Structure (v.17, #10)
Proteins Do Not Have Strong Spines After All
by Zbigniew Dauter; Alexander Wlodawer (pp. 1278-1279).
In this issue of Structure, Berkholz et al. show that the detailed backbone geometry of proteins depends on the local conformation and suggest how this information can be practically used in modeling and refining protein structures.
How ATPases Unravel a Mystery
by Nerea Gallastegui; Michael Groll (pp. 1279-1281).
Principles of intracellular protein degradation remain among the most challenging questions in cell biology. Here, we discuss Wang and colleagues' crystal structure elucidation of the intermediate domain of Mpa, a regulatory particle of Mtb proteasome, the core proteolytic machinery of Mycobacterium tuberculosis.
Structure and Signaling Mechanism of Per-ARNT-Sim Domains
by Andreas Möglich; Rebecca A. Ayers; Keith Moffat (pp. 1282-1294).
Per-ARNT-Sim (PAS) domains serve as versatile sensor and interaction modules in signal transduction proteins. PAS sensors detect chemical and physical stimuli and regulate the activity of functionally diverse effector domains. In contrast to this chemical, physical, and functional diversity, the structure of the core of PAS domains is broadly conserved and comprises a five-stranded antiparallel β sheet and several α helices. Signals originate within the conserved core and generate structural and dynamic changes predominantly within the β sheet, from which they propagate via amphipathic α-helical and coiled-coil linkers at the N or C termini of the core to the covalently attached effector domain. Effector domains are typically dimeric; their activity appears to be largely regulated by signal-dependent changes in quaternary structure and dynamics. The signaling mechanisms of PAS and other signaling domains share common features, and these commonalities can be exploited to enable structure-based design of artificial photosensors and chemosensors.
Keywords: PROTEINS; SIGNALING; CELLBIO
Discovery Through the Computational Microscope
by Eric H. Lee; Jen Hsin; Marcos Sotomayor; Gemma Comellas; Klaus Schulten (pp. 1295-1306).
All-atom molecular dynamics simulations have become increasingly popular as a tool to investigate protein function and dynamics. However, researchers are concerned about the short time scales covered by simulations, the apparent impossibility to model large and integral biomolecular systems, and the actual predictive power of the molecular dynamics methodology. Here we review simulations that were in the past both hotly disputed and considered key successes, namely of proteins with mainly mechanical functions (titin, fibrinogen, ankyrin, and cadherin). The simulation work covered shows how state-of-the-art modeling alleviates some of the prior concerns and how unrefuted discoveries are made through the “computational microscope.”
On the Relationship between Diffraction Patterns and Motions in Macromolecular Crystals
by Peter B. Moore (pp. 1307-1315).
The quality of many macromolecular crystal structures published recently has been enhanced through the use of new methods for treating the effects of molecular motion and disorder on diffraction patterns, among them a technique called translation, libration, screw-axis (TLS) parameterization. TLS parameterization rationalizes those effects in terms of domain-scale, rigid-body motions and, interestingly, the models for molecular motion that emerge when macromolecular diffraction data are analyzed this way often make sense biochemically. Here it is pointed out that all such models should be treated with caution until it is shown that they are consistent with the diffuse scatter produced by the crystals that provided the diffraction data from which they derive.
Conformation Dependence of Backbone Geometry in Proteins
by Donald S. Berkholz; Maxim V. Shapovalov; Roland L. Dunbrack Jr.; P. Andrew Karplus (pp. 1316-1325).
Protein structure determination and predictive modeling have long been guided by the paradigm that the peptide backbone has a single, context-independent ideal geometry. Both quantum-mechanics calculations and empirical analyses have shown this is an incorrect simplification in that backbone covalent geometry actually varies systematically as a function of the ϕ and Ψ backbone dihedral angles. Here, we use a nonredundant set of ultrahigh-resolution protein structures to define these conformation-dependent variations. The trends have a rational, structural basis that can be explained by avoidance of atomic clashes or optimization of favorable electrostatic interactions. To facilitate adoption of this paradigm, we have created a conformation-dependent library of covalent bond lengths and bond angles and shown that it has improved accuracy over existing methods without any additional variables to optimize. Protein structures derived from crystallographic refinement and predictive modeling both stand to benefit from incorporation of the paradigm.
Structure of the Human Dicer-TRBP Complex by Electron Microscopy
by Pick-Wei Lau; Clinton S. Potter; Bridget Carragher; Ian J. MacRae (pp. 1326-1332).
Dicer is a specialized ribonuclease that initiates RNA interference (RNAi) by cleaving double-stranded RNA (dsRNA) into small RNA fragments about 22 nucleotides long. Here, we present the three-dimensional structure of human Dicer bound to the protein TRBP at ∼20 Å resolution determined by negative-stain electron microscopy (EM) and single-particle analysis. Our analysis reveals that the Dicer-TRBP complex is an L-shaped molecule with a long edge of 150 Å and a 100 Å extension on one end. A surface trench runs the length of the long edge of the molecule, defining a putative dsRNA-binding site. Docking the crystal structure of Giardia Dicer, which represents the nuclease core of human Dicer, into the EM map suggests two possible overall molecular architectures for human Dicer. These results offer insights into the structure of Dicer proteins found in multicellular organisms and provide a conceptual framework for understanding the initiation of RNAi.
Keywords: PROTEINS; RNA
Structure of PAS-Linked Histidine Kinase and the Response Regulator Complex
by Seiji Yamada; Hiroshi Sugimoto; Miki Kobayashi; Ayako Ohno; Hiro Nakamura; Yoshitsugu Shiro (pp. 1333-1344).
We determined the structure of the complex of the sensory histidine kinase (HK) and its cognate response regulator (RR) in the two-component signal transduction system of Thermotoga maritima. This was accomplished by fitting the high-resolution structures of the isolated HK domains and the RR onto the electron density map (3.8 Å resolution) of the HK/RR complex crystal. Based on the structural information, we evaluated the roles of both interdomain and intermolecular interactions in the signal transduction of the cytosolic PAS-linked HK and RR system, in particular the O2-sensor FixL/FixJ system. The PAS-sensor domain of HK interacts with the catalytic domain of the same polypeptide chain by creating an interdomain β sheet. The interaction site between HK and RR, which was confirmed by NMR, is suitable for the intermolecular transfer reaction of the phosphoryl group, indicating that the observed interaction is important for the phosphatase activity of HK that dephosphorylates phospho-RR.
Keywords: PROTEINS; SIGNALING
Structural Basis of Novel Interactions Between the Small-GTPase and GDI-like Domains in Prokaryotic FeoB Iron Transporter
by Motoyuki Hattori; Yaohua Jin; Hiroshi Nishimasu; Yoshiki Tanaka; Masahiro Mochizuki; Toshio Uchiumi; Ryuichiro Ishitani; Koichi Ito; Osamu Nureki (pp. 1345-1355).
The FeoB family proteins are widely distributed prokaryotic membrane proteins involved in Fe2+ uptake. FeoB consists of N-terminal cytosolic and C-terminal transmembrane domains. The N-terminal region of the cytosolic domain is homologous to small GTPase (G) proteins and is considered to regulate Fe2+ uptake. The spacer region connecting the G and TM domains reportedly functions as a GDP dissociation inhibitor (GDI)–like domain that stabilizes the GDP-binding state. However, the function of the G and GDI-like domains in iron uptake remains unclear. Here, we report the structural and functional analyses of the FeoB cytosolic domain from Thermotoga maritima. The structure-based mutational analysis indicated that the interaction between the G and GDI-like domains is important for both the GDI and Fe2+ uptake activities. On the basis of these results, we propose a regulatory mechanism of Fe2+ uptake.
Keywords: PROTEINS; SIGNALING
Intrinsic Domain and Loop Dynamics Commensurate with Catalytic Turnover in an Induced-Fit Enzyme
by Omar Davulcu; Peter F. Flynn; Michael S. Chapman; Jack J. Skalicky (pp. 1356-1367).
Arginine kinase catalyzes reversible phosphoryl transfer between ATP and arginine, buffering cellular ATP concentrations. Structures of substrate-free and -bound enzyme have highlighted a range of conformational changes thought to occur during the catalytic cycle. Here, NMR is used to characterize the intrinsic backbone dynamics over multiple timescales. Relaxation dispersion indicates rigid-body motion of the N-terminal domain and flexible dynamics in the I182–G209 loop, both at millisecond rates commensurate with kcat, implying that either might be rate limiting upon catalysis. Lipari-Szabo analysis indicates backbone flexibility on the nanosecond timescale in the V308–V322 loop, while the rest of the enzyme is more rigid in this timescale. Thus, intrinsic dynamics are most prominent in regions that have been independently implicated in conformational changes. Substrate-free enzyme may sample an ensemble of different conformations, of which a subset is selected upon substrate binding, with critical active site residues appropriately configured for binding and catalysis.
The Structure of a Bacterial DUF199/WhiA Protein: Domestication of an Invasive Endonuclease
by Brett K. Kaiser; Matthew C. Clifton; Betty W. Shen; Barry L. Stoddard (pp. 1368-1376).
Proteins of the DUF199 family, present in all Gram-positive bacteria and best characterized by the WhiA sporulation control factor in Streptomyces coelicolor, are thought to act as genetic regulators. The crystal structure of the DUF199/WhiA protein from Thermatoga maritima demonstrates that these proteins possess a bipartite structure, in which a degenerate N-terminal LAGLIDADG homing endonuclease (LHE) scaffold is tethered to a C-terminal helix-turn-helix (HTH) domain. The LHE domain has lost those residues critical for metal binding and catalysis, and also displays an extensively altered DNA-binding surface as compared with homing endonucleases. The HTH domain most closely resembles related regions of several bacterial sigma70 factors that bind the −35 regions of bacterial promoters. The structure illustrates how an invasive element might be transformed during evolution into a larger assemblage of protein folds that can participate in the regulation of a complex biological pathway.
Keywords: PROTEINS; SIGNALING
Structural Insights on the Mycobacterium tuberculosis Proteasomal ATPase Mpa
by Tao Wang; Hua Li; Gang Lin; Chunyan Tang; Dongyang Li; Carl Nathan; K. Heran Darwin; Huilin Li (pp. 1377-1385).
Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPγS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved interdomain showed a five stranded double β barrel structure containing a Greek key motif. Structure and mutational analysis indicate a major role of the interdomain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome.
Keywords: PROTEINS; CELLBIO
Structural Plasticity of Eph Receptor A4 Facilitates Cross-Class Ephrin Signaling
by Thomas A. Bowden; A. Radu Aricescu; Joanne E. Nettleship; Christian Siebold; Nahid Rahman-Huq; Raymond J. Owens; David I. Stuart; E. Yvonne Jones (pp. 1386-1397).
The EphA4 tyrosine kinase cell surface receptor regulates an array of physiological processes and is the only currently known class A Eph receptor that binds both A and B class ephrins with high affinity. We have solved the crystal structure of the EphA4 ligand binding domain alone and in complex with (1) ephrinB2 and (2) ephrinA2. This set of structures shows that EphA4 has significant conformational plasticity in its ligand binding face. In vitro binding data demonstrate that it has a higher affinity for class A than class B ligands. Structural analyses, drawing on previously reported Eph receptor structures, show that EphA4 in isolation and in complex with ephrinA2 resembles other class A Eph receptors but on binding ephrinB2 assumes structural hallmarks of the class B Eph receptors. This interactive plasticity reveals EphA4 as a structural chameleon, able to adopt both A and B class Eph receptor conformations, and thus provides a molecular basis for EphA-type cross-class reactivity.
Keywords: SIGNALING; CELLBIO
Structure of IL-33 and Its Interaction with the ST2 and IL-1RAcP Receptors—Insight into Heterotrimeric IL-1 Signaling Complexes
by Andreas Lingel; Thomas M. Weiss; Marc Niebuhr; Borlan Pan; Brent A. Appleton; Christian Wiesmann; J. Fernando Bazan; Wayne J. Fairbrother (pp. 1398-1410).
Members of the interleukin-1 (IL-1) family of cytokines play major roles in host defense and immune system regulation in infectious and inflammatory diseases. IL-1 cytokines trigger a biological response in effector cells by assembling a heterotrimeric signaling complex with two IL-1 receptor chains, a high-affinity primary receptor and a low-affinity coreceptor. To gain insights into the signaling mechanism of the novel IL-1-like cytokine IL-33, we first solved its solution structure and then performed a detailed biochemical and structural characterization of the interaction between IL-33, its primary receptor ST2, and the coreceptor IL-1RAcP. Using nuclear magnetic resonance data, we obtained a model of the IL-33/ST2 complex in solution that is validated by small-angle X-ray scattering (SAXS) data and is similar to the IL-1β/IL-1R1 complex. We extended our SAXS analysis to the IL-33/ST2/IL-1RAcP and IL-1β/IL-1R1/IL-1RAcP complexes and propose a general model of the molecular architecture of IL-1 ternary signaling complexes.
Keywords: PROTEINS; MOLIMMUNO
OMP Peptides Activate the DegS Stress-Sensor Protease by a Relief of Inhibition Mechanism
by Jungsan Sohn; Robert A. Grant; Robert T. Sauer (pp. 1411-1421).
In the E. coli periplasm, C-terminal peptides of misfolded outer-membrane porins (OMPs) bind to the PDZ domains of the trimeric DegS protease, triggering cleavage of a transmembrane regulator and transcriptional activation of stress genes. We show that an active-site DegS mutation partially bypasses the requirement for peptide activation and acts synergistically with mutations that disrupt contacts between the protease and PDZ domains. Biochemical results support an allosteric model, in which these mutations, active-site modification, and peptide/substrate binding act in concert to stabilize proteolytically active DegS. Cocrystal structures of DegS in complex with different OMP peptides reveal activation of the protease domain with varied conformations of the PDZ domain and without specific contacts from the bound OMP peptide. Taken together, these results indicate that the binding of OMP peptides activates proteolysis principally by relieving inhibitory contacts between the PDZ domain and the protease domain of DegS.
Keywords: MICROBIO; SIGNALING; CELLBIO