Search

In the eukaryotic nucleus, DNA is packaged in the form of nucleosomes, each of which comprises about 147 base pairs of DNA wrapped around a histone protein octamer. The position and histone composition of nucleosomes is governed by ATP-dependent chromatin remodellers.sup.1-3 such as the 15-subunit INO80 complex.sup.4. INO80 regulates gene expression, DNA repair and replication by sliding nucleosomes, the exchange of histone H2A.Z with H2A, and the positioning of + 1 and -1 nucleosomes at promoter DNA.sup.5-8. The structures and mechanisms of these remodelling reactions are currently unknown. Here we report the cryo-electron microscopy structure of the evolutionarily conserved core of the INO80 complex from the fungus Chaetomium thermophilum bound to a nucleosome, at a global resolution of 4.3 Å and with major parts at 3.7 Å. The INO80 core cradles one entire gyre of the nucleosome through multivalent DNA and histone contacts. An Rvb1/Rvb2 AAA.sup.+ ATPase heterohexamer is an assembly scaffold for the complex and acts as a 'stator' for the motor and nucleosome-gripping subunits. The Swi2/Snf2 ATPase motor binds to nucleosomal DNA at superhelical location -6, unwraps approximately 15 base pairs, disrupts the H2A-DNA contacts and is poised to pump entry DNA into the nucleosome. Arp5 and Ies6 bind superhelical locations -2 and -3 to act as a counter grip for the motor, on the other side of the H2A-H2B dimer. The Arp5 insertion domain forms a grappler element that binds the nucleosome dyad, connects the Arp5 actin-fold and entry DNA over a distance of about 90 Å and packs against histone H2A-H2B near the 'acidic patch'. Our structure together with biochemical data.sup.8 suggests a unified mechanism for nucleosome sliding and histone editing by INO80. The motor is part of a macromolecular ratchet, persistently pumping entry DNA across the H2A-H2B dimer against the Arp5 grip until a large nucleosome translocation step occurs. The transient exposure of H2A-H2B by motor activity as well as differential recognition of H2A.Z and H2A may regulate histone exchange.Cryo-electron microscopy structures of the evolutionarily conserved core of a fungal INO80 complex bound to the nucleosomal substrate reveal the mechanism underlying nucleosome sliding and histone editing used by this ATP-dependent chromatin remodeller., Author(s): Sebastian Eustermann [sup.1] [sup.2] , Kevin Schall [sup.1] [sup.2] , Dirk Kostrewa [sup.1] [sup.2] , Kristina Lakomek [sup.1] [sup.2] , Mike Strauss [sup.3] , Manuela Moldt [sup.1] [sup.2] , [...]

Transcription of ribosomal RNA by RNA polymerase (Pol) I initiates ribosome biogenesis and regulates eukaryotic cell growth. The crystal structure of Pol I from the yeast Saccharomyces cerevisiae at 2.8 Å resolution reveals all 14 subunits of the 590-kilodalton enzyme, and shows differences to Pol II. An 'expander' element occupies the DNA template site and stabilizes an expanded active centre cleft with an unwound bridge helix. A 'connector' element invades the cleft of an adjacent polymerase and stabilizes an inactive polymerase dimer. The connector and expander must detach during Pol I activation to enable transcription initiation and cleft contraction by convergent movement of the polymerase 'core' and 'shelf' modules. Conversion between an inactive expanded and an active contracted polymerase state may generally underlie transcription. Regulatory factors can modulate the core-shelf interface that includes a 'composite' active site for RNA chain initiation, elongation, proofreading and termination., Eukaryotic genes are transcribed by Pol I, II and III (ref. 1), which synthesize ribosomal, messenger and transfer RNA, respectively. Pol I synthesizes about 60% of cellular RNA by transcribing [...]

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., Transcription of eukaryotic protein-coding genes begins with formation of the pre-initiation complex (PIC) on promoter DNA. The PIC contains Pol II and the general transcription factors, including B and the [...]

Author(s): Liudmila Andreeva [1, 2]; Bjrn Hiller [1, 2]; Dirk Kostrewa [1, 2]; Charlotte Lssig [1, 2]; Carina C. de Oliveira Mann [1, 2]; David Jan Drexler [1, 2]; Andreas [...]

In recent years, short coiled coils have been used for applications ranging from biomaterial to medical sciences. For many of these applications knowledge of the factors that control the topology of the engineered protein systems is essential. Here, we demonstrate that trimerization of short coiled coils is determined by a distinct structural motif that encompasses specific networks of surface salt bridges and optimal hydrophobic packing interactions. The motif is conserved among intracellular, extracellular, viral, and synthetic proteins and defines a universal molecular determinant for trimer formation of short coiled coils. In addition to being of particular interest for the biotechnological production of candidate therapeutic proteins, these findings may be of interest for viral drug development strategies. protein engineering | sequence-to-structure rules | protein-protein interaction | salt bridges | x-ray crystallography

Ammonium is one of the most important nitrogen sources for bacteria, fungi, and plants, but it is toxic to animals. The ammonium transport proteins (methylamine permeases/ammonium transporters/rhesus) are present in all domains of life; however, functional studies with members of this family have yielded controversial results with respect to the chemical identity (N[H.sup.+.sub.4] or N[H.sub.3]) of the transported species. We have solved the structured of wild-type AmtB from Escherichia coli in two crystal forms at 1.8- and 2.1-[Angstrom] resolution, respectively. Substrate transport occurs through a narrow mainly hydrophobic pore located at the center of each monometer of the trimeric AmtB. At the periplasmic entry, a binding site for N[H.sup.+.sub.4] is observed. Two phenylalanine side chains (F107 and F215) block access into the pore from the periplasmic side. Further into the pore, the side chains of two highly conserved histidine residues (H168 and H318) bridged by a H-bond lie adjacent, with their edges pointing into the cavity. These histidine residues may facilitate the deprotonation of an ammonium ion entering the pore. Adiabatic free energy calculations support the hypothesis that an electrostatic barrier between H168 and H318 hinders the permeation of cations but not that of the uncharged N[H.sub.3]. The structural data and energetic considerations strongly indicate that the methylamine permeases/ammonium transporters/ rhesus proteins are ammonia gas channels. Interestingly, at the cytoplasmic exit of the pore, two different conformational states are observed that might be related to the inactivation mechanism by its regulatory partner. conformational change | x-ray structure

Protein deposition as amyloid fibrils underlies many debilitating human disorders. The complexity and size of disease-related polypeptides, however, often hinders a detailed rational approach to study effects that contribute to the process of amyloid formation. We report here a simplified peptide sequence successfully designed de novo to fold into a coiled-coil conformation under ambient conditions but to transform into amyloid fibrils at elevated temperatures. We have determined the crystal structure of the coiled-coil form and propose a detailed molecular model for the peptide in its fibrillar state. The relative stabilities of the two structural forms and the kinetics of their interconversion were found to be highly sensitive to small sequence changes. The results reveal the importance of specific packing interactions on the kinetics of amyloid formation and show the potential of this exceptionally favorable system for probing details of the molecular origins of amyloid disease.

Research has been conducted on Aspergillus fumigatus. The pH activity of this bacterium and the consensus phytases have been engineered via the use of chemical modification and site-directed mutagenesis and the details are presented.

X-ray diffraction experiments enable the determination of the crystal structures of the type II restriction endonuclease EcoRV complexed with an undecameric DNA fragment and of the complex formed between the enzyme-product and Mg2+. Crystal data enable the determination of the location of water molecules in the protein-DNA interface. X-ray data facilitate the determination of location and coordination of Mg2+ binding site before and after cleavage. Structural and kinetic data help design a transition model having two metals attached to a pentavalent phosphorane group.