Portal de Periódicos da CAPES

Paper alert

2001; Elsevier BV; Volume: 9; Issue: 3 Linguagem: Inglês

10.1016/s0969-2126(01)00582-2

1878-4186

Chosen by Robert Liddington, Christin Frederick, Stephen D. Fuller, Sophie Jackson,

Amino Acid Enzymes and Metabolism

A selection of interesting papers that were published in the month before our press date in major journals most likely to report significant results in structural biology, protein and RNA folding. □ Stability, folding, dimerization, and assembly properties of the yeast prion Ure2p. Carine Thual, Luc Bousset, Anton A. Komar, Stefan Walter, Johannes Buchner, Christophe Cullin, and Ronald Melki (2001). Biochemistry 40, 1764–1773. The [URE3] factor of Saccharomyces cerevisiae propagates by a prion-like mechanism and corresponds to the loss of the function of the cellular protein Ure2. The molecular basis of the propagation of this phenotype is unknown. We recently expressed Ure2p in Escherichia coli and demonstrated that the N-terminal region of the protein is flexible and unstructured, while its C-terminal region is compactly folded. Ure2p oligomerizes in solution to form mainly dimers that assemble into fibrils [Thual et al. (1999). J. Biol. Chem. 274, 13666–13674]. To determine the role played by each domain of Ure2p in the overall properties of the protein, specifically, its stability, conformation, and capacity to assemble into fibrils, we have further analyzed the properties of Ure2p N- and C-terminal regions. We show here that Ure2p dimerizes through its C-terminal region. We also show that the N-terminal region is essential for directing the assembly of the protein into a particular pathway that yields amyloid fibrils. A full-length Ure2p variant that possesses an additional tryptophan residue in its N-terminal moiety was generated to follow conformational changes affecting this domain. Comparison of the overall conformation, folding, and unfolding properties, and the behavior upon proteolytic treatments of full-length Ure2p, Ure2pW37 variant, and Ure2p C-terminal fragment reveals that Ure2p N-terminal domain confers no additional stability to the protein. This study reveals the existence of a stable unfolding intermediate of Ure2p under conditions where the protein assembles into amyloid fibrils. Our results contradict the intramolecular interaction between the N- and C-terminal moieties of Ure2p and the single unfolding transitions reported in a number of previous studies. □ Structure of the complete extracellular domain of the common β Subunit of the human GM-CSF, IL-3, and IL-5 receptors reveals a novel dimer configuration. Paul D. Carr, Sonja E. Gustin, Alice P. Church, James M. Murphy, Sally C. Ford, David A. Mann, Donna M. Woltring, Ian Walker, David L. Ollis, and Ian G. Young (2001). Cell 104, 291–300. The receptor systems for the hemopoietic cytokines GM-CSF, IL-3, and IL-5 consist of ligand-specific α receptor subunits that play an essential role in the activation of the shared βc subunit, the major signaling entity. The authors report the structure of the complete βc extracellular domain. It has a structure unlike any class I cytokine receptor described thus far, forming a stable interlocking dimer in the absence of ligand in which the G strand of domain 1 hydrogen bonds into the corresponding β sheet of domain 3 of the dimer-related molecule. The G strand of domain 3 similarly partners with the dimer-related domain 1. □ Crystal structure of a complement control protein that regulates both pathways of complement activation and binds heparan sulfate proteoglycans. Krishna H. M. Murthy, Scott A. Smith, Vannakambadi K. Ganesh, Ken W. Judge, Nick Mullin, Paul N. Barlow, Craig M. Ogata, and Girish J. Kotwal (2001). Cell 104, 301–311. Vaccinia virus complement control protein (VCP), a homolog of mammalian regulators of complement activation, inhibits both pathways of complement activation through binding the third and fourth components. VCP possesses additional activities of significance to viral infectivity. The structure of VCP reveals a highly extended molecule with a putative heparin recognition site at its C-terminal end. A second cluster of positive charges provides a possibly overlapping binding site for both heparin and complement components. Experiments suggested by the structure indicate that VCP can bind heparin and control complement simultaneously. □ CD81 extracellular domain 3D structure: insight into the tetraspanin superfamily structural motifs. Kengo Kitadokoro, Domenico Bordo, Giuliano Galli, Roberto Petracca, Fabiana Falugi, Sergio Abrignani, Guido Grandi, and Martino Bolognesi (2001). EMBO J. 20, 12–18. Human CD81 is a transmembrane protein belonging to the tetraspanin family. The crystal structure of human CD81 large extracellular domain reveals that each subunit within the homodimeric protein displays a mushroom-like structure, composed of five α helices arranged in “stalk” and “head” subdomains. Sequence analysis of 160 tetraspanins indicates that key structural features and the new protein fold observed in the CD81 large extracellular domain are conserved within the family. It is proposed that tetraspanins may assemble at the cell surface into homo- and/or hetero-dimers through a conserved hydrophobic interface located in the stalk subdomain, while interacting with other liganding proteins, including hepatitis C virus E2, through the head subdomain. □ Structure of a genetically engineered molecular motor. Werner Kliche, Setsuko Fujita-Becker, Martin Kollmar, Dietmar J. Manstein, and F. Jon Kull (2001). EMBO J. 20, 40–46. Molecular motors move unidirectionally along polymer tracks, producing movement and force in an ATP-dependent fashion. They achieve this by amplifying small conformational changes in the nucleotide binding region into force-generating movements of larger protein domains. The authors describe the crystal structure of an artificial actin-based motor. By combining the catalytic domain of myosin II with a 130 Å conformational amplifier consisting of repeats 1 and 2 of α-actinin, they demonstrate that it is possible to genetically engineer single-polypeptide molecular motors with precisely defined lever arm lengths and specific motile properties. □ Solution structure of the PHD domain from the KAP-1 corepressor: structural determinants for PHD, RING and LIM zinc binding domains. Allan D. Capili, David C. Schultz, Frank J. Rauscher III, and Katherine L.B. Borden (2001). EMBO J. 20, 165–177. Plant homeodomain (PHD) domains are found in >400 eukaryotic proteins, many of which are transcriptional regulators. The authors report the first structural characterization of a PHD domain. The PHD domain from KAP-1 corepressor binds zinc in a cross-brace topology between antiparallel β strands reminiscent of RING domains. Using a mutational analysis, the authors define the structural features required for transcriptional repression by KAP-1 and explain naturally occurring, disease-causing mutations. □ Structural basis for the inactivation of retinoblastoma tumor suppressor by SV40 large T antigen. Hye-Yeon, Kim Byung-Yoon Ahn, and Yunje Cho (2001). EMBO J. 20, 295–304. Inactivation of the retinoblastoma (Rb) tumor suppressor by Simian virus 40 (SV40) large T antigen is one of the central features of tumorigenesis induced by SV40. Both the N-terminal J domain and the LxCxE motif of large T antigen are required for inactivation of Rb. The crystal structure of the N-terminal region (residues 7–117) of SV40 large T antigen bound to the pocket domain of Rb reveals that large T antigen contains a four-helix bundle, and residues from helices 2 and 4 and the loop containing the LxCxE motif participate in the interactions with Rb. The two central helices and a connecting loop in large T antigen have structural similarities with the J domains of the molecular chaperones DnaJ and HDJ-1, suggesting that large T antigen may use a chaperone mechanism for its biological function. □ The cellular receptor to human rhinovirus 2 binds around the 5-fold axis and not in the canyon: a structural view. Elizabeth A. Hewat, Emmanuelle Neumann, James E. Conway, R. Moser, B. Ronacher, and D. Blass (2000). EMBO J. 19, 6317–6325. The binding site of the major group of human rhinoviruses (e.g., HRV14) to their receptor, ICAM-1, has been well defined by the combination of X-ray crystallography, cEM, and biochemistry. These studies gave rise to the canyon hypothesis that further work modified by showing the degree of accessibility of residues near the receptor binding site. This paper uses cEM together with docking to explore the binding of a member of the minor group rhinoviruses (HRV2) to its receptor, the very low density lipoprotein receptor (VLDL-R). In contrast to the binding of ICAM-1 within the canyon, VLDL-R binds to the star-shaped dome on the virus 5-fold axis. The authors study binding of portions of the VLDL-R at 15 Å resolution to define the orientation of the receptor in its binding site. VLDL-R binding to HRV-2 does not cause the destabilization and expulsion of pocket factor that results from ICAM-1 binding to HRV14. The authors discuss the differences in the mechanism of virus entry and uncoating that result from the differences in position and effect of receptor binding. □ Escherichia coli RNA polymerase core and holoenzyme structures. R. D. Finn, E. V. Orlova, B. Gowen, M. Buck, and M. van Heel (2000). EMBO J. 19, 6833–6844. cEM and image reconstruction were used to determine two new structures for the RNA polymerase core (11 Å) and holoenzyme (9 Å) that serve essential roles in regulated gene expression. The authors align their E. coli core enzyme structure with the Taq core RNAp structure that was previously established by X-ray crystallography to define subunit locations. They suggest that the previously determined 27 Å E. coli core structure represents an open conformation due to the similarity with the Taq core. They interpret the substantial conformational changes between the holoenzyme and core by positioning the known structures of the subunits within the complex cEM map. They define the position of the aNTD dimer and s70 subunit within the holoenzyme but carefully indicate the limits of their ability to assign subunits in this complex structure. □ Hydrogen bonds with π-acceptors in proteins: frequencies and role in stabilizing local 3D structures. Thomas Steiner and Gertraud Koellner (2001). J. Mol. Biol. 305, 535–557. A comprehensive structural analysis of X-H···π hydrogen bonding in proteins is performed based on 592 published high-resolution crystal structures (1.6 Å). All potential donors and acceptors are considered, including acidic C-H groups. The sample contains 1311 putative X-H···π hydrogen bonds with N-H, O-H, or S-H donors, that is about 1 per 10.8 aromatic residues. By far the most efficient π-acceptor is the side-chain of Trp, which accepts one X-H···π hydrogen bond per 5.7 residues. The focus of the analysis is on recurrent structural patterns involving regular secondary structure elements. Numerous examples are found where peptide X-H···π interactions are functional in stabilization of helix termini, strand ends, strand edges, β bulges and regular turns. Side-chain X-H···π hydrogen bonds are formed in considerable numbers in α helices and β sheets. Geometrical data on various types of X-H···π hydrogen bonds are given. □ Solution structure of the human parvulin-like peptidyl prolyl cis/trans isomerase, hPar14. Tohru Terada, Mikako Shirouzu, Yasuhiro Fukumori, Fumihiro Fujimori, Yutaka Ito, Takanori Kigawa, Shigeyuki Yokoyama, and Takafumi Uchida (2001). J. Mol. Biol. 305, 917–926. The hPar14 protein is a peptidyl prolyl cis/trans isomerase and is a human parvulin homologue. The hPar14 protein shows about 30% sequence identity with the other human parvulin homologue, hPin1. Here, the solution structure of hPar14 was determined by nuclear magnetic resonance spectroscopy. The N-terminal 35 residues preceding the peptidyl prolyl isomerase domain of hPar14 are unstructured, whereas hPin1 possesses the WW domain at its N terminus. The fold of residues 36–131 of hPar14, which comprises a four-stranded β-sheet and three α-helices, is superimposable onto that of the peptidyl prolyl isomerase domain of hPin1. To investigate the interaction of hPar14 with a substrate, the backbone chemical-shift changes of hPar14 were monitored during titration with a tetra peptide. Met90, Val91, and Phe94 around the N terminus of 3 showed large chemical-shift changes. These residues form a hydrophobic patch on the molecular surface of hPar14. Two of these residues are conserved and have been shown to interact with the proline residue of the substrate in hPin1. On the other hand, hPar14 lacks the hPin1 positively charged residues (Lys63, Arg68, and Arg69), which determine the substrate specificity of hPin1 by interacting with phosphorylated Ser or Thr preceding the substrate Pro, and exhibits a different structure in the corresponding region. Therefore, the mechanism determining the substrate specificity seems to be different between hPar14 and hPin1. □ Detection of altered protein conformations in living cells. Xavier Raquet, Jörg H. Eckert, Silke Müller, and Nils Johnsson (2001). J. Mol. Biol. 305, 927–938. The maturation, conformational stability, and the rate of in vivo degradation are specific for each protein and depend on both the intrinsic features of the protein and those of the surrounding cellular environment. While synthesis and degradation can be measured in living cells, stability and maturation of proteins are more difficult to quantify. We developed the split-ubiquitin method into a tool for detecting and analyzing changes in protein conformation. The biophysical parameter that forms the basis of these measurements is the time-averaged distance between the N terminus and C terminus of a protein. Starting from three proteins of known structure, we demonstrate the feasibility of this approach, and employ it to elucidate the effect of a previously described mutation in the protein Sec62p on its conformation in living cells. □ Three-dimensional structure of Erwinia chrysanthemi pectin methylesterase reveals a novel esterase active site. John Jenkins, Olga Mayans, Drummond Smith, Kathryn Worboys, and Richard W. Pickersgill (2001). J. Mol. Biol. 305, 951–960. The authors report the crystal structure of pectin methylesterase that has neither the common/hydrolase fold nor the common catalytic Ser-His-Asp triad. The structure of the Erwinia chrysanthemi enzyme reveals the enzyme to comprise a right-handed parallel β helix as seen in the pectinolytic enzyme pectate lyase. There is no significant sequence similarity with any protein of known structure. Sequence conservation among the pectin methylesterases reveals that the active site comprises two aspartate residues and an arginine residue at a location similar to that of the active site and substrate binding cleft of pectate lyase. □ Rotamer strain energy in protein helices—quantification of a major force opposing protein folding. Simon Penel and Andrew J. Doig (2001). J. Mol. Biol. 305, 961–968. It is widely believed that the dominant force opposing protein folding is the entropic cost of restricting internal rotations. The energetic changes from restricting side chain torsional motion are more complex than simply a loss of conformational entropy, however. A second force opposing protein folding arises when a side chain in the folded state is not in its lowest energy rotamer, giving rotameric strain. Strain energy results from a dihedral angle being shifted from the most stable conformation of a rotamer when a protein folds. We calculated the energy of a side chain as a function of its dihedral angles in a poly(Ala) helix. Using these energy profiles, we quantify conformational entropy, rotameric strain energy and strain energy for all 17 amino acid residues with side-chains in α helices. We can calculate these terms for any amino acid in a helix interior in a protein, as a function of its side chain dihedral angles, and have implemented this algorithm on a web page. The mean change in rotameric strain energy on folding is 0.42 kcal mol−1 per residue and the mean strain energy is 0.64 kcal mol−1 per residue. Loss of conformational entropy opposes folding by a mean of 1.1 kcal mol−1 per residue, and the mean total force opposing restricting a side-chain into a helix is 2.2 kcal mol−1. Conformational entropy estimates alone therefore greatly underestimate the forces opposing protein folding. The introduction of strain when a protein folds should not be neglected when attempting to quantify the balance of forces affecting protein stability. Consideration of rotameric strain energy may help the use of rotamer libraries in protein design and rationalize the effects of mutations where side chain conformations change. □ A conserved HEAT domain within eIF4G directs assembly of the translation initiation machinery. Joseph Marcotrigiano, Ivan B. Lomakin, Nahum Sonenberg, Tatyana V. Pestova, Christopher U. T. Hellen, and Stephen K. Burley (2001). Mol. Cell 7, 193–203. The X-ray structure of the phylogenetically conserved middle portion of human eukaryotic initiation factor (eIF) 4GII has been determined at 2.4 Å resolution, revealing a crescent-shaped domain consisting of ten α helices arranged as five HEAT repeats. Together with the ATP-dependent RNA helicase eIF4A, this HEAT domain suffices for 48S ribosomal complex formation with a picornaviral RNA internal ribosome entry site (IRES). Structure-based site-directed mutagenesis was used to identify two adjacent features on the surface of this essential component of the translation initiation machinery that, respectively, bind eIF4A and a picornaviral IRES. The structural and biochemical results provide mechanistic insights into both cap-dependent and cap-independent translation initiation. □ Structure of the bacteriophage f29 DNA packaging motor. Alan A. Simpson, Yizihi Tao, P. G. Leimen, M. O. Badasso, Y. He, P. J. Jardine, N. Olson, M. C. Morais, S. Grimes, D. L. Anderson, T. S. Baker, and G. G. Rossmann (2000). Nature 408, 745–750. The efficient packaging of DNA by viral proheads presents a wealth of intriguing structural phenomena. The fact that the packaging occurs at the position of attachment of the 5-fold vertex to the 6-fold tail gave rise to the suggestion that the symmetry mismatch is important in the packaging mechanism. Genetic evidence demonstrated a requirement for a hexameric pRNA that associates with connector further strengthening the case for a role of symmetry mismatch. This background explains the intense interest in the structure of the central components of the packaging machinery, the connector. This paper presents the structure of the bacteriophage f29 connector at 3.5 Å resolution and uses cEM reconstructions to explore its role in packaging. The dodecameric connector is a funnel shaped, three domain structure. The structure is dominated by three α helices which run the length of the central domain connecting the predominantly β sheet end domains. The connector has an uncharged exterior, perhaps facilitating rotation, and a negatively charged channel that may ease the passage of DNA. The comparison with the symmetrized and non-symmetrized cEM reconstructions localizes the pRNA and defines the interaction of the components with the procapsid. The well defined pRNA is clearly 5-fold suggesting that it remains fixed during translocation. The DNA, connector and prohead-pRNA-ATPase complex form a set of concentric structures with 101, 12, and 5 fold symmetry, respectively. The authors propose that these function as a movable central spindle, intervening ball-race and static outer assembly during packaging. The sequential firing of each ATPase, spaced at one-fifth rotation about the DNA causes translation of the DNA by one fifth of its pitch. This mechanism uses the symmetry mismatch by allowing each connector monomer to occupy a different position on the DNA after each ATP hydrolysis. The f29 connector represents a novel type of molecular motor in which rotary motion is converted to translational movement. □ Crystal structures of SarA, a pleiotropic regulator of virulence genes in S. aureus. Maria A. Schumacher, Barry K. Hurlburt, and Richard G. Brennan (2001). Nature 409, 215–219. SarA is a transcriptional regulator of the virulence factors of Staphylococcus aureus, a major human pathogen. SarA binds to multiple AT-rich sequences of variable lengths. The crystal structure of SarA and a SarA–DNA complex show that SarA has a fold consisting of a four-helix core region and “inducible regions” comprising a β hairpin and a carboxy-terminal loop. On binding DNA, the inducible regions undergo marked conformational changes, becoming part of extended and distorted α helices, which encase the DNA. SarA recognizes an AT-rich site in which the DNA is highly overwound and adopts a D-DNA-like conformation by indirect readout. □ Projection structure of a ClC-type chloride channel at 6.5 Å resolution. Joseph A. Mindell, Merritt Maduke, Christopher Miller, and Nikolaus Grigorieff (2001). Nature 409, 219–223. Nearly all cells express ion channels of the ClC type, the only known molecular family of chloride-ion-selective channels. The authors report report the formation of two-dimensional crystals of a prokaryotic ClC channel protein reconstituted into phospholipid bilayer membranes. Cryoelectron microscopic analysis of these crystals yielded a projection structure at 6.5 Å resolution, which shows off-axis water-filled pores within the dimeric channel complex. □ Crystal structure of the transcription activator BmrR bound to DNA and a drug. Ekaterina E. Zheleznova Heldwein and Richard G. Brennan (2001). Nature 409, 378–382. The efflux of chemically diverse drugs by multidrug transporters that span the membrane is one mechanism of multidrug resistance in bacteria. The concentrations of many of these transporters are controlled by transcriptional regulators, such as BmrR in Bacillus subtilis. BmrR activates transcription of the multidrug transporter gene, bmr, in response to cellular invasion by certain lipophilic cationic compounds. Here the authors report the crystal structure of BmrR in complex with the drug tetraphenylphosphonium (TPP) and a 22-base-pair oligodeoxynucleotide encompassing the bmr promoter. The structure reveals an unexpected mechanism for transcription activation that involves localized base-pair breaking, and base sliding and realignment of the −35 and −10 operator elements. □ Arrangement of RNA and proteins in the spliceosomal U1 small nuclear ribo-nucleoprotein particle. Holger Stark, Prakash Dube, Reinhard Lührmann, and Berthold Kastner (2001). Nature 409, 539–542. In eukaryotic cells, freshly synthesized messenger RNA (pre-mRNA) contains stretches of noncoding RNA that must be excised before the RNA can be translated into protein. Their removal is catalysed by the spliceosome, a large complex formed when a number of small nuclear ribonucleoprotein particles (snRNPs) bind sequentially to the pre-mRNA. The first snRNP to bind is called U1; other snRNPs (U2, U4/U6, and U5) follow. Here the authors describe the three-dimensional structure of human U1 snRNP, determined by single-particle electron cryomicroscopy at 10 Å resolution. The reconstruction reveals a doughnut-shaped central element that accommodates the seven Sm proteins common to all snRNPs. The remaining density of the map was assigned to the other known components of U1 snRNP, providing a structural model that describes the three-dimensional arrangement of proteins and RNA in U1 snRNP. □ Evidence for cleft closure in actomyosin upon ADP release. N. Volkmann, D. Hanein, G. Ouyang, K. M. Trybus, D. J. DeRosier, and S. Lowey (2000). Nat. Struct. Biol. 7, 1147–1155. The ATP driven cycle of interaction of actin and myosin lies at the basis of muscle contraction. This paper uses comparisons of helical reconstructions of MgADP and rigor states of smooth muscle actomyosin from CEM as the starting point of a careful fitting and analysis of variance that allows them to describe the conformational changes in the actomyosin complex. The authors draw upon their analysis of variance to define the significance of their difference maps and the positions of mobility in the within the fitted molecules. Several domains were defined for motion by the comparison of the known crystal structures of S1. Allowing rigid body motions of these domains allowed fitting of the densities and generation of an atomic model of the complex. The authors interpret their atomic model to conclude that ADP release results in closing of the cleft in actomyosin. These results extend the description of the tight coupling between lever arm position and cleft closure by showing that actin binding induces novel myosin conformations. They support a mechanism in which ATP binding opens the cleft and disrupts the interface with actin leading to myosin release. □ SMN Tudor domain structure and its interaction with the Sm proteins. Philipp Selenko, Remco Sprangers, Gunter Stier, Dirk Bühler, Utz Fischer, and Michael Sattler (2001). Nat. Struct. Biol. 8, 27–31. Spinal muscular atrophy (SMA) is a common motor neuron disease that results from mutations in the Survival of Motor Neuron (SMN) gene. The SMN protein plays a crucial role in the assembly of spliceosomal duridine-rich small nuclear ribonucleoprotein (U snRNP) complexes via binding to the spliceosomal Sm core proteins. SMN contains a central Tudor domain that facilitates the SMN-Sm protein interaction. The authors have determined the three-dimensional structure of the Tudor domain of human SMN. The structure exhibits a conserved negatively charged surface that is shown to interact with the C-terminal Arg and Gly-rich tails of Sm proteins. Structural similarity between the Tudor domain and the Sm proteins suggests the presence of an additional binding interface that resembles that in hetero-oligomeric complexes of Sm proteins. □ Crystal structure of molybdopterin synthase and its evolutionary relationship to ubiquitin activation. Michael J. Rudolph, Margot M. Wuebbens, K.V. Rajagopalan, and Hermann Schindelin (2001). Nat. Struct. Biol. 8, 42–46. Molybdenum cofactor (Moco) biosynthesis is an evolutionarily conserved requisite pathway present in eubacteria, archaea, and eukaryotes, including humans. Moco contains a tricyclic pyranopterin, termed molybdopterin (MPT), that bears the cis-dithiolene group responsible for molybdenum ligation. The dithiolene group of MPT is generated by MPT synthase, which consists of a large and small subunit. The 1.45 Å resolution crystal structure of MPT synthase reveals a heterotetrameric protein in which the C terminus of each small subunit is inserted into a large subunit to form the active site. In the activated form of the enzyme this C terminus is present as a thiocarboxylate. In the structure of a covalent complex of MPT synthase, an isopeptide bond is present between the C terminus of the small subunit and a Lys side chain in the large subunit. There is strong structural similarity between the small subunit of MPT synthase and ubiquitin. □ Solution structure of ThiS and implications for the evolutionary roots of ubiquitin. Chunyu Wang, Jun Xi, Tadhg P. Begley, and Linda K. Nicholson (2001). Nat. Struct. Biol. 8, 47–51. ThiS is a sulfur carrier protein that plays a central role in thiamin biosynthesis in Escherichia coli. The authors report the solution NMR structure of ThiS, the first for this class of sulfur carrier proteins. Although ThiS shares only 14% sequence identity with ubiquitin, it possesses the ubiquitin fold. This structural homology, combined with established functional similarities involving sulfur chemistry, demonstrates that the eukaryotic ubiquitin and the prokaryotic ThiS evolved from a common ancestor. The ThiS structure reveals both hydrophobic and electrostatic surface features that are likely determinants for interactions with binding partners. □ Crystal structure of an activated response regulator bound to its target. Seok-Yong Lee, Ho S. Cho, Jeffrey G. Pelton, Dalai Yan, Robert K. Henderson, David S. King, Li-shar Huang, Sydney Kustu, Edward A. Berry, and David E. Wemmer (2001). Nat. Struct. Biol. 8, 52–56. The chemotactic regulator CheY controls the direction of flagellar rotation in Escherichia coli. The authors have determined the crystal structure of BeF3—activated CheY from E. coli in complex with an N-terminal peptide derived from its target, FliM. The structure reveals that the first seven residues of the peptide pack against CheY in an extended conformation, whereas residues 8–15 form two turns of helix that pack against CheY. The peptide binds the only region of CheY that undergoes noticeable conformational change upon activation and would most likely be sandwiched between activated CheY and the remainder of FliM to reverse the direction of flagellar rotation. □ Crystal structure of the Holliday junction resolving enzyme T7 endonuclease I. Jonathan M. Hadden, Máire A. Convery, Anne-Cécile Déclais, David M.J. Lilley, and Simon E.V. Phillips (2001). Nat. Struct. Biol. 8, 62–67. The authors have determined the crystal structure of the Holliday junction resolving enzyme T7 endonuclease I at 2.1 Å. Endonuclease I exhibits strong structural specificity for four-way DNA junctions. The structure shows that it forms a symmetric homodimer arranged in two well-separated domains. Each domain, however, is composed of elements from both subunits, and amino acid side chains from both protomers contribute to the active site. While no significant structural similarity could be detected with any other junction resolving enzyme, the active site is similar to that found in several restriction endonucleases. T7 endonuclease I therefore represents the first crystal structure of a junction resolving enzyme that is a member of the nuclease superfamily of enzymes. □ Bacterial RNA polymerase subunit ω and eukaryotic RNA polymerase subunit RPB6 are sequence, structural, and functional homologs and promote RNA polymerase assembly. Leonid Minakhin, Sechal Bhagat, Adrian Brunning, Elizabeth A. Campbell, Seth A. Darst, Richard H. Ebright and Konstantin Severinov (2001). Proc. Natl. Acad. Sci. USA 98, 892–897. Bacterial DNA-dependent RNA polymerase (RNAP) has subunit composition β'βαIαIIω. The role of ω has been unclear. The authors show that ω is homologous in sequence and structure to RPB6, an essential subunit shared in eukaryotic RNAP I, II, and III. Overproduction of ω or RPB6 suppresses equivalent assembly defects in Escherichia coli and yeast, respectively. Structural analysis of the ω-β′ interface in bacterial RNAP, and comparison with the RPB6-RPB1 interface in yeast RNAP II, confirms the structural relationship and suggests a “latching” mechanism for the role of ω and RPB6 in promoting RNAP assembly. □ Crystal structure of an initiation factor bound to the 30S ribosomal subunit. Andrew P. Carter, William M. Clemons, Jr., Ditlev E. Brodersen, Robert J. Morgan-Warren, Thomas Hartsch, Brian T. Wimberly, and V. Ramakrishnan (2001). 291, 498–501. Initiation of translation at the correct position on messenger RNA is essential for accurate protein synthesis. In prokaryotes, this process requires three initiation factors: IF1, IF2, and IF3. The authors report the crystal structure of a complex of IF1 and the 30S ribosomal subunit. Binding of IF1 occludes the ribosomal A site and flips out the functionally important bases A1492 and A1493 from helix 44 of 16S RNA, burying them in pockets in IF1. The binding of IF1 causes long-range changes in the conformation of H44 and leads to movement of the domains of 30S with respect to each other. The structure explains how localized changes at the ribosomal A site lead to global alterations in the conformation of the 30S subunit. □ Structure of the globular region of the prion protein Ure2 from the yeast Saccharomyces cerevisiae. Luc Bousset, Hassan Belrhali, Joël Janin, Ronald Melki, and Solange Morera (2001). Structure 9, 39–46. The [URE3] non-Mendelian element of the yeast S. cerevisiae is due to the propagation of a transmissible form of the protein Ure2. The infectivity of Ure2p is thought to originate from a conformational change of the normal form of the prion protein to a form that assembles into amyloid fibrils. The crystal structure of the globular region of Ure2p (residues 95–354) shows a two-domain protein forming a globular dimer. The N-terminal domain is composed of a central four strand β sheet flanked by four helices. The C-terminal domain is entirely α-helical. The fold resembles that of the glutathione S-transferases (GST). This is the first crystal structure of a prion protein. Possible mechanisms of amyloid fibril assembly are discussed. □ Molecular basis for regulatory subunit diversity in cAMP-dependent protein kinase: crystal structure of the type II regulatory subunit. Thomas C. Diller, Madhusudan, Nguyen-Huu Xuong, and Susan S. Taylor (2001). Structure 9, 73–82. Cyclic AMP binding domains possess common structural features yet are diversely coupled to different signaling modules. The authors report the first type II regulatory subunit crystal structure, from cAMP-dependent protein kinase, demonstrating that the relative orientations of the two tandem cAMP binding domains are very different from those of the type I regulatory subunit. The highly conserved phosphate binding cassette motif is coupled to nonconserved regions that link the cAMP signal to diverse structural and functional modules.