Structure (v.17, #1)
In This Issue (ix-x).
On the Structural Model of the COP9 Signalosome by Ning Wei (1-2).
In this issue, determined the subunit organization of the COP9 signalosome by mass spectrometry. The structural features uncovered from this analysis are consistent with the multifunctionality of the complex and suggest tantalizing new clues about the inner workings of the CSN.
Journey to the Ends of the Arf by James M. Gruschus; Pei-Wen Chen; Ruibai Luo; Paul A. Randazzo (2-4).
In this issue, used NMR to provide the most complete information to date on the structure of Arf1 and the role of myristate in GDP/GTP exchange. Unanticipated details lead to speculation about functions for the N and C termini of Arfs.
Pseudokinases: Functional Insights Gleaned from Structure by Alexandr P. Kornev; Susan S. Taylor (5-7).
Pseudokinases are an intriguing group inside the large protein kinase family. Lacking the highly conserved catalytic machinery, they can be still important for multiple functions in living cells. The structures of two pseudokinases VRK3 () and ROP2 (), presented in this issue of Structure, shed light on the internal machinery of these unique molecules. Structural information allows us to go beyond sequence alone in the analysis of these pseudokinases.
Discovery of New GPCR Biology: One Receptor Structure at a Time by Michael A. Hanson; Raymond C. Stevens (8-14).
G-protein-coupled receptors (GPCRs) are the largest family of proteins in the human genome. Within the last year, we have witnessed a relative explosion in the amount of structural information available for the GPCR family with two new structures of opsin in the presence and absence of transducin peptide, four new structures of β-adrenergic receptors, and a recent structure of the human adenosine A2A receptor. The new biological insight being gained, such as the highly divergent extracellular loops and areas of structural convergence within the transmembrane helices, allows us to chart a course for further investigation into this important class of membrane proteins.
Keywords: PROTEINS; SIGNALING; HUMDISEASE;
Structural Microengineers: Pathogenic Escherichia coli Redesigns the Actin Cytoskeleton in Host Cells by Neta Sal-Man; Esther Biemans-Oldehinkel; B. Brett Finlay (15-19).
Several virulent bacteria have the ability to manipulate the host cell actin cytoskeleton as part of their pathogenic strategy. These pathogens subvert the host cell actin polymerization machinery for various purposes including motility within host cells, cell-to-cell spread, and to prevent phagocytic engulfment by professional phagocytes. In contrast to intracellular pathogens, pathogenic Escherichia coli (including both enterohemorrhagic and enteropathogenic E. coli) subvert actin polymerization from an extracellular position to facilitate adherence. This review summarizes recent data on the mechanisms by which pathogenic E. coli hijack members of the Wiskott-Aldrich syndrome protein family to manipulate actin polymerization within host cells, including the novel, and surprisingly simple, mechanism recently revealed for the EspFu effector.
Keywords: PROTEINS; MICROBIO; SIGNALING;
Structural Basis for Recruitment of Rab6-Interacting Protein 1 to Golgi via a RUN Domain by Rosario Recacha; Annick Boulet; Florence Jollivet; Solange Monier; Anne Houdusse; Bruno Goud; Amir R. Khan (21-30).
Small GTPase Rab6 regulates vesicle trafficking at the level of Golgi via recruitment of numerous and unrelated effectors. The crystal structure of Rab6a(GTP) in complex with a 378-residue internal fragment of the effector Rab6IP1 was solved at 3.2 Å resolution. This Rab6IP1 region encompasses an all α-helical RUN domain followed in tandem by a PLAT domain that adopts a β sandwich fold. The structure reveals that the first and last α helices of the RUN domain mediate binding to switch I, switch II, and the interswitch region of Rab6. It represents the largest Rab-effector complex determined to date. Comparisons with the recent structure of Rab6 in complex with an unrelated effector, human golgin GCC185, reveals significant conformational changes in the conserved hydrophobic triad of Rab6. Flexibility in the switch and interswitch regions of Rab6 mediates recognition of compositionally distinct α-helical coiled coils, thereby contributing to Rab6 promiscuity in effector recruitment.
Keywords: PROTEINS; CELLBIO;
Symmetrical Modularity of the COP9 Signalosome Complex Suggests its Multifunctionality by Michal Sharon; Haibin Mao; Elisabetta Boeri Erba; Elaine Stephens; Ning Zheng; Carol V. Robinson (31-40).
The COP9 signalosome (CSN) is an eight-subunit protein complex that is found in all eukaryotes. Accumulating evidence indicates its diverse biological functions that are often linked to ubiquitin-mediated proteolysis. Here we applied an emerging mass spectrometry approach to gain insight into the structure of the CSN complex. Our results indicate that the catalytically active human complex, reconstituted in vitro, is composed of a single copy of each of the eight subunits. By forming a total of 35 subcomplexes, we are able to build a comprehensive interaction map that shows two symmetrical modules, Csn1/2/3/8 and Csn4/5/6/7, connected by interactions between Csn1-Csn6. Overall the stable modules and multiple subcomplexes observed here are in agreement with the “mini-CSN” complexes reported previously. This suggests that the propensity of the CSN complex to change and adapt its subunit composition might underlie its ability to perform multiple functions in vivo.
Keywords: SIGNALING; CELLBIO; PROTEINS;
Differences in Flexibility Underlie Functional Differences in the Ras Activators Son of Sevenless and Ras Guanine Nucleotide Releasing Factor 1 by Tanya S. Freedman; Holger Sondermann; Olga Kuchment; Gregory D. Friedland; Tanja Kortemme; John Kuriyan (41-53).
The Ras-specific nucleotide exchange factor Son of sevenless (Sos) is inactive without Ras bound to a distal allosteric site. In contrast, the catalytic domain of Ras guanine nucleotide releasing factor 1 (RasGRF1) is active intrinsically. By substituting residues from RasGRF1 into Sos, we have generated mutants of Sos with basal activity, partially relieved of their dependence on allosteric activation. We have performed molecular dynamics simulations showing how Ras binding to the allosteric site leads to a bias toward the active conformation of Sos. The trajectories show that Sos fluctuates between active and inactive conformations in the absence of Ras and that the activating mutations favor conformations of Sos that are more permissive to Ras binding at the catalytic site. In contrast, unliganded RasGRF1 fluctuates primarily among active conformations. Our results support the premise that the catalytic domain of Sos has evolved an allosteric activation mechanism that extends beyond the simple process of membrane recruitment.
Keywords: PROTEINS; SIGNALING;
Structural and Biophysical Studies of the Human IL-7/IL-7Rα Complex by Craig A. McElroy; Julie A. Dohm; Scott T.R. Walsh (54-65).
IL-7 and IL-7Rα bind the γc receptor, forming a complex crucial to several signaling cascades leading to the development and homeostasis of T and B cells. We report that the IL-7Rα ectodomain uses glycosylation to modulate its binding constants to IL-7, unlike the other receptors in the γc family. IL-7 binds glycosylated IL-7Rα 300-fold more tightly than unglycosylated IL-7Rα, and the enhanced affinity is attributed primarily to an accelerated on rate. Structural comparison of IL-7 in complex to both forms of IL-7Rα reveals that glycosylation does not participate directly in the binding interface. The SCID mutations of IL-7Rα locate outside the binding interface with IL-7, suggesting that the expressed mutations cause protein folding defects in IL-7Rα. The IL-7/IL-7Rα structures provide a window into the molecular recognition events of the IL-7 signaling cascade and provide sites to target for designing new therapeutics to treat IL-7-related diseases.
Keywords: PROTEINS; MOLIMMUNO;
The Evolutionary Mechanics of Domain Organization in Proteomes and the Rise of Modularity in the Protein World by Minglei Wang; Gustavo Caetano-Anollés (66-78).
Protein domains are compact evolutionary units of structure and function that usually combine in proteins to produce complex domain arrangements. In order to study their evolution, we reconstructed genome-based phylogenetic trees of architectures from a census of domain structure and organization conducted at protein fold and fold-superfamily levels in hundreds of fully sequenced genomes. These trees defined timelines of architectural discovery and revealed remarkable evolutionary patterns, including the explosive appearance of domain combinations during the rise of organismal lineages, the dominance of domain fusion processes throughout evolution, and the late appearance of a new class of multifunctional modules in Eukarya by fission of domain combinations. Our study provides a detailed account of the history and diversification of a molecular interactome and shows how the interplay of domain fusions and fissions defines an evolutionary mechanics of domain organization that is fundamentally responsible for the complexity of the protein world.
Structure and Membrane Interaction of Myristoylated ARF1 by Yizhou Liu; Richard A. Kahn; James H. Prestegard (79-87).
ADP-ribosylation factors (ARFs) are small (21 kDa), monomeric GTPases that are important regulators of membrane traffic. When membrane bound, they recruit soluble adaptors to membranes and trigger the assembly of coating complexes involved in cargo selection and vesicular budding. N-myristoylation is a conserved feature of all ARF proteins that is required for its biological functions, although the mechanism(s) by which the myristate acts in ARF functions is not fully understood. Here we present the structure of a myristoylated ARF1 protein, determined by solution NMR methods, and an assessment of the influence of myristoylation on association of ARF1·GDP and ARF1·GTP with lipid bilayers. A model in which myristoylation contributes to both the regulation of guanine nucleotide exchange and stable membrane association is supported.
Keywords: PROTEINS; SIGNALING;
An Atomistic View to the Gas Phase Proteome by Tim Meyer; Xavier de la Cruz; Modesto Orozco (88-95).
Extended all-atom molecular dynamics simulations on all protein metafolds have been performed to obtain a complete picture of the gas phase proteome. The structural atlas of the gas phase proteome obtained here shows an unexpected maintenance of the global and local structure and of the general deformability pattern upon transfer to the gas phase under electrospray conditions. Despite a general compression, the solution structure can be easily very well recognized from the gas phase one, and most structural details, such as secondary structure, are well preserved upon vaporization. Rehydration of the gas phase protein leads in most cases to a very fast transition from gas phase to solution structure. Overall, our massive analysis (over 4 μs in solution and over 12 μs in the gas phase) demonstrates that solution-like structures can be determined by using mass spectroscopy and related techniques to obtain fast approximations to the solution structure.
The Structure of the MAP2K MEK6 Reveals an Autoinhibitory Dimer by Xiaoshan Min; Radha Akella; Haixia He; John M. Humphreys; Susan E. Tsutakawa; Seung-Jae Lee; John A. Tainer; Melanie H. Cobb; Elizabeth J. Goldsmith (96-104).
MAP2Ks are dual-specificity protein kinases functioning at the center of three-tiered MAP kinase modules. The structure of the kinase domain of the MAP2K MEK6 with phosphorylation site mimetic aspartic acid mutations (MEK6/ΔN/DD) has been solved at 2.3 Å resolution. The structure reveals an autoinhibited elongated ellipsoidal dimer. The enzyme adopts an inactive conformation, based upon structural queues, despite the phosphomimetic mutations. Gel filtration and small-angle X-ray scattering analysis confirm that the crystallographically observed ellipsoidal dimer is a feature of MEK6/ΔN/DD and full-length unphosphorylated wild-type MEK6 in solution. The interface includes the phosphate binding ribbon of each subunit, part of the activation loop, and a rare “arginine stack” between symmetry-related arginine residues in the N-terminal lobe. The autoinhibited structure likely confers specificity on active MAP2Ks. The dimer may also serve the function in unphosphorylated MEK6 of preventing activation loop phosphorylation by inappropriate kinases.
Keywords: PROTEINS; SIGNALING;
The Crystal Structure of the N-Terminal Region of BUB1 Provides Insight into the Mechanism of BUB1 Recruitment to Kinetochores by Victor M. Bolanos-Garcia; Tomomi Kiyomitsu; Sheena D'Arcy; Dimitri Y. Chirgadze; J. Günter Grossmann; Dijana Matak-Vinkovic; Ashok R. Venkitaraman; Mitsuhiro Yanagida; Carol V. Robinson; Tom L. Blundell (105-116).
The interaction of the central mitotic checkpoint component BUB1 with the mitotic kinetochore protein Blinkin is required for the kinetochore localization and function of BUB1 in the mitotic spindle assembly checkpoint, the regulatory mechanism of the cell cycle that ensures the even distribution of chromosomes during the transition from metaphase to anaphase. Here, we report the 1.74 Å resolution crystal structure of the N-terminal region of BUB1. The structure is organized as a tandem arrangement of three divergent units of the tetratricopeptide motif. Functional assays in vivo of native and site-specific mutants identify the residues of human BUB1 important for the interaction with Blinkin and define one region of potential therapeutic interest. The structure provides insight into the molecular basis of Blinkin-specific recognition by BUB1 and, on a broader perspective, of the mechanism that mediates kinetochore localization of BUB1 in checkpoint-activated cells.
Keywords: PROTEINS; CELLBIO;
The Structure of Phosphorylase Kinase Holoenzyme at 9.9 Å Resolution and Location of the Catalytic Subunit and the Substrate Glycogen Phosphorylase by Catherine Vénien-Bryan; Slavica Jonic; Vasiliki Skamnaki; Nick Brown; Nicolas Bischler; Nikos G. Oikonomakos; Nicolas Boisset; Louise N. Johnson (117-127).
Phosphorylase kinase (PhK) coordinates hormonal and neuronal signals to initiate the breakdown of glycogen. The enzyme catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a. We present a 9.9 Å resolution structure of PhK heterotetramer (αβγδ)4 determined by cryo-electron microscopy single-particle reconstruction. The enzyme has a butterfly-like shape comprising two lobes with 222 symmetry. This three-dimensional structure has allowed us to dock the catalytic γ subunit to the PhK holoenzyme at a location that is toward the ends of the lobes. We have also determined the structure of PhK decorated with GPb at 18 Å resolution, which shows the location of the substrate near the kinase subunit. The PhK preparation contained a number of smaller particles whose structure at 9.8 Å resolution was consistent with a proteolysed activated form of PhK that had lost the α subunits and possibly the γ subunits.
Keywords: PROTEINS; SIGNALING;
Structure of the Pseudokinase VRK3 Reveals a Degraded Catalytic Site, a Highly Conserved Kinase Fold, and a Putative Regulatory Binding Site by Eric D. Scheeff; Jeyanthy Eswaran; Gabor Bunkoczi; Stefan Knapp; Gerard Manning (128-138).
About 10% of all protein kinases are predicted to be enzymatically inactive pseudokinases, but the structural details of kinase inactivation have remained unclear. We present the first structure of a pseudokinase, VRK3, and that of its closest active relative, VRK2. Profound changes to the active site region underlie the loss of catalytic activity, and VRK3 cannot bind ATP because of residue substitutions in the binding pocket. However, VRK3 still shares striking structural similarity with VRK2, and appears to be locked in a pseudoactive conformation. VRK3 also conserves residue interactions that are surprising in the absence of enzymatic function; these appear to play important architectural roles required for the residual functions of VRK3. Remarkably, VRK3 has an “inverted” pattern of sequence conservation: although the active site is poorly conserved, portions of the molecular surface show very high conservation, suggesting that they form key interactions that explain the evolutionary retention of VRK3.
Keywords: PROTEINS; SIGNALING;
ROP2 from Toxoplasma gondii: A Virulence Factor with a Protein-Kinase Fold and No Enzymatic Activity by Gilles Labesse; Muriel Gelin; Yannick Bessin; Maryse Lebrun; Julien Papoin; Rachel Cerdan; Stefan T. Arold; Jean-François Dubremetz (139-146).
The ROP2 protein and its paralogs are important virulence factors secreted into the host cell by the parasite Toxoplasma gondii. Here we describe the crystal structure of a large and soluble domain of mature ROP2, representative of the ROP2-like protein family. This is a structure of a protein-kinase fold that is devoid of catalytic residues and does not bind ATP. Various structural extensions constitute a signature of this protein family and act to maintain the protein kinase in an open conformation. Our ROP2 structure rules out a previous structural model of attachment of ROP2-like proteins to the parasitophorous vacuole membrane. We propose an alternative mode of membrane attachment implicating basic and amphiphatic helices present in the flexible N terminus of ROP2.