Structure (v.16, #9)
A GBD Uncovered: the FHOD1 N Terminus Is Formin'
by Christian Baarlink; Robert Grosse (pp. 1287-1288).
In this issue, present the structure of the N-terminal region of human Diaphanous-related formin FHOD1, providing new insights into the differential molecular mechanisms of formin regulation and activity.
A SIR-tain Acetyl Complex Is Caught by a Sulfur Trap
by Anthony A. Sauve (pp. 1289-1292).
In this issue, provide additional insight into the mechanistic properties of sirtuin enzymes by describing the structure of a thio-imidate in the active site of Thermatoga maritima Sir2, which strengthens the proposal that the enzyme directly couples NAD+ and acetyllysine oxygen to form a versatile ADPR-peptidyl-imidate intermediate.
Send for Reinforcements! Conserved Binding of Capsid Decoration Proteins
by Peter E. Prevelige Jr. (pp. 1292-1293).
In this issue of Structure, highlight the confluence of three exciting developments in structural virology: an appreciation of the evolutionary relatedness of viruses, the advances in cryo-EM technology, and the renewed interest of biophysicists in understanding the forces involved in DNA packaging and capsid stability.
RapA: Completing the Transcription Cycle?
by Sergei Nechaev; Konstantin Severinov (pp. 1294-1295).
In this issue of Structure, report a crystal structure of the Escherichia coli RapA protein, the only bacterial member of the SWI/SNF2 family (). The structure reported is also the first structure of any full-sized SWI/SNF2 family protein.
PACemakers of Proteasome Core Particle Assembly
by Paula C. Ramos; R. Jürgen Dohmen (pp. 1296-1304).
The 26S proteasome mediates ubiquitin-dependent proteolysis in eukaryotic cells. A number of studies including very recent ones have revealed that assembly of its 20S catalytic core particle is an ordered process that involves several conserved proteasome assembly chaperones (PACs). Two heterodimeric chaperones, PAC1-PAC2 and PAC3-PAC4, promote the assembly of rings composed of seven α subunits. Subsequently, β subunits join to form half-proteasome precursor complexes containing all but one of the 14 subunits. These complexes lack the β7 subunit but contain UMP1, another assembly chaperone, and in yeast, at least to some degree, the activator protein Blm10. Dimerization of two such complexes is triggered by incorporation of β7, whose C-terminal extension reaches out into the other half to stabilize the newly formed 20S particle. The process is completed by the maturation of active sites and subsequent degradation of UMP1 and PAC1-PAC2.
Accurate NMR Structures Through Minimization of an Extended Hybrid Energy
by Michael Nilges; Aymeric Bernard; Benjamin Bardiaux; Thérèse Malliavin; Michael Habeck; Wolfgang Rieping (pp. 1305-1312).
The use of generous distance bounds has been the hallmark of NMR structure determination. However, bounds necessitate the estimation of data quality before the calculation, reduce the information content, introduce human bias, and allow for major errors in the structures. Here, we propose a new rapid structure calculation scheme based on Bayesian analysis. The minimization of an extended energy function, including a new type of distance restraint and a term depending on the data quality, results in an estimation of the data quality in addition to coordinates. This allows for the determination of the optimal weight on the experimental information. The resulting structures are of better quality and closer to the X–ray crystal structure of the same molecule. With the new calculation approach, the analysis of discrepancies from the target distances becomes meaningful. The strategy may be useful in other applications—for example, in homology modeling.
The Human Formin FHOD1 Contains a Bipartite Structure of FH3 and GTPase-Binding Domains Required for Activation
by Antje Schulte; Bettina Stolp; André Schönichen; Olena Pylypenko; Alexey Rak; Oliver T. Fackler; Matthias Geyer (pp. 1313-1323).
Formins induce the nucleation and polymerization of unbranched actin filaments. They share three homology domains required for profilin binding, actin polymerization, and regulation. Diaphanous-related formins (DRFs) are activated by GTPases of the Rho/Rac family, whose interaction with the N-terminal formin domain is thought to displace a C-terminal Diaphanous-autoregulatory domain (DAD). We have determined the structure of the N-terminal domains of FHOD1 consisting of a GTPase-binding domain (GBD) and the DAD-recognition domain FH3. In contrast to the formin mDia1, the FHOD1-GBD reveals a ubiquitin superfold as found similarly in c-Raf1 or PI3 kinase. This GBD is recruited by Rac and Ras GTPases in cells and plays an essential role for FHOD1-mediated actin remodeling. The FHOD1-FH3 domain is composed of five armadillo repeats, similarly to other formins. Mutation of one residue in the predicted DAD-interaction surface efficiently activates FHOD1 in cells. These results demonstrate that DRFs have evolved different molecular solutions to govern their autoregulation and GTPase specificity.
Keywords: PROTEINS; SIGNALING
Structure of the PduU Shell Protein from the Pdu Microcompartment of Salmonella
by Christopher S. Crowley; Michael R. Sawaya; Thomas A. Bobik; Todd O. Yeates (pp. 1324-1332).
The Pdu microcompartment is a proteinaceous, subcellular structure that serves as an organelle for the metabolism of 1,2-propanediol in Salmonella enterica. It encapsulates several related enzymes within a shell composed of a few thousand protein subunits. Recent structural studies on the carboxysome, a related microcompartment involved in CO2 fixation, have concluded that the major shell proteins from that microcompartment form hexamers that pack into layers comprising the facets of the shell. Here we report the crystal structure of PduU, a protein from the Pdu microcompartment, representing the first structure of a shell protein from a noncarboxysome microcompartment. Though PduU is a hexamer like other characterized shell proteins, it has undergone a circular permutation leading to dramatic differences in the hexamer pore. In view of the hypothesis that microcompartment metabolites diffuse across the outer shell through these pores, the unique structure of PduU suggests the possibility of a special functional role.
Structure of IL-22 Bound to Its High-Affinity IL-22R1 Chain
by Brandi C. Jones; Naomi J. Logsdon; Mark R. Walter (pp. 1333-1344).
IL-22 is an IL-10 family cytokine that initiates innate immune responses against bacterial pathogens and contributes to immune disease. IL-22 biological activity is initiated by binding to a cell-surface complex composed of IL-22R1 and IL-10R2 receptor chains and further regulated by interactions with a soluble binding protein, IL-22BP, which shares sequence similarity with an extracellular region of IL-22R1 (sIL-22R1). IL-22R1 also pairs with the IL-20R2 chain to induce IL-20 and IL-24 signaling. To define the molecular basis of these diverse interactions, we have determined the structure of the IL-22/sIL-22R1 complex. The structure, combined with homology modeling and surface plasmon resonance studies, defines the molecular basis for the distinct affinities and specificities of IL-22 and IL-10 receptor chains that regulate cellular targeting and signal transduction to elicit effective immune responses.
Keywords: PROTEIN; MOLIMMUNO; MICROBIO
Structure and Disassembly of Filaments Formed by the ESCRT-III Subunit Vps24
by Sara Ghazi-Tabatabai; Suraj Saksena; Judith M. Short; Ajaybabu V. Pobbati; Dmitry B. Veprintsev; R. Anthony Crowther; Scott D. Emr; Edward H. Egelman; Roger L. Williams (pp. 1345-1356).
The ESCRT machinery mediates sorting of ubiquitinated transmembrane proteins to lysosomes via multivesicular bodies (MVBs) and also has roles in cytokinesis and viral budding. The ESCRT-III subunits are metastable monomers that transiently assemble on membranes. However, the nature of these assemblies is unknown. Among the core yeast ESCRT-III subunits, Snf7 and Vps24 spontaneously form ordered polymers in vitro. Single-particle EM reconstruction of helical Vps24 filaments shows both parallel and head-to-head subunit arrangements. Mutations of regions involved in intermolecular assembly in vitro result in cargo-sorting defects in vivo, suggesting that these homopolymers mimic interactions formed by ESCRT-III heteropolymers during MVB biogenesis. The C terminus of Vps24 is at the surface of the filaments and is not required for filament assembly. When this region is replaced by the MIT-interacting motif from the Vps2 subunit of ESCRT-III, the AAA-ATPase Vps4 can both bundle and disassemble the chimeric filaments in a nucleotide-dependent fashion.
Keywords: CELLBIO; PROTEINS
The Complete VS Ribozyme in Solution Studied by Small-Angle X-Ray Scattering
by Jan Lipfert; Jonathan Ouellet; David G. Norman; Sebastian Doniach; David M.J. Lilley (pp. 1357-1367).
We have used small-angle X-ray solution scattering to obtain ab initio shape reconstructions of the complete VS ribozyme. The ribozyme occupies an electron density envelope with an irregular shape, into which helical sections have been fitted. The ribozyme is built around a core comprising a near-coaxial stack of three helices, organized by two three-way helical junctions. An additional three-way junction formed by an auxiliary helix directs the substrate stem-loop, juxtaposing the cleavage site with an internal loop to create the active complex. This is consistent with the current view of the probable mechanism of trans-esterification in which adenine and guanine nucleobases contributed by the interacting loops combine in general acid-base catalysis.
Structural Insights into Intermediate Steps in the Sir2 Deacetylation Reaction
by William F. Hawse; Kevin G. Hoff; David G. Fatkins; Alison Daines; Olga V. Zubkova; Vern L. Schramm; Weiping Zheng; Cynthia Wolberger (pp. 1368-1377).
Sirtuin enzymes comprise a unique class of NAD+-dependent protein deacetylases. Although structures of many sirtuin complexes have been determined, structural resolution of intermediate chemical steps are needed to understand the deacetylation mechanism. We report crystal structures of the bacterial sirtuin, Sir2Tm, in complex with an S-alkylamidate intermediate, analogous to the naturally occurring O-alkylamidate intermediate, and a Sir2Tm ternary complex containing a dissociated NAD+ analog and acetylated peptide. The structures and biochemical studies reveal critical roles for the invariant active site histidine in positioning the reaction intermediate, and for a conserved phenylalanine residue in shielding reaction intermediates from base exchange with nicotinamide. The new structural and biochemical studies provide key mechanistic insight into intermediate steps of the Sir2 deacetylation reaction.
Insights into the Mode of Action of a Putative Zinc Transporter CzrB in Thermus thermophilus
by Vadim Cherezov; Nicole Höfer; Doletha M.E. Szebenyi; Olga Kolaj; J. Gerard Wall; Richard Gillilan; Vasundara Srinivasan; Christopher P. Jaroniec; Martin Caffrey (pp. 1378-1388).
The crystal structures of the cytoplasmic domain of the putative zinc transporter CzrB in the apo and zinc-bound forms reported herein are consistent with the protein functioning in vivo as a homodimer. NMR, X-ray scattering, and size-exclusion chromatography provide support for dimer formation. Full-length variants of CzrB in the apo and zinc-loaded states were generated by homology modeling with the Zn2+/H+ antiporter YiiP. The model suggests a way in which zinc binding to the cytoplasmic fragment creates a docking site to which a metallochaperone can bind for delivery and transport of its zinc cargo. Because the cytoplasmic domain may exist in the cell as an independent, soluble protein, a proposal is advanced that it functions as a metallochaperone and that it regulates the zinc-transporting activity of the full-length protein. The latter requires that zinc binding becomes uncoupled from the creation of a metallochaperone-docking site on CzrB.
Noncanonical Binding of Calmodulin to Aquaporin-0: Implications for Channel Regulation
by Steve L. Reichow; Tamir Gonen (pp. 1389-1398).
Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homotetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens, where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between AQP0 and CaM. We show that CaM binds the AQP0 C-terminal domain in a calcium-dependent manner. We demonstrate that only two CaM molecules bind a single AQP0 tetramer in a noncanonical fashion, suggesting a form of cooperativity between AQP0 monomers. Based on these results, we derive a structural model of the AQP0/CaM complex, which suggests CaM may be inhibitory to channel permeability by capping the vestibules of two monomers within the AQP0 tetramer. Finally, phosphorylation within AQP0's CaM binding domain inhibits the AQP0/CaM interaction, suggesting a temporal regulatory mechanism for complex formation.
Bacteriophage Lambda Stabilization by Auxiliary Protein gpD: Timing, Location, and Mechanism of Attachment Determined by Cryo-EM
by Gabriel C. Lander; Alex Evilevitch; Meerim Jeembaeva; Clinton S. Potter; Bridget Carragher; John E. Johnson (pp. 1399-1406).
We report the cryo-EM structure of bacteriophage lambda and the mechanism for stabilizing the 20-Å-thick capsid containing the dsDNA genome. The crystal structure of the HK97 bacteriophage capsid fits most of the T = 7 lambda particle density with only minor adjustment. A prominent surface feature at the 3-fold axes corresponds to the cementing protein gpD, which is necessary for stabilization of the capsid shell. Its position coincides with the location of the covalent cross-link formed in the docked HK97 crystal structure, suggesting an evolutionary replacement of this gene product in lambda by autocatalytic chemistry in HK97. The crystal structure of the trimeric gpD, in which the 14 N-terminal residues required for capsid binding are disordered, fits precisely into the corresponding EM density. The N-terminal residues of gpD are well ordered in the cryo-EM density, adding a strand to a beta-sheet formed by the capsid proteins and explaining the mechanism of particle stabilization.
Keywords: MICROBIO; PROTEIN
Structural Basis for DNA Recognition by FoxO1 and Its Regulation by Posttranslational Modification
by Michael M. Brent; Ruchi Anand; Ronen Marmorstein (pp. 1407-1416).
FoxO transcription factors regulate the transcription of genes that control metabolism, cellular proliferation, stress tolerance, and possibly life span. A number of posttranslational modifications within the forkhead DNA-binding domain regulate FoxO-mediated transcription. We describe the crystal structures of FoxO1 bound to three different DNA elements and measure the change in FoxO1-DNA affinity with acetylation and phosphorylation. The structures reveal additional contacts and increased DNA distortion for the highest affinity DNA site. The flexible wing 2 region of the forkhead domain was not observed in the structures but is necessary for DNA binding, and we show that p300 acetylation in wing 2 reduces DNA affinity. We also show that MST1 phosphorylation of FoxO1 prevents high-affinity DNA binding. The observation that FoxO-DNA affinity varies between response elements and with posttranslational modifications suggests that modulation of FoxO-DNA affinity is an important component of FoxO regulation in health and misregulation in disease.
Structure of RapA, a Swi2/Snf2 Protein that Recycles RNA Polymerase During Transcription
by Gary Shaw; Jianhua Gan; Yan Ning Zhou; Huijun Zhi; Priadarsini Subburaman; Rongguang Zhang; Andrzej Joachimiak; Ding Jun Jin; Xinhua Ji (pp. 1417-1427).
RapA, as abundant as σ70 in the cell, is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein with ATPase activity. It stimulates RNAP recycling during transcription. We report a structure of RapA that is also a full-length structure for the entire Swi2/Snf2 family. RapA contains seven domains, two of which exhibit novel protein folds. Our model of RapA in complex with ATP and double-stranded DNA (dsDNA) suggests that RapA may bind to and translocate on dsDNA. Our kinetic template-switching assay shows that RapA facilitates the release of sequestered RNAP from a posttranscrption/posttermination complex for transcription reinitiation. Our in vitro competition experiment indicates that RapA binds to core RNAP only but is readily displaceable by σ70. RapA is likely another general transcription factor, the structure of which provides a framework for future studies of this bacterial Swi2/Snf2 protein and its important roles in RNAP recycling during transcription.
Keywords: RNA; PROTEINS
Quaternary Structure of Flavorubredoxin as Revealed by Synchrotron Radiation Small-Angle X-Ray Scattering
by Maxim V. Petoukhov; João B. Vicente; Peter B. Crowley; Maria Arménia Carrondo; Miguel Teixeira; Dmitri I. Svergun (pp. 1428-1436).
Flavodiiron proteins (FDP) are modular enzymes which function as NO and/or O2 reductases. Although the majority is composed of two structural domains, the homolog found in Escherichia coli, flavorubredoxin, possesses an extra C-terminal module consisting of a linker and a rubredoxin (Rd) domain necessary for interprotein redox processes. In order to investigate the location of the Rd domain with respect to the flavodiiron structural core, small-angle X-ray scattering was used to construct low-resolution structural models of flavorubredoxin. Scattering patterns from the Rd domain, the FDP core, and full-length flavorubredoxin were collected. The latter two species were found to be tetrameric in solution. Ab initio shape reconstruction and rigid-body modeling indicate a peripheral location for the Rd domains, which appear to have weak contacts with the FDP core. This finding suggests that Rd behaves as an independent domain and is freely available to participate in redox reactions with protein partners.
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