Content
2011
On the specificity of antibiotics targeting the large ribosomal subunit
22.12.2011
Annals of the New York Academy of Sciences,
2011,
DOI: 10.1111/j.1749-6632.2011.06192.x,
Volume 1241, pages 1–16,
published on 22.12.2011
The peptidyltransferase center of the large ribosomal subunit is responsible for catalyzing peptide bonds. This active site is the target of a variety of diverse antibiotics, many of which are used clinically. The past decade has seen a plethora of structures of antibiotics in complex with the large ribosomal subunit, providing unprecedented insight into the mechanism of action of these inhibitors. Ten distinct antibiotics (chloramphenicol, clindamycin, linezolid, tiamulin, sparsomycin, and five macrolides) have been crystallized in complex with four distinct ribosomal species, three bacterial, and one archaeal. This review aims to compare these structures in order to provide insight into the conserved and species-specific modes of interaction for particular members of each class of antibiotics. Coupled with the wealth of biochemical data, a picture is emerging defining the specific functional states of the ribosome
that antibiotics preferentially target. Such mechanistic insight into antibiotic inhibition will be important for the development of the next generation of antimicrobial agents.
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Tailoring of Integrin Ligands: Probing the Charge Capability of the Metal Ion-Dependent Adhesion Site
19.12.2011
J. Med. Chem.,
2011,
DOI: 10.1021/jm2013826,
55 (2), pp 871–882
published on 19.12.2011
Intervention in integrin-mediated cell adhesion and integrin signaling pathways is an ongoing area of research in medicinal chemistry and drug development. One key element in integrin–ligand interaction is the coordination of the bivalent cation at the metal ion-dependent adhesion site (MIDAS) by a carboxylic acid function, a consistent feature of all integrin ligands. With the exception of the recently discovered hydroxamic acids, all bioisosteric attempts to replace the carboxylic acid of integrin ligands failed. We report that phosphinates as well as monomethyl phosphonates represent excellent isosters, when introduced into integrin antagonists for the platelet integrin αIIbβ3. The novel inhibitors exhibit in vitro and ex vivo activities in the low nanomolar range. Steric and charge requirements of the MIDAS region were unraveled, thus paving the way for an in silico prediction of ligand activity and in turn the rational design of the next generation of integrin antagonists.
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Spectroscopically Well-Characterized RGD Optical Probe as a Prerequisite for Lifetime-Gated Tumor Imaging
15.12.2011
Molecular Imaging,
2011,
DOI 10.2310/7290.2011.00018,
Vol 10, No 6, pp 469–480
published on 15.12.2011
Labeling of RGD peptides with near-infrared fluorophores yields optical probes for noninvasive imaging of tumors overexpressing alpha-v-beta3 integrins. An important prerequisite for optimum detection sensitivity in vivo is strongly absorbing and highly emissive probes with a known fluorescence lifetime. The RGD-Cy5.5 optical probe was derived by coupling Cy5.5 to a cyclic arginine–glycine–aspartic acid–D-phenylalanine–lysine (RGDfK) peptide via an aminohexanoic acid spacer. Spectroscopic properties of the probe were studied in different matrices in comparison to Cy5.5. For in vivo imaging, human glioblastoma cells were subcutaneously implanted into nude mice, and in vivo fluorescence intensity and lifetime were measured. The fluorescence quantum yield and lifetime of Cy5.5 were found to be barely affected on RGD conjugation but dramatically changed in the presence of proteins. By time domain fluorescence imaging, we demonstrated specific binding of RGD-Cy5.5 to glioblastoma xenografts in nude mice. Discrimination of unspecific fluorescence by lifetime-gated analysis further enhanced the detection sensitivity of RGD-Cy5.5-derived signals. We characterized RGD-Cy5.5 as a strongly emissive and stable probe adequate for selective targeting of alpha-v-beta3 integrins. The specificity and thus the overall detection sensitivity in vivo were optimized with lifetime gating, based on the previous determination of the probes fluorescence lifetime under application-relevant conditions.
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Assignment strategies for aliphatic protons in the solid-state in randomly protonated proteins
04.12.2011
Journal of Biomolecular NMR,
2011,
DOI: 10.1007/s10858-011-9591-4,
Volume 52, Number 1, 31-39
published on 04.12.2011
Biological solid-state nuclear magnetic resonance spectroscopy developed rapidly in the past two decades and emerged as an important tool for structural biology. Resonance assignment is an essential prerequisite for structure determination and the characterization of motional properties of a molecule. Experiments, which rely on carbon or nitrogen detection, suffer, however, from low sensitivity. Recently, we introduced the RAP (Reduced Adjoining Protonation) labeling scheme, which allows to detect backbone and sidechain protons with high sensitivity and resolution. We present here a 1H-detected 3D (H)CCH experiment for assignment of backbone and sidechain proton resonances. Resolution is significantly improved by employing simultaneous 13CO and 13Cβ J-decoupling during evolution of the 13Cα chemical shift. In total, ~90% of the 1Hα-13Cα backbone resonances of chicken α-spectrin SH3 could be assigned.
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Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins
20.11.2011
Nature Structural & Molecular Biology,
2011,
doi:10.1038/nsmb.2185,
published on 20.11.2011
Arginine dimethylation plays critical roles in the assembly of ribonucleoprotein complexes in pre-mRNA splicing and piRNA pathways. We report solution structures of SMN and SPF30 Tudor domains bound to symmetric and asymmetric dimethylated arginine (DMA) that is inherent in the RNP complexes. An aromatic cage in the Tudor domain mediates dimethylarginine recognition by electrostatic stabilization through cation-π interactions. Distinct from extended Tudor domains, dimethylarginine binding by the SMN and SPF30 Tudor domains is independent of proximal residues in the ligand. Yet, enhanced micromolar affinities are obtained by external cooperativity when multiple methylation marks are presented in arginine- and glycine-rich peptide ligands. A hydrogen bond network in the SMN Tudor domain, including Glu134 and a tyrosine hydroxyl of the aromatic cage, enhances cation-π interactions and is impaired by a mutation causing an E134K substitution associated with spinal muscular atrophy. Our structural analysis enables the design of an optimized binding pocket and the prediction of DMA binding properties of Tudor domains.
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The DARC site: a database of aligned ribosomal complexes
18.10.2011
Nucl. Acids Res.,
2011,
doi: 10.1093/nar/gkr824,
published on 18.10.2011
The ribosome is a highly dynamic machine responsible for protein synthesis within the cell. Cryo-electron microscopy (cryo-EM) and X-ray crystallography structures of ribosomal particles, alone and in complex with diverse ligands (protein factors, RNAs and small molecules), have revealed the dynamic
nature of the ribosome and provided much needed insight into translation and its regulation. In the past years, there has been exponential growth in the deposition of cryo-EM maps into
the Electron Microscopy Data Bank (EMDB) as well as atomic structures into the Protein Data Bank (PDB). Unfortunately, the deposited ribosomal particles usually have distinct orientations
with respect to one another, which complicate the comparison of the available structures. To simplify this, we have developed a Database of Aligned Ribosomal Complexes, the DARC site
(http://darcsite.genzentrum.lmu.de/darc/), which houses the available cryo-EM maps and atomic coordinates of ribosomal particles from the EMDB and PDB aligned within a common coordinate system. An easy-to-use, searchable interface allows users to access and download >130 cryo-EM maps and
>300 atomic models in the format of brix and pdb files, respectively. The aligned coordinate system substantially simplifies direct visualization of conformational changes in the ribosome, such as subunit rotation and head-swiveling, as well as direct comparison of bound ligands, such as antibiotics
or translation factors.
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The RIG-I ATPase domain structure reveals insights into ATP-dependent antiviral signalling
07.10.2011
EMBO reports,
2011,
doi:10.1038/embor.2011.190,
12, 1127 - 1134
published on 07.10.2011
RIG-I detects cytosolic viral dsRNA with 5′ triphosphates (5′-ppp-dsRNA), thereby initiating an antiviral innate immune response. Here we report the crystal structure of superfamily 2 (SF2) ATPase domain of RIG-I in complex with a nucleotide analogue. RIG-I SF2 comprises two RecA-like domains 1A and 2A and a helical insertion domain 2B, which together form a ‘C’-shaped structure. Domains 1A and 2A are maintained in a ‘signal-off’ state with an inactive ATP hydrolysis site by an intriguing helical arm. By mutational analysis, we show surface motifs that are critical for dsRNA-stimulated ATPase activity, indicating that dsRNA induces a structural movement that brings domains 1A and 2A/B together to form an active ATPase site. The structure also indicates that the regulatory domain is close to the end of the helical arm, where it is well positioned to recruit 5′-ppp-dsRNA to the SF2 domain. Overall, our results indicate that the activation of RIG-I occurs through an RNA- and ATP-driven structural switch in the SF2 domain.
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Increasing αvβ3 Selectivity of the Anti-Angiogenic Drug Cilengitide by N-Methylation
26.09.2011
Angewandte Chemie,
2011,
DOI: 10.1002/anie.201102971,
Volume 50, Issue 40, pages 9496–9500
published on 26.09.2011
The drug Cilengitide, c(RGDf(NMe)V), is a cyclic RGD pentapeptide (R=arginine, D=aspartic acid, G=glycine) currently in clinical phase III for the treatment of brain tumors and in phase II for other cancer types.[1] The antitumoral properties of this peptide are based on its antagonistic activity for pro-angiogenic integrins, such as alpha-v-beta3, alpha-v-beta5, or alpha5 beta1. However, the specific roles of these integrin subtypes in angiogenesis and cancer are not yet clear and fully understood. In this work, we present di-N-methylated analogues of the stem peptide c(RGDfV) which retain an alpha-v-beta3- binding activity in the nanomolar range but have lost most of
the activity for integrins alpha-vbeta5 and/or alpha5 beta1. Highly active and selective peptides for alpha-v-bets3 are important tools to study the specific role of this integrin in angiogenesis and cancer.
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Increasing alpha-v-beta-3 Selectivity of the Anti-Angiogenic Drug Cilengitide by N-Methylation
26.09.2011
Angewandte Chemie,
2011,
DOI: 10.1002/anie.201102971,
Volume 50, Issue 40, pages 9496–9500
published on 26.09.2011
The drug Cilengitide, c(RGDf(NMe)V), is a cyclic RGD pentapeptide (R=arginine, D=aspartic acid, G=glycine) currently in clinical phase III for the treatment of brain tumors and in phase II for other cancer types.The antitumoral properties of this peptide are based on its antagonistic activity for pro-angiogenic integrins, such as alpha-v-beta-3, alpha-v-beta-5, or alpha-5-beta-1. However, the specific roles of these integrin subtypes in angiogenesis and cancer are not yet clear and fully understood. In this work, we present di-N-methylated analogues of the stem peptide c(RGDfV) which retain an alpha-v-beta-3- binding activity in the nanomolar range but have lost most of the activity for integrins alpha-v-beta-5 and/or alpha-5-beta-1. Highly active and selective peptides for avb3 are important tools to study the specific role of this integrin in angiogenesis and cancer. Integrins are heterodimeric receptors that govern cell–cell and cell–extracellular matrix (ECM) interactions, and play crucial roles in a plethora of cellular functions.The fact that many integrins are involved in pathological processes, such as tumor angiogenesis, has stimulated their study as therapeutic targets. A number of integrin receptors recognize and bind the tripeptide sequence RGD, which is a prominent celladhesion motif present in ECM proteins. Mimicking this tripeptide sequence with RGD-peptides or peptidomimetics is hence a promising approach to target integrins involved in angiogenesis and to develop anti-cancer agents.It is known that alpha-v-beta-3 and alpha-v-beta-5 are involved in two different angiogenic pathways. Whereas angiogenesis induced by basic fibroblast growth factor (bFGF) or tumor necrosis factor-a depends on alpha-v-beta-3, angiogenesis triggered by vascular endothelial growth factor (VEGF) or transforming.
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Structure of human mitochondrial RNA polymerase
25.09.2011
Nature Letter,
2011,
478,
269 - 73
published on 25.09.2011
Transcription of the mitochondrial genome is performed by a single-subunit RNA polymerase (mtRNAP) that is distantly related to the RNAP of bacteriophage T7, the pol I family of DNA polymerases, and single-subunit RNAPs from chloroplasts1, 2, 3, 4. Whereas T7 RNAP can initiate transcription by itself, mtRNAP requires the factors TFAM and TFB2M for binding and melting promoter DNA5, 6, 7. TFAM is an abundant protein that binds and bends promoter DNA 15–40 base pairs upstream of the transcription start site, and stimulates the recruitment of mtRNAP and TFB2M to the promoter8, 9. TFB2M assists mtRNAP in promoter melting and reaches the active site of mtRNAP to interact with the first base pair of the RNA–DNA hybrid10. Here we report the X-ray structure of human mtRNAP at 2.5 Å resolution, which reveals a T7-like catalytic carboxy-terminal domain, an amino-terminal domain that remotely resembles the T7 promoter-binding domain, a novel pentatricopeptide repeat domain, and a flexible N-terminal extension. The pentatricopeptide repeat domain sequesters an AT-rich recognition loop, which binds promoter DNA in T7 RNAP, probably explaining the need for TFAM during promoter binding. Consistent with this, substitution of a conserved arginine residue in the AT-rich recognition loop, or release of this loop by deletion of the N-terminal part of mtRNAP, had no effect on transcription. The fingers domain and the intercalating hairpin, which melts DNA in phage RNAPs, are repositioned, explaining the need for TFB2M during promoter melting. Our results provide a new venue for the mechanistic analysis of mitochondrial transcription. They also indicate how an early phage-like mtRNAP lost functions in promoter binding and melting, which were provided by initiation factors in trans during evolution, to enable mitochondrial gene regulation and the adaptation of mitochondrial function to changes in the environment.
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Molecular basis of Rrn3-regulated RNA polymerase I initiation and cell growth
22.09.2011
Genes & Dev. 2011,
2011,
doi: 10.1101/gad.1736331,
published on 22.09.2011
Cell growth is regulated during RNA polymerase (Pol) I transcription initiation by the conserved factor Rrn3/TIF- IA in yeast/humans. Here we provide a structure–function analysis of Rrn3 based on a combination of structural biology with in vivo and in vitro functional assays. The Rrn3 crystal structure reveals a unique HEAT repeat fold and a surface serine patch. Phosphorylation of this patch represses human Pol I transcription, and a phospho- mimetic patch mutation prevents Rrn3 binding to Pol I in vitro and reduces cell growth and Pol I gene occupancy in vivo. Cross-linking indicates that Rrn3 binds Pol I between its subcomplexes, AC40/19 and A14/43, which faces the serine patch. The corresponding region of Pol II binds the Mediator head that cooperates with transcription factor (TF) IIB. Consistent with this, the Rrn3-binding factor Rrn7 is predicted to be a TFIIB homolog. This reveals the molecular basis of Rrn3-regulated Pol I initiation and cell growth, and indicates a general architecture of eukaryotic transcription initiation complexes.
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ATP driven structural changes of the bacterial Mre11:Rad50 catalytic head complex
21.09.2011
Nucleic Acids Research,
2011,
doi: 10.1093/nar/gkr749,
40 (2): 914-927.
published on 21.09.2011
DNA double-strand breaks (DSBs) threaten genome stability in all kingdoms of life and are linked to cancerogenic chromosome aberrations in humans. The Mre11:Rad50 (MR) complex is an evolutionarily conserved complex of two Rad50 ATPases and a dimer of the Mre11 nuclease that senses and processes DSBs and tethers DNA for repair. ATP binding and hydrolysis by Rad50 is functionally coupled to DNA-binding and tethering, but also regulates Mre11's nuclease in processing DNA ends. To understand how ATP controls the interaction between Mre11 and Rad50, we determined the crystal structure of Thermotoga maritima (Tm) MR trapped in an ATP/ADP state. ATP binding to Rad50 induces a large structural change from an open form with accessible Mre11 nuclease sites into a closed form. Remarkably, the NBD dimer binds in the Mre11 DNA-binding cleft blocking Mre11's dsDNA-binding sites. An accompanying large swivel of the Rad50 coiled coil domains appears to prepare the coiled coils for DNA tethering. DNA-binding studies show that within the complex, Rad50 likely forms a dsDNA-binding site in response to ATP, while the Mre11 nuclease module retains a ssDNA-binding site. Our results suggest a possible mechanism for ATP-dependent DNA tethering and DSB processing by MR.
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Design, Synthesis, and Functionalization of Dimeric Peptides Targeting Chemokine Receptor CXCR4
12.09.2011
J. Med. Chem.,
2011,
DOI: 10.1021/jm2009716,
54 (21), pp 7648–7662
published on 12.09.2011
The chemokine receptor CXCR4 is a critical regulator of inflammation and immune surveillance, and it is specifically implicated in cancer metastasis and HIV-1 infection. On the basis of the observation that several of the known antagonists remarkably share a C2 symmetry element, we constructed symmetric dimers with excellent antagonistic activity using a derivative of a cyclic pentapeptide as monomer. To optimize the binding affinity, we investigated the influence of the distance between the monomers and the pharmacophoric sites in the synthesized constructs. The affinity studies in combination with docking computations support a two-site binding model. In a final step, 1,4,7,10 tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was introduced as chelator for (radio-)metals, thus allowing to exploit these compounds as a new group of CXCR4-binding peptidic probes for molecular imaging and endoradiotherapeutic purposes. Both the DOTA conjugates and some of their corresponding metal complexes retain good CXCR4 affinity, and one 68Ga labeled compound was studied as PET tracer.
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Introducing Lasso Peptides as Molecular Scaffolds for Drug Design: Engineering of an Integrin Antagonist
05.09.2011
Angewandte Chemie,
2011,
DOI: 10.1002/ange.201102190,
Volume 123, Issue 37, pages 8873–8876
published on 05.09.2011
Peptides combine a high specificity for their target receptor with a low toxicity and are therefore a promising source for drug leads.However, their use has been limited because of undesirable physicochemical and pharmacokinetic properties. To overcome these obstacles protein scaffolds, such as ultrastable ribosomally assembled peptides, can be used together with synthetic chemical strategies to present biologically active peptide epitopes, as shown recently by the conversion of the cyclotide kalata B1 into a vascular
endothelial growth-factor-A antagonist. In addition, bacterial lasso peptides have been under discussion as molecular scaffolds for drug design.These ribosomally assembled peptides consist of 16–21 amino acids and share an N-terminal eight/nine-residue macrolactam ring through which the Cterminal linear tail is threaded and trapped by steric hindrance of bulky side chains.
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Structural analysis of the interaction between Hsp90 and the tumor suppressor protein p53
04.09.2011
Nature Structural & Molecular Biology,
2011,
doi:10.1038/nsmb.2114,
published on 04.09.2011
Nature Structural & Molecular Biology, online article
Nature Structural & Molecular Biology, online article
In eukaryotes, the essential dimeric molecular chaperone Hsp90 is required for the activation and maturation of specific substrates such as steroid hormone receptors, tyrosine kinases and transcription factors. Hsp90 is involved in the establishment of cancer and has become an attractive target for drug design. Here we present a structural characterization of the complex between Hsp90 and the tumor suppressor p53, a key mediator of apoptosis whose structural integrity is crucial for cell-cycle control. Using biophysical methods, we show that the human p53 DNA-binding domain interacts with multiple domains of yeast Hsp90. p53 binds to the Hsp90 C-terminal domain in its native-like state in a charge-dependent manner, but it also associates weakly with binding sites in the middle and the N-terminal domains. The fine-tuned interplay between several Hsp90 domains provides the interactions required for efficient chaperoning of p53.
Nuclear actin-related proteins take shape
03.08.2011
Bioarchitecture,
2011,
DOI: 10.4161/bioa.1.4.17643,
1(4):192-195
published on 03.08.2011
The function of nuclear actin is poorly understood. It is known to be a discrete component of several chromatin-modifying complexes. Nevertheless, filamentous forms of actin are important for various nuclear processes as well. Nuclear actin is often associated with nuclear actin-related protein Arp4 and other actin-related proteins like Arp8 in the INO80 chromatin remodeler. We recently determined the crystal structure of S. cerevisiae Arp4 that explains why Arp4 is unable to form actin like filaments and shows that it is constitutively bound to an ATP nucleotide. More interestingly, in vitro activities of Arp4 and Arp8 seem to be directed towards stabilizing monomeric actin and to integrate it stoichiometrically into the INO80 complex. Based on this activity, we discuss possible roles of nuclear Arps in chromatin modifying complexes and in regulating more general aspects of nuclear actin dynamics.
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Strategies for the structural analysis of multi-protein complexes: Lessons from the 3D-Repertoire project
02.08.2011
Journal of Structural Biology,
2011,
doi:10.1016/j.jsb.2011.03.018,
Volume 175, Issue 2, Pages 147-158
published on 02.08.2011
Structural studies of multi-protein complexes, whether by X-ray diffraction, scattering, NMR spectroscopy or electron microscopy, require stringent quality control of the component samples. The inability to produce ‘keystone’ subunits in a soluble and correctly folded form is a serious impediment to the reconstitution of the complexes. Co-expression of the components offers a valuable alternative to the expression of single proteins as a route to obtain sufficient amounts of the sample of interest. Even in cases where milligram-scale quantities of purified complex of interest become available, there is still no guarantee that good quality crystals can be obtained. At this step, protein engineering of one or more components of the complex is frequently required to improve solubility, yield or the ability to crystallize the sample. Subsequent characterization of these constructs may be performed by solution techniques such as Small Angle X-ray Scattering and Nuclear Magnetic Resonance to identify ‘well behaved’ complexes. Herein, we recount our experiences gained at protein production and complex assembly during
the European 3D Repertoire project (3DR). The goal of this consortium was to obtain structural information on multi-protein complexes from yeast by combining crystallography, electron microscopy, NMR and in silico modeling methods. We present here representative set case studies of complexes that were
produced and analyzed within the 3DR project. Our experience provides useful insight into strategies that are more generally applicable for structural analysis of protein complexes.
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Telethonin Deficiency Is Associated With Maladaptation to Biomechanical Stress in the Mammalian Heart
28.07.2011
Circulation Research,
2011,
doi: 10.1161/CIRCRESAHA.111.245787,
758-769
published on 28.07.2011
Telethonin (also known as titin-cap or t-cap) is a 19-kDa Z-disk protein with a unique beta-sheet structure, hypothesized to assemble in a palindromic way with the N-terminal portion of titin and to constitute a signalosome participating in the process of cardiomechanosensing. In addition, a variety of telethonin mutations
are associated with the development of several different diseases; however, little is known about the underlying molecular mechanisms and telethonin’s in vivo function.
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1H, 13C, 15N and 31P chemical shift assignments of a human Xist RNA A-repeat tetraloop hairpin essential for X-chromosome inactivation
21.07.2011
Biomolecular NMR Assignments,
2011,
DOI: 10.1007/s12104-011-9328-z,
published on 21.07.2011
Initiation of X-chromosome inactivation in female mammals depends on the non-coding RNA Xist. We have solved the NMR structure of a 14-nucleotide hairpin with a novel AUCG tetraloop fold from a Xist A-repeat that is essential for silencing. The 1H, 13C, 15N
and 31P chemical shift assignments are reported.
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PET Imaging of CXCR4 Receptors in Cancer by a New Optimized Ligand
20.07.2011
ChemMedChem,
2011,
DOI: 10.1002/cmdc.201100320,
Volume 6, Issue 10, pages 1789–1791
published on 20.07.2011
Nowadays, personalized medicine is considered to be of utmost importance to target the different causes of identical phenotypes. For example, cancer of the same type can significantly differ in its biochemical phenotypes and thus its molecular profile between patients. The disease-specific characterization of malignant cells at the molecular level is a prerequisite for targeted therapy and personalized treatment. Positron emission tomography (PET) and its combination with computer tomography (PET/CT) and magnetic resonance tomography
(PET/MRT) in modern hybrid systems offer the possibility to localize and quantify biochemical function by means of PET with
anatomical (CT) and morphological (MRT) information. For this
purpose, radiolabeled probes are used that target, for example,
enzyme activities, transport systems, and surface receptors with high affinity and specificity. We describe the development of the first gallium-68 (t1/2=68 min) ligand for the G-protein- coupled receptor CXCR4 and preliminary demonstrate its potential for in vivo imaging of CXCR4 expression using a mouse model with a human small-cell lung cancer xenograft. This ligand offers the possibility to be used as an initial tool for diagnosis in an approach of personalized medicine for treating CXCR4-related cancer.
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Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF
13.07.2011
Nature,
2011,
475,
408 - 11
published on 13.07.2011
Many cellular functions involve multi-domain proteins, which are composed of structurally independent modules connected by flexible linkers. Although it is often well understood how a given domain recognizes a cognate oligonucleotide or peptide motif, the dynamic interaction of multiple domains in the recognition of these ligands remains to be characterized. Here we have studied the molecular mechanisms of the recognition of the 3′-splice-site-associated polypyrimidine tract RNA by the large subunit of the human U2 snRNP auxiliary factor (U2AF65)1, 2, 3 as a key early step in pre-mRNA splicing4. We show that the tandem RNA recognition motif domains of U2AF65 adopt two remarkably distinct domain arrangements in the absence or presence of a strong (that is, high affinity) polypyrimidine tract. Recognition of sequence variations in the polypyrimidine tract RNA involves a population shift between these closed and open conformations. The equilibrium between the two conformations functions as a molecular rheostat that quantitatively correlates the natural variations in polypyrimidine tract nucleotide composition, length and functional strength to the efficiency to recruit U2 snRNP to the intron during spliceosome assembly 1, 5, 6, 7, 8. Mutations that shift the conformational equilibrium without directly affecting RNA binding modulate splicing activity accordingly. Similar mechanisms of cooperative multi-domain conformational selection may operate more generally in the recognition of degenerate nucleotide or amino acid motifs by multi-domain proteins 9, 10.
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Structure and mechanism of the Swi2/Snf2 remodeller Mot1 in complex with its substrate TBP
06.07.2011
Nature,
2011,
doi:10.1038/nature10215,
published on 06.07.2011
Swi2/Snf2-typeATPases regulate genome-associated processes such as transcription, replication and repair by catalysing the disruption, assembly or remodelling of nucleosomes or other protein–DNA complexes1,2. It has been suggested that ATP-driven motor activity
along DNA disrupts target protein–DNA interactions in the remodelling reaction3–5. However, the complex and highly specific remodelling reactions are poorly understood, mostly because of a lack of high-resolution structural information about how
remodellers bind to their substrate proteins. Mot1 (modifier of transcription 1 in Saccharomyces cerevisiae, denoted BTAF1 in
humans) is a Swi2/Snf2 enzyme that specifically displaces the TATA box binding protein (TBP) from the promoter DNA and regulates transcription globally by generating a highly dynamic
TBP pool in the cell6,7. As a Swi2/Snf2 enzyme that functions as a single polypeptide and interacts with a relatively simple substrate,
Mot1 offers an ideal systemfromwhich to gain a better understanding of thisimportant enzyme family.Toreveal howMot1 specifically
disrupts TBP–DNA complexes, we combined crystal and electron microscopy structures of Mot1–TBP from Encephalitozoon cuniculi
with biochemical studies.Here weshowthatMot1 wraps around TBP and seems to act like a bottle opener: a spring-like array of 16 HEAT(huntingtin, elongation factor 3, protein phosphatase 2Aand
lipid kinase TOR) repeats grips theDNA-distal side of TBP via loop
insertions, and the Swi2/Snf2 domain binds to upstream DNA, positioned to weaken the TBP–DNAinteraction byDNAtranslocation.
A‘latch’ subsequently blocks theDNA-binding groove ofTBP, acting as a chaperone to prevent DNA re-association and ensure
efficient promoter clearance. This work shows how a remodelling enzyme can combine both motor and chaperone activities to achieve
functional specificity using a conserved Swi2/Snf2 translocase.
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Differential Effects of Thiopeptide and Orthosomycin Antibiotics on Translational GTPases
27.05.2011
Chemistry & Biology,
2011,
DOI 10.1016/j.chembiol.2011.03.010,
Volume 18, Issue 5, 589-600
published on 27.05.2011
The ribosome is a major target in the bacterial cell for antibiotics. Here, we dissect the effects that the thiopeptide antibiotics thiostrepton (ThS) and micrococcin (MiC) as well as the orthosomycin antibiotic evernimicin (Evn) have on translational GTPases. We demonstrate that, like ThS, MiC is a translocation
inhibitor, and that the activation by MiC of the ribosome- dependent GTPase activity of EF-G is dependent on the presence of the ribosomal proteins L7/ L12 as well as the G0 subdomain of EF-G. In contrast, Evn does not inhibit translocation but is a potent inhibitor of back-translocation as well as IF2-dependent 70S-initiation complex formation. Collectively, these results shed insight not only into fundamental aspects of translation but also into the unappreciated
specificities of these classes of translational inhibitors.
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Evolution of Two Modes of Intrinsic RNA Polymerase Transcript Cleavage
27.05.2011
The Journal of Biological Chemistry,
2011,
286,
18701 - 07
published on 27.05.2011
During gene transcription, the RNA polymerase (Pol) active center can catalyze RNA cleavage. This intrinsic cleavage activity is strong for Pol I and Pol III but very weak for Pol II. The reason for this difference is unclear because the active centers of the polymerases are virtually identical. Here we show that Pol II gains strong cleavage activity when the C-terminal zinc ribbon domain (C-ribbon) of subunit Rpb9 is replaced by its counterpart from the Pol III subunit C11. X-ray analysis shows that the C-ribbon has detached from its site on the Pol II surface and is mobile. Mutagenesis indicates that the C-ribbon transiently inserts into the Pol II pore to complement the active center. This mechanism is also used by transcription factor IIS, a factor that can bind Pol II and induce strong RNA cleavage. Together with published data, our results indicate that Pol I and Pol III contain catalytic C-ribbons that complement the active center, whereas Pol II contains a non-catalytic C-ribbon that is immobilized on the enzyme surface. Evolution of the Pol II system may have rendered mRNA transcript cleavage controllable by the dissociable factor transcription factor IIS to enable promoter-proximal gene regulation and elaborate 3′-processing and transcription termination.
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Optimal 2H rf Pulses and 2H–13C Cross-Polarization Methods for Solid-State 2H MAS NMR of Perdeuterated Proteins
12.05.2011
J. Phys. Chem. Letters,
2011,
DOI: 10.1021/jz200511b,
2 (11), pp 1289–1294
published on 12.05.2011
We present a novel concept for rf pulses and optimal control designed cross-polarization experiments for quadrupolar nuclei. The methods are demonstrated for 2H CP-MAS and 2H multiple-pulse NMR of perdeuterated proteins, for which sensitivity enhancements up to an order of magnitude are presented relative to commonly used approaches. The so-called RESPIRATION rf pulses combines the concept of short broad-band pulses with generation of pulses with large flip angles through distribution of the rf pulse over several rotor echoes. This lead to close-to-ideal rf pulses, facilitating implementation of experiments relying on the ability to realize high-performance 90 and 180° pulses, as, for example, in refocused INEPT and double-to-single quantum coherence experiments, or just pulses that provide a true representation of the quadrupolar powder pattern to extract information about the structure or dynamics. The optimal control 2H → 13C CP-MAS method demonstrates transfer efficiencies up to around 85% while being extremely robust toward rf inhomogeneity and resonance offsets.
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A Conformational Switch Underlies ClpP Protease Function
04.05.2011
Angewandte Chemie,
2011,
DOI: 10.1002/anie.201100666,
Volume 50, Issue 25, pages 5749–5752
published on 04.05.2011
The barrel-shaped serine protease ClpP degrades misfolded, damaged, and regulatory proteins. Substrate proteins enter the ClpP barrel through the two axial pores, but it is unclear how the peptide products exit the barrel. Here we report the structure of ClpP from Staphylococcus aureus, which reveals a previously unobserved compressed state of the barrel. A conformational switch in the active center “handle region” results in closure of the active sites and opening of equatorial pores. Conserved residues in the handle region underlie the conformational switch and are functionally essential although they are not part of the active sites. These results are consistent with processive cycling of ClpP between an extended state with open active sites and closed equatorial pores, and a compressed state with closed active sites and open pores for product release.
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Proton-Detected Solid-State NMR Spectroscopy of Fibrillar and Membrane Proteins
02.05.2011
Angewandte Chemie,
2011,
DOI: 10.1002/anie.201008244,
Volume 50, Issue 19, pages 4508–4512
published on 02.05.2011
Structural characterization of insoluble proteins often relies on solid-state NMR spectroscopy. Perdeuteration and partial back-substitution of exchangeable protons, as proposed for crystalline model proteins, is now shown to lead to beneficial proton spectra for heterogeneous systems, such as fibrils formed by the Alzheimer's disease β-amyloid peptide Aβ40, the lipid reconstituted β-barrel membrane protein OmpG, and the α-helical membrane protein bacteriorhodopsin.
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1H, 13C and 15N chemical shift assignments of IPSEΔNLS
24.04.2011
Biomolecular NMR Assignments,
2011,
DOI: 10.1007/s12104-011-9305-6,
Volume 5, Number 2, 225-227
published on 24.04.2011
The interleukin-4-inducing principle from Schistosoma mansoni eggs (IPSE/alpha-1) is a major immunogenic component of schistosomes. It potently triggers the release of interleukin-4 from basophilic granulocytes in an IgE-dependent manner, suggesting a key function in the modulation of the host’s immune response to Schistosoma mansoni infection. Here we present the near complete
assignment of an IPSE/alpha-1 variant, IPSEDNLS, which comprises the core domain of the protein.
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Iwr1 Directs RNA Polymerase II Nuclear Import
22.04.2011
Molecular Cell,
2011,
10.1016/j.molcel.2011.02.033,
Volume 42, Issue 2, 261-266
published on 22.04.2011
RNA polymerase (Pol) II transcribes protein-coding genes in the nucleus of eukaryotic cells and consists of 12 polypeptide subunits. It is unknown how Pol II is imported into the nucleus. Here we show that Pol II nuclear import requires the protein Iwr1 and provide
evidence for cyclic Iwr1 function. Iwr1 binds Pol II in the active center cleft between the two largest subunits, maybe facilitating or sensing complete Pol II assembly in the cytoplasm. Iwr1 then uses an N-terminal bipartite nuclear localization signal that is recognized by karyopherin a to direct Pol II nuclear import. In the nucleus, Iwr1 is displaced from Pol II by transcription initiation factors and nucleic acids, enabling its export and recycling. Iwr1 function is Pol II specific, transcription independent, and apparently conserved from yeast to human.
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Cryo-EM structure of the ribosome–SecYE complex in the membrane environment
17.04.2011
Nature Structural & Molecular Biology,
2011,
doi:10.1038/nsmb.2026,
published on 17.04.2011
The ubiquitous SecY–Sec61 1 complex translocates nascent secretory proteins across cellular membranes and integrates membrane proteins into lipid bilayers. Several structures of mostly detergent-solubilized Sec complexes have been reported. Here we present a single-particle cryo-EM structure of the SecYEG complex in a membrane environment, bound to a translating ribosome, at subnanometer resolution. Using the SecYEG complex reconstituted in a so-called Nanodisc, we could trace the nascent polypeptide chain from the peptidyltransferase center into the membrane. The reconstruction allowed for the identification of ribosome–lipid interactions. The rRNA helix 59 (H59) directly contacts the lipid surface and appears to modulate the membrane in immediate vicinity to the proposed lateral gate of the protein-conducting channel (PCC). On the basis of our map and molecular dynamics simulations, we present a model of a signal anchor–gated PCC in the membrane.
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A Glycopeptide Dendrimer Inhibitor of the Galactose-Specific Lectin LecA and of Pseudomonas aeruginosa Biofilms
14.09.2011
Angewandte Chemie,
2011,
DOI: 10.1002/anie.201104342,
published on 17.04.2011
The spread of antibiotic resistant bacteria is one of the most
pressing problems in human health today. In the case of the
opportunistic pathogen Pseudomonas aeruginosa, which
causes lethal airway infections in cystic fibrosis and immunocompromised patients, the formation of biofilms plays an important role in antibiotic resistance and disease progression. Biofilm formation is mediated in part by the galactosespecific lectin LecA (PA-IL) and the fucose-specific lectin LecB (PA-IIL) as evidenced by studies with deletion
Mutants and the partial inhibitory effect of simple fucose and galactose derivatives in vitro and in vivo. Understanding the glycoconjugate–lectin interaction is a key feature in developing potent biofilm inhibitors. Capitalizing on the well-known cluster effect observed on binding of multivalent carbohydrates to lectins, we recently reported the first case of P. aeruginosa biofilm inhibition with a multivalent lectin inhibitor, the fucosylated glycopeptide dendrimer FD2 (cFuc-Lys-Pro-Leu) (Lys-Phe-Lys-Ile)2Lys-His-IleNH2, which targets LecB. Herein we report the first case of P. aeruginosa biofilm inhibition with a multivalent ligand targeting the galactose-specific lectin LecA, using the related beta-phenylgalactosyl peptide dendrimer GalAG2.
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The Effect of Multiple N-Methylation on Intestinal Permeability of Cyclic Hexapeptides
17.04.2011
molecular pharmaceutics,
2011,
8, 2,
479 - 87
published on 17.04.2011
Recent progress in peptide synthesis simplified the synthesis of multiple N-methylation of peptides. To evaluate how multiple N-methylation affects the bioavailability of peptides, a poly alanine cyclic hexapeptide library (n = 54), varying in the number of N-methyl (N-Me) groups (1−5 groups) and their position, was synthesized. The peptides were evaluated for their intestinal permeability in vitro using the Caco-2 model. Further evaluation of the transport route of chosen analogues was performed using rat excised viable intestinal tissue, a novel colorimetric liposomal model and the parallel artificial membrane permeability assay (PAMPA). While most members were found to have poor permeability (permeability coefficient, Papp < 1 × 10−6 cm/s, lower than mannitol, the marker for paracellular permeability), 10 analogues were found to have high Caco-2 permeability, (Papp > 1 × 10−5 cm/s, similar to testosterone, a marker of transcellular permeability). No correlation was found between the number of N-methylated groups and the enhanced permeability. However, 9/10 permeable peptides in the Caco-2 model included an N-Me placed adjacently to the d-Ala position. While the exact transport route was not fully characterized, the data suggests a facilitated diffusion. It can be concluded that multiple N-methylation of peptides may improve intestinal permeability, and therefore can be utilized in the design of orally available peptide-based therapeutics.
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Structural biochemistry of nuclear actin-related proteins 4 and 8 reveals their interaction with actin
15.04.2011
The EMBO Journal,
2011,
doi:10.1038/emboj.2011.118,
30, 2153 - 2166
published on 15.04.2011
Nuclear actin and actin-related proteins (Arps) are integral components of various chromatin-remodelling complexes. Actin in such nuclear assemblies does not form filaments but associates in defined complexes, for instance with Arp4 and Arp8 in the INO80 remodeller. To understand the relationship between nuclear actin and its associated Arps and to test the possibility that Arp4 and Arp8 help maintain actin in defined states, we structurally analysed Arp4 and Arp8 from Saccharomyces cerevisiae and tested their biochemical effects on actin assembly and disassembly. The solution structures of isolated Arp4 and Arp8 indicate them to be monomeric and the crystal structure of ATP–Arp4 reveals several differences to actin that explain why Arp4 does not form filaments itself. Remarkably, Arp4, assisted by Arp8, influences actin polymerization in vitro and is able to depolymerize actin filaments. Arp4 likely forms a complex with monomeric actin via the barbed end. Our data thus help explaining how nuclear actin is held in a discrete complex within the INO80 chromatin remodeller.
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Mediator head subcomplex Med11/22 contains a common helix bundle building block with a specific function in transcription initiation complex stabilization
15.04.2011
Nucl. Acids Res.,
2011,
Vol. 39 Issue 14,
6291–304
published on 15.04.2011
Mediator is a multiprotein co-activator of RNA poly- merase (Pol) II transcription. Mediator contains a conserved core that comprises the ‘head’ and ‘middle’ modules. We present here a structure– function analysis of the essential Med11/22 hetero- dimer, a part of the head module. Med11/22 forms a conserved four-helix bundle domain with C-terminal extensions, which bind the central head subunit Med17. A highly conserved patch on the bundle surface is required for stable transcription pre- initiation complex formation on a Pol II promoter in vitro and in vivo and may recruit the general tran- scription factor TFIIH. The bundle domain fold is also present in the Mediator middle module sub- complex Med7/21 and is predicted in the Mediator heterodimers Med2/3, Med4/9, Med10/14 and Med28/30. The bundle domain thus represents a common building block that has been multiplied and functionally diversified during Mediator evolu- tion in eukaryotes.
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N-Methylated sst2 Selective Somatostatin Cyclic Peptide Analogue as a Potent Candidate for Treating Neurogenic Inflammation
04.04.2011
ACS Medicinal Chemistry Letters,
2011,
DOI: 10.1021/ml200032v,
published on 04.04.2011
A focused multiply N-methylated library of a cyclic hexapeptidic somatostatin analogue: MK678 cyclo(−MeAYwKVF−) was generated, which resulted in the unexpected observation of an efficacious tetra-N-methylated analogue, cyclo(−MeAYMewMeKVMeF−) with a potent inhibitory action on sensory neuropeptide release in vitro and on acute neurogenic inflammatory response in vivo. The analogue shows selectivity toward somatostatin receptor subtype 2 (sst2). Extensive 2D NMR spectroscopy and molecular dynamics simulation revealed the solution conformation of the analogue, which can be adopted as a lead for the further structure−activity relationship studies targeting neurogenic inflammation.
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A Cytoplasmic Complex Mediates Specific mRNA Recognition and Localization in Yeast
01.04.2011
PLOS Biology,
2011,
9, 4,
published on 01.04.2011
In eukaryotes, hundreds of mRNAs are localized by specialized transport complexes. For localization, transcripts are
recognized by RNA-binding proteins and incorporated into motor-containing messenger ribonucleoprotein particles
(mRNPs). To date, the molecular assembly of such mRNPs is not well understood and most details on cargo specificity
remain unresolved. We used ASH1-mRNA transport in yeast to provide a first assessment of where and how localizing
mRNAs are specifically recognized and incorporated into mRNPs. By using in vitro–interaction and reconstitution assays, we
found that none of the implicated mRNA-binding proteins showed highly specific cargo binding. Instead, we identified the
cytoplasmic myosin adapter She3p as additional RNA-binding protein. We further found that only the complex of the RNAbinding
proteins She2p and She3p achieves synergistic cargo binding, with an at least 60-fold higher affinity for localizing
mRNAs when compared to control RNA. Mutational studies identified a C-terminal RNA-binding fragment of She3p to be
important for synergistic RNA binding with She2p. The observed cargo specificity of the ternary complex is considerably
higher than previously reported for localizing mRNAs. It suggests that RNA binding for mRNP localization generally exhibits
higher selectivity than inferred from previous in vitro data. This conclusion is fully consistent with a large body of in vivo
evidence from different organisms. Since the ternary yeast complex only assembles in the cytoplasm, specific mRNA
recognition might be limited to the very last steps of mRNP assembly. Remarkably, the mRNA itself triggers the assembly of
mature, motor-containing complexes. Our reconstitution of a major portion of the mRNA-transport complex offers new and
unexpected insights into the molecular assembly of specific, localization-competent mRNPs and provides an important step
forward in our mechanistic understanding of mRNA localization in general.
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The Mre11:Rad50 Structure Shows an ATP-Dependent Molecular Clamp in DNA Double-Strand Break Repair
01.04.2011
Cell,
2011,
DOI 10.1016/j.cell.2011.02.038,
Volume 145, Issue 1, 54-66,
published on 01.04.2011
The MR (Mre11 nuclease and Rad50 ABC ATPase) complex is an evolutionarily conserved sensor for DNA double-strand breaks, highly genotoxic lesions linked to cancer development.MRcan recognize and process DNA ends even if they are blocked and misfolded. To reveal its mechanism, we determined the crystal structure of the catalytic head of Thermotoga maritima MR and analyzed ATP-dependent conformational changes. MR adopts an open form with a central Mre11 nuclease dimer and two peripheral
Rad50 molecules, a form suited for sensing obstructed breaks. The Mre11 C-terminal helix-loophelix domain binds Rad50 and attaches flexibly to the nuclease domain, enabling large conformational changes. ATP binding to the two Rad50 subunits induces a rotation of the Mre11 helix-loop-helix and Rad50 coiled-coil domains, creating a clamp conformation with increased DNA-binding activity. The results suggest that MR is an ATP-controlled transient molecular clamp at DNA double-strand breaks.
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Structural Analysis of Large Protein Complexes Using Solvent Paramagnetic Relaxation Enhancements
25.03.2011
Angewandte Chemie,
2011,
DOI: 10.1002/anie.201007168,
Volume 50, Issue 17, pages 3993–3997
published on 25.03.2011
Understanding the function of biomolecular complexes requires their structural analysis at atomic resolution. To solve high-resolution structures by ab initio calculations typically data from NMR spectroscopy or X-ray crystallography are employed. In the latter approach, intrinsic flexibility and dynamics may prevent crystallization or introduce artificial conformations linked to crystal packing. Solution NMR spectroscopy does not suffer from such limitations, but is demanding because the adverse relaxation properties of large complexes may lead to extensive signal broadening and severe spectral overlap. Consequently, only sparse restraints can be obtained from such complexes by NMR experiments. Provided that the structures of the individual components of the complex (i.e. proteins, DNA/RNA) are available and that no large-scale conformational changes occur upon complex
formation, experimental and computational approaches can be used to obtain the quaternary arrangement of complexes.
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Structure Calculation from Unambiguous Long-Range Amide and Methyl 1H−1H Distance Restraints for a Microcrystalline Protein with MAS Solid-State NMR Spectroscopy
24.03.2011
J. Am. Chem. Soc.,,
2011,
DOI: 10.1021/ja110222h,
133 (15), pp 5905–5912
published on 24.03.2011
Magic-angle spinning (MAS) solid-state NMR becomes an increasingly important tool for the determination of structures of membrane proteins and amyloid fibrils. Extensive deuteration of the protein allows multidimensional experiments with exceptionally high sensitivity and resolution to be obtained. Here we present an experimental strategy to measure highly unambiguous spatial correlations for distances up to 13 Å. Two complementary three-dimensional experiments, or alternatively a four-dimensional experiment, yield highly unambiguous cross-peak assignments, which rely on four encoded chemical shift dimensions. Correlations to residual aliphatic protons are accessible via synchronous evolution of the 15N and 13C chemical shifts, which encode valuable amide−methyl distance restraints. On average, we obtain six restraints per residue. Importantly, 50% of all restraints correspond to long-range distances between residues i and j with |i − j| > 5, which are of particular importance in structure calculations. Using ARIA, we calculate a high-resolution structure for the microcrystalline 7.2 kDa α-spectrin SH3 domain with a backbone precision of 1.1 Å.
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Take a look at Karl-Peter Hopfner's "Sensing DNA Damage" Cell Movie
18.03.2011
Cell,,
2011,
145 Issue 1 (doi:10.1016/j.cell.2011.02.038),
54-66
published on 18.03.2011
Take a look at the new Cell movie clip of CIPSM's Karl-Peter Hopfner and group called "Sensing DMA Damage".
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Click here to watch "Sensing DMA Damage" on YouTube
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NMR and small-angle scattering-based structural analysis of protein complexes in solution
15.03.2011
Journal of Structural Biology,
2011,
doi:10.1016/j.jsb.2010.11.004,
Volume 173, Issue 3, March 2011, Pages 472-482
published on 15.03.2011
Structural analysis of multi-domain protein complexes is a key challenge in current biology and a prerequisite for understanding the molecular basis of essential cellular processes. The use of solution techniques is important for characterizing the quaternary arrangements and dynamics of domains and subunits of these complexes. In this respect solution NMR is the only technique that allows atomic- or
residue-resolution structure determination and investigation of dynamic properties of multi-domain -proteins and their complexes. As experimental NMR data for large protein complexes are sparse, it is advantageous to combine these data with additional information from other solution techniques. Here, the utility and computational approaches of combining solution state NMR with small-angle X-ray and Neutron scattering (SAXS/SANS) experiments for structural analysis of large protein complexes is reviewed. Recent progress in experimental and computational approaches of combining NMR and SAS are discussed and illustrated with recent examples from the literature. The complementary aspects of combining NMR and SAS data for studying multi-domain proteins, i.e. where weakly interacting domains are connected by flexible linkers, are illustrated with the structural analysis of the tandem RNA recognition motif (RRM) domains (RRM1-RRM2) of the human splicing factor U2AF65 bound to a nine-uridine (U9) RNA oligonucleotide.
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Architecture of the RNA polymerase–Spt4/5 complex and basis of universal transcription processivity
09.03.2011
The EMBO Journal,
2011,
30,
1302 - 10
published on 09.03.2011
Related RNA polymerases (RNAPs) carry out cellular gene
transcription in all three kingdoms of life. The universal
conservation of the transcription machinery extends to a
single RNAP-associated factor, Spt5 (or NusG in bacteria),
which renders RNAP processive and may have arisen early
to permit evolution of long genes. Spt5 associates with Spt4 to
form the Spt4/5 heterodimer. Here, we present the crystal
structure of archaeal Spt4/5 bound to the RNAP clamp
domain, which forms one side of the RNAP active centre
cleft. The structure revealed a conserved Spt5–RNAP interface
and enabled modelling of complexes of Spt4/5 counterparts
with RNAPs from all kingdoms of life, and of the
complete yeast RNAP II elongation complex with bound
Spt4/5. The N-terminal NGN domain of Spt5/NusG closes
the RNAP active centre cleft to lock nucleic acids and render
the elongation complex stable and processive. The C-terminal
KOW1 domain is mobile, but its location is restricted to a
region between the RNAP clamp and wall above the RNA exit
tunnel, where it may interact with RNA and/or other factors.
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Structure and VP16 binding of the Mediator Med25 activator interaction domain
06.03.2011
Nature Structural & Molecular Biology,
2011,
doi:10.1038/nsmb.1997,
18, 404–409 (2011)
published on 06.03.2011
Eukaryotic transcription is regulated by interactions between gene-specific activators and the coactivator complex Mediator.
Here we report the NMR structure of the Mediator subunit Med25 (also called Arc92) activator interaction domain (ACID) and analyze the structural and functional interaction of ACID with the archetypical acidic transcription activator VP16. Unlike other known activator targets, ACID forms a seven-stranded -barrel framed by three helices. The VP16 subdomains H1 and H2 bind to opposite faces of ACID and cooperate during promoter-dependent activated transcription in a in vitro system. The activator-binding ACID faces are functionally required and conserved among higher eukaryotes. Comparison with published activator structures reveals that the VP16 activation domain uses distinct interaction modes to adapt to unrelated target surfaces and folds that evolved for activator binding.
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Structural basis of RNA polymerase II backtracking, arrest and reactivation
23.02.2011
Nature,
2011,
doi:10.1038/nature09785,
471, 249–253
published on 23.02.2011
During gene transcription, RNA polymerase (Pol) II moves
forwards along DNA and synthesizes messenger RNA. However,
at certain DNA sequences, Pol II moves backwards, and such backtracking can arrest transcription. Arrested Pol II is reactivated by transcription factor IIS (TFIIS), which inducesRNAcleavage that is required for cell viability1. Pol II arrest and reactivation are involved in transcription through nucleosomes and in promoter-proximal gene regulation4–6. Here we present X-ray structures at 3.3A˚ resolution of an arrested Saccharomyces cerevisiae Pol II complex with
DNAandRNA, and of a reactivation intermediate that additionally
contains TFIIS. In the arrested complex, eight nucleotides of backtrackedRNAbind a conserved ‘backtrack site’ in the Pol II pore and funnel, trapping the active centre trigger loop and inhibiting mRNA elongation. In the reactivation intermediate, TFIIS locks the trigger loop away from backtracked RNA, displaces RNA from the backtrack site, and complements the polymerase active site with a basic and two acidic residues that may catalyse proton transfers during RNA cleavage. The active site is demarcated from the backtrack site by a ‘gating tyrosine’ residue that probably delimits backtracking. These results establish the structural basis of Pol II backtracking, arrest and reactivation, and provide a framework for analysing gene regulation during transcription elongation.
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Bacterial Inclusion Bodies of Alzheimer’s Disease b-Amyloid Peptides Can Be Employed To Study Native-Like Aggregation Intermediate States
11.02.2011
ChemBioChem,
2011,
DOI: 10.1002/cbic.201000602,
Volume 12, Issue 3, pages 407–423,
published on 11.02.2011
The structures of oligomeric intermediate states in the aggregation process of Alzheimer’s disease b-amyloid peptides have been the subject of debate for many years. Bacterial inclusion bodies contain large amounts of small heat shock proteins (sHSPs), which are highly homologous to those found in the plaques of the brains of Alzheimer’s disease patients. sHSPs break down amyloid fibril structure in vitro and induce oligomeric assemblies. Prokaryotic protein overexpression thus
mimics the conditions encountered in the cell under stress and
allows the structures of Ab aggregation intermediate states to
be investigated under native-like conditions, which is not otherwise technically possible. We show that IB40/IB42 fulfil all
the requirements to be classified as amyloids: they seed fibril
growth, are Congo red positive and show characteristic bsheet-
rich CD spectra. However, IB40 and IB42 are much less stable than fibrils formed in vitro and contain significant amounts of non-b-sheet regions, as seen from FTIR studies. Quantitative analyses of solution-state NMR H/D exchange rates show that the hydrophobic cores involving residues V18- F19-F20 adopt b-sheet conformations, whereas the C termini adopt a-helical coiled-coil structures. In the past, an a-helical intermediate-state structure has been postulated, but could not be verified experimentally. In agreement with the current literature, in which Ab oligomers are described as the most toxic state of the peptides, we find that IB42 contains SDS-resistant oligomers that are more neurotoxic than Ab42 fibrils. E. coli inclusion bodies formed by the Alzheimer’s disease bamyloid peptides Ab40 and Ab42 thus behave structurally like amyloid aggregation intermediate states and open the possibility of studying amyloids in a native-like, cellular environment.
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The ribosomal tunnel as a functional environment for nascent polypeptide folding and translational stalling
10.02.2011
Current Opinion in Structiral Biology,
2011,
21, Doi: 10.1016/j.sbi.2011.01.007,,
published on 10.02.2011
Current Opinion in Structural Biology, online article
Current Opinion in Structural Biology, online article
As the nascent polypeptide chain is being synthesized, it passes through a tunnel within the large ribosomal subunit and emerges at the solvent side where protein folding occurs. Despite the universality and conservation of dimensions of the ribosomal tunnel, a functional role for the ribosomal tunnel is only beginning to emerge: Rather than a passive conduit for the nascent chain, accumulating evidence indicates that the tunnel plays a more active role. In this article, we discuss recent structural insights into the role of the tunnel environment, and its implications for protein folding, co-translational targeting and translation regulation.
Peptides in the Ribosomal Tunnel Talk Back
04.02.2011
Molecular Cell,,
2011,
doi:10.1016/j.molcel.2011.01.017,
Volume 41, Issue 3, 247-248,
published on 04.02.2011
In this issue of Molecular Cell, Ramu et al. demonstrate that nascent peptides located within the ribosomal tunnel can talk back to the peptidyl transferase center to induce translational stalling by restricting the species of aminoacyl-tRNAs that can bind there.
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SecM-Stalled Ribosomes Adopt an Altered Geometry at the Peptidyl Transferase Center
18.01.2011
PLOS Biology,
2011,
doi:10.1371/journal.pbio.1000581,
10
published on 18.01.2011
PLOS Biology, online article
PLOS Biology, online article
As nascent polypeptide chains are synthesized, they pass through a tunnel in the large ribosomal subunit. Interaction
between specific nascent chains and the ribosomal tunnel is used to induce translational stalling for the regulation of gene
expression. One well-characterized example is the Escherichia coli SecM (secretion monitor) gene product, which induces stalling to up-regulate translation initiation of the downstream secA gene, which is needed for protein export. Although many of the key components of SecM and the ribosomal tunnel have been identified, understanding of the mechanism by which the peptidyl transferase center of the ribosome is inactivated has been lacking. Here we present a cryo-electron microscopy reconstruction of a SecM-stalled ribosome nascent chain complex at 5.6 A°. While no cascade of rRNA conformational changes is evident, this structure reveals the direct interaction between critical residues of SecM and the ribosomal tunnel. Moreover, a shift in the position of the tRNA–nascent peptide linkage of the SecM-tRNA provides a
rationale for peptidyl transferase center silencing, conditional on the simultaneous presence of a Pro-tRNAPro in the ribosomal A-site. These results suggest a distinct allosteric mechanism of regulating translational elongation by the SecM
stalling peptide.
PET Imaging of Integrin alpha-V-beta-3 Expression
17.01.2011
Theranostics,
2011,
ISSN: 1838-7640,
1:48-57
published on 17.01.2011
PET imaging of integrin alpha-v-beta-3 expression has been studied intensely by the academia and recently also by the industry. Imaging of integrin alpha-v-beta-3 expression is of great potential value, as the integrin alpha-v-beta-3 is a key player in tumor metastasis and angiogenesis. Therefore PET imaging of this target might be a suitable in-vivo biomarker of angiogenesis and metastatic potential of tumors. In this manuscript, the various strategies for PET imaging of the integrin alpha-v-beta-3 will be summarized, in-cluding monomeric and multimeric radiolabelled RGD peptides and nanoparticles. While most experiments have been performed using preclinical tumor models, more and more clinical results on PET imaging of alpha-v-beta-3 expression are available and will be discussed in detail. However, while a multitude of radiotracer strategies have been successfully evaluated for PET imaging of alpha-v-beta3, the ultimate clinical value of this new imaging biomarker still has to be evaluated in large clinical trials.
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Dynamic transcriptome analysis measures rates of mRNA synthesis and decay in yeast
04.01.2011
JMB,
2011,
doi:10.1038/msb.2010.112,
published on 04.01.2011
To obtain rates of mRNA synthesis and decay in yeast, we established dynamic transcriptome analysis (DTA). DTA combines non-perturbing metabolic RNA labeling with dynamic kinetic modeling. DTA reveals that most mRNA synthesis rates are around several transcripts per cell and cell cycle, and most mRNA half-lives range around a median of 11 min. DTA can monitor the cellular response to osmotic stress with higher sensitivity and temporal resolution than standard transcriptomics. In contrast to monotonically increasing total mRNA levels, DTA reveals three phases of the stress response. During the initial shock phase, mRNA synthesis and decay rates decrease globally, resulting in mRNA storage. During the subsequent induction phase, both rates increase for a subset of genes, resulting in production and rapid removal of stress-responsive mRNAs. During the recovery phase, decay rates are largely restored, whereas synthesis rates remain altered, apparently enabling growth at high salt concentration. Stress-induced changes in mRNA synthesis rates are predicted from gene occupancy with RNA polymerase II. DTA-derived mRNA synthesis rates identified 16 stress-specific pairs/triples of cooperative transcription factors, of which seven were known. Thus, DTA realistically monitors the dynamics in mRNA metabolism that underlie gene regulatory systems.
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Stable functionalized PEGylated quantum dots micelles with a controlled stoichiometry
03.01.2011
Chem. Commun.,
2011,
DOI: 10.1039/C0CC03753B,
1246-1248
published on 03.01.2011
Here we report the synthesis of monofunctional PEGylated amide ligands that were used to prepare bioactivable quantum dots of a 20 nm diameter with a controlled mean number of the covalently grafted ligands. They are stable in aqueous medium of high salinity including a large pH domain
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pH-Dependent Dimerization and Salt-Dependent Stabilization of the N-terminal Domain of Spider Dragline Silk-Implications for Fiber Formation
03.01.2011
Angewandte Chemie,
2011,
DOI: 10.1002/anie.201003795,
Volume 50, Issue 1, pages 310–313
published on 03.01.2011
The formation of spider dragline silk is controlled by the relatively small C- and N-terminal domains of the spidroins. The formidable and unrivaled mechanical tensile strength of spider silk fibers is a result of the carefully matched assembly of polyalanine (polyA) or poly(glycinealanine) (polyGA) repeat sequences separated by GGX or GPGXX repeats, which are thought to confer elasticity to the thread.The correct alignment of polyA/polyGA sequences to form microcrystalline structures is controlled by the pH value, salt concentration, and shear-force-induced partial unfolding of the disulfide-bridged dimeric C-terminal domain.The N-terminal domain was also shown to be important for the pH-dependent assembly of fiber.Here, we
use NMR spectroscopy and light-scattering techniques to show that the N-terminal domain of the major ampullate spider silk from Latrodectus hesperus (black widow spider) is mainly monomeric at neutral pH, as found in the spinning gland. The slight tendency to dimerize disappears under high salt conditions, as found in the gland. However, the Nterminal
domain will dimerize at the lower pH value found in the spinning duct. Hence, acidification mainly controls the assembly of the N terminus, which is important for the formation of silk fiber, while high ionic strength stabilizes the monomeric N-terminal structure. The crystal structure of the N-terminal domain shows a homodimer with an antiparallel orientation of the subunit. In addition to this picture, our NMR data provide further evidence for the regulation and functional role of this domain in forming elongated silk threads.
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Proteomic Characterization of Archaeal Ribosomes Reveals the Presence of Novel Archaeal-Specific Ribosomal Proteins
04.12.2010
JMB,
2011,
405,
1215-32
published on 04.12.2010
Protein synthesis occurs in macromolecular particles called ribosomes. All ribosomes are composed of RNA and proteins. While the protein composition of bacterial and eukaryotic ribosomes has been well-characterized, a systematic analysis of archaeal ribosomes has been lacking. Here we report the first comprehensive two-dimensional PAGE and mass spectrometry analysis of archaeal ribosomes isolated from the thermophilic Pyrobaculum aerophilum and the thermoacidophilic Sulfolobus acidocaldarius Crenarchaeota. Our analysis identified all 66 ribosomal proteins (r-proteins) of the P. aerophilum small and large subunits, as well as all but two (62 of 64; 97%) r-proteins of the S. acidocaldarius small and large subunits that are predicted genomically. Some r- proteins were identified with one or two lysine methylations and N-terminal acetylations. In addition, we identify three hypothetical proteins that appear to be bona fide r-proteins of the S. acidocaldarius large subunit. Dissociation of r- proteins from the S. acidocaldarius large subunit indicates that the novel r- proteins establish tighter interactions with the large subunit than some integral r-proteins. Furthermore, cryo electron microscopy reconstructions of the S. acidocaldarius and P. aerophilum 50S subunits allow for a tentative localization of the binding site of the novel r-proteins. This study illustrates not only the potential diversity of the archaeal ribosomes but also the necessity to experimentally analyze the archaeal ribosomes to ascertain their protein composition. The discovery of novel archaeal r-proteins and factors may be the first step to understanding how archaeal ribosomes cope with extreme environmental conditions.
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