Structure (v.18, #6)
In This Issue (v-vi).
The Lighter Side of a Sweet Reaction by Brad C. Bennett; Mark Yeager (657-659).
The unique advantage of neutrons as biological probes is the ability to visualize hydrogen atoms in macromolecules. In this issue, solved an ensemble of xylose isomerase structures by neutron crystallography, and the determination of hydrogen atom rearrangements during the catalytic cycle provides insight into the enzyme's mechanism.
Fibronectin Structure: A New Piece of the Puzzle Emerges by Richard J. Bingham; Jennifer R. Potts (660-661).
In the structure of the gelatin binding domain (GBD) of fibronectin reported by , both the zinc-mediated dimerization and the rearrangement of 8FI from the canonical FI fold are unexpected, suggesting interesting new directions for researchers in the field.
Topoisomerase IB-DNA Interactions: X Marks the Spot by Lynn Zechiedrich; Neil Osheroff (661-663).
The observation made twenty years ago that type IB topoisomerases bound DNA helix-helix juxtapositions was unexpected, given the controlled helical rotation mechanism of the enzyme. In this issue, provide an elegant structural explanation for this interaction.
From Poor Resolution to Rich Insight by Randy J. Read (664-665).
In a recent Nature paper, the stability and convergence radius of low-resolution crystal structure refinement is improved by the addition of a deformable elastic network energy, which brings in higher-resolution information and preserves local interactions ().
An Approach for De Novo Structure Determination of Dynamic Molecular Assemblies by Electron Cryomicroscopy by Bjoern Sander; Monika M. Golas; Reinhard Lührmann; Holger Stark (667-676).
Single-particle electron cryomicroscopy is a powerful method for three-dimensional (3D) structure determination of macromolecular assemblies. Here we address the challenge of determining a 3D structure in the absence of reference models. The 3D structures are determined by alignment and weighted averaging of densities obtained by native cryo random conical tilt (RCT) reconstructions including consideration of missing data. Our weighted averaging scheme (wRCT) offers advantages for potentially heterogeneous 3D densities of low signal-to-noise ratios. Sets of aligned RCT structures can also be analyzed by multivariate statistical analysis (MSA) to provide insights into snapshots of the assemblies. The approach is used to compute 3D structures of the Escherichia coli 70S ribosome and the human U4/U6.U5 tri-snRNP under vitrified unstained cryo conditions, and to visualize by 3D MSA the L7/L12 stalk of the 70S ribosome and states of tri-snRNP. The approach thus combines de novo 3D structure determination with an analysis of compositional and conformational heterogeneity.Display Omitted► Weighted averaging of cryo-EM maps suitable for unstained cryo random conical tilt ► De novo structure determination independent of starting model ► Multivariate statistical analysis of 3D maps to analyze structural variations ► 70S ribosome and human tri-snRNP structures and dynamic snapshots
Keywords: PROTEINS; RNA;
Three-Dimensional Structure of TspO by Electron Cryomicroscopy of Helical Crystals by Vladimir M. Korkhov; Carsten Sachse; Judith M. Short; Christopher G. Tate (677-687).
The 18 kDa TSPO protein is a polytopic mitochondrial outer membrane protein involved in a wide range of physiological functions and pathologies, including neurodegeneration and cancer. The pharmacology of TSPO has been extensively studied, but little is known about its biochemistry, oligomeric state, and structure. We have expressed, purified, and characterized a homologous protein, TspO from Rhodobacter sphaeroides, and reconstituted it as helical crystals. Using electron cryomicroscopy and single-particle helical reconstruction, we have determined a three-dimensional structure of TspO at 10 Å resolution. The structure suggests that monomeric TspO comprises five transmembrane α helices that form a homodimer, which is consistent with the dimeric state observed in detergent solution. Furthermore, the arrangement of transmembrane domains of individual TspO subunits indicates a possibility of two substrate translocation pathways per dimer. The structure provides the first insight into the molecular architecture of TSPO/PBR protein family that will serve as a framework for future studies.► R. sphaeroides TspO reconstituted as helical tubular crystals ► Cryo-EM and image analysis lead to structure of TspO at 10 Å resolution ► The structure confirms a dimeric five transmembrane domain architecture of TspO ► Two putative substrate translocation pathways per dimer are suggested
Keywords: PROTEINS; CELLBIO;
Metal Ion Roles and the Movement of Hydrogen during Reaction Catalyzed by D-Xylose Isomerase: A Joint X-Ray and Neutron Diffraction Study by Andrey Y. Kovalevsky; Leif Hanson; S. Zoe Fisher; Marat Mustyakimov; Sax A. Mason; V. Trevor Forsyth; Matthew P. Blakeley; David. A. Keen; Trixie Wagner; H.L. Carrell; Amy K. Katz; Jenny P. Glusker; Paul Langan (688-699).
Conversion of aldo to keto sugars by the metalloenzyme D-xylose isomerase (XI) is a multistep reaction that involves hydrogen transfer. We have determined the structure of this enzyme by neutron diffraction in order to locate H atoms (or their isotope D). Two studies are presented, one of XI containing cadmium and cyclic D-glucose (before sugar ring opening has occurred), and the other containing nickel and linear D-glucose (after ring opening has occurred but before isomerization). Previously we reported the neutron structures of ligand-free enzyme and enzyme with bound product. The data show that His54 is doubly protonated on the ring N in all four structures. Lys289 is neutral before ring opening and gains a proton after this; the catalytic metal-bound water is deprotonated to hydroxyl during isomerization and O5 is deprotonated. These results lead to new suggestions as to how changes might take place over the course of the reaction.Display Omitted► Hydrogen is located at stages of the reactions catalyzed by D-xylose isomerase ► Lys289 is neutral before sugar ring opening, and gains a proton after this ► The catalytic metal-bound water is deprotonated to hydroxyl during isomerization ► His5 remains doubly protonated throughout the reaction
Evolution in Action: N and C Termini of Subunits in Related T = 4 Viruses Exchange Roles as Molecular Switches by Jeffrey A. Speir; Derek J. Taylor; Padmaja Natarajan; Fiona M. Pringle; L. Andrew Ball; John E. Johnson (700-709).
The T = 4 tetravirus and T = 3 nodavirus capsid proteins undergo closely similar autoproteolysis to produce the N-terminal β and C-terminal, lipophilic γ polypeptides. The γ peptides and the N termini of β also act as molecular switches that determine their quasi equivalent capsid structures. The crystal structure of Providence virus (PrV), only the second of a tetravirus (the first was NωV), reveals conserved folds and cleavage sites, but the protein termini have completely different structures and the opposite functions of those in NωV. N termini of β form the molecular switch in PrV, whereas γ peptides play this role in NωV. PrV γ peptides instead interact with packaged RNA at the particle two-folds by using a repeating sequence pattern found in only four other RNA- or membrane-binding proteins. The disposition of peptide termini in PrV is closely related to those in nodaviruses, suggesting that PrV may be closer to the primordial T = 4 particle than NωV.► First crystal structure of a β tetravirus, only second from the Tetraviridae ► Evolved opposite functions for protein termini in critical virion molecular switch ► C termini contact RNA at two-folds with commingled membrane-binding motif ► More closely related to nodaviruses and primordial T = 4 than to other tetraviruses
Keywords: PROTEINS; MICROBIO; EVO_ECOL;
Zinc Induces Structural Reorganization of Gelatin Binding Domain from Human Fibronectin and Affects Collagen Binding by Marc Graille; Maurice Pagano; Thierry Rose; Michèle Reboud Ravaux; Herman van Tilbeurgh (710-718).
Fibronectin is a modular extracellular matrix protein involved in cell adhesion, cell motility, wound healing, and maintenance of cell morphology. It is composed of multiple repeats of three distinct modules: FI, FII, and FIII. Various combinations of these modules create fragments able to interact with different constituents of the extracellular matrix. Here, we present the 2.5-Å resolution crystal structure of its 45-kDa gelatin-binding domain (GBD; 6FI-1FII-2FII-7FI-8FI-9FI), which also corresponds to the C-terminal half of the migration stimulating factor, a Fn splice variant expressed in human breast cancers. GBD forms a very compact zinc-mediated homodimer, in stark contrast with previous structures of fibronectin fragments. Most remarkably, 8FI no longer adopts the canonical FI fold but is composed of two long strands that associate with 7FI and 9FI into a large β-sheet superdomain. Binding studies in solution confirmed that Zn induces conformational rearrangements and causes loss of binding of Fn-GBD to high-affinity collagen peptides. These data suggest the Zn may play a regulatory role for the cellular functions of fibronectin.► Crystal structure of the entire collagen-binding domain from human fibronectin ► FnI module changes fold and the GBD dimerizes upon zinc binding ► Zinc affects binding to collagen peptide.
Structure of the 100S Ribosome in the Hibernation Stage Revealed by Electron Cryomicroscopy by Takayuki Kato; Hideji Yoshida; Tomoko Miyata; Yasushi Maki; Akira Wada; Keiichi Namba (719-724).
In the stationary growth phase of bacteria, protein biosynthesis on ribosomes is suppressed, and the ribosomes are preserved in the cell by the formation of the 100S ribosome. The 100S ribosome is a dimer of the 70S ribosome and is formed by the binding of the ribosome modulation factor and the hibernation promoting factor. However, the binding mode between the two 70S ribosomes and the mechanism of complex formation are still poorly understood. Here, we report the structure of the 100S ribosome by electron cryomicroscopy and single-particle image analysis. The 100S ribosome purified from the cell in the stationary growth phase is composed of two transfer RNA-free 70S ribosomes, has two-fold symmetry, and is formed through interactions between their 30S subunits, where interactions between small subunit proteins, S2, S3 and S5, appear to be critical for the dimerization.Display Omitted► The 100S ribosome was purified from Escherichia coli in the stationary growth phase ► The structure was analyzed by electron cryomicroscopy and single-particle image analysis ► The 100S ribosome is composed of two tRNA-free 70S ribosomes ► The 100S ribosome is formed through contacts between 30S subunits and has two-fold symmetry
Keywords: PROTEINS; RNA;
Crystal Structure of a Bacterial Topoisomerase IB in Complex with DNA Reveals a Secondary DNA Binding Site by Asmita Patel; Lyudmila Yakovleva; Stewart Shuman; Alfonso Mondragón (725-733).
Type IB DNA topoisomerases (TopIB) are monomeric enzymes that relax supercoils by cleaving and resealing one strand of duplex DNA within a protein clamp that embraces a ∼21 DNA segment. A longstanding conundrum concerns the capacity of TopIB enzymes to stabilize intramolecular duplex DNA crossovers and form protein-DNA synaptic filaments. Here we report a structure of Deinococcus radiodurans TopIB in complex with a 12 bp duplex DNA that demonstrates a secondary DNA binding site located on the surface of the C-terminal domain. It comprises a distinctive interface with one strand of the DNA duplex and is conserved in all TopIB enzymes. Modeling of a TopIB with both DNA sites suggests that the secondary site could account for DNA crossover binding, nucleation of DNA synapsis, and generation of a filamentous plectoneme. Mutations of the secondary site eliminate synaptic plectoneme formation without affecting DNA cleavage or supercoil relaxation.Display Omitted► Structure of D. radiodurans TopIB in complex with DNA ► Secondary DNA binding site in type IB topoisomerases ► Conserved site in all type IB enzymes ► Site responsible for stabilizing intramolecular duplex DNA crossovers
Unusual Target Site Disruption by the Rare-Cutting HNH Restriction Endonuclease PacI by Betty W. Shen; Daniel F. Heiter; Siu-Hong Chan; Hua Wang; Shuang-Yong Xu; Richard D. Morgan; Geoffrey G. Wilson; Barry L. Stoddard (734-743).
The crystal structure of the rare-cutting HNH restriction endonuclease PacI in complex with its eight-base-pair target recognition sequence 5′-TTAATTAA-3′ has been determined to 1.9 Å resolution. The enzyme forms an extended homodimer, with each subunit containing two zinc-bound motifs surrounding a ββα-metal catalytic site. The latter is unusual in that a tyrosine residue likely initiates strand cleavage. PacI dramatically distorts its target sequence from Watson-Crick duplex DNA base pairing, with every base separated from its original partner. Two bases on each strand are unpaired, four are engaged in noncanonical A:A and T:T base pairs, and the remaining two bases are matched with new Watson-Crick partners. This represents a highly unusual DNA binding mechanism for a restriction endonuclease, and implies that initial recognition of the target site might involve significantly different contacts from those visualized in the DNA-bound cocrystal structures.
Crystal Structure of an Intracellular Subtilisin Reveals Novel Structural Features Unique to this Subtilisin Family by Jitka Vévodová; Michael Gamble; Georg Künze; Antonio Ariza; Eleanor Dodson; D. Dafydd Jones; Keith S. Wilson (744-755).
The intracellular subtilisin proteases (ISPs) are the only known members of the important and ubiquitous subtilisin family that function exclusively within the cell, constituting a major component of the degradome in many Gram-positive bacteria. The first ISP structure reported herein at a spacing of 1.56 Å reveals features unique among subtilisins that has enabled potential functional and physiological roles to be assigned to sequence elements exclusive to the ISPs. Unlike all other subtilisins, ISP from B. clausii is dimeric, with residues from the C terminus making a major contribution to the dimer interface by crossing over to contact the partner subunit. A short N-terminal extension binds back across the active site to provide a potential novel regulatory mechanism of intrinsic proteolytic activity: a proline residue conserved throughout the ISPs introduces a kink in the polypeptide backbone that lifts the target peptide bond out of reach of the catalytic residues.Display Omitted► The first 3D structure of an intracellular subtilisin protease ► Unique among the subtilisins because intracellular subtilisin is dimeric ► Novel regulation mechanism utilizing a “proline bulge” is revealed ► A mechanism for activation is proposed