Structure (v.18, #8)
In This Issue (v-vi).
Further Insights into the Ubiquitin Pathway: Understanding the Scarlet Letter Code by Annalisa Pastore (891-892).
Understanding the machinery that decides proteins' fate by tagging them with ubiquitin is an important goal of structural biology. have solved the structure of human E4B (or UFD2a), a U-box-containing protein that functions both as an E3 Ub ligase and as an E4 polyUb chain elongation factor.
Articulated Tubes by Linda A. Amos (892-894).
High quality images of microtubules with different numbers of protofilaments, and hence substantially different curvatures, have been reconstructed from electron microscopy (EM) data (). The data show how three versatile loops that mediate lateral interactions allow microtubules to be strong without being brittle.
RIG-I “Sees” the 5′-Triphosphate by Chao Zheng; Hao Wu (894-896).
RIG-I protects host cells against various RNA viruses by sensing viral RNAs in the cytoplasm. Crystal structures of RIG-I C-terminal domain bound to 5′-triphosphate dsRNA unveils how RIG-I recognizes the 5′-triphosphate moiety, a hallmark of viral RNAs ().
Structures of Get3, Get4, and Get5 Provide New Models for TA Membrane Protein Targeting by Peter J. Simpson; Blanche Schwappach; Henrik G. Dohlman; Rivka L. Isaacson (897-902).
The GET pathway, using several proteins (Gets 1-5 and probably Sgt2), posttranslationally conducts tail-anchored (TA) proteins to the endoplasmic reticulum (ER). At the ER, TA proteins are inserted into the lipid bilayer and then sorted and directed to their respective destinations in the secretory pathway. Until last year, there was no structural information on any of the GET components but now there are ten crystal structures of Get3 in a variety of nucleotide-bound states and conformations. The structures of Get4 and a portion of Get5 also emerged in 2010. This minireview provides a detailed comparison of the GET structures and discusses their mechanistic relevance to TA protein insertion. It also addresses the outstanding gaps in detailed molecular information on this system, including the structures of Get5, Sgt2, and the transmembrane complex comprising Get1 and Get2.
Zernike Phase Contrast Cryo-Electron Microscopy and Tomography for Structure Determination at Nanometer and Subnanometer Resolutions by Kazuyoshi Murata; Xiangan Liu; Radostin Danev; Joanita Jakana; Michael F. Schmid; Jonathan King; Kuniaki Nagayama; Wah Chiu (903-912).
Zernike phase contrast cryo-electron microscopy (ZPC-cryoEM) is an emerging technique that is capable of producing higher image contrast than conventional cryoEM. By combining this technique with advanced image processing methods, we achieved subnanometer resolution for two biological specimens: 2D bacteriorhodopsin crystal and epsilon15 bacteriophage. For an asymmetric reconstruction of epsilon15 bacteriophage, ZPC-cryoEM can reduce the required amount of data by a factor of ∼3, compared with conventional cryoEM. The reconstruction was carried out to 13 Å resolution without the need to correct the contrast transfer function. New structural features at the portal vertex of the epsilon15 bacteriophage are revealed in this reconstruction. Using ZPC cryo-electron tomography (ZPC-cryoET), a similar level of data reduction and higher resolution structures of epsilon15 bacteriophage can be obtained relative to conventional cryoET. These results show quantitatively the benefits of ZPC-cryoEM and ZPC-cryoET for structural determinations of macromolecular machines at nanometer and subnanometer resolutions.Display Omitted► ZPC-cryoEM resolves a better structure with one-third the data of conventional cryoEM ► α helices seen in maps obtained from ZPC-cryoEM of ɛ15 phage and bacteriorhodopsin ► 100 ZPC-cryoEM particle images resolve nonicosahedral portal vertex of ɛ15 phage ► Asymmetric map of ɛ15 phage computed from 50 subtomograms in a single ZPC-tomogram
Keywords: PROTEINS; CELLBIO; SYSBIO;
Structural and Functional Analysis of A-Type Ketoreductases from the Amphotericin Modular Polyketide Synthase by Jianting Zheng; Clint A. Taylor; Shawn K. Piasecki; Adrian T. Keatinge-Clay (913-922).
Complex polyketides are characterized by multiple chiral centers harboring hydroxyl and alkyl substituents. To investigate the mechanisms by which these stereocenters are set, several high-resolution structures of the ketoreductase (KR) domain from the second module of the amphotericin modular polyketide synthase (PKS) were solved. This first structural analysis of an A-type KR helps reveal how these KRs direct polyketide intermediates into their active sites from the side opposite that used by B-type KRs, resulting in a β-hydroxyl group of opposite stereochemistry. A comparison of structures obtained in the absence and presence of ligands reveals an induced fit mechanism that is important for catalysis. Activity assays of mutants of KRs from the first and second modules of the amphotericin PKS reveal the relative contributions of several active site residues toward catalysis and stereocontrol. Together, these results highlight the possibility of region-specific modification of polyketides through active site engineering of KRs.► This first A-type KR structure helps explain how KR types set stereocenters ► The complete nicotinamide cofactor is bound and its binding site described ► The binding of ligands induces conformational changes necessary for catalysis ► A stereocontrol assay reveals mutation that reverses α-substituent stereochemistry
Using NMR Chemical Shifts as Structural Restraints in Molecular Dynamics Simulations of Proteins by Paul Robustelli; Kai Kohlhoff; Andrea Cavalli; Michele Vendruscolo (923-933).
We introduce a procedure to determine the structures of proteins by incorporating NMR chemical shifts as structural restraints in molecular dynamics simulations. In this approach, the chemical shifts are expressed as differentiable functions of the atomic coordinates and used to compute forces to generate trajectories that lead to the reduction of the differences between experimental and calculated chemical shifts. We show that this strategy enables the folding of a set of proteins with representative topologies starting from partially denatured initial conformations without the use of additional experimental information. This method also enables the straightforward combination of chemical shifts with other standard NMR restraints, including those derived from NOE, J-coupling, and residual dipolar coupling measurements. We illustrate this aspect by calculating the structure of a transiently populated excited state conformation from chemical shift and residual dipolar coupling data measured by relaxation dispersion NMR experiments.Display Omitted► Chemical shift restraints in molecular dynamics simulations ► Protein structure determination using chemical shift restraints ► Prediction of chemical shifts from protein structures ► Determination of the structures of protein intermediate states from NMR restraints
Structural and Functional Studies of Igαβ and Its Assembly with the B Cell Antigen Receptor by Sergei Radaev; Zhongcheng Zou; Pavel Tolar; Khanh Nguyen; AnhThao Nguyen; Peter D. Krueger; Nicole Stutzman; Susan Pierce; Peter D. Sun (934-943).
The B cell antigen receptor (BCR) plays an essential role in all phases of B cell development. Here we show that the extracellular domains of murine and human Igβ form an I-set immunoglobulin-like structure with an interchain disulfide between cysteines on their G strands. Structural and sequence analysis suggests that Igα displays a similar fold as Igβ. An Igαβ heterodimer model was generated based on the unique disulfide-bonded Igβ dimer. Solution binding studies showed that the extracellular domains of Igαβ preferentially recognize the constant region of BCR with μ chain specificity, suggesting a role for Igαβ to enhance BCRμ chain signaling. Cluster mutations on Igα, Igβ, and a membrane-bound form of immunoglobulin (mIgM) based on the structural model identified distinct areas of potential contacts involving charged residues on both subunits of the coreceptor and the Cμ4 domain of mIgM. These studies provide the first structural model for understanding BCR function.Display Omitted► The crystal structure of B cell coreceptor Igβ ► Igαβ preferentially recognizes the μ chain of BCR ► A model for Igαβ is generated based on the dimeric Igβ structure ► Mutational analysis of both Igαβ and BCRμ identified a potential BCR/Igαβ binding site
Keywords: PROTEINS; MOLIMMUNO;
Conserved β-Hairpin Recognition by the GYF Domains of Smy2 and GIGYF2 in mRNA Surveillance and Vesicular Transport Complexes by Miriam-Rose Ash; Katja Faelber; Daniela Kosslick; Gesa Ines Albert; Yvette Roske; Michael Kofler; Michael Schuemann; Eberhard Krause; Christian Freund (944-954).
The yeast suppressor of myosin 2 protein (Smy2) interacts with mRNA-processing proteins through recognition of proline-rich sequences (PRS). Here, we describe the crystal structure of the GYF domain of Smy2 in association with a PRS from the yeast branch point binding protein (BBP/ScSF1). Complex formation requires that the β-hairpin of the central PPGL motif of the ligand is accommodated by an extended hydrophobic cleft in the domain—a specificity feature that is maintained in the human protein GIGYF2. SILAC/MS experiments in combination with PRS site inhibition show that Smy2 associates with the Ccr4-NOT deadenylase complex, whereas GIGYF2 interacts not only with mRNA surveillance factors, but also with vesicular transport proteins and Atrophin-1. GIGYF2 is shown to associate with COPII-vesicle proteins and localize to the ER and Golgi in resting cells, whereas environmental challenge drives GIGYF2 into stress granules. The current study highlights the structural basis for PRS recognition by Smy2-type GYF domains, and implicates Smy2 and GIGYF2 in both mRNA processing and the secretory pathway.► Sequence recognition by Smy2-type GYF domains requires b-hairpin formation of ligands ► Proteomic analysis reveals association of GIGYF2 with atrophin-1 and Sec31 ► Suppressor of myosin 2 interacts with processing body components
Keywords: RNA; CELLBIO;
Molecular Basis for the Association of Human E4B U Box Ubiquitin Ligase with E2-Conjugating Enzymes UbcH5c and Ubc4 by Robert C. Benirschke; James R. Thompson; Yves Nominé; Emeric Wasielewski; Nenad Juranić; Slobodan Macura; Shigetsugu Hatakeyama; Keiichi I. Nakayama; Maria Victoria Botuyan; Georges Mer (955-965).
Human E4B, also called UFD2a, is a U box-containing protein that functions as an E3 ubiquitin ligase and an E4 polyubiquitin chain elongation factor. E4B is thought to participate in the proteasomal degradation of misfolded or damaged proteins through association with chaperones. The U box domain is an anchor site for E2 ubiquitin-conjugating enzymes, but little is known of the binding mechanism. Using X-ray crystallography and NMR spectroscopy, we determined the structures of E4B U box free and bound to UbcH5c and Ubc4 E2s. Whereas previously characterized U box domains are homodimeric, we show that E4B U box is a monomer stabilized by a network of hydrogen bonds identified from scalar coupling measurements. These structural studies, complemented by calorimetry- and NMR-based binding assays, suggest an allosteric regulation of UbcH5c and Ubc4 by E4B U box and provide a molecular basis to understand how the ubiquitylation machinery involving E4B assembles.► Determined crystal and NMR structures of E4B U box demonstrating a monomeric state ► Demonstration of a hydrogen-bond network in E4B U box via NMR scalar couplings ► First 3D structure of a monomeric U box–E2 enzyme complex (E4B U box bound to UbcH5c) ► Evidence for an allosteric effect of E4B U box on UbcH5c and Ubc4 E2 enzymes
Polycomb Group Targeting through Different Binding Partners of RING1B C-Terminal Domain by Renjing Wang; Alexander B. Taylor; Belinda Z. Leal; Linda V. Chadwell; Udayar Ilangovan; Angela K. Robinson; Virgil Schirf; P. John Hart; Eileen M. Lafer; Borries Demeler; Andrew P. Hinck; Donald G. McEwen; Chongwoo A. Kim (966-975).
RING1B, a Polycomb Group (PcG) protein, binds methylated chromatin through its association with another PcG protein called Polycomb (Pc). However, RING1B can associate with nonmethylated chromatin suggesting an alternate mechanism for RING1B interaction with chromatin. Here, we demonstrate that two proteins with little sequence identity between them, the Pc cbox domain and RYBP, bind the same surface on the C-terminal domain of RING1B (C-RING1B). Pc cbox and RYBP each fold into a nearly identical, intermolecular beta sheet with C-RING1B and a loop structure which are completely different in the two proteins. Both the beta sheet and loop are required for stable binding and transcription repression. Further, a mutation engineered to disrupt binding on the Drosophila dRING1 protein prevents chromatin association and PcG function in vivo. These results suggest that PcG targeting to different chromatin locations relies, in part, on binding partners of C-RING1B that are diverse in sequence and structure.► Crystal structure of C-RING1B/cbx7 cbox complex ► cbx7 cbox beta sheet and loop are required for binding C-RING1B and gene repression ► RYBP binds to the same surface on C-RING1B as cbx7 cbox ► Drosophila RING1 protein-protein interaction mutations hinders its chromatin binding
Structural Role of the Vps4-Vta1 Interface in ESCRT-III Recycling by Dong Yang; James H. Hurley (976-984).
The ESCRT complexes are required for multivesicular body biogenesis, macroautophagy, cytokinesis, and the budding of HIV-1. The final step in the ESCRT cycle is the disassembly of the ESCRT-III lattice by the AAA+ ATPase Vps4. Vps4 assembles on its membrane-bound ESCRT-III substrate with its cofactor, Vta1. The crystal structure of the dimeric VSL domain of yeast Vta1 with the small ATPase and the βdomains of Vps4 was determined. Residues involved in structural interactions are conserved and are required for binding in vitro and for Cps1 sorting in vivo. Modeling of the Vta1 complex in complex with the lower hexameric ring of Vps4 indicates that the two-fold axis of the Vta1 VSL domain is parallel to within ∼20 degrees of the six-fold axis of the hexamer. This suggests that Vta1 might not crosslink the two hexameric rings of Vps4, but rather stabilizes an array of Vps4-Vta1 complexes for ESCRT-III disassembly.► Structure of the interacting fragments of Vps4 and Vta1 is determined ► Interface residues are required for binding in vitro and Cps1 sorting in yeast ► Vta1 probably does not crosslink upper and lower Vps4 hexameric rings ► Modeling suggests Vta1 could crosslink different Vps4 dodecamers
Keywords: CELLBIO; PROTEINS;
The RalB-RLIP76 Complex Reveals a Novel Mode of Ral-Effector Interaction by R. Brynmor Fenwick; Louise J. Campbell; Karthik Rajasekar; Sunil Prasannan; Daniel Nietlispach; Jacques Camonis; Darerca Owen; Helen R. Mott (985-995).
RLIP76 (RalBP1) is a multidomain protein that interacts with multiple small G protein families: Ral via a specific binding domain, and Rho and R-Ras via a GTPase activating domain. RLIP76 interacts with endocytosis proteins and has also been shown to behave as a membrane ATPase that transports chemotherapeutic agents from the cell. We have determined the structure of the Ral-binding domain of RLIP76 and show that it comprises a coiled-coil motif. The structure of the RLIP76-RalB complex reveals a novel mode of binding compared to the structures of RalA complexed with the exocyst components Sec5 and Exo84. RLIP76 interacts with both nucleotide-sensitive regions of RalB, and key residues in the interface have been identified using affinity measurements of RalB mutants. Sec5, Exo84, and RLIP76 bind Ral proteins competitively and with similar affinities in vitro.Display Omitted► The Ra1 binding coiled coil is the first structure available of RLIP/Ra1BP1 ► RLIP76 interacts with the switch regions of Ra1B using a novel Ra1-binging motif ► The binding to Ra1B is different from, and competitive with, exocyst component binding ► Mutational analysis shows that RLIP76 Trp-430 is a hotspot in the interface
Keywords: SIGNALING; CELLBIO;
Crystal Structures of Phd-Doc, HigA, and YeeU Establish Multiple Evolutionary Links between Microbial Growth-Regulating Toxin-Antitoxin Systems by Mark A. Arbing; Samuel K. Handelman; Alexandre P. Kuzin; Grégory Verdon; Chi Wang; Min Su; Francesca P. Rothenbacher; Mariam Abashidze; Mohan Liu; Jennifer M. Hurley; Rong Xiao; Thomas Acton; Masayori Inouye; Gaetano T. Montelione; Nancy A. Woychik; John F. Hunt (996-1010).
Bacterial toxin-antitoxin (TA) systems serve a variety of physiological functions including regulation of cell growth and maintenance of foreign genetic elements. Sequence analyses suggest that TA families are linked by complex evolutionary relationships reflecting likely swapping of functional domains between different TA families. Our crystal structures of Phd-Doc from bacteriophage P1, the HigA antitoxin from Escherichia coli CFT073, and YeeU of the YeeUWV systems from E. coli K12 and Shigella flexneri confirm this inference and reveal additional, unanticipated structural relationships. The growth-regulating Doc toxin exhibits structural similarity to secreted virulence factors that are toxic for eukaryotic target cells. The Phd antitoxin possesses the same fold as both the YefM and NE2111 antitoxins that inhibit structurally unrelated toxins. YeeU, which has an antitoxin-like activity that represses toxin expression, is structurally similar to the ribosome-interacting toxins YoeB and RelE. These observations suggest extensive functional exchanges have occurred between TA systems during bacterial evolution.► Crystal structures establish evolutionary relationships between disparate toxin-antitoxin systems ► Crystal structures of two YeeU antitoxins revealed a toxin-like protein fold ► The Phd antitoxin structure was determined in a 2:2 complex with the Doc toxin ► The crystal structure was determined of the apo HigA antitoxin from E. coli CFT073
Keywords: RNA; CELLBIO;
Mapping the Interactions between a Major Pollen Allergen and Human IgE Antibodies by Guilherme Razzera; Gabriele Gadermaier; Viviane de Paula; Marcius S. Almeida; Matthias Egger; Beatrice Jahn-Schmid; Fabio C.L. Almeida; Fatima Ferreira; Ana Paula Valente (1011-1021).
The interaction of specific IgE antibodies with allergens is a key event in the induction of allergic symptoms, thus representing an important target for therapeutic interventions in Type I allergies. We report here the solution NMR structure of Art v 1, the major mugwort pollen allergen. Art v 1 is the first protein structure with an allergenic defensin fold linked to a polyproline domain, which has not been identified in any reported allergen structure in the PDB. Moreover, the direct interaction of polyclonal IgE antibodies from an allergic patient has been mapped on the surface of an allergen for the first time. The data presented herein provide the basis for the design of tools for safe and effective vaccination against mugwort pollen allergy.► Art v 1 displays a cysteine stabilized defensin fold linked to a polyproline region ► The proline region consists of a structured part and a flexible C terminus ► Residues interacting with human IgE Abs form two patches on the defensin domain ► Epitope patches are positively charged and conserved among allergenic defensins
Keywords: MOLIMMUNO; HUMDISEASE;
Structural Basis of Interprotofilament Interaction and Lateral Deformation of Microtubules by Haixin Sui; Kenneth H. Downing (1022-1031).
The diverse functions of microtubules require stiff structures possessing sufficient lateral flexibility to enable bending with high curvature. We used cryo-electron microscopy to investigate the molecular basis for these critical mechanical properties. High-quality structural maps were used to build pseudoatomic models of microtubules containing 11–16 protofilaments, representing a wide range of lateral curvature. Protofilaments in all these microtubules were connected primarily via interprotofilament interactions between the M loops, and the H1′-S2 and H2-S3 loops. We postulate that the tolerance of the loop-loop interactions to lateral deformation provides the capacity for high-curvature bending without breaking. On the other hand, the local molecular architecture that surrounds these connecting loops contributes to the overall rigidity. Interprotofilament interactions in the seam region are similar to those in the normal helical regions, suggesting that the existence of the seam does not significantly affect the mechanical properties of microtubules.► Interprotofilament interactions are conserved in microtubules with various diameters ► Flexible loop-loop interactions enable microtubule lateral deformation and bending ► Structure surrounding the connecting loops contributes to microtubule rigidity ► Seams have little or no effect on the mechanical properties of microtubules
The Structural Basis of 5′ Triphosphate Double-Stranded RNA Recognition by RIG-I C-Terminal Domain by Cheng Lu; Hengyu Xu; C.T. Ranjith-Kumar; Monica T. Brooks; Tim Y. Hou; Fuqu Hu; Andrew B. Herr; Roland K. Strong; C. Cheng Kao; Pingwei Li (1032-1043).
RIG-I is a cytosolic sensor of viral RNA that plays crucial roles in the induction of type I interferons. The C-terminal domain (CTD) of RIG-I is responsible for the recognition of viral RNA with 5′ triphosphate (ppp). However, the mechanism of viral RNA recognition by RIG-I is still not fully understood. Here, we show that RIG-I CTD binds 5′ ppp dsRNA or ssRNA, as well as blunt-ended dsRNA, and exhibits the highest affinity for 5′ ppp dsRNA. Crystal structures of RIG-I CTD bound to 5′ ppp dsRNA with GC- and AU-rich sequences revealed that RIG-I recognizes the termini of the dsRNA and interacts with the 5′ ppp through extensive electrostatic interactions. Mutagenesis and RNA-binding studies demonstrated that similar binding surfaces are involved in the recognition of different forms of RNA. Mutations of key residues at the RNA-binding surface affected RIG-I signaling in cells.► RIG-I CTD binds 5′ triphosphate dsRNA with high affinity ► Structures of RIG-I CTD bound to 5′ triphosphate dsRNA with GC- and AU-rich sequences ► RIG-I and LGP2 CTD bind dsRNA with or without triphosphate differently ► Overlapping sets of residues are involved in binding of various forms of RNA
The Macromolecular Architecture of Extracellular Domain of αNRXN1: Domain Organization, Flexibility, and Insights into Trans-Synaptic Disposition by Davide Comoletti; Meghan T. Miller; Cy M. Jeffries; Jennifer Wilson; Borries Demeler; Palmer Taylor; Jill Trewhella; Terunaga Nakagawa (1044-1053).
Neurexins are multidomain synaptic cell-adhesion proteins that associate with multiple partnering proteins. Genetic evidence indicates that neurexins may contribute to autism, schizophrenia, and nicotine dependence. Using analytical ultracentrifugation, single-particle electron microscopy, and solution X-ray scattering, we obtained a three-dimensional structural model of the entire extracellular domain of neurexin-1α. This protein adopts a dimensionally asymmetric conformation that is monomeric in solution, with a maximum dimension of ∼170 Å. The extracellular domain of α-neurexin maintains a characteristic “Y” shape, whereby LNS domains 1–4 form an extended base of the “Y” and LNS5-6 the shorter arms. Moreover, two major regions of flexibility are present: one between EGF1 and LNS2, corresponding to splice site 1, another between LNS5 and 6. We thus provide the first structural insights into the architecture of the extracellular region of neurexin-1α, show how the protein may fit in the synaptic cleft, and how partnering proteins could bind simultaneously.► The extracellular domain of αNRXN1 is monomeric in solution ► αNRXN1 maintains a characteristic “Y” shape in solution ► Extensive flexibility is present between LNS1 and 2 and between LNS5 and 6 ► A three-dimensional structural model of the αNRXN1 is presented and discussed
Structure and Function of Sphingosine-1-Phosphate Lyase, a Key Enzyme of Sphingolipid Metabolism by Florence Bourquin; Howard Riezman; Guido Capitani; Markus G. Grütter (1054-1065).
Sphingosine-1-phosphate lyase (SPL), a key enzyme of sphingolipid metabolism, catalyzes the irreversible degradation of sphingoid base phosphates. Its main substrate sphingosine-1-phosphate (S1P) acts both extracellularly, by binding G protein-coupled receptors of the lysophospholipid receptor family, and inside the cell, as a second messenger. There, S1P takes part in regulating various cellular processes and its levels are tightly regulated. SPL is a pivotal enzyme regulating S1P intracellular concentrations and a promising drug target for the design of immunosuppressants. We structurally and functionally characterized yeast SPL (Dpl1p) and its first prokaryotic homolog, from Symbiobacterium thermophilum. The Dpl1p structure served as a basis for a very reliable model of Homo sapiens SPL. The above results, together with in vitro and in vivo studies of SPL mutants, reveal which residues are involved in activity and substrate binding and pave the way to studies aimed at controlling the activity of this pivotal enzyme.► SPL is a key enzyme of sphingolipid metabolism and a promising drug target ► We identified the first prokaryotic SPL in S. thermophilum and solved its structure ► We determined the structure of yeast SPL and of two complexes of prokaryotic SPL ► We functionally characterized the two proteins in vivo and in vitro
Keywords: PROTEINS; CELLBIO;