Structure (v.15, #10)
Flexibility of a Glutamate-Binding Domain
by Robert E. Oswald (pp. 1157-1158).
The molecular dynamics simulation of the binding domain of a glutamate receptor presented in this issue of Structure () provides insights into large-scale fluctuations of this protein that are supported by experiment and provide constraints on possible models for the function of the intact glutamate receptor.
Protease Yoga: Extreme Flexibility of a Matrix Metalloproteinase
by Christopher M. Overall; Georgina S. Butler (pp. 1159-1161).
Proteases face a daunting task in tackling the huge variety of protein folds. In this issue of Structure, Irit Sagi's laboratory exploits atomic force microscopy and SAXS to reveal the extreme flexibility of matrix metalloproteinase (MMP)-9, heralding new functional possibilities for MMPs ().
AMPK Structure and Regulation from Three Angles
by Bruce E. Kemp; Jonathan S. Oakhill; John W. Scott (pp. 1161-1163).
In this issue of Structure, report the structure of Schizosaccharomyces pombe AMPK embracing two moles of ADP by its γ subunit. This structure highlights the complexity of AMPK regulation across eukaryotes and challenges the regulatory dogma.
When One Protein Does the Job of Many
by Caroline Kisker (pp. 1163-1165).
In this issue of Structure, the high-resolution structure of the ultraviolet damage endonuclease by provides the first glimpse at a third DNA repair mechanism that efficiently repairs UV-induced DNA damage. The surprising structural homology to Endonuclease IV suggests a path for DNA binding and provides insights into DNA hydrolysis.
Modeling Experimental Image Formation for Likelihood-Based Classification of Electron Microscopy Data
by Sjors H.W. Scheres; Rafael Núñez-Ramírez; Yacob Gómez-Llorente; Carmen San Martín; Paul P.B. Eggermont; José María Carazo (pp. 1167-1177).
The coexistence of multiple distinct structural states often obstructs the application of three-dimensional cryo-electron microscopy to large macromolecular complexes. Maximum likelihood approaches are emerging as robust tools for solving the image classification problems that are posed by such samples. Here, we propose a statistical data model that allows for a description of the experimental image formation within the formulation of 2D and 3D maximum-likelihood refinement. The proposed approach comprises a formulation of the probability calculations in Fourier space, including a spatial frequency-dependent noise model and a description of defocus-dependent imaging effects. The Expectation-Maximization-like algorithms presented are generally applicable to the alignment and classification of structurally heterogeneous projection data. Their effectiveness is demonstrated with various examples, including 2D classification of top views of the archaeal helicase MCM and 3D classification of 70S E. coli ribosome and Simian Virus 40 large T-antigen projections.
Implementation of a k/k0 Method to Identify Long-Range Structure in Transition States during Conformational Folding/Unfolding of Proteins
by Lovy Pradeep; Igor Kurinov; Steven E. Ealick; Harold A. Scheraga (pp. 1178-1189).
A previously introduced kinetic-rate constant (k/k0) method, where k and k0 are the folding (unfolding) rate constants in the mutant and the wild-type forms, respectively, of a protein, has been applied to obtain qualitative information about structure in the transition state ensemble (TSE) of bovine pancreatic ribonuclease A (RNase A), which contains four native disulfide bonds. The method compares the folding (unfolding) kinetics of RNase A, with and without a covalent crosslink and tests whether the crosslinked residues are associated in the folding (unfolding) transition state (TS) of the noncrosslinked version. To confirm that the fifth disulfide bond has not introduced a significant structural perturbation, we solved the crystal structure of the V43C-R85C mutant to 1.6 Å resolution. Our findings suggest that residues Val43 and Arg85 are not associated, and that residues Ala4 and Val118 may form nonnative contacts, in the folding (unfolding) TSE of RNase A.
A Corkscrew Model for Dynamin Constriction
by Jason A. Mears; Pampa Ray; Jenny E. Hinshaw (pp. 1190-1202).
Numerous vesiculation processes throughout the eukaryotic cell are dependent on the protein dynamin, a large GTPase that constricts lipid bilayers. We have combined X-ray crystallography and cryo-electron microscopy (cryo-EM) data to generate a coherent model of dynamin-mediated membrane constriction. GTPase and pleckstrin homology domains of dynamin were fit to cryo-EM structures of human dynamin helices bound to lipid in nonconstricted and constricted states. Proteolysis and immunogold labeling experiments confirm the topology of dynamin domains predicted from the helical arrays. Based on the fitting, an observed twisting motion of the GTPase, middle, and GTPase effector domains coincides with conformational changes determined by cryo-EM. We propose a corkscrew model for dynamin constriction based on these motions and predict regions of sequence important for dynamin function as potential targets for future mutagenic and structural studies.
Keywords: PROTEINS; CELLBIO
The Free Energy Landscapes Governing Conformational Changes in a Glutamate Receptor Ligand-Binding Domain
by Albert Y. Lau; Benoît Roux (pp. 1203-1214).
Ionotropic glutamate receptors are ligand-gated transmembrane ion channels activated by the binding of glutamate. The free energy landscapes governing the opening/closing of the GluR2 S1S2 ligand-binding domain in the apo, DNQX-, and glutamate-bound forms are computed by using all-atom molecular dynamics simulations with explicit solvent, in conjunction with an umbrella sampling strategy. The apo S1S2 easily accesses low-energy conformations that are more open than observed in X-ray crystal structures. A free energy of 9–12 kcal/mol becomes available upon glutamate binding for driving conformational changes in S1S2 associated with receptor activation. Small-angle X-ray scattering profiles calculated from computed ensemble averages agree better with experimental results than profiles calculated from static X-ray crystal structures. Water molecules in the cleft may contribute to stabilizing the apo S1S2 in open conformations. Free energy landscapes were also computed for the glutamate-bound T686A and T686S S1S2 mutants, and the results elaborate on findings from experimental functional studies.
Keywords: PROTEINS; SIGNALING
Structural and Functional Characterization of the Human Protein Kinase ASK1
by Gabor Bunkoczi; Eidarus Salah; Panagis Filippakopoulos; Oleg Fedorov; Susanne Müller; Frank Sobott; Sirlester A. Parker; Haifeng Zhang; Wang Min; Benjamin E. Turk; Stefan Knapp (pp. 1215-1226).
Apoptosis signal-regulating kinase 1 (ASK1) plays an essential role in stress and immune response and has been linked to the development of several diseases. Here, we present the structure of the human ASK1 catalytic domain in complex with staurosporine. Analytical ultracentrifugation (AUC) and crystallographic analysis showed that ASK1 forms a tight dimer (Kd ∼ 0.2 μM) interacting in a head-to-tail fashion. We found that the ASK1 phosphorylation motifs differ from known ASK1 phosphorylation sites but correspond well to autophosphorylation sites identified by mass spectrometry. Reporter gene assays showed that all three identified in vitro autophosphorylation sites (Thr813, Thr838, Thr842) regulate ASK1 signaling, but site-directed mutants showed catalytic activities similar to wild-type ASK1, suggesting a regulatory mechanism independent of ASK1 kinase activity. The determined high-resolution structure of ASK1 and identified ATP mimetic inhibitors will provide a first starting point for the further development of selective inhibitors.
Keywords: PROTEINS; SIGNALING
Insights into the Structure and Domain Flexibility of Full-Length Pro-Matrix Metalloproteinase-9/Gelatinase B
by Gabriel Rosenblum; Philippe E. Van den Steen; Sidney R. Cohen; J. Günter Grossmann; Jessica Frenkel; Rotem Sertchook; Nelle Slack; Richard W. Strange; Ghislain Opdenakker; Irit Sagi (pp. 1227-1236).
The multidomain zinc endopeptidase matrix metalloproteinase-9 (MMP-9) is a recognized therapeutic target in autoimmune diseases, vascular pathologies, and cancer. Despite its importance, structural characterization of full-length pro-MMP-9 is incomplete. Here, we report the structural model of full-length pro-MMP-9 and, in particular, the molecular character of its unique proline-rich and heavily O-glycosylated (OG) domain. Using a powerful combination of small-angle X-ray scattering and single-molecule imaging, we demonstrate that pro-MMP-9 possesses an elongated structure with two terminal globular domains connected by an unstructured OG domain. Image analysis highlights the flexibility of the OG domain, implicating its role in the varied enzyme conformations and in facilitating independent movements of the terminal domains. This may endorse recognition, binding, and processing of substrates, ligands, as well as receptors and marks this domain as an additional target for the design of selective regulators.
Keywords: PROTEINS; CELLBIO
Structural Biology of RNA Polymerase III: Mass Spectrometry Elucidates Subcomplex Architecture
by Kristina Lorenzen; Alessandro Vannini; Patrick Cramer; Albert J.R. Heck (pp. 1237-1245).
RNA polymerases (Pol) II and III synthesize eukaryotic mRNAs and tRNAs, respectively. The crystal structure of the 12 subunit Pol II is known, but only limited structural information is available for the 17 subunit Pol III. Using mass spectrometry (MS), we correlated masses of Pol II complexes with the Pol II structure. Analysis of Pol III showed that the complete enzyme contains a single copy of each subunit and revealed a 15 subunit form lacking the Pol III-specific subcomplex C53/37. DMSO treatment dissociated the C17/25 heterodimer of Pol III, confirming a peripheral location as its counterpart in Pol II. Tandem MS revealed the Pol III-specific subunits C82 and C34 dissociating as a heterodimer. C11 was retained, arguing against a stable trimeric subcomplex, C53/37/11. These data suggest that Pol III consists of a 10 subunit Pol II-like core; the peripheral heterodimers C17/25, C53/37, and C82/34; and subunit C31, which bridges between C82/34, C17/25, and the core.
Structure of the Yeast WD40 Domain Protein Cia1, a Component Acting Late in Iron-Sulfur Protein Biogenesis
by Vasundara Srinivasan; Daili J.A. Netz; Holger Webert; Judita Mascarenhas; Antonio J. Pierik; Hartmut Michel; Roland Lill (pp. 1246-1257).
The WD40-repeat protein Cia1 is an essential, conserved member of the cytosolic iron-sulfur (Fe/S) protein assembly (CIA) machinery in eukaryotes. Here, we report the crystal structure of Saccharomyces cerevisiae Cia1 to 1.7 Å resolution. The structure folds into a β propeller with seven blades pseudo symmetrically arranged around a central axis. Structure-based sequence alignment of Cia1 proteins shows that the WD40 propeller core elements are highly conserved. Site-directed mutagenesis of amino acid residues in loop regions with high solvent accessibility identified that the conserved top surface residue R127 performs a critical function: the R127 mutant cells grew slowly and were impaired in cytosolic Fe/S protein assembly. Human Ciao1, which reportedly interacts with the Wilms' tumor suppressor, WT1, is structurally similar to yeast Cia1. We show that Ciao1 can functionally replace Cia1 and support cytosolic Fe/S protein biogenesis. Hence, our structural and biochemical studies indicate the conservation of Cia1 function in eukaryotes.
Partial Agonists Activate PPARγ Using a Helix 12 Independent Mechanism
by John B. Bruning; Michael J. Chalmers; Swati Prasad; Scott A. Busby; Theodore M. Kamenecka; Yuanjun He; Kendall W. Nettles; Patrick R. Griffin (pp. 1258-1271).
Binding to helix 12 of the ligand-binding domain of PPARγ is required for full agonist activity. Previously, the degree of stabilization of the activation function 2 (AF-2) surface was thought to correlate with the degree of agonism and transactivation. To examine this mechanism, we probed structural dynamics of PPARγ with agonists that induced graded transcriptional responses. Here we present crystal structures and amide H/D exchange (HDX) kinetics for six of these complexes. Amide HDX revealed each ligand induced unique changes to the dynamics of the ligand-binding domain (LBD). Full agonists stabilized helix 12, whereas intermediate and partial agonists did not at all, and rather differentially stabilized other regions of the binding pocket. The gradient of PPARγ transactivation cannot be accounted for solely through changes to the dynamics of AF-2. Thus, our understanding of allosteric signaling must be extended beyond the idea of a dynamic helix 12 acting as a molecular switch.
A Conformational Transition State Accompanies Tryptophan Activation by B. stearothermophilus Tryptophanyl-tRNA Synthetase
by Maryna Kapustina; Violetta Weinreb; Li Li; Brian Kuhlman; Charles W. Carter Jr. (pp. 1272-1284).
B. stearothermophilus tryptophanyl-tRNA synthetase catalysis proceeds via high-energy protein conformations. Unliganded MD trajectories of the pretransition-state complex with Mg2+•ATP and the (post) transition-state analog complex with adenosine tetraphosphate relax rapidly in opposite directions, the former regressing, the latter progressing along the structural reaction coordinate. The two crystal structures (rmsd 0.7 Å) therefore lie on opposite sides of a conformational free-energy maximum as the chemical transition state forms. SNAPP analysis illustrates the complexity of the associated long-range conformational coupling. Switching interactions in four nonpolar core regions are locally isoenergetic throughout the transition. Different configurations, however, propagate their effects to unfavorable, longer-range interactions at the molecular surface. Designed mutation shows that switching interactions enhance the rate, perhaps by destabilizing the ground state immediately before the transition state and limiting nonproductive diffusion before and after the chemical transition state, thereby reducing the activation entropy. This paradigm may apply broadly to energy-transducing enzymes.
Keywords: PROTEINS; RNA
Structural Insight into AMPK Regulation: ADP Comes into Play
by Xiangshu Jin; Robert Townley; Lawrence Shapiro (pp. 1285-1295).
The AMP-activated protein kinase (AMPK), a sensor of cellular energy status found in all eukaryotes, responds to changes in intracellular adenosine nucleotide levels resulting from metabolic stresses. Here we describe crystal structures of a heterotrimeric regulatory core fragment from Schizosaccharomyces pombe AMPK in complex with ADP, ADP/AMP, ADP/ATP, and 5-aminoimidazole-4-carboxamide 1-β- D-ribofuranotide (AICAR phosphate, or ZMP), a well-characterized AMPK activator. Prior crystallographic studies had revealed a single site in the γ subunit that binds either ATP or AMP within Bateman domain B. Here we show that ZMP binds at this site, mimicking the binding of AMP. An analogous site in Bateman domain A selectively accommodates ADP, which binds in a distinct manner that also involves direct ligation to elements from the β subunit. These observations suggest a possible role for ADP in regulating AMPK response to changes in cellular energy status.
Structure and Substrate Specificity of an SspB Ortholog: Design Implications for AAA+ Adaptors
by Peter Chien; Robert A. Grant; Robert T. Sauer; Tania A. Baker (pp. 1296-1305).
AAA+ proteases are frequently regulated by adaptors that modulate spatial and temporal control of protein turnover. Caulobacter crescentus is an α-proteobacterium which requires protein degradation by the AAA+ ClpXP protease for cell-cycle progression, and contains an adaptor (SspBα) that binds ssrA-tagged proteins and targets them to ClpXP. Here we determine the tag-binding specificity and crystal structure of SspBα. Despite poor sequence homology, the overall SspBα fold resembles orthologs from other bacteria. However, several structural features are specific to the SspBα subfamily, including the dimerization interface, binding surfaces optimized for ssrA-tag delivery, and residues in the tag-binding groove that act as selectivity gatekeepers for substrate recognition. Mutagenesis of these residues broadens specificity, creating a promiscuous adaptor that recognizes an expanded substrate repertoire. These results highlight general features of adaptor-mediated substrate recognition and shed light on design principles that underlie adaptor function.
Structural Basis of EZH2 Recognition by EED
by Zhifu Han; Xinmiao Xing; Min Hu; Yin Zhang; Peiyuan Liu; Jijie Chai (pp. 1306-1315).
The WD-repeat domain is a highly conserved recognition module in eukaryotes involved in diverse cellular processes. It is still not well understood how the bottom of a WD-repeat domain recognizes its binding partners. The WD-repeat-containing protein EED is one component of the PRC2 complex that possesses histone methyltransferase activity required for gene repression. Here we report the crystal structure of EED in complex with a 30 residue peptide from EZH2. The structure reveals that the peptide binds to the bottom of the WD-repeat domain of EED. The structural determinants of EZH2-EED interaction are present not only in EZH2 and EZH1 but also in its Drosophila homolog E(Z), suggesting that the recognition of ESC by E(Z) in Drosophila employs similar structural motifs. Structure-based mutagenesis identified critical residues from both EED and EZH2 for their interaction. The structure presented here may provide a template for understanding of how WD-repeat proteins recognize their interacting proteins.
Keywords: PROTEINS; SIGNALING
Crystal Structure of the DNA Repair Enzyme Ultraviolet Damage Endonuclease
by Keti Paspaleva; Ellen Thomassen; Navraj S. Pannu; Shigenori Iwai; Geri F. Moolenaar; Nora Goosen; Jan Pieter Abrahams (pp. 1316-1324).
The ultraviolet damage endonuclease (UVDE) performs the initial step in an alternative excision repair pathway of UV-induced DNA damage, nicking immediately adjacent to the 5′ phosphate of the damaged nucleotides. Unique for a single-protein DNA repair endonuclease, it can detect different types of damage. Here we show that Thermus thermophilus UVDE shares some essential structural features with Endo IV, an enzyme from the base excision repair pathway that exclusively nicks at abasic sites. A comparison between the structures indicates how DNA is bound by UVDE, how UVDE may recognize damage, and which of its residues are involved in catalysis. Furthermore, the comparison suggests an elegant explanation of UVDE's potential to recognize different types of damage. Incision assays including point mutants of UVDE confirmed the relevance of these conclusions.
A Structural Code for Discriminating between Transcription Signals Revealed by the Feast/Famine Regulatory Protein DM1 in Complex with Ligands
by Hideyasu Okamura; Katsushi Yokoyama; Hideaki Koike; Mitsugu Yamada; Ai Shimowasa; Mamiko Kabasawa; Tsuyoshi Kawashima; Masashi Suzuki (pp. 1325-1338).
Feast/famine regulatory proteins (FFRPs) comprise the largest group of archaeal transcription factors. Crystal structures of an FFRP, DM1 from Pyrococcus, were determined in complex with isoleucine, which increases the association state of DM1 to form octamers, and with selenomethionine, which decreases it to maintain dimers under some conditions. Asp39 and Thr/Ser at 69–71 were identified as being important for interaction with the ligand main chain. By analyzing residues surrounding the ligand side chain, partner ligands were identified for various FFRPs from Pyrococcus, e.g., lysine facilitates homo-octamerization of FL11, and arginine facilitates hetero-octamerization of FL11 and DM1. Transcription of the fl11 gene and lysine synthesis are regulated by shifting the equilibrium between association states of FL11 and by shifting the equilibrium toward association with DM1, in response to amino acid availability. With FFRPs also appearing in eubacteria, the origin of such regulation can be traced back to the common ancestor of all extant organisms, serving as a prototype of transcription regulations, now highly diverged.