Structure (v.16, #3)
In This Issue (ix-x).
Activity versus Peroxisomal Targeting of PerCR by Michael E. Baker; Suresh Subramani (331-332).
Peroxisomal carbonyl reductase (PerCR), a tetrameric enzyme, enters peroxisomes when expressed in human cells, but not when PerCR tetramers are introduced into these cells. The PerCR crystal structure () yields insights that explain these data.
A Trigger Squeezed by Charles Eigenbrot (332-334).
add new weight to the very highly detailed understanding of how extracellular ligand binding-induced dimerization of a receptor tyrosine kinase is first manifested inside the cell.
DNA-PKcs at 7Å: Insights for DNA Repair by Noriko Shimazaki; Michael R. Lieber (334-336).
In this issue of Structure, the Stewart laboratory and their collaborators have provided a markedly improved cryo-EM reconstruction of DNA-PKcs (). The new level of detail heightens interest in integrating the understanding of nonhomologous DNA end joining.
Terminal Regions of β-Catenin Come into View by Cara J. Gottardi; Mark Peifer (336-338).
β-catenin is remarkably multifunctional, acting in adhesion, cytoskeletal regulation, and Wnt signaling. In this issue, present the full-length structure of β-catenin, providing a clearer picture of how these terminal regions modulate β-catenin activities.
Deubiquitination of Lys63-Linkage by a CYLD UBP by Yigong Shi (338-340).
In the recent issue of Molecular Cell, report structural and biochemical characterization of the catalytic core domain of CYLD, a member of the UBP family of deubiquitinating enzymes.
Mammalian DNA Methyltransferases: A Structural Perspective by Xiaodong Cheng; Robert M. Blumenthal (341-350).
The methylation of mammalian DNA, primarily at CpG dinucleotides, has long been recognized to play a major role in controlling gene expression, among other functions. Given their importance, it is surprising how many basic questions remain to be answered about the proteins responsible for this methylation and for coordination with the parallel chromatin-marking system that operates at the level of histone modification. This article reviews recent studies on, and discusses the resulting biochemical and structural insights into, the DNA nucleotide methyltransferase (Dnmt) proteins 1, 3a, 3a2, 3b, and 3L.
Keywords: DNA; PROTEINS;
Microscale Fluorescent Thermal Stability Assay for Membrane Proteins by Alexander I. Alexandrov; Mauro Mileni; Ellen Y.T. Chien; Michael A. Hanson; Raymond C. Stevens (351-359).
Systematic efforts to understand membrane protein stability under a variety of different solution conditions are not widely available for membrane proteins, mainly due to technical problems stemming from the presence of detergents necessary to keep the proteins in the solubilized state and the background that such detergents usually generate during biophysical characterization. In this report, we introduce an efficient microscale fluorescent stability screen using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) for stability profiling of membrane proteins under different solution and ligand conditions. The screen uses the chemical reactivity of the native cysteines embedded in the protein interior as a sensor for the overall integrity of the folded state. The thermal information gained by thorough investigation of the protein stability landscape can be effectively used to guide purification and biophysical characterization efforts including crystallization. To evaluate the method, three different protein families were analyzed, including the Apelin G protein-coupled receptor (APJ).
Crystal Structure of an Intact Type II DNA Topoisomerase: Insights into DNA Transfer Mechanisms by Marc Graille; Lionel Cladière; Dominique Durand; François Lecointe; Danièle Gadelle; Sophie Quevillon-Cheruel; Patrice Vachette; Patrick Forterre; Herman van Tilbeurgh (360-370).
DNA topoisomerases resolve DNA topological problems created during transcription, replication, and recombination. These ubiquitous enzymes are essential for cell viability and are highly potent targets for the development of antibacterial and antitumoral drugs. Type II enzymes catalyze the transfer of a DNA duplex through another one in an ATP-dependent mechanism. Because of its small size and sensitivity to antitumoral drugs, the archaeal DNA topoisomerase VI, a type II enzyme, is an excellent model for gaining further understanding of the organization and mechanism of these enzymes. We present the crystal structure of intact DNA topoisomerase VI bound to radicicol, an inhibitor of human topo II, and compare it to the conformation of the apo-protein as determined by small-angle X-ray scattering in solution. This structure, combined with a wealth of experimental data gathered on these enzymes, allows us to propose a structural model for the two-gate DNA transfer mechanism.
Colicin N Binds to the Periphery of Its Receptor and Translocator, Outer Membrane Protein F by Thomas G. Baboolal; Matthew J. Conroy; Katrina Gill; Helen Ridley; Virak Visudtiphole; Per A. Bullough; Jeremy H. Lakey (371-379).
Colicins kill Escherichia coli after translocation across the outer membrane. Colicin N displays an unusually simple translocation pathway, using the outer membrane protein F (OmpF) as both receptor and translocator. Studies of this binary complex may therefore reveal a significant component of the translocation pathway. Here we show that, in 2D crystals, colicin is found outside the porin trimer, suggesting that translocation may occur at the protein-lipid interface. The major lipid of the outer leaflet interface is lipopolysaccharide (LPS). It is further shown that colicin N binding displaces OmpF-bound LPS. The N-terminal helix of the pore-forming domain, which is not required for pore formation, rearranges and binds to OmpF. Colicin N also binds artificial OmpF dimers, indicating that trimeric symmetry plays no part in the interaction. The data indicate that colicin is closely associated with the OmpF-lipid interface, providing evidence that this peripheral pathway may play a role in colicin transmembrane transport.
Keywords: PROTEINS; SIGNALING;
Regulation of Enzyme Localization by Polymerization: Polymer Formation by the SAM Domain of Diacylglycerol Kinase δ1 by Bryan T. Harada; Mary Jane Knight; Shin-ichi Imai; Feng Qiao; Ranjini Ramachander; Michael R. Sawaya; Mari Gingery; Fumio Sakane; James U. Bowie (380-387).
The diacylglycerol kinase (DGK) enzymes function as regulators of intracellular signaling by altering the levels of the second messengers, diacylglycerol and phosphatidic acid. The DGK δ and η isozymes possess a common protein-protein interaction module known as a sterile α-motif (SAM) domain. In DGK δ, SAM domain self-association inhibits the translocation of DGK δ to the plasma membrane. Here we show that DGK δ SAM forms a polymer and map the polymeric interface by a genetic selection for soluble mutants. A crystal structure reveals that DGKSAM forms helical polymers through a head-to-tail interaction similar to other SAM domain polymers. Disrupting polymerization by polymer interface mutations constitutively localizes DGK δ to the plasma membrane. Thus, polymerization of DGK δ regulates the activity of the enzyme by sequestering DGK δ in an inactive cellular location. Regulation by dynamic polymerization is an emerging theme in signal transduction.
Molecular Basis for Peroxisomal Localization of Tetrameric Carbonyl Reductase by Nobutada Tanaka; Ken-ichi Aoki; Shuhei Ishikura; Makoto Nagano; Yorishige Imamura; Akira Hara; Kazuo T. Nakamura (388-397).
Pig heart peroxisomal carbonyl reductase (PerCR) belongs to the short-chain dehydrogenase/reductase family, and its sequence comprises a C-terminal SRL tripeptide, which is a variant of the type 1 peroxisomal targeting signal (PTS1) Ser-Lys-Leu. PerCR is imported into peroxisomes of HeLa cells when the cells are transfected with vectors expressing the enzyme. However, PerCR does not show specific targeting when introduced into the cells with a protein transfection reagent. To understand the structural basis for peroxisomal localization of PerCR, we determined the crystal structure of PerCR. Our data revealed that the C-terminal PTS1 of each subunit of PerCR was involved in intersubunit interactions and was buried in the interior of the tetrameric molecule. These findings indicate that the PTS1 receptor Pex5p in the cytosol recognizes the monomeric form of PerCR whose C-terminal PTS1 is exposed, and that this PerCR is targeted into the peroxisome, thereby forming a tetramer.
Keywords: MICROBIO; CELLBIO;
Structural Dynamics of an Isolated Voltage-Sensor Domain in a Lipid Bilayer by Sudha Chakrapani; Luis G. Cuello; D. Marien Cortes; Eduardo Perozo (398-409).
A strong interplay between the voltage-sensor domain (VSD) and the pore domain (PD) underlies voltage-gated channel functions. In a few voltage-sensitive proteins, the VSD has been shown to function without a canonical PD, although its structure and oligomeric state remain unknown. Here, using EPR spectroscopy, we show that the isolated VSD of KvAP can remain monomeric in a reconstituted bilayer and retain a transmembrane conformation. We find that water-filled crevices extending deep into the membrane around S3, a scaffold conducive to transport of protons/cations, are intrinsic to the VSD. Differences in solvent accessibility in comparison to the full-length KvAP allowed us to define an interacting footprint of the PD on the VSD. This interaction is centered around S1 and S2 and suggests a rotation of 70°–100° relative to Kv1.2-Kv2.1 chimera. Sequence-conservation patterns in Kv channels, Hv channels, and voltage-sensitive phosphatases reveal several near-universal features suggesting a common molecular architecture for all VSDs.
Keywords: PROTEINS; MOLNEURO;
Crystal Structures of β-Neurexin 1 and β-Neurexin 2 Ectodomains and Dynamics of Splice Insertion Sequence 4 by Jesko Koehnke; Xiangshu Jin; Nikola Trbovic; Phinikoula S. Katsamba; Julia Brasch; Goran Ahlsen; Peter Scheiffele; Barry Honig; Arthur G. Palmer; Lawrence Shapiro (410-421).
Presynaptic neurexins (NRXs) bind to postsynaptic neuroligins (NLs) to form Ca2+-dependent complexes that bridge neural synapses. β-NRXs bind NLs through their LNS domains, which contain a single site of alternative splicing (splice site 4) giving rise to two isoforms: +4 and Δ. We present crystal structures of the Δ isoforms of the LNS domains from β-NRX1 and β-NRX2, crystallized in the presence of Ca2+ ions. The Ca2+-binding site is disordered in the β-NRX2 structure, but the 1.7 Å β-NRX1 structure reveals a single Ca2+ ion, ∼12 Å from the splice insertion site, with one coordinating ligand donated by a glutamic acid from an adjacent β-NRX1 molecule. NMR studies of β-NRX1+4 show that the insertion sequence is unstructured, and remains at least partially disordered in complex with NL. These results raise the possibility that β-NRX insertion sequence 4 may function in roles independent of neuroligin binding.
Keywords: PROTEINS; SIGNALING;
Regulation of Neurexin 1β Tertiary Structure and Ligand Binding through Alternative Splicing by Kaiser C. Shen; Dorota A. Kuczynska; Irene J. Wu; Beverly H. Murray; Lauren R. Sheckler; Gabby Rudenko (422-431).
Neurexins and neuroligins play an essential role in synapse function, and their alterations are linked to autistic spectrum disorder. Interactions between neurexins and neuroligins regulate inhibitory and excitatory synaptogenesis in vitro through a “splice-insert signaling code.” In particular, neurexin 1β carrying an alternative splice insert at site SS#4 interacts with neuroligin 2 (found predominantly at inhibitory synapses) but much less so with other neuroligins (those carrying an insert at site B and prevalent at excitatory synapses). The structure of neurexin 1β+SS#4 reveals dramatic rearrangements to the “hypervariable surface,” the binding site for neuroligins. The splice insert protrudes as a long helix into space, triggers conversion of loop β10-β11 into a helix rearranging the binding site for neuroligins, and rearranges the Ca2+-binding site required for ligand binding, increasing its affinity. Our structures reveal the mechanism by which neurexin 1β isoforms acquire neuroligin splice isoform selectivity.
Keywords: PROTEINS; SIGNALING;
Structure of the Oligosaccharyl Transferase Complex at 12 Å Resolution by Hua Li; Manasi Chavan; Hermann Schindelin; William J. Lennarz; Huilin Li (432-440).
Oligosaccharyl transferase (OT) catalyzes the transfer of a lipid-linked oligosaccharide to the nascent polypeptide emerging from the translocon. Currently, there is no structural information on the membrane-embedded OT complex, which consists of eight different polypeptide chains. We report a 12 Å resolution cryo-electron microscopy structure of OT from yeast. We mapped the locations of four essential OT subunits through a maltose-binding protein fusion strategy. OT was found to have a large domain in the lumenal side of endoplasmic reticulum where the catalysis occurs. The lumenal domain mainly comprises the catalytic Stt3p, the donor substrate-recognizing Wbp1p, and the acceptor substrate-recognizing Ost1p. A prominent groove was observed between these subunits, and we propose that the nascent polypeptide from the translocon threads through this groove while being scanned by the Ost1p subunit for the presence of the glycosylation sequon.
De Novo Backbone Trace of GroEL from Single Particle Electron Cryomicroscopy by Steven J. Ludtke; Matthew L. Baker; Dong-Hua Chen; Jiu-Li Song; David T. Chuang; Wah Chiu (441-448).
In this work, we employ single-particle electron cryo-microscopy (cryo-EM) to reconstruct GroEL to ∼4 Å resolution with both D7 and C7 symmetry. Using a newly developed skeletonization algorithm and secondary structure element identification in combination with sequence-based secondary structure prediction, we demonstrate that it is possible to achieve a de novo Cα trace directly from a cryo-EM reconstruction. The topology of our backbone trace is completely accurate, though subtle alterations illustrate significant differences from existing crystal structures. In the map with C7 symmetry, the seven monomers in each ring are identical; however, the subunits have a subtly different structure in each ring, particularly in the equatorial domain. These differences include an asymmetric salt bridge, density in the nucleotide-binding pocket of only one ring, and small shifts in α helix positions. This asymmetric conformation is different from previous asymmetric structures, including GroES-bound GroEL, and may represent a “primed state” in the chaperonin pathway.
Molecular Basis of Fibrin Clot Elasticity by Bernard B.C. Lim; Eric H. Lee; Marcos Sotomayor; Klaus Schulten (449-459).
Blood clots must be stiff to stop hemorrhage yet elastic to buffer blood's shear forces. Upsetting this balance results in clot rupture and life-threatening thromboembolism. Fibrin, the main component of a blood clot, is formed from molecules of fibrinogen activated by thrombin. Although it is well known that fibrin possesses considerable elasticity, the molecular basis of this elasticity is unknown. Here, we use atomic force microscopy (AFM) and steered molecular dynamics (SMD) to probe the mechanical properties of single fibrinogen molecules and fibrin protofibrils, showing that the mechanical unfolding of their coiled-coil α helices is characterized by a distinctive intermediate force plateau in the systems' force-extension curve. We relate this plateau force to a stepwise unfolding of fibrinogen's coiled α helices and of its central domain. AFM data show that varying pH and calcium ion concentrations alters the mechanical resilience of fibrinogen. This study provides direct evidence for the coiled α helices of fibrinogen to bring about fibrin elasticity.
Keywords: PROTEINS; CELLBIO;
Mechanism of Activation and Inhibition of the HER4/ErbB4 Kinase by Chen Qiu; Mary K. Tarrant; Sung Hee Choi; Aruna Sathyamurthy; Ron Bose; Sudeep Banjade; Ashutosh Pal; William G. Bornmann; Mark A. Lemmon; Philip A. Cole; Daniel J. Leahy (460-467).
HER4/ErbB4 is a ubiquitously expressed member of the EGF/ErbB family of receptor tyrosine kinases that is essential for normal development of the heart, nervous system, and mammary gland. We report here crystal structures of the ErbB4 kinase domain in active and lapatinib-inhibited forms. Active ErbB4 kinase adopts an asymmetric dimer conformation essentially identical to that observed to be important for activation of the EGF receptor/ErbB1 kinase. Mutagenesis studies of intact ErbB4 in Ba/F3 cells confirm the importance of this asymmetric dimer for activation of intact ErbB4. Lapatinib binds to an inactive form of the ErbB4 kinase in a mode equivalent to its interaction with the EGF receptor. All ErbB4 residues contacted by lapatinib are conserved in the EGF receptor and HER2/ErbB2, which lapatinib also targets. These results demonstrate that key elements of kinase activation and inhibition are conserved among ErbB family members.
Cryo-EM Structure of the DNA-Dependent Protein Kinase Catalytic Subunit at Subnanometer Resolution Reveals α Helices and Insight into DNA Binding by Dewight R. Williams; Kyung-Jong Lee; Jian Shi; David J. Chen; Phoebe L. Stewart (468-477).
The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) regulates the nonhomologous end joining pathway for repair of double-stranded DNA (dsDNA) breaks. Here, we present a 7Å resolution structure of DNA-PKcs determined by cryo-electron microscopy single-particle reconstruction. This structure is composed of density rods throughout the molecule that are indicative of α helices and reveals structural features not observed in lower resolution EM structures. Docking of homology models into the DNA-PKcs structure demonstrates that up to eight helical HEAT repeat motifs fit well within the density. Surprisingly, models for the kinase domain can be docked into either the crown or base of the molecule at this resolution, although real space refinement suggests that the base location is the best fit. We propose a model for the interaction of DNA with DNA-PKcs in which one turn of dsDNA enters the central channel and interacts with a resolved α-helical protrusion.
Keywords: DNA; SIGNALING;
Crystal Structure of a Full-Length β-Catenin by Yi Xing; Ken-Ichi Takemaru; Jing Liu; Jason D. Berndt; Jie J. Zheng; Randall T. Moon; Wenqing Xu (478-487).
β-catenin plays essential roles in cell adhesion and Wnt signaling, while deregulation of β-catenin is associated with multiple diseases including cancers. Here, we report the crystal structures of full-length zebrafish β-catenin and a human β-catenin fragment that contains both the armadillo repeat and the C-terminal domains. Our structures reveal that the N-terminal region of the C-terminal domain, a key component of the C-terminal transactivation domain, forms a long α helix that packs on the C-terminal end of the armadillo repeat domain, and thus forms part of the β-catenin superhelical core. The existence of this helix redefines our view of interactions of β-catenin with some of its critical partners, including ICAT and Chibby, which may form extensive interactions with this C-terminal domain α helix. Our crystallographic and NMR studies also suggest that the unstructured N-terminal and C-terminal tails interact with the ordered armadillo repeat domain in a dynamic and variable manner.
A Helical String of Alternately Connected Three-Helix Bundles for the Cell Wall-Associated Adhesion Protein Ebh from Staphylococcus aureus by Yoshikazu Tanaka; Sou Sakamoto; Makoto Kuroda; Shuichiro Goda; Yong-Gui Gao; Kouhei Tsumoto; Yuzuru Hiragi; Min Yao; Nobuhisa Watanabe; Toshiko Ohta; Isao Tanaka (488-496).
The 1.1 MDa cell-wall-associated adhesion protein of staphylococci, Ebh, consists of several distinct regions, including a large central region with 52 imperfect repeats of 126 amino acid residues. We investigated the structure of this giant molecule by X-ray crystallography, circular dichroism (CD) spectrometry, and small-angle X-ray scattering (SAXS). The crystal structure of two repeats showed that each repeat consists of two distinct three-helix bundles, and two such repeats are connected along the long axis, resulting in a rod-like structure that is 120 Å in length. CD and SAXS analyses of the samples with longer repeats suggested that each repeat has an identical structure, and that such repeats are connected tandemly to form a rod-like structure in solution, the length of which increased proportionately with the number of repeating units. On the basis of these results, it was proposed that Ebh is a 320 nm rod-like molecule with some plasticity at module junctions.
Keywords: MICROBIO; CELLBIO;