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Structure (v.14, #2)

The Most Complex Pseudouridylase by Yi-Tao Yu (pp. 167-168).
In a recent issue of Molecular Cell, report the crystal structure of a complex containing archaeal Cbf5, Nop10 and Gar1, critical components of box H/ACA RNPs. These RNPs constitute the most complex pseudouridylases yet discovered.

A Lock on Formins by Wenqing Xu (pp. 168-169).
Formin proteins control the dynamics of unbranched actin filaments and are regulated by intramolecular interactions. In this issue of Structure, reveal the structure of a central component of the interaction that locks Diaphanous-related formins in the inactive state.

Selectivity and Promiscuity in Eph Receptors by Anna-Pavlina G. Haramis; Anastassis Perrakis (pp. 169-171).
In this issue of Structure, report a structural and thermodynamic analysis of EphB4 in complex with an antagonistic peptide; the insight into Eph-ephrin interaction suggests determinants for Eph receptor specificity. These findings will contribute to the development of EphB4 antagonists for therapeutic applications.

Decapper Comes into Focus by Vincent Shen; Megerditch Kiledjian (pp. 171-172).
In this issue of Structure, report structures of the Xenopus X29 Nudix decapping protein, including homodimer structures in complex with cap nucleotides. These structures reveal insights into the mechanism of cap substrate recognition and predict an RNA binding path on the protein surface.

Structure of the Catalytic and Ubiquitin-Associated Domains of the Protein Kinase MARK/Par-1 by Saravanan Panneerselvam; Alexander Marx; Eva-Maria Mandelkow; Eckhard Mandelkow (pp. 173-183).
The Ser/Thr kinase MARK2 phosphorylates tau protein at sites that cause detachment from microtubules in Alzheimer neurofibrillary degeneration. Homologs of MARK2 include Par-1 in C. elegans and Drosophila, which generates embryonic polarity. We report the X-ray structure of the catalytic and ubiquitin-associated domains (UBA) of human MARK2. The activity was altered by mutations in the ATP binding site and/or activation loop. The catalytic domain shows the small and large lobes typical of kinases. The substrate cleft is in an inactive, open conformation in the inactivated and the wild-type structure. The UBA domain is attached via a taut linker to the large lobe of the kinase domain and leans against a hydrophobic patch on the small lobe. The UBA structure is unusual because the orientation of its third helix is inverted, relative to previous structures. Possible implications of the structure for the regulation of kinase activity are discussed.

The Crystal Structure of Aspergillus fumigatus Cyclophilin Reveals 3D Domain Swapping of a Central Element by Andreas Limacher; Daniel P. Kloer; Sabine Flckiger; Gerd Folkers; Reto Crameri; Leonardo Scapozza (pp. 185-195).
The crystal structure of Aspergillus fumigatus cyclophilin (Asp f 11) was solved by the multiwavelength anomalous dispersion method and was refined to a resolution of 1.85 Å with R and Rfree values of 18.9% and 21.4%, respectively. Many cyclophilin structures have been solved to date, all showing the same monomeric conformation. In contrast, the structure of A. fumigatus cyclophilin reveals dimerization by 3D domain swapping and represents one of the first proteins with a swapped central domain. The domain-swapped element consists of two β strands and a subsequent loop carrying a conserved tryptophan. The tryptophan binds into the active site, inactivating cis- trans isomerization. This might be a means of biological regulation. The two hinge loops leave the protein prone to misfolding. In this context, alternative forms of 3D domain swapping that can lead to N- or C-terminally swapped dimers, oligomers, and aggregates are discussed.

Modeling a Self-Avoiding Chromatin Loop: Relation to the Packing Problem, Action-at-a-Distance, and Nuclear Context by Michal Bon; Davide Marenduzzo; Peter R. Cook (pp. 197-204).
There is now convincing evidence that genomes are organized into loops, and that looping brings distant genes together so that they can bind to local concentrations of polymerases in “factories? or “hubs.? As there remains no systematic analysis of how looping affects the probability that a gene can access binding sites in such factories/hubs, we used an algorithm that we devised and Monte Carlo methods to model a DNA or chromatin loop as a semiflexible (self-avoiding) tube attached to a sphere; we examine how loop thickness, rigidity, and contour length affect where particular segments of the loop lie relative to binding sites on the sphere. Results are compared with those obtained with the traditional model of an (infinitely thin) freely jointed chain. They provide insights into the packing problem (how long genomes are packed into small nuclei), and action-at-a-distance (how firing of one origin or gene can prevent firing of an adjacent one).

Complete Reaction Cycle of a Cocaine Catalytic Antibody at Atomic Resolution by Xueyong Zhu; Tobin J. Dickerson; Claude J. Rogers; Gunnar F. Kaufmann; Jenny M. Mee; Kathleen M. McKenzie; Kim D. Janda; Ian A. Wilson (pp. 205-216).
Antibody 7A1 hydrolyzes cocaine to produce nonpsychoactive metabolites ecgonine methyl ester and benzoic acid. Crystal structures of 7A1 Fab′ and six complexes with substrate cocaine, the transition state analog, products ecgonine methyl ester and benzoic acid together and individually, as well as heptaethylene glycol have been analyzed at 1.5–2.3 Å resolution. Here, we present snapshots of the complete cycle of the cocaine hydrolytic reaction at atomic resolution. Significant structural rearrangements occur along the reaction pathway, but they are generally limited to the binding site, including the ligands themselves. Several interacting side chains either change their rotamers or alter their mobility to accommodate the different reaction steps. CDR loop movements (up to 2.3 Å) and substantial side chain rearrangements (up to 9 Å) alter the shape and size (∼320–500 Å3) of the antibody active site from “open? to “closed? to “open? for the substrate, transition state, and product states, respectively.

Flexibility of Thiamine Diphosphate Revealed by Kinetic Crystallographic Studies of the Reaction of Pyruvate-Ferredoxin Oxidoreductase with Pyruvate by Christine Cavazza; Carlos Contreras-Martel; Laetitia Pieulle; Eric Chabrire; E. Claude Hatchikian; Juan C. Fontecilla-Camps (pp. 217-224).
Pyruvate-ferredoxin oxidoreductases (PFOR) are unique among thiamine pyrophosphate (ThDP)-containing enzymes in giving rise to a rather stable cofactor-based free-radical species upon the decarboxylation of their first substrate, pyruvate. We have obtained snapshots of unreacted and partially reacted (probably as a tetrahedral intermediate) pyruvate-PFOR complexes at different time intervals. We conclude that pyruvate decarboxylation involves very limited substrate-to-product movements but a significant displacement of the thiazolium moiety of ThDP. In this respect, PFOR seems to differ substantially from other ThDP-containing enzymes, such as transketolase and pyruvate decarboxylase. In addition, exposure of PFOR to oxygen in the presence of pyruvate results in significant inhibition of catalytic activity, both in solution and in the crystals. Examination of the crystal structure of inhibited PFOR suggests that the loss of activity results from oxime formation at the 4′ amino substituent of the pyrimidine moiety of ThDP.

Crystal Structure of Group A Streptococcus Mac-1: Insight into Dimer-Mediated Specificity for Recognition of Human IgG by Johnson Agniswamy; Michal J. Nagiec; Mengyao Liu; Peter Schuck; James M. Musser; Peter D. Sun (pp. 225-235).
Group A Streptococcus secretes cysteine proteases named Mac-1 and Mac-2 that mediate host immune evasion by targeting both IgG and Fc receptors. Here, we report the crystal structures of Mac-1 and its catalytically inactive C94A mutant in two different crystal forms. Despite the lack of sequence homology, Mac-1 adopts the canonical papain fold. Alanine mutations at the active site confirmed the critical residues involved in a papain-like catalytic mechanism. Mac-1 forms a symmetric dimer in both crystal forms and displays the unique dimer interface among papain superfamily members. Mutations at the dimer interface resulted in a significant reduction in IgG binding and catalysis, suggesting that the dimer contributes to both IgG specificity and enzyme cooperativity. A tunnel observed at the dimer interface constitutes a target for designing potential Mac-1-specific antimicrobial agents. The structures also offer insight into the functional difference between Mac-1 and Mac-2.

Ligand-Induced Domain Rearrangement of Fatty Acid β-Oxidation Multienzyme Complex by Daisuke Tsuchiya; Nobutaka Shimizu; Momoyo Ishikawa; Yoshikazu Suzuki; Kosuke Morikawa (pp. 237-246).
The quaternary structure of a fatty acid β-oxidation multienzyme complex, catalyzing three sequential reactions, was investigated by X-ray crystallographic and small-angle X-ray solution scattering analyses. X-ray crystallography revealed an intermediate structure of the complex among the previously reported structures. However, the theoretical scattering curves calculated from the crystal structures remarkably disagree with the experimental profiles. Instead, an ensemble of the atomic models, which were all calculated by rigid-body optimization, reasonably explained the experimental data. These structures significantly differ from those in the crystals, but they maintain the substrate binding pocket at the domain boundary. Comparisons among these structures indicated that binding of 3-hydroxyhexadecanoyl-CoA or nicotinamide adenine dinucleotide induces domain rearrangements in the complex. The conformational changes suggest the structural events occurring during the chain reaction catalyzed by the multienzyme complex.

Antiparallel Four-Stranded Coiled Coil Specified by a 3-3-1 Hydrophobic Heptad Repeat by Yiqun Deng; Jie Liu; Qi Zheng; David Eliezer; Neville R. Kallenbach; Min Lu (pp. 247-255).
Coiled-coil sequences in proteins commonly share a seven-amino acid repeat with nonpolar side chains at the first ( a) and fourth ( d) positions. We investigate here the role of a 3-3-1 hydrophobic repeat containing nonpolar amino acids at the a, d, and g positions in determining the structures of coiled coils using mutants of the GCN4 leucine zipper dimerization domain. When three charged residues at the g positions in the parental sequence are replaced by nonpolar alanine or valine side chains, stable four-helix structures result. The X-ray crystal structures of the tetramers reveal antiparallel, four-stranded coiled coils in which the a, d, and g side chains interlock in a combination of knobs-into-knobs and knobs-into-holes packing. Interfacial interactions in a coiled coil can therefore be prescribed by hydrophobic-polar patterns beyond the canonical 3-4 heptad repeat. The results suggest that the conserved, charged residues at the g positions in the GCN4 leucine zipper can impart a negative design element to disfavor thermodynamically more stable, antiparallel tetramers.

Structure of the Autoinhibitory Switch in Formin mDia1 by Azin G. Nezami; Florence Poy; Michael J. Eck (pp. 257-263).
Diaphanous-related formins (DRFs) regulate the nucleation and polymerization of unbranched actin filaments. The activity of DRFs is inhibited by an intramolecular interaction between their N-terminal regulatory region and a conserved C-terminal segment termed the Diaphanous autoinhibitory domain (DAD). Binding of GTP bound Rho to the mDia1 N terminus releases this autoinhibitory restraint. Here, we describe the crystal structure of the DAD segment of mDia1 in complex with the relevant N-terminal fragment, termed the DID domain. The structure reveals that the DAD segment forms an amphipathic helix that binds a conserved, concave surface on the DID domain. Comparison with the structure of the mDia1 N terminus bound to RhoC suggests that release of the autoinhibitory DAD interaction is accomplished largely by Rho-induced restructuring of the adjacent GTPase binding subdomain (GBD), but also by electrostatic repulsion and a small, direct steric occlusion of the DAD binding cleft by Rho itself.

The Backrub Motion: How Protein Backbone Shrugs When a Sidechain Dances by Ian W. Davis; W. Bryan Arendall III; David C. Richardson; Jane S. Richardson (pp. 265-274).
Surprisingly, the frozen structures from ultra-high-resolution protein crystallography reveal a prevalent, but subtle, mode of local backbone motion coupled to much larger, two-state changes of sidechain conformation. This “backrub? motion provides an influential and common type of local plasticity in protein backbone. Concerted reorientation of two adjacent peptides swings the central sidechain perpendicular to the chain direction, changing accessible sidechain conformations while leaving flanking structure undisturbed. Alternate conformations in sub-1 Å crystal structures show backrub motions for two-thirds of the significant Cβ shifts and 3% of the total residues in these proteins (126/3882), accompanied by two-state changes in sidechain rotamer. The Backrub modeling tool is effective in crystallographic rebuilding. For homology modeling or protein redesign, backrubs can provide realistic, small perturbations to rigid backbones. For large sidechain changes in protein dynamics or for single mutations, backrubs allow backbone accommodation while maintaining H bonds and ideal geometry.

Structural and Functional Aspects of the Sensor Histidine Kinase PrrB from Mycobacterium tuberculosis by Elzbieta Nowak; Santosh Panjikar; J. Preben Morth; Rositsa Jordanova; Dmitri I. Svergun; Paul A. Tucker (pp. 275-285).
We describe the solution structures of two- and three-domain constructs of the sensor histidine kinase PrrB from Mycobacterium tuberculosis, which allow us to locate the HAMP linker relative to the ATP binding and dimerization domains. We show that the three-domain construct is active both for autophosphorylation and for phosphotransfer to the cognate response regulator, PrrA. We also describe the high-resolution crystal structure of the catalytic domain alone, and we show that, in solution, it binds ATP. The conformational flexibility of this domain is discussed and related to other structural information.

A Versatile Conformational Switch Regulates Reactivity in Human Branched-Chain α-Ketoacid Dehydrogenase by Mischa Machius; R. Max Wynn; Jacinta L. Chuang; Jun Li; Ronald Kluger; Daria Yu; Diana R. Tomchick; Chad A. Brautigam; David T. Chuang (pp. 287-298).
The dehydrogenase/decarboxylase (E1b) component of the 4 MD human branched-chain α-ketoacid dehydrogenase complex (BCKDC) is a thiamin diphosphate (ThDP)-dependent enzyme. We have determined the crystal structures of E1b with ThDP bound intermediates after decarboxylation of α-ketoacids. We show that a key tyrosine residue in the E1b active site functions as a conformational switch to reduce the reactivity of the ThDP cofactor through interactions with its thiazolium ring. The intermediates do not assume the often-postulated enamine state, but likely a carbanion state. The carbanion presumably facilitates the second E1b-catalyzed reaction, involving the transfer of an acyl moiety from the intermediate to a lipoic acid prosthetic group in the transacylase (E2b) component of the BCKDC. The tyrosine switch further remodels an E1b loop region to promote E1b binding to E2b. Our results illustrate the versatility of the tyrosine switch in coordinating the catalytic events in E1b by modulating the reactivity of reaction intermediates.

Crystal Structure of the Cytoplasmic Domain of the Chloride Channel ClC-0 by Sebastian Meyer; Raimund Dutzler (pp. 299-307).
Ion channels are frequently organized in a modular fashion and consist of a membrane-embedded pore domain and a soluble regulatory domain. A similar organization is found for the ClC family of Cl channels and transporters. Here, we describe the crystal structure of the cytoplasmic domain of ClC-0, the voltage-dependent Cl channel from T. marmorata. The structure contains a folded core of two tightly interacting cystathionine β-synthetase (CBS) subdomains. The two subdomains are connected by a 96 residue mobile linker that is disordered in the crystals. As revealed by analytical ultracentrifugation, the domains form dimers, thereby most likely extending the 2-fold symmetry of the transmembrane pore. The structure provides insight into the organization of the cytoplasmic domains within the ClC family and establishes a framework for guiding future investigations on regulatory mechanisms.

Keywords: CELLBIO

Molecular Basis for Phosphorylation-Dependent, PEST-Mediated Protein Turnover by Maria M. Garca-Alai; Mariana Gallo; Marcelo Salame; Diana E. Wetzler; Alison A. McBride; Maurizio Paci; Daniel O. Cicero; Gonzalo de Prat-Gay (pp. 309-319).
Proteasomal-mediated rapid turnover of proteins is often modulated by phosphorylation of PEST sequences. The E2 protein from papillomavirus participates in gene transcription, DNA replication, and episomal genome maintenance. Phosphorylation of a PEST sequence located in a flexible region accelerates its degradation. NMR analysis of a 29 amino acid peptide fragment derived from this region shows pH-dependent polyproline II and α helix structures, connected by a turn. Phosphorylation, in particular that at serine 301, disrupts the overall structure, and point mutations have either stabilizing or destabilizing effects. There is an excellent correlation between the thermodynamic stability of different peptides and the half-life of E2 proteins containing the same mutations in vivo. The structure around the PEST region appears to have evolved a marginal stability that is finely tunable by phosphorylation. Thus, conformational stability, rather than recognition of a phosphate modification, modulates the degradation of this PEST sequence by the proteasome machinery.

Structure and Thermodynamic Characterization of the EphB4/Ephrin-B2 Antagonist Peptide Complex Reveals the Determinants for Receptor Specificity by Jill E. Chrencik; Alexei Brooun; Michael I. Recht; Michelle L. Kraus; Mitchell Koolpe; Anand R. Kolatkar; Richard H. Bruce; Georg Martiny-Baron; Hans Widmer; Elena B. Pasquale; Peter Kuhn (pp. 321-330).
The Eph receptor tyrosine kinases and their ligands, the ephrins, regulate numerous biological processes in developing and adult tissues and have been implicated in cancer progression and in pathological forms of angiogenesis. We report the crystal structure of the EphB4 receptor in complex with a highly specific antagonistic peptide at a resolution of 1.65 . The peptide is situated in a hydrophobic cleft of EphB4 corresponding to the cleft in EphB2 occupied by the ephrin-B2 G-H loop, consistent with its antagonistic properties. Structural analysis identifies several residues within the EphB4 binding cleft that likely determine the ligand specificity of this receptor, while isothermal titration calorimetry experiments with truncated forms of the peptide define the amino acid residues of the peptide that are critical for receptor binding. These studies reveal structural features that will aid drug discovery initiatives to develop EphB4 antagonists for therapeutic applications.

Crystal Structures of U8 snoRNA Decapping Nudix Hydrolase, X29, and Its Metal and Cap Complexes by J. Neel Scarsdale; Brenda A. Peculis; H. Tonie Wright (pp. 331-343).
X29, a 25 kDa Nudix hydrolase from Xenopus laevis that cleaves 5′ caps from U8 snoRNA, crystallizes as a homodimeric apoenzyme. Manganese binds crystals of apo-X29 to form holo-X29 only in the presence of nucleot(s)ide. Structural changes in X29 on nucleo-t(s)ide-assisted Mn+2 uptake account for the observed cooperativity of metal binding. Structures of X29 with GTP or m7GpppA show a different mode of ligand binding from that of other cap binding proteins and suggest a possible three- or four-metal Nudix reaction mechanism. The X29 dimer has no known RNA binding motif, but its striking surface dipolarity and unique structural features create a plausible RNA binding channel on the positive face of the protein. Because U8 snoRNP is essential for accumulation of mature 5.8S and 28S rRNA in vertebrate ribosome biogenesis, and cap structures are required for U8 stability in vivo, X29 could profoundly influence this fundamental cellular pathway.

Structure and Specific RNA Binding of ADAR2 Double-Stranded RNA Binding Motifs by Richard Stefl; Ming Xu; Lenka Skrisovska; Ronald B. Emeson; Frdric H.-T. Allain (pp. 345-355).
Adenosine deaminases that act on RNA (ADARs) site-selectively modify adenosines to inosines within RNA transcripts, thereby recoding genomic information. How ADARs select specific adenosine moieties for deamination is poorly understood. Here, we report NMR structures of the two double-stranded RNA binding motifs (dsRBMs) of rat ADAR2 and an NMR chemical shift perturbation study of the interaction of the two dsRBMs with a 71 nucleotide RNA encoding the R/G site of the GluR-B. We have identified the protein and the RNA surfaces involved in complex formation, allowing us to present an NMR-based model of the complex. We have found that dsRBM1 recognizes a conserved pentaloop, whereas dsRBM2 recognizes two bulged bases adjacent to the editing site, demonstrating RNA structure-dependent recognition by the ADAR2 dsRBMs. In vitro mutagenesis studies with both the protein and the RNA further support our structural findings.

Structural Basis for Sulfur Relay to RNA Mediated by Heterohexameric TusBCD Complex by Tomoyuki Numata; Shuya Fukai; Yoshiho Ikeuchi; Tsutomu Suzuki; Osamu Nureki (pp. 357-366).
Uridine at wobble position 34 of tRNALys, tRNAGlu, and tRNAGln is exclusively modified into 2-thiouridine (s2U), which is crucial for both precise codon recognition and recognition by the cognate aminoacyl-tRNA synthetases. Recent Escherichia coli genetic studies revealed that the products of five novel genes, tusABCDE, function in the s2U modification. Here, we solved the 2.15 crystal structure of the E. coli TusBCD complex, a sulfur transfer mediator, forming a heterohexamer composed of a dimer of the heterotrimer. Structure-based sequence alignment suggested two putative active site Cys residues, Cys79 (in TusC) and Cys78 (in TusD), which are exposed on the hexameric complex. In vivo mutant analyses revealed that only Cys78, in the TusD subunit, participates in sulfur transfer during the s2U modification process. Since the single Cys acts as a catalytic residue, we proposed that TusBCD mediates sulfur relay via a putative persulfide state of the TusD subunit.

Crystal Structure of the Human Retinitis Pigmentosa 2 Protein and Its Interaction with Arl3 by Karin Khnel; Stefan Veltel; Ilme Schlichting; Alfred Wittinghofer (pp. 367-378).
The crystal structure of human retinitis pigmentosa 2 protein (RP2) was solved to 2.1 Å resolution. It consists of an N-terminal β helix and a C-terminal ferredoxin-like α/β domain. RP2 is functionally and structurally related to the tubulin-specific chaperone cofactor C. Seven of nine known RP2 missense mutations identified in patients are located in the β helix domain, and most of them cluster to the hydrophobic core and are likely to destabilize the protein. Two residues, Glu138 and the catalytically important Arg118, are solvent-exposed and form a salt bridge, indicating that Glu138 might be critical for positioning Arg118 for catalysis. RP2 is a specific effector protein of Arl3. The N-terminal 34 residues and β helix domain of RP2 are required for this interaction. The abilitities of RP2 to bind Arl3 and cause retinitis pigmentosa seem to be correlated, since both the R118H and E138G mutants show a drastically reduced affinity to Arl3.

A Phosphorylation-Induced Conformation Change in Dematin Headpiece by Zhenghui Gordon Jiang; C. James McKnight (pp. 379-387).
Dematin is an actin binding protein from the junctional complex of the erythrocyte cytoskeleton. The protein has two actin binding sites and bundles actin filaments in vitro. This actin bundling activity is reversibly regulated by phosphorylation in the carboxyl terminal “headpiece? domain (DHP). DHP is a typical villin-type headpiece actin binding motif and contains a flexible N-terminal loop and an α-helical C-terminal subdomain that is phosphorylated at Ser74. The NMR structure of a Ser74-to-Glu mutant (DHPs74e) closely mimics the conformation of phosphorylated DHP. The negative charge at Ser74 does not alter the conformation of the C-terminal subdomain, but attracts the N-terminal loop toward the C terminus, changing the orientation of the N-terminal subdomain. NMR relaxation studies also indicate reduced mobility in the N-terminal loop in DHPs74e. Thus, phosphorylation in DHP serves as a switch controlling the conformational state of DHP and the actin bundling activity of dematin.
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