Structure (v.19, #9)

In This Issue (v-vi).

Ribonuclease J: How to Lead a Double Life by Jamie Richards; Joel G. Belasco (1201-1203).
New structures of RNase J reported by Dorléans et al. and Newman et al. in this issue of Structure suggest how an enzyme whose identical subunits each contain a single buried active site can function as both a 5′ exonuclease and an endonuclease.

Trapping Small Caffeine in a Large GPCR Pocket by Fei Xu; Raymond C. Stevens (1204-1207).
Three new X-ray structures of a thermostabilized A2A adenosine receptor in complex with different antagonists reported by Doré et al. in this issue have advanced the understanding of molecular recognition for this receptor.

Assembling Good Amyloid: Some Structures at Last by Daniel E. Otzen (1207-1209).
Taylor et al. report the crystal structure of CsgC, a redox-active member of E. coli's curli-producing Csg operon. The outer membrane protein CsgG is a potential redox-substrate and might exist in an octameric structure with a periplasmatic CsgC binding site, highlighting a potential role for disulfide bonds in curli production.

Symmetry-Restrained Flexible Fitting for Symmetric EM Maps by Kwok-Yan Chan; James Gumbart; Ryan McGreevy; Jean M. Watermeyer; B. Trevor Sewell; Klaus Schulten (1211-1218).
Many large biological macromolecules have inherent structural symmetry, being composed of a few distinct subunits, repeated in a symmetric array. These complexes are often not amenable to traditional high-resolution structural determination methods, but can be imaged in functionally relevant states using cryo-electron microscopy (cryo-EM). A number of methods for fitting atomic-scale structures into cryo-EM maps have been developed, including the molecular dynamics flexible fitting (MDFF) method. However, quality and resolution of the cryo-EM map are the major determinants of a method's success. In order to incorporate knowledge of structural symmetry into the fitting procedure, we developed the symmetry-restrained MDFF method. The new method adds to the cryo-EM map-derived potential further restraints on the allowed conformations of a complex during fitting, thereby improving the quality of the resultant structure. The benefit of using symmetry-based restraints during fitting, particularly for medium to low-resolution data, is demonstrated for three different systems.► Structural symmetry information can be incorporated into flexible fitting ► Symmetry-based restraints improve quality of fitted structures ► Benefits are most pronounced for lower resolution data

The Architecture of CopA from Archeaoglobus fulgidus Studied by Cryo-Electron Microscopy and Computational Docking by Gregory S. Allen; Chen-Chou Wu; Tim Cardozo; David L. Stokes (1219-1232).
CopA uses ATP to pump Cu+ across cell membranes. X-ray crystallography has defined atomic structures of several related P-type ATPases. We have determined a structure of CopA at 10 Å resolution by cryo-electron microscopy of a new crystal form and used computational molecular docking to study the interactions between the N-terminal metal-binding domain (NMBD) and other elements of the molecule. We found that the shorter-chain lipids used to produce these crystals are associated with movements of the cytoplasmic domains, with a novel dimer interface and with disordering of the NMBD, thus offering evidence for the transience of its interaction with the other cytoplasmic domains. Docking identified a binding site that matched the location of the NMBD in our previous structure by cryo-electron microscopy, allowing a more detailed view of its binding configuration and further support for its role in autoinhibition.► Dramatic changes in crystal morphology of CopA were obtained with short-chain lipids ► Movements of cytoplasmic domains suggest transition between E2 and E1 conformations ► Disordering of the NMBD illustrates its transient interaction with other domains ► Best docking result matches previous structure, supporting model of autoinhibition

RNA Tertiary Interactions in a Riboswitch Stabilize the Structure of a Kink Turn by Kersten T. Schroeder; Peter Daldrop; David M.J. Lilley (1233-1240).
The kink turn is a widespread RNA motif that introduces an acute kink into the axis of duplex RNA, typically comprising a bulge followed by a G⋅A and A⋅G pairs. The kinked conformation is stabilized by metal ions, or the binding of proteins including L7Ae. We now demonstrate a third mechanism for the stabilization of k-turn structure, involving tertiary interactions within a larger RNA structure. The SAM-I riboswitch contains an essential standard k-turn sequence that kinks a helix so that its terminal loop can make a long-range interaction. We find that some sequence variations in the k-turn within the riboswitch do not prevent SAM binding, despite preventing the folding of the k-turn in isolation. Furthermore, two crystal structures show that the sequence-variant k-turns are conventionally folded within the riboswitch. This study shows that the folded structure of the k-turn can be stabilized by tertiary interactions within a larger RNA structure.Display Omitted► The folded conformation of a kink turn is stabilized by long-range tertiary interactions in RNA ► The SAM-I riboswitch contains a k-turn, that folds in the presence of Mg2+ ions in isolation ► Nucleotide substitution in the G•A pairs of the k-turn prevent folding in isolation, but some can be stabilized in the context of the riboswitch, allowing binding of SAM ligand ► The crystal structure of a SAM-I riboswitch with a variant k-turn provides the first example of a k-turn with an A•A pair at the 2b•2n position

Unusual, Dual Endo- and Exonuclease Activity in the Degradosome Explained by Crystal Structure Analysis of RNase J1 by Joseph A. Newman; Lorraine Hewitt; Cecilia Rodrigues; Alexandra Solovyova; Colin R. Harwood; Richard J. Lewis (1241-1251).
RNase J is an essential enzyme in Bacillus subtilis with unusual dual endonuclease and 5′-to-3′ exonuclease activities that play an important role in the maturation and degradation of mRNA. RNase J is also a component of the recently identified “degradosome” of B. subtilis. We report the crystal structure of RNase J1 from B. subtilis to 3.0 Å resolution, analysis of which reveals it to be in an open conformation suitable for binding substrate RNA. RNase J is a member of the β-CASP family of zinc-dependent metallo-β-lactamases. We have exploited this similarity in constructing a model for an RNase J1:RNA complex. Analysis of this model reveals candidate-stacking interactions with conserved aromatic side chains, providing a molecular basis for the observed enzyme activity. Comparisons of the B. subtilis RNase J structure with related enzymes reveal key differences that provide insights into conformational changes during catalysis and the role of the C-terminal domain.► We report the crystal structure of RNase J1 from B. subtilis to 3.0 Å resolution ► The open form of the structure indicates that large changes occur during catalysis ► By homology to other structures, a model for RNA binding has been built ► The RNA-bound model explains the unusual dual exo- and endonuclease activities

Molecular Basis for the Recognition and Cleavage of RNA by the Bifunctional 5′–3′ Exo/Endoribonuclease RNase J by Audrey Dorléans; Inés Li de la Sierra-Gallay; Jérémie Piton; Léna Zig; Laetitia Gilet; Harald Putzer; Ciarán Condon (1252-1261).
RNase J is a key member of the β-CASP family of metallo-β-lactamases involved in the maturation and turnover of RNAs in prokaryotes. The B. subtilis enzyme possesses both 5′-3′ exoribonucleolytic and endonucleolytic activity, an unusual property for a ribonuclease. Here, we present the crystal structure of T. thermophilus RNase J bound to a 4 nucleotide RNA. The structure reveals an RNA-binding channel that illustrates how the enzyme functions in 5′-3′ exoribonucleolytic mode and how it can function as an endonuclease. A second, negatively charged tunnel leads from the active site, and is ideally located to evacuate the cleaved nucleotide in 5′-3′ exonucleolytic mode. We show that B. subtilis RNase J1, which shows processive behavior on long RNAs, behaves distributively for substrates less than 5 nucleotides in length. We propose a model involving the binding of the RNA to the surface of the β-CASP domain to explain the enzyme's processive action.Display Omitted► Structure of RNase J bound to RNA ► Explanation of enzyme's dual endo and 5′-3′ exonuclease function ► Identification of potential evacuation tunnel for cleaved nucleotide ► Structural basis for RNase J's processivity

Structural Basis of Substrate Methylation and Inhibition of SMYD2 by Andrew D. Ferguson; Nicholas A. Larsen; Tina Howard; Hannah Pollard; Isabelle Green; Christie Grande; Tony Cheung; Renee Garcia-Arenas; Scott Cowen; Jiaquan Wu; Robert Godin; Huawei Chen; Nicholas Keen (1262-1273).
Protein lysine methyltransferases are important regulators of epigenetic signaling. These enzymes catalyze the transfer of donor methyl groups from S-adenosylmethionine to specific acceptor lysines on histones, leading to changes in chromatin structure and transcriptional regulation. These enzymes also methylate nonhistone protein substrates, revealing an additional mechanism to regulate cellular physiology. The oncogenic protein SMYD2 represses the functional activities of the tumor suppressor proteins p53 and Rb, making it an attractive drug target. Here we report the discovery of AZ505, a potent and selective inhibitor of SMYD2 that was identified from a high throughput chemical screen. We also present the crystal structures of SMYD2 with p53 substrate and product peptides, and notably, in complex with AZ505. This substrate competitive inhibitor is bound in the peptide binding groove of SMYD2. These results have implications for the development of SMYD2 inhibitors, and indicate the potential for developing novel therapies targeting this target class.► The oncogenic protein SMYD2 represses the functional activities of the tumor suppressor proteins p53 and Rb, making it an attractive drug target ► We report the discovery of AZ505, a potent and selective inhibitor of SMYD2 that was identified from a high throughput chemical screen. We also present the crystal structures of SMYD2 with p53 substrate and product peptides, and notably, in complex with AZ505 ► This substrate competitive inhibitor is bound in the peptide binding groove of SMYD2 ► These results have implications for the development of SMYD2 inhibitors, and indicate the potential for developing novel therapies targeting this target class

Quantitative Analysis of the Interaction Strength and Dynamics of Human IgG4 Half Molecules by Native Mass Spectrometry by Rebecca J. Rose; Aran F. Labrijn; Ewald T.J. van den Bremer; Stefan Loverix; Ignace Lasters; Patrick H.C. van Berkel; Jan G.J. van de Winkel; Janine Schuurman; Paul W.H.I. Parren; Albert J.R. Heck (1274-1282).
Native mass spectrometry (MS) is a powerful technique for studying noncovalent protein-protein interactions. Here, native MS was employed to examine the noncovalent interactions involved in homodimerization of antibody half molecules (HL) in hinge-deleted human IgG4 (IgG4Δhinge). By analyzing the concentration dependence of the relative distribution of monomer HL and dimer (HL)2 species, the apparent dissociation constant (KD) for this interaction was determined. In combination with site-directed mutagenesis, the relative contributions of residues at the CH3-CH3 interface to this interaction could be characterized and corresponding KD values quantified over a range of 10−10–10−4 M. The critical importance of this noncovalent interaction in maintaining the intact dimeric structure was also proven for the full-length IgG4 backbone. Using time-resolved MS, the kinetics of the interaction could be measured, reflecting the dynamics of IgG4 HL exchange. Hence, native MS has provided a quantitative view of local structural features that define biological properties of IgG4.Display Omitted► Mass spectrometry analysis of noncovalent association of antibody heavy chains ► Binding affinity determined for panel of IgG4 constructs with single-point mutations ► Specific interactions between CH3 domains define stability of IgG4 dimeric structure ► Noncovalent interaction strength correlates with Fab-arm exchange kinetics

Structure of the Adenosine A2A Receptor in Complex with ZM241385 and the Xanthines XAC and Caffeine by Andrew S. Doré; Nathan Robertson; James C. Errey; Irene Ng; Kaspar Hollenstein; Ben Tehan; Edward Hurrell; Kirstie Bennett; Miles Congreve; Francesca Magnani; Christopher G. Tate; Malcolm Weir; Fiona H. Marshall (1283-1293).
Methylxanthines, including caffeine and theophylline, are among the most widely consumed stimulant drugs in the world. These effects are mediated primarily via blockade of adenosine receptors. Xanthine analogs with improved properties have been developed as potential treatments for diseases such as Parkinson's disease. Here we report the structures of a thermostabilized adenosine A2A receptor in complex with the xanthines xanthine amine congener and caffeine, as well as the A2A selective inverse agonist ZM241385. The receptor is crystallized in the inactive state conformation as defined by the presence of a salt bridge known as the ionic lock. The complete third intracellular loop, responsible for G protein coupling, is visible consisting of extended helices 5 and 6. The structures provide new insight into the features that define the ligand binding pocket of the adenosine receptor for ligands of diverse chemotypes as well as the cytoplasmic regions that interact with signal transduction proteins.Display Omitted► Structure of adenosine A2A receptor in ground state conformation with ionic lock ► Complete third intracellular loop present consists of extended helices 5 and 6 ► Complexes with xanthines and ZM241385 show overlapping binding interactions ► Thermostabilization enables a structure bound with the low affinity ligand caffeine

Structural Basis for Complex Formation between Human IRSp53 and the Translocated Intimin Receptor Tir of Enterohemorrhagic E. coli by Jens C. de Groot; Kai Schlüter; Yvonne Carius; Claudia Quedenau; Didier Vingadassalom; Jan Faix; Stefanie M. Weiss; Joachim Reichelt; Christine Standfuß-Gabisch; Cammie F. Lesser; John M. Leong; Dirk W. Heinz; Konrad Büssow; Theresia E.B. Stradal (1294-1306).
Actin assembly beneath enterohemorrhagic E. coli (EHEC) attached to its host cell is triggered by the intracellular interaction of its translocated effector proteins Tir and EspFU with human IRSp53 family proteins and N-WASP. Here, we report the structure of the N-terminal I-BAR domain of IRSp53 in complex with a Tir-derived peptide, in which the homodimeric I-BAR domain binds two Tir molecules aligned in parallel. This arrangement provides a protein scaffold linking the bacterium to the host cell's actin polymerization machinery. The structure uncovers a specific peptide-binding site on the I-BAR surface, conserved between IRSp53 and IRTKS. The Tir Asn-Pro-Tyr (NPY) motif, essential for pedestal formation, is specifically recognized by this binding site. The site was confirmed by mutagenesis and in vivo-binding assays. It is possible that IRSp53 utilizes the NPY-binding site for additional interactions with as yet unknown partners within the host cell.Display Omitted► The crystal structure of human IRSp53 in complex with the EHEC effector Tir ► The I-BAR domain of IRSp53 harbors an unreported binding site for NPY motifs ► Two Tir molecules are bound in parallel and contribute to a larger scaffold ► I-BAR residues essential for this interaction are validated by mutational analysis

Atomic Resolution Insights into Curli Fiber Biogenesis by Jonathan D. Taylor; Yizhou Zhou; Paula S. Salgado; Ardan Patwardhan; Matt McGuffie; Tillmann Pape; Grzegorz Grabe; Elisabeth Ashman; Sean C. Constable; Peter J. Simpson; Wei-chao Lee; Ernesto Cota; Matthew R. Chapman; Steve J. Matthews (1307-1316).
Bacteria produce functional amyloid fibers called curli in a controlled, noncytotoxic manner. These extracellular fimbriae enable biofilm formation and promote pathogenicity. Understanding curli biogenesis is important for appreciating microbial lifestyles and will offer clues as to how disease-associated human amyloid formation might be ameliorated. Proteins encoded by the curli specific genes (csgA-G) are required for curli production. We have determined the structure of CsgC and derived the first structural model of the outer-membrane subunit translocator CsgG. Unexpectedly, CsgC is related to the N-terminal domain of DsbD, both in structure and oxido-reductase capability. Furthermore, we show that CsgG belongs to the nascent class of helical outer-membrane macromolecular exporters. A cysteine in a CsgG transmembrane helix is a potential target of CsgC, and mutation of this residue influences curli assembly. Our study provides the first high-resolution structural insights into curli biogenesis.Display Omitted► CsgC is related to the redox-active N-terminal domain of DsbD ► Outer-membrane transporter CsgG inserts into the membrane via an α-helical oligomer ► The transmembrane domain of CsgG is vital for pore forming and curli assembly ► CsgC appears to affect CsgG pore behavior and biofilm formation

Crystal Structure of cGMP-Dependent Protein Kinase Reveals Novel Site of Interchain Communication by Brent W. Osborne; Jian Wu; Caitlin J. McFarland; Christian K. Nickl; Banumathi Sankaran; Darren E. Casteel; Virgil L. Woods; Alexandr P. Kornev; Susan S. Taylor; Wolfgang R. Dostmann (1317-1327).
The cGMP-dependent protein kinase (PKG) serves as an integral component of second messenger signaling in a number of biological contexts including cell differentiation, memory, and vasodilation. PKG is homodimeric and large conformational changes accompany cGMP binding. However, the structure of PKG and the molecular mechanisms associated with protomer communication following cGMP-induced activation remain unknown. Here, we report the 2.5 Å crystal structure of a regulatory domain construct (aa 78–355) containing both cGMP binding sites of PKG Iα. A distinct and segregated architecture with an extended central helix separates the two cGMP binding domains. Additionally, a previously uncharacterized helical domain (switch helix) promotes the formation of a hydrophobic interface between protomers. Mutational disruption of this interaction in full-length PKG implicates the switch helix as a critical site of dimer communication in PKG biology. These results offer new structural insight into the mechanism of allosteric PKG activation.► This presents the first crystal structure of tandem cGMP binding domains ► A novel subdomain promotes unexpected protomer-protomer communication in PKG 78–355 ► The two cGMP-binding domains remain extended despite occupation of the A-domain ► A C117-C195 disulfide bond uncouples communication between the two CNB domains

Characterization of the Structure and Function of Escherichia coli DegQ as a Representative of the DegQ-like Proteases of Bacterial HtrA Family Proteins by Xiao-chen Bai; Xi-jiang Pan; Xiao-jing Wang; Yun-ying Ye; Lei-fu Chang; Dong Leng; Jianlin Lei; Sen-Fang Sui (1328-1337).
HtrA family proteins play a central role in protein quality control in the bacterial periplasmic space. DegQ-like proteases, a group of bacterial HtrA proteins, are characterized by a short LA loop as compared with DegP-like proteases, and are found in many bacterial species. As a representative of the DegQ-like proteases, we report that Escherichia coli DegQ exists in vivo primarily as a trimer (substrate-free) or dodecamer (substrate-containing). Biochemical analysis of DegQ dodecamers revealed that the major copurified protein substrate is OmpA. Importantly, wild-type DegQ exhibited a much lower proteolytic activity, and thus higher chaperone-like activity, than DegP. Furthermore, using cryo-electron microscopy we determined high-resolution structures of DegQ 12- and 24-mers in the presence of substrate, thus revealing the structural mechanism by which DegQ moderates its proteolytic activity.Display Omitted► Wild-type DegQ exists primarily as a trimer and dodecamer in vivo ► Wild-type DegQ exhibits lower proteolytic and higher chaperone-like activities ► High-resolution cryo-EM structures of DegQ 12- and 24-mers ► A groove-like structure near catalytic center may moderate DegQ proteolytic activity

Biophysical and Computational Studies of Membrane Penetration by the GRP1 Pleckstrin Homology Domain by Craig N. Lumb; Ju He; Yi Xue; Phillip J. Stansfeld; Robert V. Stahelin; Tatiana G. Kutateladze; Mark S.P. Sansom (1338-1346).
The pleckstrin homology (PH) domain of the general receptor for phosphoinositides 1 (GRP1) exhibits specific, high-affinity, reversible binding to phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) at the plasma membrane, but the nature and extent of the interaction between this bound complex and the surrounding membrane environment remains unclear. Combining equilibrium and nonequilibrium molecular dynamics (MD) simulations, NMR spectroscopy, and monolayer penetration experiments, we characterize the membrane-associated state of GRP1-PH. MD simulations show loops flanking the binding site supplement the interaction with PI(3,4,5)P3 through multiple contacts with the lipid bilayer. NMR data show large perturbations in chemical shift for these loop regions on binding to PI(3,4,5)P3-containing DPC micelles. Monolayer penetration experiments and further MD simulations demonstrate that mutating hydrophobic residues to polar residues in the flanking loops reduces membrane penetration. This supports a “dual-recognition” model of binding, with specific GRP1-PH-PI(3,4,5)P3 interactions supplemented by interactions of loop regions with the lipid bilayer.Display Omitted► Experiment and simulation to investigate the membrane-bound state of GRP1-PH ► GRP1-PH interacts with surrounding lipids in addition to PI(3,4,5)P3 ► Three loop regions near the binding site exhibit protein-lipid contacts ► This suggests a “dual recognition” model of PH binding to membranes