Structure (v.13, #10)
Two-Step Pathway to Aminoacylated tRNA by John J. Perona (1397-1398).
The 3.0 Å crystal structure of a tRNA-dependent amidotransferase from the hyperthermophilic archaeon Pyrococcus abyssi (; in this issue of Structure) provides the first detailed insight into how cells lacking canonical tRNA synthetases nonetheless carry out protein synthesis with high fidelity.
One Channel: Open and Closed by Janice L. Robertson; Benoît Roux (1398-1400).
report structural information about the prokaryotic KirBac3.1 inward rectifier family K+ channel from Magnetospirillum magnetotacticum. These results from two-dimensional electron cryomicroscopy (EM) shed light on the gating mechanism of members of the Kir channel family.
Bending in the Right Direction by Trevor Siggers,; Tonya Silkov,; Barry Honig (1400-1401).
report the success of a new Monte Carlo algorithm in describing sequence-specific DNA bending. The approach offers the possibility of improved treatments of “indirect readout” effects in the prediction of transcription factor binding sites.
YjjX: From Structure “Tu” Function by Zihe Rao (1401-1402).
show by structural analysis that YjjX, a hypothetical protein in E. coli, is an ITPase/XTPase and suggest that it may play dual roles in prokaryotic translational regulation and oxidative cell stress response.
Structure and Function of Argonaute Proteins by Traci M. Tanaka Hall (1403-1408).
Argonaute (Ago) family proteins are multidomain proteins expressed in prokaryotic and eukaryotic organisms. In eukaryotes, Ago proteins are most well known for their roles in RNA silencing. In prokaryotes, the functions of Ago proteins are unknown, but based on their similarity to eukaryotic Ago proteins, they could be involved in nucleic acid-directed regulatory pathways related to RNA silencing. Recent structural and biochemical studies have shed new light on the function of this family of proteins. These studies reveal how these proteins recognize and cleave RNA and suggest a function for prokaryotic family members.
Raman Optical Activity: A Tool for Protein Structure Analysis by Fujiang Zhu; Neil W. Isaacs; Lutz Hecht; Laurence D. Barron (1409-1419).
On account of its sensitivity to chirality, Raman optical activity (ROA), measured here as the intensity of a small, circularly polarized component in the scattered light using unpolarized incident light, is a powerful probe of protein structure and behavior. Protein ROA spectra provide information on secondary and tertiary structures of polypeptide backbones, backbone hydration, and side chain conformations, and on structural elements present in unfolded states. This article describes the ROA technique and presents ROA spectra, recorded with a commercial instrument of novel design, of a selection of proteins to demonstrate how ROA may be used to readily distinguish between the main classes of protein structure. A principal component analysis illustrates how the many structure-sensitive bands in protein ROA spectra are favorable for applying pattern recognition techniques to determine structural relationships between different proteins.
Structural Basis for tRNA-Dependent Amidotransferase Function by Emmanuelle Schmitt; Michel Panvert; Sylvain Blanquet; Yves Mechulam (1421-1433).
Besides direct charging of tRNAs by aminoacyl-tRNA synthetases, indirect routes also ensure attachment of some amino acids onto tRNA. Such routes may explain how new amino acids entered into protein synthesis. In archaea and in most bacteria, tRNAGln is first misaminoacylated by glutamyl-tRNA synthetase. Glu-tRNAGln is then matured into Gln-tRNAGln by a tRNA-dependent amidotransferase. We report the structure of a tRNA-dependent amidotransferase—that of GatDE from Pyrococcus abyssi. The 3.0 Å resolution crystal structure shows a tetramer with two GatD molecules as the core and two GatE molecules at the periphery. The fold of GatE cannot be related to that of any tRNA binding enzyme. The ammonium donor site on GatD and the tRNA site on GatE are markedly distant. Comparison of GatD and L-asparaginase structures shows how the motion of a β hairpin region containing a crucial catalytic threonine may control the overall reaction cycle of GatDE.
Intermediate States of Ribonuclease III in Complex with Double-Stranded RNA by Jianhua Gan; Joseph E. Tropea; Brian P. Austin; Donald L. Court; David S. Waugh; Xinhua Ji (1435-1442).
Bacterial ribonuclease III (RNase III) can affect RNA structure and gene expression in either of two ways: as a processing enzyme that cleaves double-stranded (ds) RNA, or as a binding protein that binds but does not cleave dsRNA. We previously proposed a model of the catalytic complex of RNase III with dsRNA based on three crystal structures, including the endonuclease domain of RNase III with and without bound metal ions and a dsRNA binding protein complexed with dsRNA. We also reported a noncatalytic assembly observed in the crystal structure of an RNase III mutant, which binds but does not cleave dsRNA, complexed with dsRNA. We hypothesize that the RNase III•dsRNA complex can exist in two functional forms, a catalytic complex and a noncatalytic assembly, and that in between the two forms there may be intermediate states. Here, we present four crystal structures of RNase III complexed with dsRNA, representing possible intermediates.
Crystal Structure of Human Taspase1, a Crucial Protease Regulating the Function of MLL by Javed A. Khan; Ben M. Dunn; Liang Tong (1443-1452).
Taspase1 catalyzes the proteolytic processing of the mixed lineage leukemia (MLL) nuclear protein, which is required for maintaining Hox gene expression patterns. Chromosomal translocations of the MLL gene are associated with leukemia in infants. Taspase1, a threonine aspartase, is a member of the type 2 asparaginase family, but is the only protease in this family. We report here the crystal structures of human activated Taspase1 and its proenzyme, as well as the characterization of the effects of mutations in the active site region using a newly developed fluorogenic assay. The structure of Taspase1 has significant differences from other asparaginases, especially near the active site. Mutation of the catalytic nucleophile, Thr234, abolishes autocatalytic processing in cis but does not completely block proteolysis in trans. The structure unexpectedly showed the binding of a chloride ion in the active site, and our kinetic studies confirm that chlorides ions are inhibitors of this enzyme at physiologically relevant concentrations.
Structural Basis for Glycogen Recognition by AMP-Activated Protein Kinase by Galina Polekhina; Abhilasha Gupta; Bryce J.W. van Denderen; Susanne C. Feil; Bruce E. Kemp; David Stapleton; Michael W. Parker (1453-1462).
AMP-activated protein kinase (AMPK) coordinates cellular metabolism in response to energy demand as well as to a variety of stimuli. The AMPK β subunit acts as a scaffold for the α catalytic and γ regulatory subunits and targets the AMPK heterotrimer to glycogen. We have determined the structure of the AMPK β glycogen binding domain in complex with β-cyclodextrin. The structure reveals a carbohydrate binding pocket that consolidates all known aspects of carbohydrate binding observed in starch binding domains into one site, with extensive contact between several residues and five glucose units. β-cyclodextrin is held in a pincer-like grasp with two tryptophan residues cradling two β-cyclodextrin glucose units and a leucine residue piercing the β-cyclodextrin ring. Mutation of key β-cyclodextrin binding residues either partially or completely prevents the glycogen binding domain from binding glycogen. Modeling suggests that this binding pocket enables AMPK to interact with glycogen anywhere across the carbohydrate’s helical surface.
Two Different Conformational States of the KirBac3.1 Potassium Channel Revealed by Electron Crystallography by Anling Kuo; Carmen Domene; Louise N. Johnson; Declan A. Doyle; Catherine Vénien-Bryan (1463-1472).
Potassium channels allow the selective flow of K+ ions across membranes. In response to external gating signals, the potassium channel can move reversibly through a series of structural conformations from a closed to an open state. 2D crystals of the inwardly rectifying K+ channel KirBac3.1 from Magnetospirillum magnetotacticum have been captured in two distinct conformations, providing “snap shots” of the gating process. Analysis by electron cryomicroscopy of these KirBac3.1 crystals has resulted in reconstructed images in projection at 9 Å resolution. Kir channels are tetramers of four subunits arranged as dimers of dimers. Each subunit has two transmembrane helices (inner and outer). In one crystal form, the pore is blocked; in the other crystal form, the pore appears open. Modeling based on the KirBac1.1 (closed) crystal structure shows that opening of the ion conduction pathway could be achieved by bending of the inner helices and significant movements of the outer helices.
Metalloproteomics: High-Throughput Structural and Functional Annotation of Proteins in Structural Genomics by Wuxian Shi; Chenyang Zhan; Alexander Ignatov; Babu A. Manjasetty; Nebojsa Marinkovic; Michael Sullivan; Raymond Huang; Mark R. Chance (1473-1486).
A high-throughput method for measuring transition metal content based on quantitation of X-ray fluorescence signals was used to analyze 654 proteins selected as targets by the New York Structural GenomiX Research Consortium. Over 10% showed the presence of transition metal atoms in stoichiometric amounts; these totals as well as the abundance distribution are similar to those of the Protein Data Bank. Bioinformatics analysis of the identified metalloproteins in most cases supported the metalloprotein annotation; identification of the conserved metal binding motif was also shown to be useful in verifying structural models of the proteins. Metalloproteomics provides a rapid structural and functional annotation for these sequences and is shown to be ∼95% accurate in predicting the presence or absence of stoichiometric metal content. The project’s goal is to assay at least 1 member from each Pfam family; approximately 500 Pfam families have been characterized with respect to transition metal content so far.
Functional Plasticity in the Substrate Binding Site of β-Secretase by Alemayehu A. Gorfe; Amedeo Caflisch (1487-1498).
The aspartic protease β-secretase (BACE) cleaves the amyloid precursor protein into a 42 residue β-peptide, which is the principal biochemical marker of Alzheimer’s disease. Multiple explicit-water molecular dynamics simulations of the apo and inhibitor bound structures of BACE indicate that both open- and closed-flap conformations are accessible at room temperature and should be taken into account for inhibitor design. Correlated motion is observed within each of the two lobes of BACE, as well as for the interfacial region. A self-inhibited conformation with the side chain of Tyr71 occupying the S1 pocket is present in some of the unbound simulations. The reversible loss of the side chain hydrogen bond between the catalytic Asp32 and Ser35, due to the concomitant reorientation of the Ser35 hydroxyl group and a water molecule conserved in pepsin-like enzymes, provides further evidence for the suggestion that Ser35 assists in proton acceptance and release by Asp32 during catalysis.
Structural and Energetic Origins of Sequence-Specific DNA Bending: Monte Carlo Simulations of Papillomavirus E2-DNA Binding Sites by Remo Rohs; Heinz Sklenar; Zippora Shakked (1499-1509).
DNA bending is an important structural feature for indirect readout in protein-DNA recognition. The binding of papillomavirus E2 transcription factors to their DNA binding sites is associated with DNA bending, providing an attractive model system to study the origins of sequence-specific DNA bending. The consensus E2 target is of the general form ACCGN4CGGT with a variable four base pair region. We applied a new all-atom Monte Carlo (MC) algorithm that combines effective sampling with fast conformational equilibration. The resulting MC ensembles resemble the corresponding high-resolution crystal structures very well. Distinct bending is observed for the E2-DNA binding site with a central AATT linker in contrast to an essentially straight DNA with a central ACGT linker. Contributions of specific base pair steps to the overall bending are shown in terms of local structural parameters. The analysis of conformational substates provides new insights into the energetic origins of intrinsic DNA bending.
Identification of an ITPase/XTPase in Escherichia coli by Structural and Biochemical Analysis by Jimin Zheng; Vinay Kumar Singh; Zongchao Jia (1511-1520).
Inosine triphosphate (ITP) and xanthosine triphosphate (XTP) are formed upon deamination of ATP and GTP as a result of exposure to chemical mutagens and oxidative damage. Nucleic acid synthesis requires safeguard mechanisms to minimize undesired lethal incorporation of ITP and XTP. Here, we present the crystal structure of YjjX, a protein of hitherto unknown function. The three-dimensional fold of YjjX is similar to those of Mj0226 from Methanococcus janschii, which possesses nucleotidase activity, and of Maf from Bacillus subtilis, which can bind nucleotides. Biochemical analyses of YjjX revealed it to exhibit specific phosphatase activity for inosine and xanthosine triphosphates and have a possible interaction with elongation factor Tu. The enzymatic activity of YjjX as an inosine/xanthosine triphosphatase provides evidence for a plausible protection mechanism by clearing the noncanonical nucleotides from the cell during oxidative stress in E. coli.
Retroviral Matrix Domains Share Electrostatic Homology: Models for Membrane Binding Function throughout the Viral Life Cycle by Paul S. Murray; Zhaohui Li; Jiyao Wang; Chris L. Tang; Barry Honig; Diana Murray (1521-1531).
The matrix domain (MA) of Gag polyproteins performs multiple functions throughout the retroviral life cycle. MA structures have an electropositive surface patch that is implicated in membrane association. Here, we use computational methods to demonstrate that electrostatic control of membrane binding is a central characteristic of all retroviruses. We are able to explain a wide range of experimental observations and provide a level of quantitative and molecular detail that has been inaccessible to experiment. We further predict that MA may exist in a variety of oligomerization states and propose mechanistic models for the effects of phosphoinositides and phosphorylation. The calculations provide a conceptual model for how non-myristoylated and myristoylated MAs behave similarly in assembly and disassembly. Hence, they provide a unified quantitative picture of the structural and energetic origins of the entire range of MA function and thus enhance, extend, and integrate previous observations on individual stages of the process.
Inter- and Intramolecular Determinants of the Specificity of Single-Stranded DNA Binding and Cleavage by the F Factor Relaxase by Chris Larkin; Saumen Datta; Matthew J. Harley; Brian J. Anderson; Alexandra Ebie; Victoria Hargreaves; Joel F. Schildbach (1533-1544).
The TraI protein of conjugative plasmid F factor binds and cleaves a single-stranded region of the plasmid prior to transfer to a recipient. TraI36, an N-terminal TraI fragment, binds ssDNA with a subnanomolar KD and remarkable sequence specificity. The structure of the TraI36 Y16F variant bound to ssDNA reveals specificity determinants, including a ssDNA intramolecular 3 base interaction and two pockets within the protein’s binding cleft that accommodate bases in a knob-into-hole fashion. Mutagenesis results underscore the intricate design of the binding site, with the greatest effects resulting from substitutions for residues that both contact ssDNA and stabilize protein structure. The active site architecture suggests that the bound divalent cation, which is essential for catalysis, both positions the DNA by liganding two oxygens of the scissile phosphate and increases the partial positive charge on the phosphorus to enhance nucleophilic attack.
Features of Reovirus Outer Capsid Protein μ1 Revealed by Electron Cryomicroscopy and Image Reconstruction of the Virion at 7.0 Å Resolution by Xing Zhang; Yongchang Ji; Lan Zhang; Stephen C. Harrison; Dan C. Marinescu; Max L. Nibert; Timothy S. Baker (1545-1557).
Reovirus is a useful model for addressing the molecular basis of membrane penetration by one of the larger nonenveloped animal viruses. We now report the structure of the reovirus virion at ∼7.0 Å resolution as obtained by electron cryomicroscopy and three-dimensional image reconstruction. Several features of the myristoylated outer capsid protein μ1, not seen in a previous X-ray crystal structure of the μ1-σ3 heterohexamer, are evident in the virion. These features appear to be important for stabilizing the outer capsid, regulating the conformational changes in μ1 that accompany perforation of target membranes, and contributing directly to membrane penetration during cell entry.
Crystal Structures of the Mnk2 Kinase Domain Reveal an Inhibitory Conformation and a Zinc Binding Site by Ralf Jauch; Stefan Jäkel; Catharina Netter; Kay Schreiter; Babette Aicher; Herbert Jäckle; Markus C. Wahl (1559-1568).
Human mitogen-activated protein kinases (MAPK)-interacting kinases 1 and 2 (Mnk1 and Mnk2) target the translational machinery by phosphorylation of the eukaryotic initiation factor 4E (eIF4E). Here, we present the 2.1 Å crystal structure of a nonphosphorylated Mnk2 fragment that encompasses the kinase domain. The results show Mnk-specific features such as a zinc binding motif and an atypical open conformation of the activation segment. In addition, the ATP binding pocket contains an Asp-Phe-Asp (DFD) in place of the canonical magnesium binding Asp-Phe-Gly (DFG) motif. The phenylalanine of this motif sticks into the ATP binding pocket and blocks ATP binding as observed with inhibitor bound and, thus, inactive p38 kinase. Replacement of the DFD by the canonical DFG motif affects the conformation of Mnk2, but not ATP binding and kinase activity. The results suggest that the ATP binding pocket and the activation segment of Mnk2 require conformational switches to provide kinase activity.
Human DNA Polymerase ι Incorporates dCTP Opposite Template G via a G.C+ Hoogsteen Base Pair by Deepak T. Nair; Robert E. Johnson; Louise Prakash; Satya Prakash; Aneel K. Aggarwal (1569-1577).
Human DNA polymerase ι (hPolι), a member of the Y family of DNA polymerases, differs in remarkable ways from other DNA polymerases, incorporating correct nucleotides opposite template purines with a much higher efficiency and fidelity than opposite template pyrimidines. We present here the crystal structure of hPolι bound to template G and incoming dCTP, which reveals a G.C+ Hoogsteen base pair in a DNA polymerase active site. We show that the hPolι active site has evolved to favor Hoogsteen base pairing, wherein the template sugar is fixed in a cavity that reduces the C1′-C1′ distance across the nascent base pair from ∼10.5 Å in other DNA polymerases to 8.6 Å in hPolι. The rotation of G from anti to syn is then largely in response to this curtailed C1′-C1′ distance. A G.C+ Hoogsteen base pair suggests a specific mechanism for hPolι’s ability to bypass N2-adducted guanines that obstruct replication.