Structure (v.17, #3)

In This Issue (ix-x).

Ancient tRNA Synthetase Meets Modern Structural Biology by Paul Schimmel; Min Guo (315-317).
Use of heavy atoms to calculate the phases in protein X-ray crystallography has now been extended to include site-specific in vivo incorporation of 3-iodo-tyrosine, enabled by the use of an archaeal aminoacyl-tRNA synthetase (Sakamoto et al., 2009).

Tri- to be Mono- for Bacterial mRNA Decay by Sophie Bail; Megerditch Kiledjian (317-319).
Messing et al. (2009) report the homodimeric structure of the Bdellovibrio bacteriovorus RppH pyrophosphohydrolase, which hydrolyzes the mRNA 5′ triphosphate to initiate bacterial mRNA decay. These structures reveal insights into BdRppH substrate recognition and analogies to eukaryotic decapping enzymes.

Mauldin et al. (2009) use NMR to show that drug binding can break up collective protein motions necessary for function. We discuss their findings in the context of drug discovery in pharmaceutical research.

The ubiquitin-like modifier NEDD8 promotes substrate ubiquitination via its covalent linkage to ubiquitin cullin-RING ligases. This depends on a NEDD8-specific sequential enzymatic cascade of E1, E2, and E3 enzymes. The identification of a second E2 enzyme for nedd8ylation recently reported in Molecular Cell (Huang et al., 2009) reveals novel insights into the molecular mechanism of target cullin selection.

In plants, unorthodox multisubunit RNA polymerases (RNAPs) play key roles in small interfering RNA (siRNA) genesis and function. In a recent issue of Molecular Cell, Ream et al. (2009) established a 12-subunit composition for Arabidopsis RNAPIV and RNAPV. Subunit and sequence divergence between RNAPIV-V and RNAPI-III suggests significant functional deviation of these intriguing RNAPs.

Toward Structural Elucidation of the γ-Secretase Complex by Huilin Li; Michael S. Wolfe; Dennis J. Selkoe (326-334).
γ-Secretase is an intramembrane protease complex that mediates the Notch signaling pathway and the production of amyloid β-proteins. As such, this enzyme has emerged as an important target for development of novel therapeutics for Alzheimer disease and cancer. Great progress has been made in the identification and characterization of the membrane complex and its biological functions. One major challenge now is to illuminate the structure of this fascinating and important protease at atomic resolution. Here, we review recent progress on biochemical and biophysical probing of the structure of the four-component complex and discuss obstacles and potential pathways toward elucidating its detailed structure.

Genetic Encoding of 3-Iodo-l-Tyrosine in Escherichia coli for Single-Wavelength Anomalous Dispersion Phasing in Protein Crystallography by Kensaku Sakamoto; Kazutaka Murayama; Kenji Oki; Fumie Iraha; Miyuki Kato-Murayama; Masahiro Takahashi; Kazumasa Ohtake; Takatsugu Kobayashi; Seiki Kuramitsu; Mikako Shirouzu; Shigeyuki Yokoyama (335-344).
We developed an Escherichia coli cell-based system to generate proteins containing 3-iodo-l-tyrosine at desired sites, and we used this system for structure determination by single-wavelength anomalous dispersion (SAD) phasing with the strong iodine signal. Tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii was engineered to specifically recognize 3-iodo-l-tyrosine. The 1.7 Å crystal structure of the engineered variant, iodoTyrRS-mj, bound with 3-iodo-l-tyrosine revealed the structural basis underlying the strict specificity for this nonnatural substrate; the iodine moiety makes van der Waals contacts with 5 residues at the binding pocket. Ecoli cells expressing iodoTyrRS-mj and the suppressor tRNA were used to incorporate 3-iodo-l-tyrosine site specifically into the ribosomal protein N-acetyltransferase from Thermus thermophilus. The crystal structure of this enzyme with iodotyrosine was determined at 1.8 and 2.2 Å resolutions by SAD phasing at CuKα and CrKα wavelengths, respectively. The native structure, determined by molecular replacement, revealed no significant structural distortion caused by iodotyrosine incorporation.
Keywords: PROTEINS;

Reverse Micelles in Integral Membrane Protein Structural Biology by Solution NMR Spectroscopy by Joseph M. Kielec; Kathleen G. Valentine; Charles R. Babu; A. Joshua Wand (345-351).
Integral membrane proteins remain a significant challenge to structural studies by solution NMR spectroscopy. This is due not only to spectral complexity, but also because the effects of slow molecular reorientation are exacerbated by the need to solubilize the protein in aqueous detergent micelles. These assemblies can be quite large and require deuteration for optimal use of the TROSY effect. In principle, another approach is to employ reverse micelle encapsulation to solubilize the protein in a low-viscosity solvent in which the rapid tumbling of the resulting particle allows for use of standard triple-resonance methods. The preparation of such samples of membrane proteins is difficult. Using a 54 kDa construct of the homotetrameric potassium channel KcsA, we demonstrate a strategy that employs a hybrid surfactant to transfer the protein to the reverse micelle system.
Keywords: PROTEINS;

Kinase Domain Insertions Define Distinct Roles of CLK Kinases in SR Protein Phosphorylation by Alex N. Bullock; Sanjan Das; Judit É. Debreczeni; Peter Rellos; Oleg Fedorov; Frank H. Niesen; Kunde Guo; Evangelos Papagrigoriou; Ann L. Amos; Suhyung Cho; Benjamin E. Turk; Gourisankar Ghosh; Stefan Knapp (352-362).
Splicing requires reversible phosphorylation of serine/arginine-rich (SR) proteins, which direct splice site selection in eukaryotic mRNA. These phosphorylation events are dependent on SR protein (SRPK) and cdc2-like kinase (CLK) families. SRPK1 phosphorylation of splicing factors is restricted by a specific docking interaction whereas CLK activity is less constrained. To understand functional differences between splicing factor targeting kinases, we determined crystal structures of CLK1 and CLK3. Intriguingly, in CLKs the SRPK1 docking site is blocked by insertion of a previously unseen helix αH. In addition, substrate docking grooves present in related mitogen activating protein kinases (MAPKs) are inaccessible due to a CLK specific β7/8-hairpin insert. Thus, the unconstrained substrate interaction together with the determined active-site mediated substrate specificity allows CLKs to complete the functionally important hyperphosphorylation of splicing factors like ASF/SF2. In addition, despite high sequence conservation, we identified inhibitors with surprising isoform specificity for CLK1 over CLK3.
Keywords: SIGNALING;

Mapping the Initiator Binding Taf2 Subunit in the Structure of Hydrated Yeast TFIID by Gabor Papai; Manish K. Tripathi; Christine Ruhlmann; Sebastiaan Werten; Corinne Crucifix; P. Anthony Weil; Patrick Schultz (363-373).
The general transcription factor TFIID is a large multisubunit complex required for the transcription of most protein-encoding genes by RNA polymerase II. Taking advantage of a TFIID preparation partially depleted in the initiator-binding Taf2p subunit, we determined the conformational and biochemical variations of the complex by electron tomography and cryo-electron microscopy of single molecules. Image analysis revealed the extent of conformational flexibility of the complex and the selection of the most homogeneous TFIID subpopulation allowed us to determine an improved structural model at 23Å resolution. This study also identified two subpopulations of Taf2p-containing and Taf2p-depleted TFIID molecules. By comparing these two TFIID species we could infer the position of Taf2p, which was confirmed by immunolabeling using a subunit-specific antibody. Mapping the position of this crucial subunit in the vicinity of Taf1p and of TBP sheds new light on its role in promoter recognition.
Keywords: PROTEINS; DNA;

Structural Basis for Binding of RNA and Cofactor by a KsgA Methyltransferase by Chao Tu; Joseph E. Tropea; Brian P. Austin; Donald L. Court; David S. Waugh; Xinhua Ji (374-385).
Among methyltransferases, KsgA and the reaction it catalyzes are conserved throughout evolution. However, the specifics of substrate recognition by the enzyme remain unknown. Here we report structures of Aquifex aeolicus KsgA, in its ligand-free form, in complex with RNA, and in complex with both RNA and S-adenosylhomocysteine (SAH, reaction product of cofactor S-adenosylmethionine), revealing critical structural information on KsgA-RNA and KsgA-SAH interactions. Moreover, the structures show how conformational changes that occur upon RNA binding create the cofactor-binding site. There are nine conserved functional motifs (motifs I–VIII and X) in KsgA. Prior to RNA binding, motifs I and VIII are flexible, each exhibiting two distinct conformations. Upon RNA binding, the two motifs become stabilized in one of these conformations, which is compatible with the binding of SAH. Motif X, which is also stabilized upon RNA binding, is directly involved in the binding of SAH.
Keywords: PROTEINS; RNA;

The arduous task of rationally designing small-molecule enzyme inhibitors is complicated by the inherent flexibility of the protein scaffold. To gain insight into the changes in dynamics associated with small-molecule-based inhibition, we have characterized, using NMR spectroscopy, Escherichia coli dihydrofolate reductase in complex with two drugs: methotrexate and trimethoprim. The complexes allowed the intrinsic dynamic effects of drug binding to be revealed within the context of the “closed” structural ensemble. Binding of both drugs results in an identical decoupling of global motion on the micro- to millisecond timescale. Consistent with a change in overall dynamic character, the drugs' perturbations to pico- to nanosecond backbone and side-chain methyl dynamics are also highly similar. These data show that the inhibitors simultaneously modulate slow concerted switching and fast motions at distal regions of dihydrofolate reductase, providing a dynamic link between the substrate binding site and distal loop residues known to affect catalysis.
Keywords: PROTEINS;

Rhomboid Protease Dynamics and Lipid Interactions by Ana-Nicoleta Bondar; Coral del Val; Stephen H. White (395-405).
Intramembrane proteases, which cleave transmembrane (TM) helices, participate in numerous biological processes encompassing all branches of life. Several crystallographic structures of Escherichia coli GlpG rhomboid protease have been determined. In order to understand GlpG dynamics and lipid interactions in a native-like environment, we have examined the molecular dynamics of wild-type and mutant GlpG in different membrane environments. The irregular shape and small hydrophobic thickness of the protein cause significant bilayer deformations that may be important for substrate entry into the active site. Hydrogen-bond interactions with lipids are paramount in protein orientation and dynamics. Mutations in the unusual L1 loop cause changes in protein dynamics and protein orientation that are relayed to the His-Ser catalytic dyad. Similarly, mutations in TM5 change the dynamics and structure of the L1 loop. These results imply that the L1 loop has an important regulatory role in proteolysis.

Hybrid Structural Model of the Complete Human ESCRT-0 Complex by Xuefeng Ren; Daniel P. Kloer; Young C. Kim; Rodolfo Ghirlando; Layla F. Saidi; Gerhard Hummer; James H. Hurley (406-416).
The human Hrs and STAM proteins comprise the ESCRT-0 complex, which sorts ubiquitinated cell surface receptors to lysosomes for degradation. Here we report a model for the complete ESCRT-0 complex based on the crystal structure of the Hrs-STAM core complex, previously solved domain structures, hydrodynamic measurements, and Monte Carlo simulations. ESCRT-0 expressed in insect cells has a hydrodynamic radius of RH = 7.9 nm and is a 1:1 heterodimer. The 2.3 Å crystal structure of the ESCRT-0 core complex reveals two domain-swapped GAT domains and an antiparallel two-stranded coiled-coil, similar to yeast ESCRT-0. ESCRT-0 typifies a class of biomolecular assemblies that combine structured and unstructured elements, and have dynamic and open conformations to ensure versatility in target recognition. Coarse-grained Monte Carlo simulations constrained by experimental RH values for ESCRT-0 reveal a dynamic ensemble of conformations well suited for diverse functions.
Keywords: CELLBIO;

Core Structure of Amyloid Fibrils Formed by Residues 106–126 of the Human Prion Protein by Patrick Walsh; Karen Simonetti; Simon Sharpe (417-426).
Peptides comprising residues 106–126 of the human prion protein (PrP) exhibit many features of the full-length protein. PrP(106–126) induces apoptosis in neurons, forms fibrillar aggregates, and can mediate the conversion of native cellular PrP (PrPC) to the scrapie form (PrPSc). Despite a wide range of biochemical and biophysical studies on this peptide, including investigation of its propensity for aggregation, interactions with cell membranes, and PrP-like toxicity, the structure of amyloid fibrils formed by PrP(106–126) remains poorly defined. In this study we use solid-state nuclear magnetic resonance to define the secondary and quaternary structure of PrP(106–126) fibrils. Our results reveal that PrP(106–126) forms in-register parallel β sheets, stacked in an antiparallel fashion within the mature fibril. The close intermolecular contacts observed in the fibril core provide a rational for the sequence-dependent behavior of PrP(106–126), and provide a basis for further investigation of its biological properties.
Keywords: PROTEIN;

Three-Dimensional Structure of AAA ATPase Vps4: Advancing Structural Insights into the Mechanisms of Endosomal Sorting and Enveloped Virus Budding by Michael John Landsberg; Parimala Rao Vajjhala; Rosalba Rothnagel; Alan Leslie Munn; Ben Hankamer (427-437).
Vps4 is a AAA ATPase that mediates endosomal membrane protein sorting. It is also a host factor hijacked by a diverse set of clinically important viruses, including HIV and Ebola, to facilitate viral budding. Here we present the three-dimensional structure of the hydrolysis-defective Vps4pE233Q mutant. Single-particle analysis, multiangle laser light scattering, and the docking of independently determined atomic models of Vps4 monomers reveal a complex with C6 point symmetry, distinguishing between a range of previously suggested oligomeric states (8–14 subunits). The 3D reconstruction also reveals a tail-to-tail subunit organization between the two rings of the complex and identifies the location of domains critical to complex assembly and interaction with partner proteins. Our refined Vps4 structure is better supported by independent lines of evidence than those previously proposed, and provides insights into the mechanism of endosomal membrane protein sorting and viral envelope budding.

Structural and Biochemical Studies on Procaspase-8: New Insights on Initiator Caspase Activation by Nadine Keller; Jiří Mareš; Oliver Zerbe; Markus G. Grütter (438-448).
Caspases are proteases with an active-site cysteine and aspartate specificity in their substrates. They are involved in apoptotic cell death and inflammation, and dysfunction of these enzymes is directly linked to a variety of diseases. Caspase-8 initiates an apoptotic pathway triggered by external stimuli. It was previously characterized in its active inhibitor bound state by crystallography. Here we present the solution structure of the monomeric unprocessed catalytic domain of the caspase-8 zymogen, procaspase-8, showing for the first time the position of the linker and flexibility of the active site forming loops. Biophysical studies of carefully designed mutants allowed disentangling dimerization and processing, and we could demonstrate lack of activity of monomeric uncleaved procaspase-8 and of a processed but dimerization-incompetent mutant. The data provide experimental support in so-far unprecedented detail, and reveal why caspase-8 (and most likely other initiator caspases) needs the dimerization platform during activation.
Keywords: PROTEIN;

Nuclear pore complexes (NPCs) are selectively gated pathways between nucleoplasm and cytoplasm. Whereas small molecules can diffuse freely through NPCs, large molecules (>40 kD) can pass only when bound to transport receptors. The NPC central channel is filled with disordered proteins, rich in phenylalanine-glycine (FG) repeats, referred to as FG-nups. Our simulations, carried out at coarse-grained and all-atom levels, show that arrays of FG-nups tethered to a planar surface, at an FG-repeat density found in the NPC, form dynamic brush-like structures of multiprotein bundles, whereas individual FG-nups form dynamic globular structures. More than half of the FG-repeats are found on the surface of the bundles, offering a favorable environment for transport receptors. Binding to FG-repeats and a sliding motion of NTF2 induced by binding and unbinding to phenylalanines were observed when adding this transport receptor into one of the brush-like structures.
Keywords: PROTEINS; RNA;

Cryo-Electron Tomography of Homophilic Adhesion Mediated by the Neural Cell Adhesion Molecule L1 by Yongning He; Grant J. Jensen; Pamela J. Bjorkman (460-471).
The neural cell adhesion molecule L1 participates in homophilic interactions important for axon guidance and neuronal development. The structural details of homophilic adhesion mediated by L1 and other immunoglobulin superfamily members containing an N-terminal horseshoe arrangement of four immunoglobulin-like domains are unknown. Here we used cryo-electron tomography to study liposomes to which intact or truncated forms of the L1 ectodomain were attached. Tomographic reconstructions revealed an adhesion interface with a regular and repeating pattern consistent with interactions between paired horseshoes contributed by L1 proteins from neighboring liposomes. The characteristics of the pattern changed when N-linked carbohydrates were altered by removing sialic acids or converting from complex to high mannose or oligomannose glycans, suggesting a regulatory role for carbohydrates in L1-mediated homophilic adhesion. Using the results from tomograms and crystal structures of L1-related molecules, we present a structural model for L1-mediated homophilic adhesion that depends on protein-protein, protein-carbohydrate, and carbohydrate-carbohydrate interactions.
Keywords: CELLBIO;

Structure and Biological Function of the RNA Pyrophosphohydrolase BdRppH from Bdellovibrio bacteriovorus by Simon A.J. Messing; Sandra B. Gabelli; Quansheng Liu; Helena Celesnik; Joel G. Belasco; Silvia A. Piñeiro; L. Mario Amzel (472-481).
Until recently, the mechanism of mRNA decay in bacteria was thought to be different from that of eukaryotes. This paradigm changed with the discovery that RppH (ORF176/NudH/YgdP), an Escherichia coli enzyme that belongs to the Nudix superfamily, is an RNA pyrophosphohydrolase that initiates mRNA decay by cleaving pyrophosphate from the 5′-triphosphate. Here we report the 1.9 Å resolution structure of the Nudix hydrolase BdRppH from Bdellovibrio bacteriovorus, a bacterium that feeds on other Gram-negative bacteria. Based on the structure of the enzyme alone and in complex with GTP-Mg2+, we propose a mode of RNA binding similar to that of the nuclear decapping enzyme from Xenopus laevis, X29. In additional experiments, we show that BdRppH can indeed function in vitro and in vivo as an RNA pyrophosphohydrolase. These findings set the basis for the identification of possible decapping enzymes in other bacteria.