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Structure (v.15, #9)


Cyclic Nucleotide-Regulated Ion Channels: Spotlight on Symmetry by Justin W. Taraska; William N. Zagotta (pp. 1023-1024).
In this issue of Structure, report the 16 Å EM structure of the prokaryotic cyclic nucleotide-regulated K+ channel MloK1. This structure reveals that the channel is arranged as a four-fold symmetric tetramer.

How to Get All “A”s in Polyadenylation by Corey R. Mandel; Liang Tong (pp. 1024-1026).
In an elegant study in this issue of Structure, report the crystal structure of yeast poly(A) polymerase in a ternary complex with its substrate MgATP and the elongating poly(A) tail, providing molecular insights into the mechanism of polyadenylation.

Another Window into Disordered Protein Function by A. Keith Dunker (pp. 1026-1028).
Multiple crystal structures of the same proteins often have specific regions that switch between structure and disorder. In this issue of Structure, show that these “dual personality fragments” are distinct from both structured and disordered protein and are functionally important.

Caught in the Act: an ABC Transporter on the Move by David Parcej; Robert Tampé (pp. 1028-1030).
The latest structure of the vitamin B12 permease reported in Science by Hvorup and colleagues represents an unexpected, asymmetric state of an ABC transporter, and indicates that the translocation cycle may be even more complex than previously thought ().

Structure and Function of a Chlorella Virus-Encoded Glycosyltransferase by Ying Zhang; Ye Xiang; James L. Van Etten; Michael G. Rossmann (pp. 1031-1039).
Paramecium bursaria chlorella virus-1 encodes at least five putative glycosyltransferases that are probably involved in the synthesis of the glycan components of the viral major capsid protein. The 1.6 Å crystal structure of one of these glycosyltransferases (A64R) has a mixed α/β fold containing a central, six-stranded β sheet flanked by α helices. Crystal structures of A64R, complexed with UDP, CMP, or GDP, established that only UDP bound to A64R in the presence of Mn2+, consistent with its high structural similarity to glycosyltransferases which utilize UDP as the sugar carrier. The structure of the complex of A64R, UDP-glucose, and Mn2+ showed that the largest conformational change occurred when hydrogen bonds were formed with the ligands. Unlike UDP-glucose, UDP-galactose and UDP-GlcNAc did not bind to A64R, suggesting a selective binding of UDP-glucose. Thus, UDP-glucose is most likely the sugar donor for A64R, consistent with glucose occurring in the virus major capsid protein glycans.

Keywords: PROTEINS


Ensemble Refinement of Protein Crystal Structures: Validation and Application by Elena J. Levin; Dmitry A. Kondrashov; Gary E. Wesenberg; George N. Phillips Jr. (pp. 1040-1052).
X-ray crystallography typically uses a single set of coordinates and B factors to describe macromolecular conformations. Refinement of multiple copies of the entire structure has been previously used in specific cases as an alternative means of representing structural flexibility. Here, we systematically validate this method by using simulated diffraction data, and we find that ensemble refinement produces better representations of the distributions of atomic positions in the simulated structures than single-conformer refinements. Comparison of principal components calculated from the refined ensembles and simulations shows that concerted motions are captured locally, but that correlations dissipate over long distances. Ensemble refinement is also used on 50 experimental structures of varying resolution and leads to decreases in Rfree values, implying that improvements in the representation of flexibility observed for the simulated structures may apply to real structures. These gains are essentially independent of resolution or data-to-parameter ratio, suggesting that even structures at moderate resolution can benefit from ensemble refinement.

Keywords: PROTEINS


The Structure of the Prokaryotic Cyclic Nucleotide-Modulated Potassium Channel MloK1 at 16 Å Resolution by Po-Lin Chiu; Matthew D. Pagel; James Evans; Hui-Ting Chou; Xiangyan Zeng; Bryant Gipson; Henning Stahlberg; Crina M. Nimigean (pp. 1053-1064).
The gating ring of cyclic nucleotide-modulated channels is proposed to be either a two-fold symmetric dimer of dimers or a four-fold symmetric tetramer based on high-resolution structure data of soluble cyclic nucleotide-binding domains and functional data on intact channels. We addressed this controversy by obtaining structural data on an intact, full-length, cyclic nucleotide-modulated potassium channel, MloK1, from Mesorhizobium loti, which also features a putative voltage-sensor. We present here the 3D single-particle structure by transmission electron microscopy and the projection map of membrane-reconstituted 2D crystals of MloK1 in the presence of cAMP. Our data show a four-fold symmetric arrangement of the CNBDs, separated by discrete gaps. A homology model for full-length MloK1 suggests a vertical orientation for the CNBDs. The 2D crystal packing in the membrane-embedded state is compatible with the S1-S4 domains in the vertical “up” state.

Keywords: MOLNEURO; SIGNALING; CELLBIO


Structural Insights into the Interaction of Insulin-like Growth Factor 2 with IGF2R Domain 11 by Christopher Williams; Dellel Rezgui; Stuart N. Prince; Oliver J. Zaccheo; Emily J. Foulstone; Briony E. Forbes; Raymond S. Norton; John Crosby; A. Bassim Hassan; Matthew P. Crump (pp. 1065-1078).
The insulin-like growth factor II/mannose-6-phosphate receptor (IGF2R) mediates trafficking of mannose-6-phosphate (M6P)-containing proteins and the mitogenic hormone IGF2. IGF2R also plays an important role as a tumor suppressor, as mutation is frequently associated with human carcinogenesis. IGF2 binds to domain 11, one of 15 extracellular domains on IGF2R. The crystal structure of domain 11 and the solution structure of IGF2 have been reported, but, to date, there has been limited success when using crystallography to study the interaction of IGFs with their binding partners. As an approach to investigate the interaction between IGF2 and IGF2R, we have used heteronuclear NMR in combination with existing mutagenesis data to derive models of the domain 11-IGF2 complex by using the program HADDOCK. The models reveal that the molecular interaction is driven by critical hydrophobic residues on IGF2 and IGF2R, while a ring of flexible, charged residues on IGF2R may modulate binding.

Functional Linkages Can Reveal Protein Complexes for Structure Determination by Sul-Min Kim; Peter M. Bowers; Debnath Pal; Michael Strong; Thomas C. Terwilliger; Markus Kaufmann; David Eisenberg (pp. 1079-1089).
In the study of protein complexes, is there a computational method for inferring which combinations of proteins in an organism are likely to form a crystallizable complex? Here we attempt to answer this question, using the Protein Data Bank (PDB) to assess the usefulness of inferred functional protein linkages from the Prolinks database. We find that of the 242 nonredundant prokaryotic protein complexes shared between the current PDB and Prolinks, 44% (107/242) contain proteins linked at high confidence by one or more methods of computed functional linkages. Similarly, high-confidence linkages detect 47% of known Escherichia coli protein complexes, with 45% accuracy. Together these findings suggest that functional linkages will be useful in defining protein complexes for structural studies, including for structural genomics. We offer a database of inferred linkages corresponding to likely protein complexes for some 629,952 pairs of proteins in 154 prokaryotes and archaea.

Keywords: PROTEINS


Fragile X Mental Retardation Syndrome: Structure of the KH1-KH2 Domains of Fragile X Mental Retardation Protein by Roberto Valverde; Irina Pozdnyakova; Tommi Kajander; Janani Venkatraman; Lynne Regan (pp. 1090-1098).
Fragile X syndrome is the most common form of inherited mental retardation in humans, with an estimated prevalence of about 1 in 4000 males. Although several observations indicate that the absence of functional Fragile X Mental Retardation Protein (FMRP) is the underlying basis of Fragile X syndrome, the structure and function of FMRP are currently unknown. Here, we present an X-ray crystal structure of the tandem KH domains of human FMRP, which reveals the relative orientation of the KH1 and KH2 domains and the location of residue Ile304, whose mutation to Asn is associated with a particularly severe incidence of Fragile X syndrome. We show that the Ile304Asn mutation both perturbs the structure and destabilizes the protein.

Keywords: PROTEINS


Cryo-EM Study of the Pseudomonas Bacteriophage φKZ by Andrei Fokine; Anthony J. Battisti; Valorie D. Bowman; Andrei V. Efimov; Lidia P. Kurochkina; Paul R. Chipman; Vadim V. Mesyanzhinov; Michael G. Rossmann (pp. 1099-1104).
The φKZ virus is one of the largest known bacteriophages. It infects Pseudomonas aeruginosa, which is frequently pathogenic in humans, and, therefore, has potential for phage therapy. The φKZ virion consists of an ∼1450 Å diameter icosahedral head and an ∼2000 Å long contractile tail. The structure of the φKZ tail has been determined using cryo-electron microscopy. The φKZ tail is much longer than that of bacteriophage T4. However, the helical parameters of their contractile sheaths, surrounding their tail tubes, are comparable. Although there is no recognizable sequence similarity between the φKZ and T4 tail sheath proteins, they are similar in size and shape, suggesting that they evolved from a common ancestor. The φKZ baseplate is significantly larger than that of T4 and has a flatter shape. Nevertheless, φKZ, similar to T4, has a cell-puncturing device in the middle of its baseplate.

Study of Recombinant Antibody Fragments and PAI-1 Complexes Combining Protein-Protein Docking and Results from Site-Directed Mutagenesis by Hector Novoa de Armas; Maarten Dewilde; Koen Verbeke; Marc De Maeyer; Paul J. Declerck (pp. 1105-1116).
Elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) have been correlated with cardiovascular diseases such as myocardial infarction and venous thrombosis. PAI-1 has also been shown to play an important role in tumor development, diabetes, and obesitas. Monoclonal antibodies MA-8H9D4 and MA-56A7C10, and their single-chain variable fragments (scFv), exhibit PAI-1-neutralizing properties. In this study, a rigid-body docking approach is used to predict the binding geometry of two distinct conformations of PAI-1 (active and latent) in complex with these antibody fragments. Resulting models were initially refined by using the dead-end elimination algorithm. Different filtering criteria based on the mutagenesis studies and structural considerations were applied to select the final models. These were refined by using the slow-cooling torsion-angle dynamic annealing protocol. The docked structures reveal the respective epitopes and paratopes and their potential interactions. This study provides crucial information that is necessary for the rational development of low-molecular weight PAI-1 inhibitors.

Keywords: PROTEINS


Mechanism of Poly(A) Polymerase: Structure of the Enzyme-MgATP-RNA Ternary Complex and Kinetic Analysis by Paul B. Balbo; Andrew Bohm (pp. 1117-1131).
We report the 1.8 Å structure of yeast poly(A) polymerase (PAP) trapped in complex with ATP and a five residue poly(A) by mutation of the catalytically required aspartic acid 154 to alanine. The enzyme has undergone significant domain movement and reveals a closed conformation with extensive interactions between the substrates and all three polymerase domains. Both substrates and 31 buried water molecules are enclosed within a central cavity that is open at both ends. Four PAP mutants were subjected to detailed kinetic analysis, and studies of the adenylyltransfer (forward), pyrophosphorolysis (reverse), and nucleotidyltransfer reaction utilizing CTP for the mutants are presented. The results support a model in which binding of both poly(A) and the correct nucleotide, MgATP, induces a conformational change, resulting in formation of a stable, closed enzyme state. Thermodynamic considerations of the data are discussed as they pertain to domain closure, substrate specificity, and catalytic strategies utilized by PAP.

Keywords: RNA


A Structural Characterization of Human SCO2 by Lucia Banci; Ivano Bertini; Simone Ciofi-Baffoni; Ioannis P. Gerothanassis; Iliana Leontari; Manuele Martinelli; Shenlin Wang (pp. 1132-1140).
Human Sco2 is a mitochondrial membrane-bound protein involved in copper supply for the assembly of cytochrome c oxidase in eukaryotes. Its precise action is not yet understood. We report here a structural and dynamic characterization by NMR of the apo and copper(I) forms of the soluble fragment. The structural and metal binding features of human Cu(I)Sco2 are similar to the more often studied Sco1 homolog, although the dynamic properties and the conformational disorder are quite different when the apo forms and the copper(I)-loaded forms of the two proteins are compared separately. Such differences are accounted for in terms of the different physicochemical properties in strategic protein locations. The misfunction of the known pathogenic mutations is discussed on the basis of the obtained structure.

Keywords: PROTEINS


Between Order and Disorder in Protein Structures: Analysis of “Dual Personality” Fragments in Proteins by Ying Zhang; Boguslaw Stec; Adam Godzik (pp. 1141-1147).
In their natural environment, three-dimensional structures of proteins undergo significant fluctuations and are often partially or completely disordered. This phenomenon recently became the focus of much attention, as many proteins, especially from higher organisms, were shown to contain large intrinsically disordered regions. Such disordered regions may become ordered only under very specific circumstances, if at all, and can be recognized by specific amino acid composition and sequence signatures. Here, we suggest that the balance between order and disorder is much more subtle in that many regions are very close to the order/disorder boundary. Specifically, analysis of redundant sets of experimental models of protein structures, where emphasis is put on comparison of structures of identical proteins solved in different conditions and functional states, shows hundreds of fragments captured in two states: ordered and disordered. We show that such fragments, which we call here “dual personality” (DP) fragments, have distinctive features that differentiate them from both regularly folded and intrinsically disordered fragments. We hypothesize, and show on several examples, that such fragments are often targets of regulation, either by allostery or posttranslational modifications.

Keywords: PROTEIN


Bombyx mori Pheromone-Binding Protein Binding Nonpheromone Ligands: Implications for Pheromone Recognition by Catherine Lautenschlager; Walter S. Leal; Jon Clardy (pp. 1148-1154).
Insect pheromone-binding proteins (PBPs) transport sex pheromones through the aqueous layer surrounding G protein-coupled receptors that initiate signaling events leading to mating. This PBP-receptor system strongly discriminates between ligands with subtle structural differences, but it has proved difficult to distinguish the degree of discrimination of the PBP from that of the G protein-coupled receptor. The three-dimensional structures of the PBP of Bombyx mori, the silkworm moth, both with and without its cognate ligand bombykol ([ E, Z]-10,12-hexadecadienol), have been determined by X-ray crystallography and NMR. In this paper, the structures of the same binding protein with bound iodohexadecane and bell pepper odorant were determined at 1.9 and 2.0 Å, respectively. These structures illustrate the remarkable plasticity in the ligand binding site of the PBP, but suggest the protein might still act as a filter during pheromone signal processing.

Keywords: PROTEINS; SIGNALING

Structure of BeF3-Modified Response Regulator PleD: Implications for Diguanylate Cyclase Activation, Catalysis, and Feedback Inhibition by Paul Wassmann; Carmen Chan; Ralf Paul; Andreas Beck; Heiko Heerklotz; Urs Jenal; Tilman Schirmer (pp. 1155-1155).
Multiprotein Expression Strategy for Structural Biology of Eukaryotic Complexes by Daniel J. Fitzgerald; Christiane Schaffitzel; Philipp Berger; Ralf Wellinger; Christoph Bieniossek; Timothy J. Richmond; Imre Berger (pp. 1156-1156).
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