Structure (v.19, #1)

In This Issue (v-vi).

Cytokine Signaling Exposed by Stevan R. Hubbard (1-2).
In this issue, provide images, obtained by electron microscopy, of a complete cytokine signaling complex, offering clues into the mechanism by which binding of cytokines to their cognate receptors triggers trans-phosphorylation and activation of Janus kinases.

EROS: Better than SAXS! by Sichun Yang; Benoît Roux (3-4).
Revealing the three-dimensional organization of large dynamic protein complexes in solution is challenging. To tackle this problem, design a method combining small angle X-ray scattering (SAXS) data with the results of computer simulations. Their study offers new insights into the conformational transition induced by salt that occurs in an endosome-associated ESCRT-III CHMP3 domain.

DARK Apoptosome Secrets Come to Light by Chu-Chiao Wu; Shawn B. Bratton (4-6).
In this issue of Structure, utilize biochemical approaches to reconstitute an active Drosophila apoptosome, as well as cryo-electron microscopy to generate an improved model for this conserved caspase-activating complex.

Homing endonucleases are microbial DNA-cleaving enzymes that mobilize their own reading frames by generating double strand breaks at specific genomic invasion sites. These proteins display an economy of size, and yet recognize long DNA sequences (typically 20 to 30 base pairs). They exhibit a wide range of fidelity at individual nucleotide positions in a manner that is strongly influenced by host constraints on the coding sequence of the targeted gene. The activity of these proteins leads to site-specific recombination events that can result in the insertion, deletion, mutation, or correction of DNA sequences. Over the past fifteen years, the crystal structures of representatives from several homing endonuclease families have been solved, and methods have been described to create variants of these enzymes that cleave novel DNA targets. Engineered homing endonucleases proteins are now being used to generate targeted genomic modifications for a variety of biotech and medical applications.

Benchmarking Membrane Protein Detergent Stability for Improving Throughput of High-Resolution X-ray Structures by Yo Sonoda; Simon Newstead; Nien-Jen Hu; Yilmaz Alguel; Emmanuel Nji; Konstantinos Beis; Shoko Yashiro; Chiara Lee; James Leung; Alexander D. Cameron; Bernadette Byrne; So Iwata; David Drew (17-25).
Obtaining well-ordered crystals is a major hurdle to X-ray structure determination of membrane proteins. To facilitate crystal optimization, we investigated the detergent stability of 24 eukaryotic and prokaryotic membrane proteins, predominantly transporters, using a fluorescent-based unfolding assay. We have benchmarked the stability required for crystallization in small micelle detergents, as they are statistically more likely to lead to high-resolution structures. Using this information, we have been able to obtain well-diffracting crystals for a number of sodium and proton-dependent transporters. By including in the analysis seven membrane proteins for which structures are already known, AmtB, GlpG, Mhp1, GlpT, EmrD, NhaA, and LacY, it was further possible to demonstrate an overall trend between protein stability and structural resolution. We suggest that by monitoring membrane protein stability with reference to the benchmarks described here, greater efforts can be placed on constructs and conditions more likely to yield high-resolution structures.Display Omitted► Benchmarked the stability required for crystallization in small sized detergents ► Membrane protein stability is inherent to the protein rather than detergent specific ► Membrane proteins stable in LDAO are more likely to yield well-diffracting crystals ► Eukaryotic membrane proteins are 3-fold less stable in 12M than prokaryotic proteins

Portrayal of Complex Dynamic Properties of Sugarcane Defensin 5 by NMR: Multiple Motions Associated with Membrane Interaction by Viviane Silva de Paula; Guilherme Razzera; Eliana Barreto-Bergter; Fabio C.L. Almeida; Ana Paula Valente (26-36).
Defensins are essentially ancient natural antibiotics with potent activity extending from lower organisms to humans. Sd5 is a recently described antifungal defensin that appears to be the result of a recent gain of function. We reported here the solution NMR structure of Sd5 and characterized the backbone dynamics in the free state and in the presence of membrane models. 15N relaxation dispersion measurements indicate intrinsic conformational exchange processes, showing two clear distinct k ex, 490 and 1800 s-1. These multiple motions may be related to transient twisting or breathing of the α helix and β sheet. The stages of membrane recognition and disruption by Sd5 over a large timescale range were mapped and demonstrated that Sd5 in solution sampled an ensemble of different conformations, of which a subset is selected upon membrane binding. Defensins share similar structures, but we demonstrated here that their dynamics can be extremely diverse.Display Omitted► Sd5 displays a cysteine stabilized fold linked to an unstructured C-terminal region ► Interactions of Sd5 with membrane were studied by NMR, a binding model was built ► Sd5 binds to membrane models via a conformational selection mechanism

Helix 11 Dynamics Is Critical for Constitutive Androstane Receptor Activity by Edward Wright; Scott A. Busby; Sarah Wisecarver; Jeremy Vincent; Patrick R. Griffin; Elias J. Fernandez (37-44).
The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Hydrogen/deuterium exchange (HDX) data indicate that the CAR activation function 2 (AF-2) is more stable in CAR(TCPOBOP):RXR and CAR(meclizine):RXR than in CAR:RXR. HDX kinetics also show significant differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Unlike CAR(meclizine):RXR, CAR(TCPOBOP):RXR shows a higher overall stabilization that extends into RXR. We identify residues 339–345 in CAR as an allosteric regulatory site with a greater magnitude reduction in exchange kinetics in CAR(TCPOBOP):RXR than CAR(meclizine):RXR. Accordingly, assays with mutations on CAR at leucine-340 and leucine-343 confirm this region as an important determinant of CAR activity.► Helix H11 dynamics is critical for NR transactivation ► H11 dynamics is inversely proportional to transactivation and coactivator binding ► H11 dynamics can be modulated by mutagenesis ► H11 dynamics is differentially modulated by agonists

Structural Snapshots of Full-Length Jak1, a Transmembrane gp130/IL-6/IL-6Rα Cytokine Receptor Complex, and the Receptor-Jak1 Holocomplex by Patrick J. Lupardus; Georgios Skiniotis; Amanda J. Rice; Christoph Thomas; Suzanne Fischer; Thomas Walz; K. Christopher Garcia (45-55).
The shared cytokine receptor gp130 signals as a homodimer or heterodimer through activation of Janus kinases (Jaks) associated with the receptor intracellular domains. Here, we reconstitute, in parts and whole, the full-length gp130 homodimer in complex with the cytokine interleukin-6 (IL-6), its alpha receptor (IL-6Rα) and Jak1, for electron microscopy imaging. We find that the full-length gp130 homodimer complex has intimate interactions between the trans- and juxtamembrane segments of the two receptors, appearing to form a continuous connection between the extra- and intracellular regions. 2D averages and 3D reconstructions of full-length Jak1 reveal a three lobed structure comprising FERM-SH2, pseudokinase, and kinase modules possessing extensive intersegmental flexibility that likely facilitates allosteric activation. Single-particle imaging of the gp130/IL-6/IL-6Rα/Jak1 holocomplex shows Jak1 associated with the membrane proximal intracellular regions of gp130, abutting the would-be inner leaflet of the cell membrane. Jak1 association with gp130 is enhanced by the presence of a membrane environment.► First structural information on a full-length Janus Kinase (JAK) ► First structure of full-length transmembrane gp130/IL-6 receptor complex ► Novel elucidation of the juxtamembrane and TM regions of a single-pass receptor ► Structure of JAK reveals basis of possible allsoteric activations

Structural Characterization of the Multidomain Regulatory Protein Rv1364c from Mycobacterium tuberculosis by Jack King-Scott; Petr V. Konarev; Santosh Panjikar; Rositsa Jordanova; Dmitri I. Svergun; Paul A. Tucker (56-69).
The open reading frame rv1364c of Mycobacterium tuberculosis, which regulates the stress-dependent σ factor, σF, has been analyzed structurally and functionally. Rv1364c contains domains with sequence similarity to the RsbP/RsbW/RsbV regulatory system of the stress-response σ factor of Bacillus subtilis. Rv1364c contains, sequentially, a PAS domain (which shows sequence similarity to the PAS domain of the B. subtilis RsbP protein), an active phosphatase domain, a kinase (anti-σF like) domain and a C-terminal anti-σF antagonist like domain. The crystal structures of two PAS domain constructs (at 2.3 and 1.6 Å) and a phosphatase/kinase dual domain construct (at 2.6 Å) are described. The PAS domain is shown to bind palmitic acid but to have 100 times greater affinity for palmitoleic acid. The full-length protein can exist in solution as both monomer and dimer. We speculate that a switch between monomer and dimer, possibly resulting from fatty acid binding, affects the accessibility of the serine of the C-terminal, anti-σF antagonist domain for dephosphorylation by the phosphatase domain thus indirectly altering the availability of σF.Display Omitted► Structure of the PAS domain of Mtb Rv1364c with and without bound palmitic acid ► Preferential binding of palmitoleic over palmitic acid ► Structure of the dual phosphatase/kinase domain construct ► Solution structures of monomeric and dimeric forms

Potassium channels are membrane proteins that selectively conduct K+ across cellular membranes. The narrowest part of their pore, the selectivity filter, is responsible for distinguishing K+ from Na+, and can also act as a gate through a mechanism known as C-type inactivation. It has been proposed that a conformation of the KcsA channel obtained by crystallization in presence of low concentration of K+ (PDB 1K4D) could correspond to the C-type inactivated state. Here, we show using molecular mechanics simulations that such conformation has little ion-binding affinity and that ions do not contribute to its stability. The simulations suggest that, in this conformation, the selectivity filter is mostly occupied by water molecules. Whether such ion-free state of the KcsA channel is physiologically accessible and representative of the inactivated state of eukaryotic channels remains unclear.Display Omitted► Both K+ and Na+ are unstable in the low-[K+] structure of KcsA ► The low-[K+] structure is stable when no ion is present ► The canonical KcsA structure adopts the low-[K+] conformation when ions are removed ► The selectivity filter of the low-[K+] structure is mostly filled with water molecules

Molecular Insights into γδ T Cell Costimulation by an Anti-JAML Antibody by Petra Verdino; Deborah A. Witherden; M. Sharon Ferguson; Adam L. Corper; André Schiefner; Wendy L. Havran; Ian A. Wilson (80-89).
γδ T cells bridge innate and adaptive immunity and function in immunosurveillance, immunoregulation, tumor cell recognition, and as first line of defense against microbial infection. Costimulation of epithelial γδ T cell activation by the JAML receptor can be induced by interaction with its endogenous ligand CAR or by binding of the stimulatory antibody HL4E10. We, therefore, determined the crystal structure of the JAML-HL4E10 Fab complex at 2.95 Å resolution. HL4E10 binds the membrane-proximal domain of JAML through hydrophobic interactions that account for nanomolar affinity and long half-life, contrasting with the fast kinetics and micromolar affinity of the hydrophilic CAR interaction with the membrane-distal JAML domain. Thus, despite different binding sites and mechanisms, JAML interaction with these two disparate ligands leads to the same functional outcome, namely JAML triggering and induction of cell signaling. Several characteristics of the HL4E10 antibody might then be harnessed in therapeutic applications, such as promoting healing of acute or chronic wounds.► mAb HL4E10 emulates the natural ligand CAR in activity, but not in interaction with JAML ► The hydrophobic HL410-JAML interface confers nanomolar affinity and long half-life ► HL4E10-induced clustering of JAML recruits PI3K and induces γδ T cell proliferation ► HL4E10 antibody has favorable characteristics for therapeutic applications

Mass Spectrometry Reveals Stable Modules in holo and apo RNA Polymerases I and III by Laura A. Lane; Carlos Fernández-Tornero; Min Zhou; Nina Morgner; Denis Ptchelkine; Ulrich Steuerwald; Argyris Politis; Doris Lindner; Jelena Gvozdenovic; Anne-Claude Gavin; Christoph W. Müller; Carol V. Robinson (90-100).
RNA polymerases are essential enzymes which transcribe DNA into RNA. Here, we obtain mass spectra of the cellular forms of apo and holo eukaryotic RNA polymerase I and III, defining their composition under different solution conditions. By recombinant expression of subunits within the initiation heterotrimer of Pol III, we derive an interaction network and couple this data with ion mobility data to define topological restraints. Our data agree with available structural information and homology modeling and are generally consistent with yeast two hybrid data. Unexpectedly, elongation complexes of both Pol I and III destabilize the assemblies compared with their apo counterparts. Increasing the pH and ionic strength of apo and holo forms of Pol I and Pol III leads to formation of at least ten stable subcomplexes for both enzymes. Uniquely for Pol III many subcomplexes contain only one of the two largest catalytic subunits. We speculate that these stable subcomplexes represent putative intermediates in assembly pathways.Display Omitted► Stable modules defined for eukaryotic RNA polymerases ► Subunit contacts and subcomplex topologies within RNA polymerases ► Comparison of the stability of Pol I and Pol III elongation complexes ► Mass spectrometry of multisubunit, protein-nucleic acid macromolecules

Structural and Biochemical Insights into MLL1 Core Complex Assembly by Vanja Avdic; Pamela Zhang; Sylvain Lanouette; Adam Groulx; Véronique Tremblay; Joseph Brunzelle; Jean-François Couture (101-108).
Histone H3 Lys-4 methylation is predominantly catalyzed by a family of methyltransferases whose enzymatic activity depends on their interaction with a three-subunit complex composed of WDR5, RbBP5, and Ash2L. Here, we report that a segment of 50 residues of RbBP5 bridges the Ash2L C-terminal domain to WDR5. The crystal structure of WDR5 in ternary complex with RbBP5 and MLL1 reveals that both proteins binds peptide-binding clefts located on opposite sides of WDR5′s β-propeller domain. RbBP5 engages in several hydrogen bonds and van der Waals contacts within a V-shaped cleft formed by the junction of two blades on WDR5. Mutational analyses of both the WDR5 V-shaped cleft and RbBP5 residues reveal that the interactions between RbBP5 and WDR5 are important for the stimulation of MLL1 methyltransferase activity. Overall, this study provides the structural basis underlying the formation of the WDR5-RbBP5 subcomplex and further highlight the crucial role of WDR5 in scaffolding the MLL1 core complex.► A 50-residue segment of RbBP5 bridges the Ash2L C-terminal domain to WDR5 ► The structure demonstrates how WDR5 binds RbBP5 and MLL1 ► Binding MLL1 and RbBP5 is mediated by two independent peptide-binding clefts ► Two hydrophobic residues are linchpin in conferring binding of RbBP5 to WDR5

SAXS Ensemble Refinement of ESCRT-III CHMP3 Conformational Transitions by Bartosz Różycki; Young C. Kim; Gerhard Hummer (109-116).
We developed and implemented an ensemble-refinement method to study dynamic biomolecular assemblies with intrinsically disordered segments. Data from small angle X-ray scattering (SAXS) experiments and from coarse-grained molecular simulations were combined by using a maximum-entropy approach. The method was applied to CHMP3 of ESCRT-III, a protein with multiple helical domains separated by flexible linkers. Based on recent SAXS data by Lata et al. (J. Mol. Biol. 378, 818, 2008), we constructed ensembles of CHMP3 at low- and high-salt concentration to characterize its closed autoinhibited state and open active state. At low salt, helix α5 is bound to the tip of helices α1 and α2, in excellent agreement with a recent crystal structure. Helix α6 remains free in solution and does not appear to be part of the autoinhibitory complex. The simulation-based ensemble refinement is general and effectively increases the resolution of SAXS beyond shape information to atomically detailed structures.Display Omitted► Ensemble-refinement of multiprotein complexes with intrinsically disordered segments ► Combination of small angle X-ray scattering and Monte Carlo simulations ► Structures of ESCRT-III CHMP3 in the activated and autoinhibited states ► Large-scale protein motions

Separation-of-Function Mutants Unravel the Dual-Reaction Mode of Human 8-Oxoguanine DNA Glycosylase by Bjørn Dalhus; Monika Forsbring; Ina Høydal Helle; Erik Sebastian Vik; Rune Johansen Forstrøm; Paul Hoff Backe; Ingrun Alseth; Magnar Bjørås (117-127).
7,8-Dihydro-8-oxoguanine (8oxoG) is a major mutagenic base lesion formed when reactive oxygen species react with guanine in DNA. The human 8oxoG DNA glycosylase (hOgg1) recognizes and initiates repair of 8oxoG. hOgg1 is acknowledged as a bifunctional DNA glycosylase catalyzing removal of the damaged base followed by cleavage of the backbone of the intermediate abasic DNA (AP lyase/β-elimination). When acting on 8oxoG-containing DNA, these two steps in the hOgg1 catalysis are considered coupled, with Lys249 implicated as a key residue. However, several lines of evidence point to a concurrent and independent monofunctional hydrolysis of the N-glycosylic bond being the in vivo relevant reaction mode of hOgg1. Here, we present biochemical and structural evidence for the monofunctional mode of hOgg1 by design of separation-of-function mutants. Asp268 is identified as the catalytic residue, while Lys249 appears critical for the specific recognition and final alignment of 8oxoG during the hydrolysis reaction.Display Omitted► Human Ogg1 is primarily a monofunctional DNA glycosylase ► Asp268 in hOgg1 is the catalytic residue for base hydrolysis ► Lys249 in hOgg1 is critical for specific recognition of 8oxoG during base hydrolysis

Structure of the Drosophila Apoptosome at 6.9 Å Resolution by Shujun Yuan; Xinchao Yu; Maya Topf; Loretta Dorstyn; Sharad Kumar; Steven J. Ludtke; Christopher W. Akey (128-140).
The Drosophila Apaf-1 related killer forms an apoptosome in the intrinsic cell death pathway. In this study we show that Dark forms a single ring when initiator procaspases are bound. This Dark-Dronc complex cleaves DrICE efficiently; hence, a single ring represents the Drosophila apoptosome. We then determined the 3D structure of a double ring at ∼6.9 Å resolution and created a model of the apoptosome. Subunit interactions in the Dark complex are similar to those in Apaf-1 and CED-4 apoptosomes, but there are significant differences. In particular, Dark has “lost” a loop in the nucleotide-binding pocket, which opens a path for possible dATP exchange in the apoptosome. In addition, caspase recruitment domains (CARDs) form a crown on the central hub of the Dark apoptosome. This CARD geometry suggests that conformational changes will be required to form active Dark-Dronc complexes. When taken together, these data provide insights into apoptosome structure, function, and evolution.► A 6.9 Å model provides insights into assembly and function of NOD proteins ► The Dark apoptosome is constructed like AAA+ ATPase rings but with novel features ► Active site CARDs are displayed as an octagonal crown on the ground state apoptosome ► Dark has lost a loop in the nucleotide-binding site, creating an open path to water ► A comparison of Dark, Apaf-1, and CED-4 apoptosomes reveals evolutionary changes