigate the atomic features of the protein-membrane interaction. The simulations yield approximately the same PIP(2)-triggered change in tilt angle observed by EPR. Moreover, the simulations predict a PIP(2):C2 stoichiometry approaching 2:1 at high PIP(2) mole density. Direct binding measurements titrating the C2 domain with PIP(2) in lipid bilayers yield a 1:1 stoichiometry at moderate mole densities, and a saturating 2:1 stoichiometry at high PIP(2) mole densities. Thus, experiment confirms the target lipid stoichiometry predicted by EPR-guided molecular dynamics simulations. Potential biological implications of the observed docking geometries and PIP(2) stoichiometries are discussed.
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Molecular basis for the structural stability of an enclosed beta barrel loop.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 22 PMID: 20655928
Authors: Tian, P. - Bernstein, H. D.
Journal: J Mol Biol
We present molecular dynamics (MD) simulation studies of the structural stability of an enclosed loop in the beta domain of the E. coli O157:H7 autotransporter EspP. Our investigation revealed that in addition to its excellent resistance to thermal perturbations, EspP loop5(L5) also has remarkable mechanical stability against pulling forces along the membrane norm. These findings are consistent with the experimental report that EspP L5 helps to maintain the permeability barrier in the outer membrane(OM). In contrast to major secondary structure elements of globular proteins such as ubiquitin, whose resistance to thermal and mechanical perturbations depend mainly on backbone hydrogen bonds and hydrophobic interactions, the structural stability of EspP L5 can be attributed mainly to geometric constraints and side chain interactions dominated by hydrogen bonds. Examination of the B factors from available high resolution structures of membrane embedded beta barrels (MEBBs) indicates that most of the enclosed loops have stable structures. This finding suggests that loops stabilized by geometric constraints and side chain interactions might be used more generally to restrict beta barrel channels for various functional purposes.
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Structural Conservation of Components in the Amino Acid Sensing Branch of the TOR Pathway in Yeast and Mammals.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 22 PMID: 20655927
Authors: Kogan, K. - Spear, E. D. - Kaiser, C. A. - Fass, D.
Journal: J Mol Biol
The highly conserved Rag family GTPases have a role in reporting amino acid availability to the TOR signaling complex, which regulates cell growth and metabolism in response to environmental cues. The yeast Rag proteins, Gtr1p and Gtr2p, were shown in multiple independent studies to interact with the membrane-associated proteins Gse1p (Ego3p) and Gse2p (Ego1p). However, mammalian orthologs of Gse1p and Gse2p could not be identified. We determined the crystal structure of Gse1p and found it to match the fold of two mammalian proteins, MP1 and p14, which form a heterodimeric complex that had been assigned a scaffolding function in mitogen-activated protein (MAP) kinase pathways. The significance of this structural similarity is validated by the recent identification of a physical and functional association between mammalian Rag proteins and MP1/p14. Taken together, these findings reveal that key components of the TOR signaling pathway are structurally conserved between yeast and mammals, despite divergence of sequence to a degree that thwarts detection through simple homology searches.
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Cysteine to serine mutants dramatically reorder the active site of human ABO(H) blood group B glycosyltransferase without affecting activity: Structural insights into cooperative substrate binding.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 22 PMID: 20655926
Authors: Schuman, B. - Persson, M. - Landry, R. C. - Polakowski, R. - Weadge, J. T. - Seto, N. O. - Borisova, S. N. - Palcic, M. M. - Evans, S. V.
Journal: J Mol Biol
A common feature in the structures of GT-A fold type glycosyltransferases is a mobile polypeptide loop that has been observed to participate in substrate recognition and enclose the active site upon substrate binding. This is the case for the human ABO(H) blood group B glycosyltransferase GTB, where amino acid residues 177-195 display significantly higher levels of disorder in the unliganded state than the fully-liganded state. Structural studies of mutant enzymes GTB/C80S/C196S and GTB/C80S/C196S/C209S at resolutions ranging from 1.93 A to 1.40 A display the opposite trend, where the unliganded structures show nearly complete ordering of the mobile loop residues that is lost upon substrate binding. In the liganded states of the mutant structures, while the UDP moiety of the donor molecule is observed to bind in the expected location, the galactose moiety is observed to bind in a conformation significantly different than observed for the wild type chimeric structures. Although this would be expected to impede catalytic turnover, the kinetics of the transfer reaction are largely unaffected. These structures demonstrate that the enzymes bind the donor in a conformation more similar to the dominant solution rotamer and facilitate its gyration into the catalytically competent form. Further, by preventing active site closure, these structures provide a basis for recently observed cooperativity in substrate binding. Finally, the mutation of C80S introduces a fully-occupied UDP binding site at the enzyme dimer interface that is observed to be dependent on the binding of H antigen acceptor analog.
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Structure and Engineering of L-Arabinitol 4-Dehydrogenase from Neurospora crassa.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 21 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 to L-xylulose with concomitant NAD(+)reduction. It is an essential enzyme in the development of recombinant organisms that convert L-arabinose to fuels and chemicals using the fungal L-arabinose catabolic pathway. Here we report the crystal structure of LAD from the filamentous fungus Neurospora crassa (ncLAD) at 2.6 A resolution. In addition, we created a number of site-directed variants of ncLAD that are capable of utilizing NADP(+)as a cofactor, yielding the first example of an LAD with almost completely switched cofactor specificity. This work represents the first structural data on any LAD and provides a molecular basis for understanding the existent literature on the substrate 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.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 21 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 HIV-1 virus with model membranes in order to elucidate how FP leads to fusion of HIV and T-cell membranes. FP constructs were (i) wildtype 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) DOPC, (iii) DOPC/30(mole%)cholesterol, (iv) diC22:1PC and (v) diC22:1PC/ 30(mole%)cholesterol. Diffuse synchrotron low-angle x-ray scattering (LAXS) 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 DOPE which further indicates that FP23 penetrates into the hydrocarbon region rather than residing in the interfacial headgroup region. LAXS data also yielded bending moduli K(C), a measure of membrane stiffness, and wide-angle x-ray scattering (WAXS) 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 fusion peptide 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.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 20 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.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 20 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 WT-like activity in channel formation, N-terminus translocation, and cytotoxicity assays. Moreover, membrane permeabilization caused by 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.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 20 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 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 3.2A 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 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.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 20 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 fH and fB. 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 the Hsp104 Protein-remodeling Activity in an Hsp70/Hsp40-dependent Manner.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 20 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 up-stream and down-stream functions for the Hsp70 system have been reported, but it remains unclear how Hsp70/Hsp40 is coupled to the Hsp104 protein remodeling activity. Hsp104 is a multi-domain 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 3D 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 the Hsp104 ATPase and substrate translocating activities. Remarkably, replacing the Hsp104 M-domain against that of bacterial ClpB, and vice versa, switches the species-specificity so that our chimeras now cooperate with the non-cognate Hsp70/DnaK chaperone system. Our results demonstrate that the M-domain controls the Hsp104 protein remodeling activities in an Hsp70/Hsp40-dependent manner, which is required to unleash the Hsp104 protein disaggregating activity.
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Residue-resolved stability of full-consensus ankyrin repeat proteins probed by NMR.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 20 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 (1)H, (15)N, (13)C triple labeled proteins. To remove further ambiguities, paramagnetic spin labels were attached to either end of these elongated proteins which attenuate the resonances of the spatially closest residues. Deuterium exchange experiments of DARPins with 2 and 3 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 indicate a very stable core structure in the DARPins which 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 unique binding mode of the Cellulosomal CBM4 from Clostridium thermocellum Cellobiohydrolase A.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 20 PMID: 20654622
Authors: Alahuhta, M. - Xu, Q. - Bomble, Y. J. - Brunecky, R. - Adney, W. S. - Ding, S. Y. - Himmel, M. E. - Lunin, V. V.
Journal: J Mol Biol
The crystal structure of the CBM4-Ig fused domain from the cellulosomal cellulase, cellobiohydrolase A (CbhA) of Clostridium thermocellum, was solved in complex with cellobiose at 2.11 A resolution. This is the first cellulosomal CBM4 crystal structure reported to date. It is similar to the previously solved non-cellulosomal soluble oligosaccharide binding CBM4 structures. However, this new structure possesses a significant feature- a binding site peptide loop with a tryptophan (Trp118) residing midway in the loop. Based on sequence alignment, this structural feature might be common in all cellulosomal Clostridial CBM4 modules. Our results indicate that the C. thermocellum CbhA CBM4 also has an extended binding pocket that can optimally bind to cellodextrins containing five or more sugar units. Molecular dynamics simulations and experimental binding studies with the Trp118Ala mutant suggest that Trp118 contributes to the binding and possibly the orientation of the module to soluble cellodextrins. Furthermore, the binding cleft aromatic residues, Trp68 and Tyr110, play a crucial role in binding to bacterial microcrystalline cellulose (BMCC), amorphous cellulose and soluble oligodextrins. The binding to BMCC is in disagreement with the structural features of the binding pocket, which does not support binding to the flat surface of crystalline cellulose, suggesting that CBM4 binds the amorphous part or the cellulose "whiskers" of BMCC. We propose that Clostridial CBM4s have possibly evolved to bind the free chain ends of crystalline cellulose in addition to their ability to bind soluble cellodextrins.
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Structure-based prediction of the peptide sequence space recognized by natural and synthetic PDZ domains.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 20 PMID: 20654621
Authors: Smith, C. A. - Kortemme, T.
Journal: J Mol Biol
Protein-protein recognition, frequently mediated by members of large families of interaction domains, is one of the cornerstones of biological function. Here we present a computational, structure-based method to predict the sequence space of peptides recognized by PDZ domains, one of the largest families of recognition proteins. As a test set, we use the considerable amount of recent phage display data that describe the peptide recognition preferences for 169 naturally occurring and engineered PDZ domains. For both wild-type PDZ domains and single point mutants, we find that 70-80% of the most frequently observed amino acids by phage display are predicted within the top 5 ranked amino acids. Phage display frequently identified recognition preferences for amino acids different from those present in the original crystal structure. Notably, in about half of these cases, our algorithm correctly captures these preferences, indicating that it can predict mutations that increase binding affinity relative to the starting structure. We also find that we can computationally recapitulate specificity changes upon mutation, a key test for successful forward design of protein-protein interface specificity. Across all evaluated datasets, we find that incorporation backbone sampling improves accuracy substantially, irrespective of using a crystal or NMR structure as the starting conformation. Finally, we report successful prediction of several amino acid specificity changes from blind tests in the DREAM4 peptide recognition domain specificity prediction challenge. Because the foundational methods developed here are structure based, these results suggest they can be more generally applied to specificity prediction and redesign of other protein-protein interfaces that have structural information but lack phage display data.
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The Impact of Viral RNA on Assembly Pathway Selection.
Journal of Molecular Biology - 16 hours 19 min ago
Publication 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.
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The role of the T7 Gp2 inhibitor of host RNA polymerase in phage development.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 19 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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RMCE-multiplexing: Versatile extensions of the Flp-Recombinase-Mediated Cassette-Exchange technology.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 19 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 sites (FRTs) represent unique targets in all eukaryotic genomes that have been investigated, and that recombinase-mediated cassette echange (Flp-RMCE) is not hampered by the occurence 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 RMCE, i-a self-recognition capacity comparable to a pair of FRT wildtype sites (FRT x FRT, abbreviated F x F) and ii - a negligible crossinteraction if part of a set of heterospecific sites (F x F). We apply a two-step strategy to explore various newly created FRT spacer mutans for these properties. As a result of our screening steps we identify combinations of sites, which 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.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 17 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.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 17 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.
Journal of Molecular Biology - 16 hours 19 min ago
Publication Date: 2010 Jul 17 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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