Content
2010
Architecture of the RNA polymerase II–TFIIF complex revealed by cross-linking and mass spectrometry
11.01.2011
The EMBO Journal,
2010,
doi:10.1038/emboj.2009.401,
717 - 726
published on 11.01.2011
Higher-order multi-protein complexes such as RNA polymerase II (Pol II) complexes with transcription initiation factors are often not amenable to X-ray structure determination. Here, we show that protein cross-linking coupled to mass spectrometry (MS) has now sufficiently advanced as a tool to extend the Pol II structure to a 15-subunit, 670 kDa complex of Pol II with the initiation factor TFIIF at peptide resolution. The N-terminal regions of TFIIF subunits Tfg1 and Tfg2 form a dimerization domain that binds the Pol II lobe on the Rpb2 side of the active centre cleft near downstream DNA. The C-terminal winged helix (WH) domains of Tfg1 and Tfg2 are mobile, but the Tfg2 WH domain can reside at the Pol II protrusion near the predicted path of upstream DNA in the initiation complex. The linkers between the dimerization domain and the WH domains in Tfg1 and Tfg2 are located to the jaws and protrusion, respectively. The results suggest how TFIIF suppresses non-specific DNA binding and how it helps to recruit promoter DNA and to set the transcription start site. This work establishes cross-linking/MS as an integrated structure analysis tool for large multi-protein complexes.
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Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites
01.12.2010
Nature,
2010,
468,
713 - 16
published on 01.12.2010
The elongation cycle of protein synthesis involves the delivery of aminoacyl-transfer RNAs to the aminoacyl-tRNA-binding site (A site) of the ribosome, followed by peptide-bond formation and translocation of the tRNAs through the ribosome to reopen the A site1, 2. The translocation reaction is catalysed by elongation factor G (EF-G) in a GTP-dependent manner3. Despite the availability of structures of various EF-G–ribosome complexes, the precise mechanism by which tRNAs move through the ribosome still remains unclear. Here we use multiparticle cryoelectron microscopy analysis to resolve two previously unseen subpopulations within Thermus thermophilus EF-G–ribosome complexes at subnanometre resolution, one of them with a partly translocated tRNA. Comparison of these substates reveals that translocation of tRNA on the 30S subunit parallels the swivelling of the 30S head and is coupled to unratcheting of the 30S body. Because the tRNA maintains contact with the peptidyl-tRNA-binding site (P site) on the 30S head and simultaneously establishes interaction with the exit site (E site) on the 30S platform, a novel intra-subunit ‘pe/E’ hybrid state is formed. This state is stabilized by domain IV of EF-G, which interacts with the swivelled 30S-head conformation. These findings provide direct structural and mechanistic insight into the ‘missing link’ in terms of tRNA intermediates involved in the universally conserved translocation process.
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Structure and DNA-binding activity of the Pyrococcus furiosus SMC protein hinge domain
29.11.2010
Proteins,
2010,
Volume 79, Issue 2, pages 558–568,
DOI: 10.1002/prot.22903
published on 29.11.2010
Structural Maintenance of Chromosomes (SMC) proteins are essential for a wide range of processes including chromosome structure and dynamics, gene regulation, and DNA repair. While bacteria and archaea have one SMC protein that forms a homodimer, eukaryotes possess three distinct SMC complexes, consisting of heterodimeric pairs of six
different SMC proteins. SMC holocomplexes additionally
contain several specific regulatory subunits. The bacterial SMC complex is required for chromosome condensation and segregation. In eukaryotes, this function is carried out by the condensin (SMC2–SMC4) complex. SMC proteins consist of
N-terminal and C-terminal domains that fold back onto each other to create an ATPase ‘‘head’’ domain, connected to a central ‘‘hinge’’ domain via a long coiled-coil region. The hinge domain mediates dimerization of SMC proteins and binds DNA. This activity implicates a direct involvement of the hinge
domain in the action of SMC proteins on DNA. We studied the SMC hinge domain from the thermophilic archaeon Pyrococcus furiosus. Its crystal structure shows that the SMC hinge domain fold is largely conserved between archaea and bacteria as well as eukarya. Like the eukaryotic condensin
hinge domain, the P. furiosus SMC hinge domain preferentially binds single-stranded DNA (ssDNA), but its affinity for DNA is weaker than that of its eukaryotic counterpart, and point mutations reveal that its DNA-binding surface is more confined. The ssDNA-binding activity of its hinge domain might
play a role in the DNA-loading process of the prokaryotic SMC complex during replication.
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CIPSM's Daniel Wilson received the EMBO Young Investigators Award for his work on new antibiotics
23.11.2010
EMBO,
2010,
published on 23.11.2010
Today, the European Molecular Biology Organization
(EMBO) announced the selection of 21 of Europeʼs most talented young researchers as 2010 beneficiaries of the
EMBO Young Investigator Programme. We congratulate CIPSM's Daniel Wilson to be one of them. Daniel received the EMBO Young Investigators Award for his development and research of new antiobiotics that regulate the ribosome.
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RNA polymerase II–TFIIB structure and mechanism of transcription initiation
19.11.2010
Nature,
2010,
462,
323 - 330
published on 19.11.2010
To initiate gene transcription, RNA polymerase II (Pol II) requires the transcription factor IIB (B). Here we present the crystal structure of the complete Pol II–B complex at 4.3 Å resolution, and complementary functional data. The results indicate the mechanism of transcription initiation, including the transition to RNA elongation. Promoter DNA is positioned over the Pol II active centre cleft with the 'B-core' domain that binds the wall at the end of the cleft. DNA is then opened with the help of the 'B-linker' that binds the Pol II rudder and clamp coiled-coil at the edge of the cleft. The DNA template strand slips into the cleft and is scanned for the transcription start site with the help of the 'B-reader' that approaches the active site. Synthesis of the RNA chain and rewinding of upstream DNA displace the B-reader and B-linker, respectively, to trigger B release and elongation complex formation.
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Reversible and Controllable Nanolocomotion of an RNA-Processing Machinery
17.11.2010
Nano Letters,
2010,
10,
5123-30
published on 17.11.2010
Molecular motors have inspired many avenues of research for nanotechnology but most molecular motors studied so far allow only unidirectional movement. The archaeal RNA-exosome is a reversible motor that can either polymerize or degrade an RNA strand, depending on the chemical environments. We developed a single molecule fluorescence assay to analyze the real time locomotion of this nanomachine on RNA. Despite the multimeric structure, the enzyme followed the Michaelis-Menten kinetics with the maximum speed of ∼3 nucleotides/s, showing that the three catalytic cylinders do not fire cooperatively. We also demonstrate rapid directional switching on demand by fluidic control. When the two reaction speeds are balanced on average, the enzyme shows a memory of the previous reaction it catalyzed and stochastically switches between primarily polymerizing and primarily degrading behaviors. The processive, reversible, and controllable locomotion propelled by this nanomachine has a promising potential in environmental sensing, diagnostic, and cargo delivery applications.
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Localization of eukaryote-specific ribosomal proteins in a 5.5-Å cryo-EM map of the 80S eukaryotic ribosome
16.11.2010
PNAS,
2010,
vol. 107 no. 46 19754-19759,
doi: 10.1073/pnas.1010005107
published on 16.11.2010
Protein synthesis in all living organisms occurs on ribonucleoprotein
particles, called ribosomes. Despite the universality of this
process, eukaryotic ribosomes are significantly larger in size than
their bacterial counterparts due in part to the presence of 80 r
proteins rather than 54 in bacteria. Using cryoelectron microscopy
reconstructions of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution, together with a 6.1-Å map of a translating Saccharomyces cerevisiae 80S ribosome, we have localized and modeled 74∕80 (92.5%) of the ribosomal proteins, encompassing 12 archaeal/eukaryote-specific small subunit proteins as well as the complete complement of the ribosomal proteins of the eukaryotic large subunit. Near-complete atomic models of the 80S ribosome provide insights into the structure, function, and evolution of the eukaryotic translational apparatus.
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Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-Å resolution
16.11.2010
PNAS,
2010,
doi: 10.1073/pnas.1009999107,
vol. 107 no. 46 19748-19753
published on 16.11.2010
Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryoelectron microscopy and single-particle reconstruction, we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution. This map, together with a 6.1-Å map of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ∼98% of the rRNA. Accurate assignment of the rRNA expansion segments (ES) and variable regions has revealed unique ES–ES and r-protein–ES interactions, providing insight into the structure and evolution of the eukaryotic ribosome.
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Mechanism of replication blocking and bypass of Y-family polymerase Eta by bulky acetylaminofluorene DNA adducts
12.11.2010
PNAS,
2010,
vol. 107 no. 48 20720-20725,
doi: 10.1073/pnas.1008894107
published on 12.11.2010
Heterocyclic aromatic amines produce bulky C8 guanine lesions in
vivo, which interfere and disrupt DNA and RNA synthesis. These
lesions are consequently strong replication blocks. In addition
bulky adducts give rise to point and frameshift mutations. The
translesion synthesis (TLS) DNA polymerase Eta is able to bypass
slowly C8 bulky adduct lesions such as the widely studied 2-aminofluorene- dG and its acetylated analogue mainly in an error-free manner. Replicative polymerases are in contrast fully blocked by the acetylated lesion. Here, we show that TLS efficiency of Pol Eta depends critically on the size of the bulky adduct forming the lesion. Based on the crystal structure, we show why the bypass reaction is so difficult and we provide a model for the bypass reaction. In our model, TLS is accomplished without rotation of the lesion into the anti conformation as previously thought.
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The transcription elongation factor Bur1-Bur2 interacts with replication protein A and maintains genome stability during replication stress
12.11.2010
The Journal of Biological Chemistry,
2010,
doi: 10.1074/jbc.M110.193292
published on 12.11.2010
Multiple DNA associated processes such as DNA repair, replication and recombination are crucial for the maintenance of genome integrity. Here, we show a novel interaction between the transcription elongation factor Bur1-Bur2 and replication protein A (RPA), the eukaryotic single-stranded DNA binding protein with functions in DNA repair, recombination, and replication. Bur1 interacts via its C-terminal domain with RPA, and bur1-ΔC mutants show a deregulated DNA damage response accompanied by increased sensitivity to DNA damage and replication stress as well as increased levels of persisting Rad52 foci. Interestingly, the DNA damage sensitivity of an rfa1 mutant is suppressed by bur1 mutation further underscoring a functional link between these two protein complexes. Taken together, the transcription elongation factor Bur1-Bur2 interacts with RPA and maintains genome integrity during DNA replication stress.
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NCO-sP(EO-stat-PO) surface coatings preserve biochemical properties of RGD peptides
10.11.2010
International Journal of Molecular Medicine,
2010,
27, 1,
139 - 145
published on 10.11.2010
We have previously reported that star shaped poly(ethylene oxide-stat-propylene oxide) macromers with 80% EO content and isocyanate functional groups at the distal ends [NCO-sP(EO-stat-PO)] can be used to generate coatings that are non-adhesive but easily functionalized for specific cell adhesion. In the present study, we investigated whether the NCO-sP(EO-stat-PO) surfaces maintain peptide configuration-specific cell-surface interactions or if differences between dissimilar binding molecules are concealed by the coating. To this end, we have covalently immobilized both linear-RGD peptides (gRGDsc) and cyclic-RGD (RGDfK) peptides in such coatings. Subsequently, SaOS-2 or human multipotent mesenchymal stromal cells (MSC) were seeded on these substrates. Cell adhesion, spreading and survival was observed for up to 30 days. The time span for cell adherence was not different on linear and cyclic RGD peptides, but was shorter in comparison to the unmodified glass surface. MSC proliferation on cyclic RGDfK modified coatings was 4 times higher than on films functionalized by linear gRGDsc sequences, underlining that the NCO-sP(EO-stat-PO) film preserves the configuration-specific biochemical peptide properties. Under basal conditions, MSC expressed osteogenic marker genes after 14 days on cyclic RGD peptides, but not on linear RGD peptides or the unmodified glass surfaces.
Our results indicate specific effects of these adhesion peptides on MSC biology and show that this coating system is useful for selective testing of cellular interactions with adhesive ligands.
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NES consensus redefined by structures of PKI-type and Rev-type nuclear export signals bound to CRM1
24.10.2010
Nature Structural & Molecular Biology,
2010,
doi:10.1038/nsmb.1931,
1367–1376
published on 24.10.2010
Nature Structural & Molecular Biology, online article
Nature Structural & Molecular Biology, online article
Classic nuclear export signals (NESs) confer CRM1-dependent nuclear export. Here we present crystal structures of the RanGTP−CRM1 complex alone and bound to the prototypic PKI or HIV-1 Rev NESs. These NESs differ markedly in the spacing of their key hydrophobic (Φ) residues, yet CRM1 recognizes them with the same rigid set of five Φ pockets. The different Φ spacings are compensated for by different conformations of the bound NESs: in the case of PKI, an alpha-helical conformation, and in the case of Rev, an extended conformation with a critical proline docking into a Φ pocket. NMR analyses of CRM1-bound and CRM1-free PKI NES suggest that CRM1 selects NES conformers that pre-exist in solution. Our data lead to a new structure-based NES consensus, and explain why NESs differ in their affinities for CRM1 and why supraphysiological NESs bind the exportin so tightly.
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Optimized Measurement Temperature Gives Access to the Solution Structure of a 49 kDa Homohexameric Beta - Propeller
20.10.2010
JACS,
2010,
132 (44), pp 15692–15698,
DOI: 10.1021/ja1064608
published on 20.10.2010
Ph1500 is a homohexameric, two-domain protein of unknown function from the hyperthermophilic archaeon Pyrococcus horikoshii. The C-terminal hexamerization domain (Ph1500C) is of particular interest, as it lacks sequence homology to proteins of known structure. However, it resisted crystallization for X-ray analysis, and proteins of this size (49 kDa) present a considerable challenge to NMR structure determination
in solution. We solved the high-resolution structure of Ph1500C, exploiting the hyperthermophilic nature of the protein to minimize unfavorable relaxation properties by high-temperature measurement. Thus, the side chain assignment (97%) and structure determination became possible at full proton density. To our knowledge, Ph1500C is the largest protein for which this has been achieved. To minimize detrimental fast water exchange of amide protons at increased temperature, we employed a strategy where the temperature was optimized separately for backbone and side chain experiments.
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Structural Basis for Translational Stalling by Human Cytomegalovirus and Fungal Arginine Attenuator Peptide
08.10.2010
Specific regulatory nascent chains establish direct interactions with the ribosomal tunnel, leading to translational stalling. Despite a wealth of biochemical data, structural insight into the mechanism of translational stalling in eukaryotes is still lacking. Here we use cryo-electron microscopy to visualize eukaryotic ribosomes stalled during the translation of two diverse regulatory peptides: the fungal arginine attenuator peptide (AAP) and the human cytomegalovirus (hCMV) gp48 upstream open reading frame 2 (uORF2). The C terminus of the AAP appears to be compacted adjacent to the peptidyl transferase center (PTC). Both nascent chains interact with ribosomal proteins L4 and L17 at tunnel constriction in a distinct fashion. Significant changes at the PTC were observed: the eukaryotic-specific loop of ribosomal protein L10e establishes direct contact with the CCA end of the peptidyl-tRNA (P-tRNA), which may be critical for silencing of the PTC during translational stalling. Our findings provide direct structural insight into two distinct eukaryotic stalling processes.
A tandem SH2 domain in transcription elongation factor Spt6 binds the phosphorylated RNA polymerase II CTD
06.10.2010
The Journal of Biological Chemistry,
2010,
doi: 10.1074/jbc.M110.144568,
41597-41603
published on 06.10.2010
The Journal of Biological Chemistry, online article
The Journal of Biological Chemistry, online article
Spt6 is an essential transcription elongation factor and histone chaperone that binds the C-terminal repeat domain (CTD) of RNA
polymerase (Pol) II. We show here that Spt6 contains a tandem SH2 domain with a novel structure and CTD-binding mode. The
tandem SH2 domain binds to a serine 2-phosphorylated CTD peptide in vitro, whereas its N-terminal SH2 subdomain, which we previously characterized, does not. CTD binding requires a positively charged crevice in the C-terminal SH2 subdomain, which lacks the canonical phospho-binding pocket of SH2 domains and had previously escaped detection. The tandem SH2 domain is apparently required for transcription elongation in vivo, as its deletion in cells is lethal in the presence of 6-azauracil.
Molecular Basis of RNA Polymerase III Transcription Repression by Maf1
01.10.2010
RNA polymerase III (Pol III) transcribes short RNAs required for cell growth. Under stress conditions, the conserved protein Maf1 rapidly represses Pol III transcription. We report the crystal structure of Maf1 and cryo-electron microscopic structures of Pol III, an active Pol III-DNA-RNA complex, and a repressive Pol III-Maf1 complex. Binding of DNA and RNA causes ordering of the Pol III-specific subcomplex C82/34/31 that is required for transcription initiation. Maf1 binds the Pol III clamp and rearranges C82/34/31 at the rim of the active center cleft. This impairs recruitment of Pol III to a complex of promoter DNA with the initiation factors Brf1 and TBP and thus prevents closed complex formation. Maf1 does however not impair binding of a DNA-RNA scaffold and RNA synthesis. These results explain how Maf1 specifically represses transcription initiation from Pol III promoters and indicate that Maf1 also prevents reinitiation by binding Pol III during transcription elongation.
Polymeric Substrates with Tunable Elasticity and Nanoscopically Controlled Biomolecule Presentation
13.09.2010
Langmuir,
2010,
pp 15472–15480,
DOI: 10.1021/la103065x
published on 13.09.2010
Despite tremendous progress in recent years, nanopatterning of hydrated polymeric systems such as hydrogels still represents a major challenge. Here, we employ block copolymer nanolithography to arrange gold nanoparticles on a
solid template, followed by the transfer of the pattern to a polymeric hydrogel. In the next step, these nanoparticles serve
as specific anchor points for active biomolecules. We demonstrate the engineering of poly(ethylene glycol) hydrogel
surfaces with respect to elasticity, nanopatterning, and functionalization with biomolecules. For the first time,
biomolecule arrangement on the nanometer scale and substrate stiffness can be varied independently from each other. Young’s moduli, a measure of the compliance of the substrates, can be tuned over 4 orders of magnitude, including the values for all of the different tissues found in the human body. Structured hydrogels can be used to pattern any histidinetagged protein as exemplified for his-protein A as an acceptor for immunoglobulin. When cell-adhesion-promoting
peptide cRGDfK is selectively coupled to gold nanoparticles, the surfaces provide cues for cell-surface interaction and allow for the study of the modulation of cellular adhesion by the mechanical properties of the environment. Therefore, these substrates represent a unique multipurpose platform for studying receptor/ligand interactions with adhering cells,
mechanotransduction, and cell-adhesion-dependent signaling.
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Uniform transitions of the general RNA polymerase II transcription complex
05.09.2010
Nature Structural & Molecular Biology,
2010,
17,
doi:10.1038/nsmb.1903
published on 05.09.2010
We present genome-wide occupancy profiles for RNA polymerase (Pol) II, its phosphorylated forms and transcription factors in proliferating yeast. Pol II exchanges initiation factors for elongation factors during a 5′ transition that is completed 150 nucleotides downstream of the transcription start site (TSS). The resulting elongation complex is composed of all the elongation factors and shows high levels of Ser7 and Ser5 phosphorylation on the C-terminal repeat domain (CTD) of Pol II. Ser2 phosphorylation levels increase until 600–1,000 nucleotides downstream of the TSS and do not correlate with recruitment of Spt6 and Pcf11, which bind the Ser2-phosphorylated CTD in vitro. This indicates CTD-independent recruitment mechanisms and CTD masking in vivo. Elongation complexes are productive and disassemble in a two-step 3′ transition. Paf1, Spt16 (part of the FACT complex), and the CTD kinases Bur1 and Ctk1 exit upstream of the polyadenylation site, whereas Spt4, Spt5, Spt6, Spn1 (also called Iws1) and Elf1 exit downstream. Transitions are uniform and independent of gene length, type and expression.
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Hsp12 Is an Intrinsically Unstructured Stress Protein that Folds upon Membrane Association and Modulates Membrane Function
27.08.2010
Hsp12 of S. cerevisiae is upregulated several 100-fold in response to stress. Our phenotypic analysis showed that this protein is important for survival of a variety of stress conditions, including high temperature. In the absence of Hsp12, we observed changes in cell morphology under stress conditions. Surprisingly, in the cell, Hsp12 exists both as a soluble cytosolic protein and associated to the plasma membrane. The in vitro analysis revealed that Hsp12, unlike all other Hsps studied so far, is completely unfolded; however, in the presence of certain lipids, it adopts a helical structure. The presence of Hsp12 does not alter the overall lipid composition of the plasma membrane but increases membrane stability.
RNA Polymerase I Contains a TFIIF-Related DNA-Binding Subcomplex
27.08.2010
Molecular Cell,
2010,
39, 4,
583 - 94
published on 27.08.2010
The eukaryotic RNA polymerases Pol I, II, and III use different promoters to transcribe different classes of genes. Promoter usage relies on initiation factors, including TFIIF and TFIIE, in the case of Pol II. Here, we show that the Pol I-specific subunits A49 and A34.5 form a subcomplex that binds DNA and is related to TFIIF and TFIIE. The N-terminal regions of A49 and A34.5 form a dimerization module that stimulates polymerase-intrinsic RNA cleavage and has a fold that resembles the TFIIF core. The C-terminal region of A49 forms a “tandem winged helix” (tWH) domain that binds DNA with a preference for the upstream promoter nontemplate strand and is predicted in TFIIE. Similar domains are predicted in Pol III-specific subunits. Thus, Pol I/III subunits that have no counterparts in Pol II are evolutionarily related to Pol II initiation factors and may have evolved to mediate promoter specificity and transcription processivity.
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Structural Basis for Adenylate Kinase Activity in ABC ATPases
13.08.2010
Journal of Molecular Biology,
2010,
401, 2,
265 - 73
published on 13.08.2010
Journal of Molecular Biology, online article
Journal of Molecular Biology, online article
ATP-binding cassette (ABC) enzymes are involved in diverse biological processes ranging from transmembrane transport to chromosome cohesion and DNA repair. They typically use ATP hydrolysis to conduct energy-dependent biological reactions. However, the cystic fibrosis transmembrane conductance regulator and the DNA repair protein Rad50 can also catalyze the adenylate kinase reaction (ATP + AMP ↔ 2ADP). To clarify and provide a mechanistic basis for the adenylate kinase activity of ABC enzymes, we report the crystal structure of the nucleotide-binding domain of the Pyrococcus furiosus structural maintenance of chromosome protein (pfSMCnbd) in complex with the adenylate kinase inhibitor P1,P5-di(adenosine-5′)pentaphosphate. We show that pfSMCnbd possesses reverse adenylate kinase activity. Our results suggest that in adenylate kinase reactions, ATP binds to its canonical binding site while AMP binds to the Q-loop glutamine and a hydration water of the Mg2+ ion. Furthermore, mutational analysis indicates that adenylate kinase reaction occurs in the engaged pfSMCnbd dimer and requires the Signature motif for phosphate transfer. Our results explain how ATP hydrolysis and adenylate kinase reactions can be catalyzed by the same functional motifs within the structural framework of ABC enzymes. Thus, adenylate kinase activity is likely to be a latent activity in many ABC enzymes.
Structure of an atypical Tudor domain in the Drosophila Polycomblike protein
28.07.2010
Protein Science,
2010,
DOI: 10.1002/pro.476,
11
published on 28.07.2010
Protein Science, online article
Protein Science, online article
Post-translational modifications of histone tails are among the most prominent epigenetic marks and play a critical role in transcriptional control at the level of chromatin. The Polycomblike (Pcl) protein is part of a histone methyltransferase complex (Pcl-PRC2) responsible for high levels of histone H3 K27 trimethylation. Studies in Drosophila larvae suggest that Pcl is required for anchoring Pcl-PRC2 at target genes, but how this is achieved is unknown. Pcl comprises a Tudor domain and two PHD fingers. These domains are known to recognize methylated lysine or arginine residues and could contribute to targeting of Pcl-PRC2. Here, we report an NMR structure of the Tudor domain from Drosophila Pcl (Pcl-Tudor) and binding studies with putative ligands. Pcl-Tudor contains an atypical, incomplete aromatic cage that does not interact with known Tudor domain ligands, such as methylated lysines or arginines. Interestingly, human Pcl orthologs exhibit a complete aromatic cage, suggesting that they may recognize methylated lysines. Structural comparison with other Tudor domains suggests that Pcl-Tudor may engage in intra- or intermolecular interactions through an exposed hydrophobic surface patch.
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Improvement of drug-like properties of peptides: the somatostatin paradigm
15.07.2010
informa healthcare,
2010,
Vol. 5, No. 7 , Pages 655-671,
doi:10.1517/17460441.2010.493935
published on 15.07.2010
Importance of the field: Peptides are promising candidates as therapeutic agents due to their wide involvement in physiological processes. However, their often non-selective activity and their poor drug-like properties, mainly their inherent low stability to enzymatic degradation and poor oral bioavailability, limit their clinical potential. Somatostatin is a peptide hormone involved in many different biological functions. The role of its five different
receptor subtypes and their interplay in medicinal processes is only partially understood. In addition, it suffers from poor drug-like properties.
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Structural Basis for Homodimerization of the Src-associated during Mitosis, 68-kDa Protein (Sam68) Qua1 Domain
06.07.2010
Sam68 (Src-associated during mitosis, 68 kDa) is a prototypical member of the STAR (signal transducer and activator of RNA) family of RNA-binding proteins. STAR proteins bind mRNA targets and modulate cellular processes such as cell cycle regulation and tissue development in response to extracellular signals. Sam68 has been shown to modulate alternative splicing of the pre-mRNAs of CD44 and Bcl-xL, which are linked to tumor progression and apoptosis. Sam68 and other STAR proteins recognize bipartite RNA sequences and are thought to function as homodimers. However, the structural and functional roles of the self-association are not known. Here, we present the solution structure of the Sam68 Qua1 homodimerization domain. Each monomer consists of two antiparallel α-helices connected by a short loop. The two subunits are arranged perpendicular to each other in an unusual four-helix topology. Mutational analysis of Sam68 in vitro and in a cell-based assay revealed that the Qua1 domain and residues within the dimerization interface are essential for alternative splicing of a CD44 minigene. Together, our results indicate that the Qua1 homodimerization domain is required for regulation of alternative splicing by Sam68.
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Probing Translation with Small-Molecule Inhibitors
25.06.2010
Cell,
2010,
DOI 10.1016/j.chembiol.2010.06.003,
Volume 17, Issue 6, 633-645
published on 25.06.2010
The translational apparatus of the bacterial cell remains one of the principal targets of antibiotics for the clinical treatment of infection worldwide. Since the introduction of specific translation inhibitors into clinical practice in the late 1940s, intense efforts have been made to understand their precise mechanisms of action. Such research has often revealed significant and sometimes unexpected insights into many fundamental
aspects of the translation mechanism. Central to progress in this area, high-resolution crystal structures of the bacterial ribosome identifying the sites of antibiotic binding are now available, which, together with recent developments in single-molecule and fast-kinetic approaches, provide an integrated view of the dynamic translation process. Assays employing these approaches and focusing on specific steps of the overall translation process are amenable for drug screening. Such assays, coupled with structural studies, have the potential not only to accelerate the discovery of novel and effective antimicrobial agents, but also to refine our understanding of the mechanisms of translation. Antibiotics often stabilize specific functional states of the ribosome and therefore allow distinct translation steps to be dissected in molecular detail.
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The archaeo-eukaryotic primase of plasmid pRN1 requires a helix bundle domain for faithful primer synthesis
28.05.2010
Nucleic Acids Research,
2010,
doi: 10.1093/nar/gkq447,
Vol. 38, No. 19 6707–6718
published on 28.05.2010
Nucleic Acids Research, online article
Nucleic Acids Research, online article
The plasmid pRN1 encodes for a multifunctional replication protein with primase, DNA polymerase and helicase activity. The minimal region required for primase activity encompasses amino-acid residues 40–370. While the N-terminal part of that minimal region (residues 47–247) folds into the prim/pol domain and bears the active site, the structure and function of the C-terminal part (residues 248–370) is unknown. Here we show that the C-terminal part of the minimal region folds into a compact domain with six helices and is stabilized by a disulfide bond. Three helices superimpose well with the C-terminal domain of the primase of the bacterial broad host range plasmid RSF1010. Structure-based site-directed mutagenesis shows that the C-terminal helix of the helix bundle domain is required for primase activity although it is distant to the active site in the crystallized conformation. Furthermore, we identified mutants of the C-terminal domain, which are defective in template binding, dinucleotide formation and conformation change prior to DNA extension.
Interplay between the Ribosomal Tunnel, Nascent Chain, and Macrolides Influences Drug Inhibition
27.05.2010
Chemistry & Biology,
2010,
17 (DOI 10.1016/j.chembiol.2010.04.008),
504-14
published on 27.05.2010
Accumulating evidence suggests that, during translation, nascent chains can form specific interactions with ribosomal exit tunnel to regulate translation and promote initial folding events. The clinically important macrolide antibiotics bind within the exit tunnel and inhibit translation by preventing progression of the nascent chain and inducing peptidyl-tRNA drop-off. Here, we have synthesized amino acid–and peptide-containing macrolides, which are used to demonstrate that distinct amino acids and peptides can establish interaction with components of the ribosomal tunnel and enhance the ribosomebinding and inhibitory properties of the macrolide
drugs, consistent with the concept that the exit tunnel is not simply a Teflon-like channel. Surprisingly, we find that macrolide antibiotics do not inhibit translation of all nascent chains similarly, but rather
exhibit polypeptide-specific inhibitory effects, providing a change to our general mechanistic understanding of macrolide inhibition.
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Nucleocytoplasmic shuttling of the La motif-containing protein Sro9 might link its nuclear and cytoplasmic functions
21.05.2010
RNA,
2010,
doi: 10.1261/rna.2089110,
published on 21.05.2010
Diverse steps in gene expression are tightly coupled. Curiously, the La-motif-containing protein Sro9 has been shown to play a role in transcription and translation. Here, we show that Sro9 interacts with nuclear and cytoplasmic protein complexes involved in gene expression. In addition, Sro9 shuttles between nucleus and cytoplasm and is exported from the nucleus in an mRNA export-dependent manner. Importantly, Sro9 is recruited to transcribed genes. However, whole genome expression analysis shows that loss of Sro9 function does not greatly change the level of specific transcripts indicating that Sro9 does not markedly affect their synthesis and/or stability. Taken together, Sro9 might bind to the mRNP already during transcription and accompany the mature mRNP to the cytoplasm where it modulates translation of the mRNA.
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Towards molecular systems biology of gene transcription and regulation
19.05.2010
Biological Chemistry,
2010,
DOI 10.1515/BC.2010.094,
Vol. 391, pp. 731–735
published on 19.05.2010
Ten years after the determination of the RNA polymerase II structure, the basic mechanism of mRNA synthesis during gene transcription is known. In the future, the initiation and regulation of transcription must be studied with a combination of structural biology, biochemistry, functional genomics, and computational methods. In this article, the efforts of our laboratory to move from an integrated structural biology of gene transcription towards molecular systems biology of gene regulation are reviewed.
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Multiple N-Methylation of MT-II Backbone Amide Bonds Leads to Melanocortin Receptor Subtype hMC1R Selectivity: Pharmacological and Conformational Studies
14.05.2010
Journal of the American Chemical Society,
2010,
132, 23,
8115 - 28
published on 14.05.2010
JACS, online article
JACS, online article
Multiple N-methylation is a novel technology to improve bioavailability of peptides and increase
receptor subtype selectivity. This technique has been applied here to the superpotent but nonselective
cyclic peptide MT-II. A library of all possible 31 backbone N-methylated derivatives has been synthesized
and tested for binding and activation at melanocortin receptor subtypes 1, 3, 4, and 5. It turned out that
selectivity is improved with every introduced N-methyl group, resulting in several N-methylated selective
and potent agonists for the hMC1R. The most potent of these derivatives is N-methylated on four out of
five amide bonds in the cyclic structure. Its solution structure indicates a strongly preferred backbone
conformation that resembles other R-MSH analogs but possesses much less flexibility and in addition distinct
differences in the spatial arrangement of individual amino acid side chains.
A conserved spider silk domain acts as a molecular switch that controls fibre assembly
13.05.2010
Nature,
2010,
465,
doi:10.1038/nature08936
published on 13.05.2010
A huge variety of proteins are able to form fibrillar structures,
especially at high protein concentrations. Hence, it is surprising
that spider silk proteins can be stored in a soluble form at high
concentrations and transformed into extremely stable fibres on
demand. Silk proteins are reminiscent of amphiphilic block copolymers containing stretches of polyalanine and glycine-rich
polar elements forming a repetitive core flanked by highly conserved non-repetitive amino-terminal and carboxy-terminal
domains. The N-terminal domain comprises a secretion signal,
but further functions remain unassigned. The C-terminal domain
was implicated in the control of solubility and fibre formation
initiated by changes in ionic composition8,9 and mechanical stimuli known to align the repetitive sequence elements and promote b-sheet formation. However, despite recent structural data little is known about this remarkable behaviour in molecular detail. Here we present the solution structure of the C-terminal domain of a spider dragline silk protein and provide evidence that the structural state of this domain is essential for controlled switching between the storage and assembly forms of silk proteins. In addition, the C-terminal domain also has a role in the alignment of secondary structural features formed by the repetitive elements in the backbone of spider silk proteins, which is known to be important for the mechanical properties of the fibre.
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Structural Analysis of Protein Interfaces from 13C Direct-Detected Paramagnetic Relaxation Enhancements
12.05.2010
The measurement of 13C directed-detected paramagnetic relaxation enhancements (PREs) on spin-labeled proteins combines the efficacy of PREs for the detection of long-range distance information with the favorable sensitivity and resolution of 13C direct-detected experiments. The 13C PREs provide long-range distance restraints to map binding interfaces in proteins and protein complexes and are especially useful for studies of high-molecular weight perdeuterated molecules.
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Cilengitide: The First Anti-Angiogenic Small Molecule Drug Candidate. Design, Synthesis and Clinical Evaluation
01.05.2010
Anti-Cancer Agents in Medicinal Chemistry,
2010,
10,
753 - 68
published on 01.05.2010
Cilengitide, a cyclic RGD pentapeptide, is currently in clinical phase III for treatment of glioblastomas and in phase II for
several other tumors. This drug is the first anti-angiogenic small molecule targeting the integrins alphavbeta3, alphavbeta5 and alpha5beta1. It was developed
by us in the early 90s by a novel procedure, the spatial screening. This strategy resulted in c(RGDfV), the first superactive alphavbeta3 inhibitor
(100 to 1000 times increased activity over the linear reference peptides), which in addition exhibited high selectivity against the platelet
receptor alphaIIbbeta3. This cyclic peptide was later modified by N-methylation of one peptide bond to yield an even greater antagonistic
activity in c(RGDf(NMe)V). This peptide was then dubbed Cilengitide and is currently developed as drug by the company Merck-Serono (Germany). This article describes the chemical development of Cilengitide, the biochemical background of its activity and a short review about the
present clinical trials. The positive anti-angiogenic effects in cancer treatment can be further increased by combination with “classical” anti-cancer therapies. Several clinical trials in this direction are under investigation.
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Structure and RNA recognition by the snRNA and snoRNA transport factor PHAX
29.04.2010
Small nuclear and small nucleolar RNAs (snRNAs and snoRNAs) are critical components of snRNPs and snoRNPs and play an essential role in the maturation of, respectively, mRNAs and rRNAs within the nucleus of eukaryotic cells. Complex and specific pathways exist for the assembly of snRNPs and snoRNPs, involving, for instance, nucleocytoplasmic transport of snRNAs and intranuclear transport between compartments of snoRNAs. The phosphorylated adaptor for nuclear export (PHAX) is required for nuclear export of snRNAs in metazoans and also involved in the intranuclear transport of snoRNAs to Cajal bodies. PHAX contains a conserved single-stranded nucleic acid binding domain (RNA_GG_bind domain) with no sequence homology with any other known RNA-binding module. Here, we report NMR and X-ray crystallography studies that elucidate the structural basis for RNA recognition by the PHAX RNA-binding domain (PHAX-RBD). The crystal structure of the RNA_GG_bind domain from the parasite Cryptosporidium parvum (Cp RBD) forms well-folded dimers in solution in the absence of any ligand. The human PHAX-RBD is monomeric and only adopts a tertiary fold upon RNA binding. The PHAX-RBD represents a novel helical fold and binds single-stranded RNA with micromolar affinity without sequence specificity. RNA recognition by human PHAX-RBD is consistent with mutational analysis that affects RNA binding and PHAX-mediated nuclear export. Our data suggest that the PHAX-RBD mediates auxiliary RNA contacts with the snRNA and snoRNA substrates that are required for transport and/or substrate release.
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Quantitative analysis of processive RNA degradation by the archaeal RNA exosome
14.04.2010
Nucleic Acids Research,
2010,
5166-5176,
doi: 10.1093/nar/gkq238
published on 14.04.2010
RNA exosomes are large multisubunit assemblies involved in controlled RNA processing. The archaeal exosome possesses a heterohexameric processing chamber with three RNase-PH-like active sites, capped by Rrp4- or Csl4-type subunits containing RNA-binding domains. RNA degradation by RNA exosomes has not been studied in a quantitative manner because of the complex kinetics involved, and exosome features contributing to efficient RNA degradation remain unclear. Here we derive a quantitative kinetic model for degradation of a model substrate by the archaeal exosome. Markov Chain Monte Carlo methods for parameter estimation allow for the comparison of reaction kinetics between different exosome variants and substrates. We show that long substrates are degraded in a processive and short RNA in a more distributive manner and that the cap proteins influence degradation speed. Our results, supported by small angle X-ray scattering, suggest that the Rrp4-type cap efficiently recruits RNA but prevents fast RNA degradation of longer RNAs by molecular friction, likely by RNA contacts to its unique KH-domain. We also show that formation of the RNase-PH like ring with entrapped RNA is not required for high catalytic efficiency, suggesting that the exosome chamber evolved for controlled processivity, rather than for catalytic chemistry in RNA decay.
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The Impact of Amino Acid Side Chain Mutations in Conformational Design of Peptides and Proteins
31.03.2010
Chemistry - A European Journal,
2010,
16, 18,
5385 - 90
published on 31.03.2010
Chemistry - A European Journal, online article
Chemistry - A European Journal, online article
Local energetic effects of amino acid replacements are often considered to have only a moderate influence on the backbone conformation of proteins or peptides. As these effects are difficult to determine experimentally, no comparison has yet been performed. However, knowledge of the influence of side chain mutations is essential in protein homology modeling and in optimizing biologically active peptide ligands in medicinal chemistry. Furthermore, the tool of N-methylation of peptides is of increasing importance for the design of peptidic drugs to gain oral availability or receptor selectivity. However, N-methylation is often accompanied by considerable population of cis-peptide bond structures, resulting in completely different conformations compared with the parent peptide. To retain a favored structure, it might be important to understand the effect of different side chains on the backbone conformation and to enable the introduction of an N-methylation at the right position without disturbing a biologically active conformation. In order to detect even small energetic effects due to side chain mutations, we employed a trick to investigate the structural equilibrium of a selected cyclic pentapeptide in which two conformations are equally populated. Very small energetic differences between both conformations could easily be determined experimentally by identifying shifts in the population of both isomers.
Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif
02.03.2010
Nucleic Acids Research,
2010,
doi: 10.1093/nar/gkq135,
4040–4051
published on 02.03.2010
Spt5 is the only known RNA polymerase-associated factor that is conserved in all three domains of life. We have solved the structure of the Methanococcus jannaschii Spt4/5 complex by X-ray crystallography, and characterized its function and interaction with the archaeal RNAP in a wholly recombinant in vitro transcription system. Archaeal Spt4 and Spt5 form a stable complex that associates with RNAP independently of the DNA–RNA scaffold of the elongation complex. The association of Spt4/5 with RNAP results in a stimulation of transcription processivity, both in the absence and the presence of the non-template strand. A domain deletion analysis reveals the molecular anatomy of Spt4/5 the Spt5 Nus-G N-terminal (NGN) domain is the effector domain of the complex that both mediates
the interaction with RNAP and is essential for its elongation activity. Using a mutagenesis approach, we have identified a hydrophobic pocket on the Spt5 NGN domain as binding site for RNAP, and recipro- cally the RNAP clamp coiled-coil motif as binding site for Spt4/5.
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Cell Adhesion Strength Is Controlled by Intermolecular Spacing of Adhesion Receptors
17.02.2010
Biophys J,
2010,
98,
543-51
published on 17.02.2010
Spatial patterning of biochemical cues on the micro- and nanometer scale controls numerous cellular processes such as spreading, adhesion, migration, and proliferation. Using force microscopy we show that the lateral spacing of individual integrin receptor-ligand bonds determines the strength of cell adhesion. For spacings R90 nm, focal contact formation was inhibited and the detachment forces as well as the stiffness of the cell body were significantly decreased compared to spacings %50 nm. Analyzing cell detachment at the subcellular level revealed that rupture forces of focal contacts increase with loading rate as predicted by a theoretical model for adhesion clusters. Furthermore, we show that the weak link between the intra- and extracellular space is at the intracellular side of a focal contact. Our results show that cells can amplify small differences in adhesive cues to large differences in cell adhesion strength.
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Asymmetric Activation of the Hsp90 Dimer by Its Cochaperone Aha1
12.02.2010
Mol. Cell,
2010,
37(3),
344-354, doi:10.1016/j.molcel.2010.01.006
published on 12.02.2010
Molecular Cell, online article
Molecular Cell, online article
The chaperone Hsp90 is an ATP-dependent, dimeric molecular machine regulated by several cochaperones, including inhibitors and the unique ATPase activator Aha1. Here, we analyzed the mechanism of the Aha1-mediated acceleration of Hsp90 ATPase activity and identified the interaction surfaces of both proteins using multidimensional NMR techniques. For maximum activation of Hsp90, the two domains of Aha1 bind to sites in the middle and N-terminal domains of Hsp90 in a sequential manner. This binding induces the kinetically unfavored N terminally dimerized state of Hsp90, which primes for the hydrolysis-competent conformation. Surprisingly, this activation mechanism is asymmetric. The presence of one Aha1 molecule per Hsp90 dimer is sufficient to bridge the two subunits and to fully stimulate Hsp90 ATPase activity. This seems to functionalize the two subunits of the Hsp90 dimer in different ways, in that one subunit can be used for conformational ATPase regulation and the other for substrate protein processing.
An Efficient Protocol for NMR-Spectroscopy-Based Structure Determination of Protein Complexes in Solution
10.02.2010
Angew. Chem.,
2010,
122(11),
2011 - 2014, doi:10.1002/ange.200906147
published on 10.02.2010
Angewandte Chemie, online article
Angewandte Chemie, online article
Puzzle-Arbeit: Eine effiziente, allgemein anwendbare Methode zur Bestimmung der Struktur von Proteinkomplexen und Mehrdomänenproteinen in Lösung mithilfe der NMR-Spektroskopie wird vorgestellt. Ausgehend von den bekannten hochaufgelösten Strukturen einzelner Domänen oder Untereinheiten wird die Domänenanordnung des Gesamtsystems aus NMR-Daten abgeleitet, die auch für hochmolekulare Komplexe zugänglich sind.
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α-Helical nascent polypeptide chains visualized within distinct regions of the ribosomal exit tunnel
07.02.2010
Nature Structural & Molecular Biology,
2010,
17,
313-17
published on 07.02.2010
Nature Structural & Molecular Biology, online article
Nature Structural & Molecular Biology, online article
As translation proceeds, the nascent polypeptide chain passes through a tunnel in the large ribosomal subunit. Although this ribosomal exit tunnel was once thought only to be a passive conduit for the growing nascent chain, accumulating evidence suggests that it may in fact play a more active role in regulating translation and initial protein folding events. Here we have determined single-particle cryo–electron microscopy reconstructions of eukaryotic 80S ribosomes containing nascent chains with high α-helical propensity located within the exit tunnel. The maps enable direct visualization of density for helices as well as allowing the sites of interaction with the tunnel wall components to be elucidated. In particular regions of the tunnel, the nascent chain adopts distinct conformations and establishes specific contacts with tunnel components, both ribosomal RNA and proteins, that have been previously implicated in nascent chain–ribosome interaction.
Structure and DNA binding activity of the mouse condensin hinge domain highlight common and diverse features of SMC proteins
05.02.2010
Nucleic Acids Research,
2010,
doi: 10.1093/nar/gkq038
published on 05.02.2010
Structural Maintenance of Chromosomes (SMC) proteins are vital for a wide range of processes including chromosome structure and dynamics, gene regulation and DNA repair. Eukaryotes have three SMC complexes, consisting of heterodimeric pairs of six different SMC proteins along with
several specific regulatory subunits. In addition to their other functions, all three SMC complexes play distinct roles in DNA repair. Cohesin (SMC1–SMC3) is involved in DNA double-strand break repair, condensin (SMC2–SMC4) participates in singlestrand break (SSB) repair, and the SMC5–SMC6
complex functions in various DNA repair pathways. SMC proteins consist of N- and C-terminal domains that fold back onto each other to create an ATPase ‘head’ domain, connected to a central ‘hinge’ domain via long coiled-coils. The hinge domain mediates dimerization of SMC proteins and binds
DNA, but it is not clear to what purpose this activity serves. We studied the structure and function of the condensin hinge domain from mouse. While the SMC hinge domain structure is
largely conserved from prokaryotes to eukaryotes, its function seems to have diversified throughout the course of evolution. The condensin hinge domain preferentially binds single-stranded DNA. We propose that this activity plays a role in the
SSB repair function of the condensin complex.
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Preparation and topology of the Mediator middle module
31.01.2010
Nucleic Acids Research,
2010,
doi: 10.1093/nar/gkq029,
published on 31.01.2010
Mediator is the central coactivor complex required for regulated transcription by RNA polymerase (Pol) II. Mediator consists of 25 subunits arranged in the head, middle, tail and kinase modules. Structural and functional studies of Mediator are limited by the availability of protocols for the prep- aration of recombinant modules. Here, we describe protocols for obtaining pure endogenous and recombinant complete Mediator middle module from Saccharomyces cerevisiae that consists of seven subunits: Med1, 4, 7, 9, 10, 21 and 31. Native
mass spectrometry reveals that all subunits are present in equimolar stoichiometry. Ion-mobility mass spectrometry, limited proteolysis, light scattering and small-angle X-ray scattering all indicate a high degree of intrinsic flexibility and an elongated shape of the middle module. Protein–protein interaction assays combined with previously published data suggest that the Med7 and Med4 subunits serve as a binding platform to form the three
heterodimeric subcomplexes, Med7N/21, Med7C/ 31 and Med4/9. The subunits, Med1 and Med10, which bridge to the Mediator tail module, bind to both Med7 and Med4.
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BclxL Changes Conformation upon Binding to Wild-type but Not Mutant p53 DNA Binding Domain
29.01.2010
The Journal of Biological Chemistry,
2010,
285,
3439 - 3450
published on 29.01.2010
p53 can induce apoptosis through mitochondrial membrane
permeabilization by interaction of its DNA binding
region with the anti-apoptotic proteins BclxL and Bcl2. However, little is known about the action of p53 at the mitochondria in molecular detail. By using NMR spectroscopy and fluorescence polarization we characterized the binding of wildtype
and mutant p53 DNA binding domains to BclxL and show
that the wild-type p53 DNA binding domain leads to structural changes in the BH3 binding region of BclxL, whereas mutants fail to induce such effects due to reduced affinity. This was probed by induced chemical shift and residual dipolar coupling data. These data imply that p53 partly achieves its pro-apoptotic function at the mitochondria by facilitating interaction between BclxL and BH3-only proteins in an allosteric mode of action. Furthermore, we characterize for the first time the binding behavior of Pifithrin-μ, a specific small molecule inhibitor of
the p53-BclxL interaction, and present a structural model of the protein-ligand complex. A rather unusual behavior is revealed whereby Pifithrin-μ binds to both sides of the protein-protein complex. These data should facilitate the rational design of more potent specific BclxL-p53 inhibitors.
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Coupled chaperone action in folding and assembly of hexadecameric Rubisco
14.01.2010
Nature,
2010,
doi:10.1038/nature08651,
Volume 463, 197-202
published on 14.01.2010
Nature, online article
Nature, online article
Form I Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), a complex of eight large (RbcL) and eight small (RbcS) subunits, catalyses the fixation of atmospheric CO2 in photosynthesis. The limited catalytic efficiency of Rubisco has sparked extensive efforts to re-engineer the enzyme with the goal of enhancing agricultural productivity. To facilitate such efforts we analysed the formation of cyanobacterial form I Rubisco by in vitro reconstitution and cryo-electron microscopy. We show that RbcL subunit folding by the GroEL/GroES chaperonin is tightly coupled with assembly mediated by the chaperone RbcX2. RbcL monomers remain partially unstable and retain high affinity for GroEL until captured by RbcX2. As revealed by the structure of a RbcL8–(RbcX2)8 assembly intermediate, RbcX2 acts as a molecular staple in stabilizing the RbcL subunits as dimers and facilitates RbcL8 core assembly. Finally, addition of RbcS results in RbcX2 release and holoenzyme formation. Specific assembly chaperones may be required more generally in the formation of complex oligomeric structures when folding is closely coupled to assembly.
Imaging of integrin avb3 expression in patients with malignant glioma by [18F] Galacto-RGD positron emission tomography
11.12.2009
Neuro-Oncology,
2009,
6,
861-70
published on 11.12.2009
Inhibitors targeting the integrin avb3 are promising new
agents currently tested in clinical trials for supplemental
therapy of glioblastoma multiforme (GBM). The
aim of our study was to evaluate 18F-labeled glycosylated
Arg-Gly-Asp peptide ([18F]Galacto-RGD) PET for
noninvasive imaging of avb3 expression in patients with
GBM, suggesting eligibility for this kind of additional
treatment. Patients with suspected or recurrent GBM
were examined with [18F]Galacto-RGD PET. Standardized
uptake values (SUVs) of tumor hotspots, galea, and
blood pool were derived by region-of-interest analysis.
[18F]Galacto-RGD PET images were fused with cranial
MR images for image-guided surgery. Tumor samples
taken from areas with intense tracer accumulation in the
[18F]Galacto-RGD PET images and were analyzed histologically
and immunohistochemically for avb3 integrin
expression. While normal brain tissue did not show significant
tracer accumulation (mean SUV, 0.09 6 0.04), GBMs demonstrated significant but heterogeneous tracer
uptake, with a maximum in the highly proliferating
and infiltrating areas of tumors (mean SUV, 1.6 6 0.5).
Immunohistochemical staining was prominent in tumor
microvessels as well as glial tumor cells. In areas of highly
proliferating glial tumor cells, tracer uptake (SUVs)
in the [18F]Galacto-RGD PET images correlated with
immunohistochemical avb3 integrin expression of corresponding
tumor samples. These data suggest that [18F]
Galacto-RGD PET successfully identifies avb3 expression
in patients with GBM and might be a promising tool for
planning and monitoring individualized cancer therapies
targeting this integrin.
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The Transmembrane Structure of Integrin aIIbb3: Significance for Signal Transduction
13.07.2009
Angewandte Chemie,
2010,
48 Issue 36,
6590-3
published on 13.07.2009
The formation of multicellular organisms requires concerted
action by cells, which alter their adhesive and migratory
behaviors. Cell adhesion and migration are tightly regulated
by intra- and extracellular signals, which are conveyed
through the cellular membrane by specialized receptors
known as integrins. Integrins are the starting point of a
variety of signaling cascades and are involved in a multitude
of physiological events important to multicellular organism
morphogenesis, ranging from cell adhesion to migration,
apoptosis, and angiogenesis as well as pathophysiological
behaviors such as those found in cancer metastasis. The
transmembrane (TM) domains of integrins are at the center
of integrin signaling. Recently, a structure of the TM
domains of the aIIbb3 integrin has been reported that sheds
light on the signal transduction mechanism of integrins. Integrins are essential TM proteins that couple the extracellular matrix to the cytoskeleton. They consist of noncovalently bound heterodimers, in which each subunit (a and b) contains one TM helix. With eighteen a and eight b subunit types identified, there are 24 known distinct heterodimer combinations with partially overlapping yet specific function.
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Force-induced destabilization of focal adhesions at defined integrin spacings on nanostructured surfaces
12.05.2009
The American Physical Society,
2010,
DOI: 10.1103/PhysRevE.81.051914,
7 pages
published on 12.05.2009
Focal adhesions are the anchoring points of cells to surfaces and are responsible for a large number of surface sensing processes. Nanopatterning studies have shown physiological changes in fibroblasts as a result of decreasing density of external binding ligands. The most striking of these changes is a decreased ability to form mature focal adhesions when lateral ligand distances exceed 76 nm. These changes are usually examined in the context of protein signaling and protein interactions. We show a physical explanation based on the balance between the forces acting on individual ligand connections and the reaction kinetics of those ligands. We propose three stability regimes for focal adhesions as a function of ligand spacing and applied stress: a stable regime, an unstable regime in which a large fraction of unbound protein causes adhesion disintegration, and a regime in which the applied force is too high to form an adhesion structure.
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