Content
2012
Translation Elongation Factor EF-P Alleviates Ribosome Stalling at Polyproline Stretches
13.12.2012
Science,
2012,
DOI: 10.1126/science.1228985,
Vol. 339 no. 6115 pp. 82-85
published on 13.12.2012
Science, online article
Science, online article
Translation elongation factor P (EF-P) is critical for virulence in bacteria. EF-P is present in all bacteria and orthologous to archaeal and eukaryotic initiation factor 5A, yet the biological function has so far remained enigmatic. Here, we demonstrate that EF-P is an elongation factor that enhances translation of polyproline-containing proteins: In the absence of EF-P, ribosomes stall at polyproline stretches, whereas the presence of EF-P alleviates the translational stalling. Moreover, we demonstrate the physiological relevance of EF-P to fine-tune the expression of the polyproline-containing pH receptor CadC to levels necessary for an appropriate stress response. Bacterial, archaeal, and eukaryotic cells have hundreds to thousands of polyproline-containing proteins of diverse function, suggesting that EF-P and a/eIF-5A are critical for copy-number adjustment of multiple pathways across all kingdoms of life.
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Funktionalisierung αvβ3- oder α5β1-selektiver Integrinantagonisten für die Oberflächenbeschichtung: ein Hilfsmittel zur Unterscheidung von Integrinsubtypen in vitro
12.12.2012
Angewandte Chemie,
2012,
DOI: 10.1002/ange.201206370,
Volume 125, Issue 5, pages 1612–1616
published on 12.12.2012
Investigating the different functions of distinct surface receptors is essential to understand the complex interactions between cells and their extracellular environment. Cells use specific transmembrane receptors of the integrin family to anchor and respond to extracellular matrix (ECM) proteins. In doing so, integrins are capable of regulating cell migration, survival, cell cycle progression, and differentiation, which are essential tasks for the development of all multicellular organisms. Integrins are often classified according to their binding specificity for extracellular ligands.
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Structure, phosphorylation and U2AF65 binding of the N-terminal domain of splicing factor 1 during 30-splice site recognition
21.11.2012
Nucleic Acids Research,
2012,
doi:10.1093/nar/gks1097,
Vol. 41, No. 2 ,1343–1354
published on 21.11.2012
Nucleic Acids Research, online article
Nucleic Acids Research, online article
Recognition of the 30-splice site is a key step in premRNA
splicing and accomplished by a dynamic complex comprising splicing factor 1 (SF1) and the U2 snRNP auxiliary factor 65-kDa subunit (U2AF65). Both proteins mediate protein–protein and protein–RNA interactions for cooperative RNA-binding during spliceosome assembly. Here, we report the solution structure of a novel helix-hairpin domain in the N-terminal region of SF1 (SF1NTD). The nuclear magnetic resonance- and small-angle X-ray scattering-derived structure of a complex of the SF1NTD with the C-terminal U2AF homology motif domain of U2AF65 (U2AF65UHM) reveals that, in addition to the known U2AF65UHM–SF1 interaction, the helix-hairpin domain forms a secondary, hydrophobic interface with U2AF65UHM, which locks the orientation of the two subunits. Mutational analysis shows that the helix hairpin is essential for cooperative formation of the ternary SF1–U2AF65–RNA complex. We further show that tandem serine phosphorylation of a conserved Ser80-Pro81-Ser82- Pro83 motif rigidifies a long unstructured linker
in the SF1 helix hairpin. Phosphorylation does not significantly alter the overall conformations of SF1, SF1–U2AF65 or the SF1–U2AF65–RNA complexes, but slightly enhances RNA complexes, but slightly enhances RNA binding. Our results indicate that the helix-hairpin domain of SF1 is required for cooperative 30-splice site recognition presumably by stabilizing a unique quaternary arrangement of the SF1–U2AF65–RNA
complex.
N-Methylierung von Peptiden und Proteinen: ein wichtiges Element für die Regulation biologischer Funktionen
19.11.2012
Angewandte Chemie,
2012,
DOI: 10.1002/ange.201205674,
Volume 125, Issue 1, pages 268–283
published on 19.11.2012
N-Methylation is one of the simplest chemical modifications often occurring in peptides and proteins of prokaryotes and higher eukaryotes. Over years of evolution, nature has employed N-methylation of peptides as an ingenious technique to modulate biological function, often as a mode of survival through the production of antibiotics. This small structural change can not only mobilize large protein complexes (as in the histone methylation), but also inhibits the action of enzymes by selective recognition of protein–protein interaction surfaces. In recent years through the advancement in synthetic approaches, the potential of N-methylation has begun to be revealed, not only in modulating biological activity and selectivity as well as pharmacokinetic properties of peptides, but also in delivering novel drugs. Herein, we summarize the current knowledge of the versatility of N-methylation in modulating biological, structural, and pharmacokinetic properties of peptides.
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Optimal degree of protonation for 1H detection of aliphatic sites in randomly deuterated proteins as a function of the MAS frequency
24.10.2012
J Biomol NMR,
2012,
DOI 10.1007/s10858-012-9659-9,
Volume 54, Issue 2, pp 155-168
published on 24.10.2012
The 1H dipolar network, which is the major obstacle for applying proton detection in the solid-state, can be reduced by deuteration, employing the RAP (Reduced Adjoining Protonation) labeling scheme, which yields random protonation at non-exchangeable sites. We present here a systematic study on the optimal degree of random sidechain protonation in RAP samples as a function of the MAS (magic angle spinning) frequency. In particular, we compare 1H sensitivity and linewidth of a microcrystalline protein, the SH3 domain of chicken α-spectrin, for samples, prepared with 5–25 % H2O in the E. coli growth medium, in the MAS frequency range of 20–60 kHz. At an external field of 19.96 T (850 MHz), we find that using a proton concentration between 15 and 25 % in the M9 medium yields the best compromise in terms of sensitivity and resolution, with an achievable average 1H linewidth on the order of 40–50 Hz. Comparing sensitivities at a MAS frequency of 60 versus 20 kHz, a gain in sensitivity by a factor of 4–4.5 is observed in INEPT-based 1H detected 1D 1H,13C correlation experiments. In total, we find that spectra recorded with a 1.3 mm rotor at 60 kHz have almost the same sensitivity as spectra recorded with a fully packed 3.2 mm rotor at 20 kHz, even though ~20× less material is employed. The improved sensitivity is attributed to 1H line narrowing due to fast MAS and to the increased efficiency of the 1.3 mm coil.
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Structural basis for TetM-mediated tetracycline resistance
01.10.2012
PNAS,
2012,
doi: 10.1073/pnas.1208037109,
published on 01.10.2012
Ribosome protection proteins (RPPs) confer tetracycline resistance by binding to the ribosome and chasing the drug from its binding site. The current model for the mechanism of action of RPPs proposes that drug release is indirect and achieved via conformational changes within the drug-binding site induced upon binding of the RPP to the ribosome. Here we report a cryo-EM structure of the RPP TetM in complex with the 70S ribosome at 7.2-Å resolution. The structure reveals the contacts of TetM with the ribosome, including interaction between the conserved and functionally critical C-terminal extension of TetM and the decoding center of the small subunit. Moreover, we observe direct interaction between domain IV of TetM and the tetracycline binding site and identify residues critical for conferring tetracycline resistance. A model is presented whereby TetM directly dislodges tetracycline to confer resistance.
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Structural Properties of EGCG-Induced, Nontoxic Alzheimer's Disease Aβ Oligomers
24.08.2012
Journal of Molecular Biology,
2012,
doi:10.1016/j.jmb.2012.01.013,
Volume 421, Issues 4–5, Pages 517–524
published on 24.08.2012
The green tea compound epigallocatechin-3-gallate (EGCG) inhibits Alzheimer's disease β-amyloid peptide (Aβ) neurotoxicity. Solution-state NMR allows probing initial EGCG–Aβ interactions. We show that EGCG-induced Aβ 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 Aβ. The C-terminal part of the Aβ peptide (residues 22–39) adopts a β-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 Aβ 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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A Conformationally Frozen Peptoid Boosts CXCR4 Affinity and Anti-HIV Activity
06.08.2012
Angewandte Chemie,
2012,
DOI: 10.1002/anie.201202090,
Volume 51, Issue 32, pages 8110–8113
published on 06.08.2012
There can be only one: Using a peptoid motif obtained by shifting the arginine side chain of a pentapeptide previously developed by Fujii et al. to the neighboring nitrogen atom restricts the conformational freedom and yields a conformationally homogeneous peptide (see picture) with a 100-fold higher binding affinity to the chemokine receptor CXCR4 in the picomolar range. Its efficiency to inhibit HIV-1 infections is also demonstrated.
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Behavior of primary human osteoblasts on trimmed and sandblasted Ti6Al4V surfaces functionalized with integrin avb3-selective cyclic RGD peptides
24.07.2012
Journal of Biomedical Materials Research,
2012,
DOI: 10.1002/jbm.a.34303,
published on 24.07.2012
It is well known that functionalization of surfaces with cell adhesive peptides mimicking the integrin binding motif of extracellular matrix proteins is a feasible approach to improve osseointegration of implant materials. Also, modification of the surface properties of the material (e.g., roughness) strongly influences cell behavior. However, these two approaches are rarely studied together. This study addressed the hypothesis that the combination of peptide functionalization and surface roughness will have an enhancing effect on the adhesion process of osteoblasts. To test this hypothesis, a series of αvβ3-selective cyclic RGD peptides were prepared and immobilized on trimmed (Sa = 0.74 μm, smooth) and sandblasted (Sa = 3.24 μm, rough) Ti6Al4V disks. Effects of these surface modifications were evaluated with respect to integrin αvβ3-mediated adhesive capacity, cell morphology, and spreading of primary human osteoblasts. After 3 h of incubation, osteoblasts adhered more strongly on sandblasted than on trimmed noncoated Ti6Al4V surfaces. Their attachment efficiency was further enhanced in the presence of RGD peptides. However, peptide functionalization had a relatively stronger impact on osteoblast attachment on trimmed surfaces compared with sandblasted surfaces. Cell morphology after 3 h of culture was exclusively altered by surface topography. RGD coating was critical for osteoblast spreading on both trimmed and sandblasted materials after 1 h of incubation but it showed almost negligible effects after 3 h. The results of this study provide evidence that the alliance of RGD coating and surface topography on Ti6Al4V positively influences osteoblast adhesion and spreading, especially at very early adhesion times.
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CTD Tyrosine Phosphorylation Impairs Termination Factor Recruitment to RNA Polymerase II
29.06.2012
Science,
2012,
DOI: 10.1126/science.1219651,
Vol. 336 no. 6089 pp. 1723-1725
published on 29.06.2012
In different phases of the transcription cycle, RNA polymerase (Pol) II recruits various factors via its C-terminal domain (CTD), which consists of conserved heptapeptide repeats with the sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. We show that the CTD of transcribing yeast Pol II is phosphorylated at Tyr1, in addition to Ser2, Thr4, Ser5, and Ser7. Tyr1 phosphorylation stimulates binding of elongation factor Spt6 and impairs recruitment of termination factors Nrd1, Pcf11, and Rtt103. Tyr1 phosphorylation levels rise downstream of the transcription start site and decrease before the polyadenylation site, largely excluding termination factors from gene bodies. These results show that CTD modifications trigger and block factor recruitment and lead to an extended CTD code that explains transcription cycle coordination on the basis of differential phosphorylation of Tyr1, Ser2, and Ser5.
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Intestinal Permeability of Cyclic Peptides: Common Key Backbone Motifs Identified
27.06.2012
J. Am. Chem. Soc.,
2012,
DOI: 10.1021/ja303200d,
134 (29), pp 12125–12133
published on 27.06.2012
Insufficient oral bioavailability is considered as a key limitation for the widespread development of peptides as therapeutics. While the oral bioavailability of small organic compounds is often estimated from simple rules, similar rules do not apply to peptides, and even the high oral bioavailability that is described for a small number of peptides is not well understood. Here we present two highly Caco-2 permeable template structures based on a library of 54 cyclo(-d-Ala-Ala5-) peptides with different N-methylation patterns. The first (all-trans) template structure possesses two β-turns of type II along Ala6-d-Ala1 and Ala3-Ala4 and is only found for one peptide with two N-methyl groups at d-Ala1 and Ala6 [(NMe(1,6)]. The second (single-cis) template possesses a characteristic cis peptide bond preceding Ala5, which results in type VI β-turn geometry along Ala4-Ala5. Although the second template structure is found in seven peptides carrying N-methyl groups on Ala5, high Caco-2 permeability is only found for a subgroup of two of them [NMe(1,5) and NMe(1,2,4,5)], suggesting that N-methylation of d-Ala1 is a prerequisite for high permeability of the second template structure. The structural similarity of the second template structure with the orally bioavailable somatostatin analog cyclo(-Pro-Phe-NMe-d-Trp-NMe-Lys-Thr-NMe-Phe-), and the striking resemblance with both β-turns of the orally bioavailable peptide cyclosporine A, suggests that the introduction of bioactive sequences on the highly Caco-2 permeable templates may result in potent orally bioavailable drug candidates.
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Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples
25.06.2012
PNAS,
2012,
doi: 10.1073/pnas.1203013109,
published on 25.06.2012
We have developed an approach for determining NMR structures of proteins over 20 kDa that utilizes sparse distance restraints obtained using transverse relaxation optimized spectroscopy experiments on perdeuterated samples to guide RASREC Rosetta NMR structure calculations. The method was tested on 11 proteins ranging from 15 to 40 kDa, seven of which were previously unsolved. The RASREC Rosetta models were in good agreement with models obtained using traditional NMR methods with larger restraint sets. In five cases X-ray structures were determined or were available, allowing comparison of the accuracy of the Rosetta models and conventional NMR models. In all five cases, the Rosetta models were more similar to the X-ray structures over both the backbone and side-chain conformations than the “best effort” structures determined by conventional methods. The incorporation of sparse distance restraints into RASREC Rosetta allows routine determination of high-quality solution NMR structures for proteins up to 40 kDa, and should be broadly useful in structural biology.
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A Movie of RNA Polymerase II Transcription
22.06.2012
Cell,
2012,
DOI 10.1016/j.cell.2012.06.006,
Volume 149, Issue 7, 1431-1437
published on 22.06.2012
We provide here a molecular movie that captures key aspects of RNA polymerase II initiation and elongation. To create the movie, we combined structural snapshots of the initiation-elongation transition and of elongation, including nucleotide addition, translocation, pausing, proofreading, backtracking, arrest, reactivation, and inhibition. The movie reveals open questions about the mechanism of transcription and provides a useful teaching tool.
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Cell adhesion and proliferation on RGD-modified recombinant spider silk proteins
22.06.2012
Biomaterials,
2012,
http://dx.doi.org/10.1016/j.biomaterials.2012.05.069,
Volume 33, Issue 28, Pages 6650–6659
published on 22.06.2012
Due to the biocompatibility and biodegradability as well as the mechanical properties of the fibers, spider silk has become an attractive material for researchers regarding biomedical applications. In this study, the engineered recombinant spider silk protein eADF4(C16) was modified with the integrin recognition sequence RGD by a genetic (fusing the amino acid sequence GRGDSPG) as well as a chemical approach (using the cyclic peptide c(RGDfK)). Both modified silk proteins were processed into films, and thereafter characterized concerning secondary structure, water contact angle and surface roughness. No influence of the RGD-modifications on any of these film properties could be detected. However, attachment and proliferation of BALB/3T3 mouse fibroblasts were significantly improved on films made of the RGD-modified silk proteins. Interestingly, the genetically created hybrid protein (with a linear RGD sequence) showed similar or slightly better cell adhesion properties as the silk protein chemically modified with the cyclic RGD peptide.
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MC EMiNEM Maps the Interaction Landscape of the Mediator
21.06.2012
PLoS Computational Biology,
2012,
doi:10.1371/journal.pcbi.1002568,
Volume 8 , Issue 6
published on 21.06.2012
The Mediator is a highly conserved, large multiprotein complex that is involved essentially in the regulation of eukaryotic mRNA transcription. It acts as a general transcription factor by integrating regulatory signals from gene-specific activators or repressors to the RNA Polymerase II. The internal network of interactions between Mediator subunits that conveys these signals is largely unknown. Here, we introduce MC EMiNEM, a novel method for the retrieval of functional dependencies between proteins that have pleiotropic effects on mRNA transcription. MC EMiNEM is based on Nested Effects Models (NEMs), a class of probabilistic graphical models that extends the idea of hierarchical clustering. It combines mode-hopping Monte Carlo (MC) sampling with an Expectation-Maximization (EM) algorithm for NEMs to increase sensitivity compared to existing methods. A meta-analysis of four Mediator perturbation studies in Saccharomyces cerevisiae, three of which are unpublished, provides new insight into the Mediator signaling network. In addition to the known modular organization of the Mediator subunits, MC EMiNEM reveals a hierarchical ordering of its internal information flow, which is putatively transmitted through structural changes within the complex. We identify the N-terminus of Med7 as a peripheral entity, entailing only local structural changes upon perturbation, while the C-terminus of Med7 and Med19 appear to play a central role. MC EMiNEM associates Mediator subunits to most directly affected genes, which, in conjunction with gene set enrichment analysis, allows us to construct an interaction map of Mediator subunits and transcription factors.
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Dynamics in multi-domain protein recognition of RNA
20.06.2012
Structural Biology,
2012,
doi.org/10.1016/j.sbi.2012.03.013,
Volume 22, Issue 3, Pages 287–296
published on 20.06.2012
Protein–RNA interactions play essential roles in gene regulation and RNA metabolism. While high-resolution structures have revealed principles of RNA recognition by individual RNA binding domains (RBDs), the presence of multiple RBDs in many eukaryotic proteins suggests additional modes of RNA recognition by combination and cooperation of these interactions. Recent structures, together with biochemical and biophysical studies have revealed novel principles of RNA recognition by multi-domain proteins. These examples highlight an important role for dynamics in RNA recognition, with mechanisms including fly-casting and conformational selection, and advocate the use of solution techniques for their analysis.
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Structure of Mre11–Nbs1 complex yields insights into ataxia-telangiectasia–like disease mutations and DNA damage signaling
17.06.2012
Nature Structural & Molecular Biology,
2012,
doi:10.1038/nsmb.2323,
19; 693-700
published on 17.06.2012
The Mre11–Rad50–Nbs1 (MRN) complex tethers, processes and signals DNA double-strand breaks, promoting genomic stability. To understand the functional architecture of MRN, we determined the crystal structures of the Schizosaccharomyces pombe Mre11 dimeric catalytic domain alone and in complex with a fragment of Nbs1. Two Nbs1 subunits stretch around the outside of the nuclease domains of Mre11, with one subunit additionally bridging and locking the Mre11 dimer via a highly conserved asymmetrical binding motif. Our results show that Mre11 forms a flexible dimer and suggest that Nbs1 not only is a checkpoint adaptor but also functionally influences Mre11-Rad50. Clinical mutations in Mre11 are located along the Nbs1-interaction sites and weaken the Mre11-Nbs1 interaction. However, they differentially affect DNA repair and telomere maintenance in Saccharomyces cerevisiae, potentially providing insight into their different human disease pathologies.
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Lys34 of translation elongation factor EF-P is hydroxylated by YfcM
17.06.2012
Nature Chemical Biology,
2012,
doi:10.1038/nchembio.1001,
published on 17.06.2012
Lys34 of the conserved translation elongation factor P (EF-P) is post-translationally lysinylated by YjeK and YjeA—a modification that is critical for bacterial virulence. Here we show that the currently accepted Escherichia coli EF-P modification pathway is incomplete and lacks a final hydroxylation step mediated by YfcM, an enzyme distinct from deoxyhypusine hydroxylase that catalyzes the final maturation step of eukaryotic initiation factor 5A, the eukaryotic EF-P homolog.
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EMBO welcomes 3 CIPSM scientists as new members
09.05.2012
EMBO,
2012,
published on 09.05.2012
The 3 life scientists Ulrike Gaul, Andreas Ladurner and Michael Sattler from CIPSM were today recognised by EMBO for their excellence in research. EMBO elects new members annually on the basis of scientific excellence. The selected researchers will help shape the direction of the life sciences in Europe and beyond by their involvement with the activities of the organization. EMBO Members provide scientific input such as acting on advisory editorial boards of the four scientific journals of the organization, serving on selection committees for EMBO Programmes and giving general advice to the scientific community.
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An Asymmetric Dimer as the Basic Subunit in Alzheimer’s Disease Amyloid β Fibrils
08.05.2012
Angewandte Chemie,
2012,
DOI: 10.1002/anie.201200965,
Volume 51, Issue 25, pages 6136–6139,
published on 08.05.2012
Aggregation of monomeric amyloid beta peptides (ABeta) into soluble oligomers and insoluble fibrils is one of the major pathological hallmarks of Alzheimer_s disease (AD). In the past few years, magic angle spinning (MAS) solid-state NMR spectroscopy has enabled considerable progress in the structural characterization of amyloid aggregates, and a number of structural models have been suggested. Amyloid samples fibrilize in a large variety of morphologies. Differences in fibril morphology have been attributed to differences in peptide conformation. These variations in the molecular structures of Abeta fibrils might be responsible for the variations in the toxicity and deposition patterns of fibrils in AD.[5] Whereas previous NMR studies implied that in all cases a U-shaped peptide fold forms the fundamental structural unit of the fibril, cryo electron microscopy studies suggested that different Abeta fibril morphologies can be constructed from a dimeric arrangement of two conformationally distinct peptide molecules in each molecular layer of the protofilament. To date, however, there are no solid-state NMR data that supports the existence of such a structural subunit in Abeta fibrils.
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Swi2/Snf2 remodelers: hybrid views on hybrid molecular machines
16.04.2012
Structural Biology,
2012,
DOI 10.1016/j.sbi.2012.02.007,
Volume 22, Issue 2, Pages 225-233
published on 16.04.2012
Swi2/Snf2 (switch/sucrose non-fermentable) enzymes form a large and diverse class of proteins and multiprotein assemblies that remodel nucleic acid:protein complexes, using the energy of ATP hydrolysis. The core Swi2/Snf2 type ATPase domain belongs to the ‘helicase and NTP driven nucleic acid translocase’ superfamily 2 (SF2). It serves as a motor that functionally and structurally interacts with different targeting domains and functional modules to drive a plethora of remodeling activities in chromatin structure and dynamics, transcription regulation and DNA repair. Recent progress on the interaction of Swi2/Snf2 enzymes and multiprotein assemblies with their substrate nucleic acids and proteins, using hybrid structural biology methods, illuminates mechanisms for complex chemo-mechanical remodeling reactions. For Mot1, a hybrid mechanism of remodeler and chaperone emerged.
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Mechanism of Translesion Transcription by RNA Polymerase II and Its Role in Cellular Resistance to DNA Damage
13.04.2012
Molecular Cell,
2012,
DOI 10.1016/j.molcel.2012.02.006,
46,18–29,
published on 13.04.2012
UV-induced cyclobutane pyrimidine dimers (CPDs) in the template DNA strand stall transcription elongation by RNA polymerase II (Pol II). If the nucleotide excision repair machinery does not promptly remove the CPDs, stalled Pol II creates a roadblock for DNA replication and subsequent rounds of transcription. Here we present evidence that Pol II has an intrinsic capacity for translesion synthesis (TLS) that enables bypass of the CPD with or without repair. Translesion synthesis depends on the trigger loop and bridge
helix, the two flexible regions of the Pol II subunit Rpb1 that participate in substrate binding, catalysis, and translocation. Substitutions in Rpb1 that promote lesion bypass in vitro increase UV resistance in vivo, and substitutions that inhibit lesion bypass decrease cell survival after UV irradiation. Thus, translesion transcription becomes essential for cell survival upon accumulation of the unrepaired CPD lesions in genomic DNA.
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Structural and Functional Organization of the Ska Complex, a Key Component of the Kinetochore-Microtubule Interface
05.04.2012
Molecular Cell,
2012,
DOI 10.1016/j.molcel.2012.03.005,
Volume 46, Issue 3, 274-286,
published on 05.04.2012
The Ska complex is an essential mitotic component required for accurate cell division in human cells. It is composed of three subunits that function together to establish stable kinetochore-microtubule interactions in concert with the Ndc80 network. We show that the structure of the Ska core complex is a W-shaped dimer of coiled coils, formed by intertwined interactions between Ska1, Ska2, and Ska3. The C-terminal domains of Ska1 and Ska3 protrude at each end of the homodimer, bind microtubules in vitro when connected to the central core, and are essential in vivo. Mutations disrupting the central coiled coil or the dimerization interface result in chromosome congression failure followed by cell death. The Ska complex is thus endowed with bipartite and cooperative tubulin-binding properties at the ends of a 350 Å-long molecule. We discuss how this symmetric architecture might complement and stabilize the Ndc80-microtubule attachments with analogies to the yeast Dam1/DASH complex.
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Comparative Dynamic Transcriptome Analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation
30.03.2012
Genome Res.,
2012,
doi: 10.1101/gr.130161.111,
published on 30.03.2012
To monitor eukaryotic mRNA metabolism, we developed comparative Dynamic Transcriptome Analysis (cDTA). cDTA provides absolute rates of mRNA synthesis and decay in Saccharomyces cerevisiae (Sc) cells with the use of Schizosaccharomyces pombe (Sp) as internal standard. cDTA uses non-perturbing metabolic labeling that supersedes conventional methods for mRNA turnover analysis. cDTA reveals that Sc and Sp transcripts that encode orthologous proteins have similar synthesis rates, whereas decay rates are five-fold lower in Sp, resulting in similar mRNA concentrations despite the larger Sp cell volume. cDTA of Sc mutants reveals that a eukaryote can buffer mRNA levels. Impairing transcription with a point mutation in RNA polymerase (Pol) II causes decreased mRNA synthesis rates as expected, but also decreased decay rates. Impairing mRNA degradation by deleting deadenylase subunits of the Ccr4-Not complex causes decreased decay rates as expected, but also decreased synthesis rates. Extended kinetic modeling reveals mutual feedback between mRNA synthesis and degradation that may be achieved by a factor that inhibits synthesis and enhances degradation.
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Substrate Transport Activation Is Mediated through Second Periplasmic Loop of Transmembrane Protein MalF in Maltose Transport Complex of Escherichia coli
26.03.2012
The Journal of Biological Chemistry,
2012,
doi: 10.1074/jbc.M112.340679,
287, 17040-17049.
published on 26.03.2012
In a recent study we described the second periplasmic loop P2 of the transmembrane protein MalF (MalF-P2) of the maltose ATP-binding cassette transporter (MalFGK2-E) as an important element in the recognition of substrate by the maltose-binding protein MalE. In this study, we focus on MalE and find that MalE undergoes a structural rearrangement after addition of MalF-P2. Analysis of residual dipolar couplings (RDCs) shows that binding of MalF-P2 induces a semiopen state of MalE in the presence and absence of maltose, whereas maltose is retained in the binding pocket. These data are in agreement with paramagnetic relaxation enhancement experiments. After addition of MalF-P2, an increased solvent accessibility for residues in the vicinity of the maltose-binding site of MalE is observed. MalF-P2 is thus not only responsible for substrate recognition, but also directly involved in activation of substrate transport. The observation that substrate-bound and substrate-free MalE in the presence of MalF-P2 adopts a similar semiopen state hints at the origin of the futile ATP hydrolysis of MalFGK2-E.
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Dynamic Architecture of a Minimal RNA Polymerase II Open Promoter Complex
15.03.2012
Molecular Cell,
2012,
DOI 10.1016/j.molcel.2012.02.008,
published on 15.03.2012
The open promoter complex (OC) is a central intermediate during transcription initiation that contains a DNA bubble. Here, we employ single-molecule Förster resonance energy transfer experiments and Nano-Positioning System analysis to determine the three-dimensional architecture of a minimal OC consisting of promoter DNA, including a TATA box and an 11-nucleotide mismatched region around the transcription start site, TATA box-binding protein (TBP), RNA polymerase (Pol) II, and general transcription factor (TF)IIB and TFIIF. In this minimal OC, TATA-DNA and TBP reside above the Pol II cleft between clamp and protrusion domains. Downstream DNA is dynamically loaded into and unloaded from the Pol II cleft at a timescale of seconds. The TFIIB core domain is displaced from the Pol II wall, where it is located in the closed promoter complex. These results reveal large overall structural changes during the initiation-elongation transition, which are apparently accommodated by the intrinsic flexibility of TFIIB.
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Crosslinking-MS analysis reveals RNA polymerase I domain architecture and basis of rRNA cleavage
06.03.2012
Nucleic Acids Research,
2012,
doi:10.1093/nar/gks220,
1–11
published on 06.03.2012
RNA polymerase (Pol) I contains a 10-subunit catalytic core that is related to the core of Pol II and includes subunit A12.2. In addition, Pol I contains the heterodimeric subcomplexes A14/43 and A49/34.5, which are related to the Pol II subcomplex Rpb4/7 and the Pol II initiation factor TFIIF, respectively. Here we used lysine-lysine crosslinking, mass spectrometry (MS) and modeling based on five crystal structures, to extend the previous homology model of the Pol I core, to confirm the location of A14/43 and to position A12.2 and A49/34.5 on the core. In the resulting model of Pol I, the C-terminal ribbon (C-ribbon) domain of A12.2 reaches the active site via the polymerase pore, like the C-ribbon of the Pol II cleavage factor TFIIS, explaining why the intrinsic RNA cleavage activity of Pol I is strong, in contrast to the weak cleavage activity of Pol II. The A49/34.5 dimerization module resides on the polymerase lobe, like TFIIF, whereas the A49 tWH domain resides above the cleft, resembling parts of TFIIE. This indicates that Pol I and also Pol III are distantly related to a Pol II–TFIIS–TFIIF–TFIIE complex.
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Radiolabelled RGD peptides for imaging and therapy
03.03.2012
Nuclear Medicine & Molecular Imaging,
2012,
DOI 10.1007/s00259-011-2028-1,
Volume 39, Issue 1 Supplement, pp 126-138
published on 03.03.2012
Imaging of angiogenesis has become increasingly important with the rising use of targeted antiangiogenic therapies like bevacizumab (Avastin). Non-invasive assessment of angiogenic activity is in this respect interesting, e.g. for response assessment of such targeted antiangiogenic therapies. One promising approach of angiogenesis imaging is imaging of specific molecular markers of the angiogenic cascade like the integrin αvβ3. For molecular imaging of integrin expression, the use of radiolabelled peptides is still the only approach that has been successfully translated into the clinic. In this review we will summarize the current data on imaging of αvβ3 expression using radiolabelled RGD peptides with a focus on tracers already in clinical use. A perspective will be presented on the future clinical use of radiolabelled RGD peptides including an outlook on potential applications for radionuclide therapy
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Structural basis of highly conserved ribosome recycling in eukaryotes and archaea
23.02.2012
Nature,
2012,
doi:10.1038/nature10829,
482, 501-506
published on 23.02.2012
Ribosome-driven protein biosynthesis is comprised of four phases: initiation, elongation, termination and recycling. In bacteria, ribosome recycling requires ribosome recycling factor and elongation factor G, and several structures of bacterial recycling complexes have been determined. In the eukaryotic and archaeal kingdoms, however, recycling involves the ABC-type ATPase ABCE1 and little is known about its structural basis. Here we present cryo-electron microscopy reconstructions of eukaryotic and archaeal ribosome recycling complexes containing ABCE1 and the termination factor paralogue Pelota. These structures reveal the overall binding mode of ABCE1 to be similar to canonical translation factors. Moreover, the iron–sulphur cluster domain of ABCE1 interacts with and stabilizes Pelota in a conformation that reaches towards the peptidyl transferase centre, thus explaining how ABCE1 may stimulate peptide-release activity of canonical termination factors. Using the mechanochemical properties of ABCE1, a conserved mechanism in archaea and eukaryotes is suggested that couples translation termination to recycling, and eventually to re-initiation.
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Solid-phase-assisted synthesis of targeting peptide–PEG–oligo(ethane amino)amides for receptor-mediated gene delivery
23.02.2012
Organic & Biomolecular Chemistry,
2012,
DOI: 10.1039/C2OB06907E,
10, 3258-3268
published on 23.02.2012
In the forthcoming era of cancer gene therapy, efforts will be devoted to the development of new efficient and non-toxic gene delivery vectors. In this regard, the use of Fmoc/Boc-protected oligo(ethane amino)acids as building blocks for solid-phase-supported assembly represents a novel promising approach towards fully controlled syntheses of effective gene vectors. Here we report on the synthesis of defined polymers containing the following: (i) a plasmid DNA (pDNA) binding domain of eight succinoyl-tetraethylenpentamine (Stp) units and two terminal cysteine residues; (ii) a central polyethylene glycol (PEG) chain (with twenty-four oxyethylene units) for shielding; and (iii) specific peptides for targeting towards cancer cells. Peptides B6 and c(RGDfK), which bind transferrin receptor and αvβ3 integrin, respectively, were chosen because of the high expression of these receptors in many tumoral cells. This study shows the feasibility of designing these kinds of fully controlled vectors and their success for targeted pDNA-based gene transfer.
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Conservation between the RNA Polymerase I, II, and III Transcription Initiation Machineries
23.02.2012
Molecular Cell,
2012,
DOI 10.1016/j.molcel.2012.01.023,
published on 23.02.2012
Recent studies of the three eukaryotic transcription machineries revealed that all initiation complexes share a conserved core. This core consists of the RNA polymerase (I, II, or III), the TATA box-binding protein (TBP), and transcription factors TFIIB, TFIIE, and TFIIF (for Pol II) or proteins structurally and functionally related to parts of these factors (for Pol I and Pol III). The conserved core initiation complex stabilizes the open DNA promoter complex and directs initial RNA synthesis. The periphery of the core initiation complex is decorated by additional polymerase-specific factors that account for functional differences in promoter recognition and opening, and gene class-specific regulation. This review outlines the similarities and differences between these important molecular machines.
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Target- and Resistance-Based Mechanistic Studies with TP-434, a Novel Fluorocycline Antibiotic
21.02.2012
Antimicrobial Agents and Chemotherapy,
2012,
doi:10.1128/AAC.06187-11,
May 2012 vol. 56 no. 5 2559-2564
published on 21.02.2012
TP-434 is a novel, broad-spectrum fluorocycline antibiotic with activity against bacteria expressing major antibiotic resistance mechanisms, including tetracycline-specific efflux and ribosomal protection. The mechanism of action of TP-434 was assessed using both cell-based and in vitro assays. In Escherichia coli cells expressing recombinant tetracycline resistance genes, the MIC of TP-434 (0.063 μg/ml) was unaffected by tet(M), tet(K), and tet(B) and increased to 0.25 and 4 μg/ml in the presence of tet(A) and tet(X), respectively. Tetracycline, in contrast, was significantly less potent (MIC ≥ 128 μg/ml) against E. coli cells when any of these resistance mechanisms were present. TP-434 showed potent inhibition in E. coli in vitro transcription/translation (50% inhibitory concentration [IC50] = 0.29 ± 0.09 μg/ml) and [3H]tetracycline ribosome-binding competition (IC50 = 0.22 ± 0.07 μM) assays. The antibacterial potencies of TP-434 and all other tetracycline class antibiotics tested were reduced by 4- to 16-fold, compared to that of the wild-type control strain, against Propionibacterium acnes strains carrying a 16S rRNA mutation, G1058C, a modification that changes the conformation of the primary binding site of tetracycline in the ribosome. Taken together, the findings support the idea that TP-434, like other tetracyclines, binds the ribosome and inhibits protein synthesis and that this activity is largely unaffected by the common tetracycline resistance mechanisms.
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A structural perspective on Mediator function
15.02.2012
Science Direct,
2012,
DOI 10.1016/j.molcel.2012.01.023,
24:1–9
published on 15.02.2012
Gene transcription by RNA polymerase II requires the multiprotein coactivator complex Mediator. Mediator was identified two decades ago, but its molecular mechanisms remain poorly understood, because structural studies are hampered by its large size, modularity, and flexibility. Here we collect all available structural data on Mediator and discuss their functional implications. Progress was made in understanding the interactions of Mediator with gene-specific transcriptional regulators and the general transcription machinery. However, around 80% of the Mediator structure remains unknown and details on the Mediator–Pol II interface are lacking. In the future, an integrated structural biology approach may unravel the functional architecture of Mediator-regulated promoter assemblies and holds the promise of understanding a key mechanism of gene regulation.
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hnRNP A1 Proofreads 30 Splice Site Recognition by U2AF
10.02.2012
Molecular Cell,
2012,
10.1016/j.molcel.2011.11.033,
Volume 45, Issue 3, 314-329
published on 10.02.2012
One of the earliest steps in metazoan pre-mRNA splicing involves binding of U2 snRNP auxiliary factor (U2AF) 65 KDa subunit to the polypyrimidine (Py) tract and of the 35 KDa subunit to the invariant AG dinucleotide at the intron 3′ end. Here we use in vitro and in vivo depletion, as well as reconstitution assays using purified components, to identify hnRNP A1 as an RNA binding protein that allows U2AF to discriminate between pyrimidine-rich RNA sequences followed or not by a 3′ splice site AG. Biochemical and NMR data indicate that hnRNP A1 forms a ternary complex with the U2AF heterodimer on AG-containing/uridine-rich RNAs, while it displaces U2AF from non-AG-containing/uridine-rich RNAs, an activity that requires the glycine-rich domain of hnRNP A1. Consistent with the functional relevance of this activity for splicing, proofreading assays reveal a role for hnRNP A1 in U2AF-mediated recruitment of U2 snRNP to the pre-mRNA.
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Synthesis of N-methylated cyclic peptides
09.02.2012
Nature Protocols,
2012,
doi:10.1038/nprot.2011.450,
7, 432–444
published on 09.02.2012
This protocol presents a detailed description of the synthesis of N-methylated cyclic peptides. N-methylation is a powerful technique to modulate the physicochemical properties of peptides by introducing one or more methyl groups into the peptidic amide bonds. Together with peptide cyclization, this procedure confers unprecedented pharmacokinetic properties to the peptides, including metabolic stability, membrane permeability and even oral bioavailability. Here we describe two simplified methods of N-methylation of linear peptides on solid supports, which can be performed in less than 2 h and are applicable to any amino acid. Finally, we also describe two methods of peptide cyclization, which can be used to obtain the N-methylated cyclic peptide and which are not limited to specific peptide sequences.
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Processive RNA decay by the exosome
01.02.2012
RNA Biology,
2011,
doi: 10.4161/rna.8.1.14067,
Volume 8:1, pages: 55-60
published on 01.02.2012
RNA exosomes are large multi-subunit protein complexes involved in controlled and processive 3' to 5' RNA degradation. Exosomes form large molecular chambers and harbor multiple nuclease sites as well as RNA
binding regions. This makes a quantitative kinetic analysis of RNA degradation with reliable parameter and error
estimates challenging. For instance, recent quantitative biochemical assays revealed that degradation speed and
efficiency depend on various factors, such as the type of RNA binding caps and the RNA length. We propose the
combination of a differential equation model with Bayesian Markov Chain Monte Carlo (MCMC) sampling for a more robust and reliable analysis of such complex kinetic systems. Using the exosome as a paradigm, it is shown
that conventional “best fit” approaches to parameter estimation are outperformed by the MCMC method. The
parameter distribution returned by MCMC sampling allows for a reliable and meaningful comparison of the data
from different time series. In the case of the exosome, we find that the cap structures of the exosome have a direct
effect on the recruitment and degradation of RNA, and that these effects are RNA length-dependent. The described
approach can be widely applied to any processive reaction with a similar kinetics like the XRN1-dependent RNA
degradation, RNA/DNA synthesis by polymerases and protein synthesis by the ribosome.
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Characterization of Membrane Proteins in Isolated Native Cellular Membranes by Dynamic Nuclear Polarization Solid-State NMR Spectroscopy without Purification and Reconstitution
09.01.2012
Angewandte Chemie,
2012,
DOI: 10.1002/anie.201104987,
Volume 51, Issue 2, pages 432–435
published on 09.01.2012
Structural information is key for understanding biological processes. Insoluble proteins, like membrane proteins and amyloid fibrils, are a large class of proteins that are underrepresented in the protein data bank (PDB). As of today, only 7% of all entries in the PDB refer to either a membrane protein or an amyloid fibril structure (membrane protein: 4994 entries; amyloid fibril: 67 entries; total number of entries: 70,303; http://www.rcsb.org/pdb/home/home.do). Given the fact that many drugs target membrane proteins, involved in signal transduction,[1] structural information is highly desirable for a better understanding of the underlying biochemical mechanisms.
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Synthesis and Conformational Analysis of Efrapeptins
09.01.2012
Chemistry,
2012,
DOI: 10.1002/chem.201102134,
Volume 18, Issue 2, pages 478–487
published on 09.01.2012
The efrapeptin family of peptide antibiotics produced by the fungus Tolypocladium niveum, and the neo-efrapeptins from the fungus Geotrichum candidumare inhibitors of F1-ATPase with promising antitumor, antimalaria, and insecticidal activity. They are rich in Cα-dialkyl amino acids (Aib, Iva, Acc) and contain one β-alanine and several pipecolic acid residues. The C-terminus bears an unusual heterocyclic cationic cap. The efrapeptins C–G and three analogues of efrapeptin C were synthesized using α-azido carboxylic acids as masked amino acid derivatives. All compounds display inhibitory activity toward F1-ATPase. The conformation in solution of the peptides was investigated with electronic CD spectroscopy, FT-IR spectroscopy, and VCD spectroscopy. All efrapeptins and most efrapeptin analogues were shown to adopt helical conformations in solution. In the case of efrapeptin C, VCD spectra proved that a 310-helix prevails. In addition, efrapeptin C was conformationally studied in detail with NMR and molecular modeling. Besides NOE distance restraints, residual dipolar couplings (RDC) observed upon partial alignment with stretched PDMS gels were used for the conformational analysis and confirmed the 310-helical conformation.
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Chromodomains read the arginine code of post-translational targeting
08.01.2012
Nature Structural & Molecular Biology,
2011,
doi:10.1038/nsmb.2196,
19, 260–263
published on 08.01.2012
Chromodomains typically recruit protein complexes to chromatin and read the epigenetic histone code by recognizing lysine methylation in histone tails. We report the crystal structure of the chloroplast signal recognition particle (cpSRP) core from Arabidopsis thaliana, with the cpSRP54 tail comprising an arginine-rich motif bound to the second chromodomain of cpSRP43. A twinned aromatic cage reads out two neighboring nonmethylated arginines and adapts chromodomains to a non-nuclear function in post-translational targeting.
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The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop
06.01.2012
The EMBO Journal,
2012,
doi:10.1038/emboj.2011.472,
published on 06.01.2012
The EMBO Journal, online article
The EMBO Journal, online article
Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90- binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous a-helical folds as determined by NMR. These, and especially
DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A– TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal
domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1–DP1 module may serve as an Hsp70–client delivery system for the TPR2A–TPR2B–DP2 segment, which is required for client activation in vivo.
Solution Structure and Molecular Interactions of Lamin B Receptor Tudor Domain
06.01.2012
The Journal of Biological Chemistry,
2012,
VOL. 287, NO. 2, DOI 10.1074/jbc.M111.281303,
pp. 1032–1042
published on 06.01.2012
Lamin B receptor (LBR) is a polytopic protein of the nuclear envelope thought to connect the inner nuclear membrane with the underlying nuclear lamina and peripheral heterochromatin. To better understand the function of this protein, we have examined in detail its nucleoplasmic region, which is predicted to harbor a Tudor domain (LBR-TD). Structural analysis by multidimensional NMR spectroscopy establishes that LBR-TD indeed adopts a classical beta-barrel Tudor fold in solution, which, however, features an incomplete aromatic cage. Removal of LBR-TD renders LBR more mobile at the plane of the nuclear envelope, but the isolated module does not bind to nuclear lamins, heterochromatin proteins (MeCP2), and nucleosomes, nor does it associate with methylated Arg/Lys residues through its aromatic cage. Instead, LBR-TD exhibits tight and stoichiometric binding to the “histone-fold” region of unassembled, free histone H3, suggesting an interesting role in histone assembly. Consistent with such a role, robust binding to native nucleosomes is observed whenLBR-TDis extended toward its carboxyl terminus, to include an area rich in Ser-Arg residues. The Ser-Arg region, alone or in combination with LBR-TD, binds both unassembled and assembled H3/H4 histones, suggesting that the TD/RS interface may operate as a “histone chaperone-like platform.
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Structural and Mechanistic Implications of Metal-Binding in the Small Heat-Shock Protein αB-Crystallin
06.01.2012
Journal of Biological Chemistry,
2012,
DOI 10.1074/jbc.M111.309047,
VOL. 287, NO. 2, pp. 1128–1138,
published on 06.01.2012
The human small heat-shock protein alphaB-crystallin (alphaB) rescues misfolded proteins from irreversible aggregation during
cellular stress. Binding of Cu(II) was shown to modulate the
oligomeric architecture and the chaperone activity of alphaB. However, the mechanistic basis of this stimulation is so far not
understood. We provide here first structural insights into this
Cu(II)-mediated modulation of chaperone function using NMR
spectroscopy and other biophysical approaches. We show that
the alpha-crystallin domain is the elementary Cu(II)-binding unit
specifically coordinating one Cu(II) ion with picomolar binding
affinity. Putative Cu(II) ligands are His83, His104, His111, and
Asp109 at the dimer interface. These loop residues are conserved among different metazoans, but also for human alphaA-crystallin, HSP20, and HSP27. The involvement of Asp109 has direct implications for dimer stability, because this residue forms a salt bridge with the disease-related Arg120 of the neighboring monomer. Furthermore, we observe structural reorganization of strands beta2-beta3 triggered by Cu(II) binding. This N-terminal region is known to mediate both the intermolecular arrangement in_B oligomers and the binding of client proteins. In the presence of Cu(II), the size and the heterogeneity of alphaB multimers are increased. Atthesametime, Cu(II) increases the chaperone activity
of alphaB toward the lens-specific protein betaL-crystallin. We therefore suggest that Cu(II) binding unblocks potential client binding sites and alters quaternary dynamics of both the dimeric building block as well as the higher order assemblies of alphaB.
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Ultra-high resolution in MAS solid-state NMR of perdeuterated proteins: Implications for structure and dynamics
05.01.2012
Journal of Magnetic Resonance,
2012,
http://dx.doi.org/10.1016/j.jmr.2011.12.017,
Volume 216,Pages 1–12
published on 05.01.2012
High resolution proton spectra are obtained in MAS solid-state NMR in case samples are prepared using perdeuterated protein and D2O in the recrystallization buffer. Deuteration reduces drastically 1H, 1H dipolar interactions and allows to obtain amide proton line widths on the order of 20 Hz. Similarly, high-resolution proton spectra of aliphatic groups can be obtained if specifically labeled precursors for biosynthesis of methyl containing side chains are used, or if limited amounts of H2O in the bacterial growth medium is employed. This review summarizes recent spectroscopic developments to access structure and dynamics of biomacromolecules in the solid-state, and shows a number of applications to amyloid fibrils and membrane proteins.
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Sensing of viral nucleic acids by RIG-I: From translocation to translation
01.01.2012
European Journal of Cell Biology,
2012,
doi:10.1016/j.ejcb.2011.01.015,
Volume 91, Issue 1, January 2012, Pages 78–85
published on 01.01.2012
The innate immune system is a first layer of defense against infection by pathogens. It responds to pathogens by activating host defense mechanisms via interferon and inflammatory cytokine expression. Pathogen associated molecular patterns (PAMPs) are sensed by specific pattern recognition receptors. Among those, the ATP dependent helicase related RIG-I like receptors RIG-I, MDA5 and LGP2 sense the presence of viral RNA in the cytoplasm of host cells. While the precise PAMPs and functions of MDA5 or LGP2 are still unclear, RIG-I senses predominantly viral RNA containing a 5´-triphosphate along with dsRNA regions. Here we review our current knowledge of how these PAMPs are sensed and integrated by RIG-I, and how RIG-I’s innate immune function can be used in translational medical approaches.
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