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Structure (v.18, #2)

In This Issue (pp. ix-x).

Werner Helicase Wings DNA Binding by Kelly A. Hoadley; James L. Keck (pp. 149-151).
In this issue of Structure, Kitano et al. describe the structure of the DNA-bound winged-helix domain from the Werner helicase. This structure of a RecQ/DNA complex offers insights into the DNA-unwinding mechanisms of RecQ family helicases.

TPR Motifs: Hallmarks of a New Polysaccharide Export Scaffold by Chris Whitfield; Iain L. Mainprize (pp. 151-153).
Bacteria produce a remarkable range of surface and secreted polysaccharides. Two pathways have been defined for the biosynthesis and export of capsular polysaccharides and exopolysaccharides in Gram-negative bacteria. A structure of AlgK described in this issue provides structural insight into a third previously unrecognized pathway associated with important biopolymers ().

Structure of D-AKAP2:PKA RI Complex: Insights into AKAP Specificity and Selectivity by Ganapathy N. Sarma; Francis S. Kinderman; Choel Kim; Sventja von Daake; Lirong Chen; Bi-Cheng Wang; Susan S. Taylor (pp. 155-166).
A-kinase anchoring proteins (AKAPs) regulate cyclic AMP-dependent protein kinase (PKA) signaling in space and time. Dual-specific AKAP 2 (D-AKAP2) binds to the dimerization/docking (D/D) domain of both RI and RII regulatory subunits of PKA with high affinity. Here we have determined the structures of the RIα D/D domain alone and in complex with D-AKAP2. The D/D domain presents an extensive surface for binding through a well-formed N-terminal helix, and this surface restricts the diversity of AKAPs that can interact. The structures also underscore the importance of a redox-sensitive disulfide in affecting AKAP binding. An unexpected shift in the helical register of D-AKAP2 compared to the RIIα:D-AKAP2 complex structure makes the mode of binding to RIα novel. Finally, the comparison allows us to deduce a molecular explanation for the sequence and spatial determinants of AKAP specificity.► Crystal structures of the PKA RI isoform alone and in complex with D-AKAP2 ► Structures reveal the importance of a redox-sensitive disulfide in AKAP binding ► Unexpected shift in D-AKAP2 helical register compared to RII:D-AKAP2 structure ► Differential binding mode helps decipher the code for isoform-specific AKAP docking


Comparison of the Structures and Peptide Binding Specificities of the BRCT Domains of MDC1 and BRCA1 by Stephen J. Campbell; Ross A. Edwards; J.N. Mark Glover (pp. 167-176).
The tandem BRCT domains of BRCA1 and MDC1 facilitate protein signaling at DNA damage foci through specific interactions with serine-phosphorylated protein partners. The MDC1 BRCT binds pSer-Gln-Glu-Tyr-COO at the C terminus of the histone variant γH2AX via direct recognition of the C-terminal carboxylate, while BRCA1 recognizes pSer-X-X-Phe motifs either at C-terminal or internal sites within target proteins. Using fluorescence polarization binding assays, we show that while both BRCTs prefer a free main chain carboxylate at the +3 position, this preference is much more pronounced in MDC1. Crystal structures of BRCA1 and MDC1 bound to tetrapeptide substrates reveal differences in the environment of conserved arginines (Arg1699 in BRCA1 and Arg1933 in MDC1) that determine the relative affinity for peptides with –COO versus –CO-NH2 termini. A mutation in MDC1 that induces a more BRCA1-like conformation relaxes the binding specificity, allowing the mutant to bind phosphopeptides lacking a –COO terminus.► BRCA1 and MDC1 are essential to the DNA damage response through interactions with phosphorylated binding partners ► MDC1 has a stronger preference for a free carboxylate tail than BRCA1 ► A reciprocal mutation dramatically reduces the preference of MDC1 for a free carboxylate tail, but does not affect the specificity of BRCA1 ► Preference is determined by the nature of a binding pocket 3 residues C-terminal to the phosphorylated residue


Structural Basis for DNA Strand Separation by the Unconventional Winged-Helix Domain of RecQ Helicase WRN by Ken Kitano; Sun-Yong Kim; Toshio Hakoshima (pp. 177-187).
The RecQ family of DNA helicases including WRN ( We rner syndrome protein) and BLM ( Bloo m syndrome protein) protects the genome against deleterious changes. Here we report the cocrystal structure of the Rec Q C-terminal (RQC) domain of human WRN bound to a DNA duplex. In the complex, the RQC domain specifically interacted with a blunt end of the duplex and, surprisingly, unpaired a Watson-Crick base pair in the absence of an ATPase domain. The β wing, an extended hairpin motif that is characteristic of winged-helix motifs, was used as a “separating knife” to wedge between the first and second base pairs, whereas the recognition helix, a principal component of helix-turn-helix motifs that are usually embedded within DNA grooves, was unprecedentedly excluded from the interaction. Our results demonstrate a function of the winged-helix motif central to the helicase reaction, establishing the first structural paradigm concerning the DNA structure-specific activities of the RecQ helicases.

Keywords: DNA

The Structural Basis of Peptide-Protein Binding Strategies by Nir London; Dana Movshovitz-Attias; Ora Schueler-Furman (pp. 188-199).
Peptide-protein interactions are very prevalent, mediating key processes such as signal transduction and protein trafficking. How can peptides overcome the entropic cost involved in switching from an unstructured, flexible peptide to a rigid, well-defined bound structure? A structure-based analysis of peptide-protein interactions unravels that most peptides do not induce conformational changes on their partner upon binding, thus minimizing the entropic cost of binding. Furthermore, peptides display interfaces that are better packed than protein-protein interfaces and contain significantly more hydrogen bonds, mainly those involving the peptide backbone. Additionally, “hot spot” residues contribute most of the binding energy. Finally, peptides tend to bind in the largest pockets available on the protein surface. Our study is based on peptiDB, a new and comprehensive data set of 103 high-resolution peptide-protein complex structures. In addition to improved understanding of peptide-protein interactions, our findings have direct implications for the structural modeling, design, and manipulation of these interactions.► Most peptides do not induce conformational changes on their partner upon binding ► Peptide-protein interfaces are better packed and contain more hydrogen bonds ► Binding is mediated by peptide hotspots that contribute most of the binding energy ► Peptides tend to bind in the largest pockets or holes on the protein surface.

Keywords: PROTEINS

SusG: A Unique Cell-Membrane-Associated α-Amylase from a Prominent Human Gut Symbiont Targets Complex Starch Molecules by Nicole M. Koropatkin; Thomas J. Smith (pp. 200-215).
SusG is an α-amylase and part of a large protein complex on the outer surface of the bacterial cell and plays a major role in carbohydrate acquisition by the animal gut microbiota. Presented here, the atomic structure of SusG has an unusual extended, bilobed structure composed of amylase at one end and an unprecedented internal carbohydrate-binding motif at the other. Structural studies further demonstrate that the carbohydrate-binding motif binds maltooligosaccharide distal to, and on the opposite side of, the amylase catalytic site. SusG has an additional starch-binding site on the amylase domain immediately adjacent to the active cleft. Mutagenesis analysis demonstrates that these two additional starch-binding sites appear to play a role in catabolism of insoluble starch. However, elimination of these sites has only a limited effect, suggesting that they may have a more important role in product exchange with other Sus components.► SusG is an outer membrane α-amylase with an extended bi-lobed structure ► One end is the α-amylase and the other is a novel starch-binding domain ► A second accessory starch-binding site lies immediately adjacent to the active site ► The starch-binding sites are likely key for interacting with other Sus proteins.


Structure of the Saccharomyces cerevisiae Cet1-Ceg1 mRNA Capping Apparatus by Meigang Gu; Kanagalaghatta R. Rajashankar; Christopher D. Lima (pp. 216-227).
The 5′ guanine-N7 cap is the first cotranscriptional modification of messenger RNA. In Saccharomyces cerevisiae, the first two steps in capping are catalyzed by the RNA triphosphatase Cet1 and RNA guanylyltransferase Ceg1, which form a complex that is directly recruited to phosphorylated RNA polymerase II (RNAP IIo), primarily via contacts between RNAP IIo and Ceg1. A 3.0 Å crystal structure of Cet1-Ceg1 revealed a 176 kDa heterotetrameric complex composed of one Cet1 homodimer that associates with two Ceg1 molecules via interactions between the Ceg1 oligonucleotide binding domain and an extended Cet1 WAQKW amino acid motif. The WAQKW motif is followed by a flexible linker that would allow Ceg1 to achieve conformational changes required for capping while maintaining interactions with both Cet1 and RNAP IIo. The impact of mutations as assessed through genetic analysis in S. cerevisiae is consonant with contacts observed in the Cet1-Ceg1 structure.

Keywords: RNA

Crystal Structure of a Legionella pneumophila Ecto -Triphosphate Diphosphohydrolase, A Structural and Functional Homolog of the Eukaryotic NTPDases by Julian P. Vivian; Patrice Riedmaier; Honghua Ge; Jérôme Le Nours; Fiona M. Sansom; Matthew C.J. Wilce; Emma Byres; Manisha Dias; Jason W. Schmidberger; Peter J. Cowan; Anthony J.F. d'Apice; Elizabeth L. Hartland; Jamie Rossjohn; Travis Beddoe (pp. 228-238).
Many pathogenic bacteria have sophisticated mechanisms to interfere with the mammalian immune response. These include the disruption of host extracellular ATP levels that, in humans, is tightly regulated by the nucleoside triphosphate diphosphohydrolase family (NTPDases). NTPDases are found almost exclusively in eukaryotes, the notable exception being their presence in some pathogenic prokaryotes. To address the function of bacterial NTPDases, we describe the structures of an NTPDase from the pathogen Legionella pneumophila ( Lpg1905/Lp1NTPDase) in its apo state and in complex with the ATP analog AMPPNP and the subtype-specific NTPDase inhibitor ARL 67156. Lp1NTPDase is structurally and catalytically related to eukaryotic NTPDases and the structure provides a basis for NTPDase-specific inhibition. Furthermore, we demonstrate that the activity of Lp1NTPDase correlates directly with intracellular replication of Legionella within macrophages. Collectively, these findings provide insight into the mechanism of this enzyme and highlight its role in host-pathogen interactions.


Structural Interactions between Collagen and Proteoglycans Are Elucidated by Three-Dimensional Electron Tomography of Bovine Cornea by Philip N. Lewis; Christian Pinali; Robert D. Young; Keith M. Meek; Andrew J. Quantock; Carlo Knupp (pp. 239-245).
Interactions between collagens and proteoglycans help define the structure and function of extracellular matrices. The cornea, which contains proteoglycans with keratan sulphate or chondroitin/dermatan sulphate glycosaminoglycan chains, is an excellent model system in which to study collagen-proteoglycan structures and interactions. Here, we present the first three-dimensional electron microscopic reconstructions of the cornea, and these include corneas from which glycosaminoglycans have been selectively removed by enzymatic digestion. Our reconstructions show that narrow collagen fibrils associate with sulphated proteoglycans that appear as extended, variable-length linear structures. The proteoglycan network appears to tether two or more collagen fibrils, and thus organize the matrix with enough spatial specificity to fulfill the requirements for corneal transparency. Based on the data, we propose that the characteristic pseudohexagonal fibril arrangement in cornea is controlled by the balance of a repulsive force arising from osmotic pressure and an attractive force due to the thermal motion of the proteoglycans.► Electron tomography shows corneal matrix structure in 3D ► Collagen-proteoglycan interactions are more complex than previously believed ► Proteoglycans determine collagen interfibrillar spacing ► Proteoglycans likely co-associate in an antiparallel manner


Crystal Structure of the p53 Core Domain Bound to a Full Consensus Site as a Self-Assembled Tetramer by Yongheng Chen; Raja Dey; Lin Chen (pp. 246-256).
Recent studies suggest that p53 binds predominantly to consensus sites composed of two decameric half-sites with zero spacing in vivo. Here we report the crystal structure of the p53 core domain bound to a full consensus site as a tetramer at 2.13Å resolution. Comparison with previously reported structures of p53 dimer:DNA complexes and a chemically trapped p53 tetramer:DNA complex reveals that DNA binding by the p53 core domain is a cooperative self-assembling process accompanied by structural changes of the p53 dimer and DNA. Each p53 monomer interacts with its two neighboring subunits through two different protein-protein interfaces. The DNA is largely B-form and shows no discernible bend, but the central base-pairs between the two half-sites display a significant slide. The extensive protein-protein and protein-DNA interactions explain the high cooperativity and kinetic stability of p53 binding to contiguous decameric sites and the conservation of such binding-site configuration in vivo.


Direct Observation of Distinct A/P Hybrid-State tRNAs in Translocating Ribosomes by John F. Flanagan IV; Olivier Namy; Ian Brierley; Robert J.C. Gilbert (pp. 257-264).
Transfer RNAs (tRNAs) link the genetic code in the form of messenger RNA (mRNA) to protein sequence. Translocation of tRNAs through the ribosome from aminoacyl (A) site to peptidyl (P) site and from P site to exit site is catalyzed in eukaryotes by the translocase elongation factor 2 (EF-2) and in prokaryotes by its homolog EF-G. During tRNA movement one or more “hybrid” states (A/P) is occupied, but molecular details of them and of the translocation process are limited. Here we show by cryo-electron microscopy that a population of mammalian ribosomes stalled at an mRNA pseudoknot structure contains structurally distorted tRNAs in two different A/P hybrid states. In one (A/P′), the tRNA is in contact with the translocase EF-2, which induces it. In the other (A/P″), the translocase is absent. The existence of these alternative A/P intermediate states has relevance to our understanding of the mechanics and kinetics of translocation.


AlgK Is a TPR-Containing Protein and the Periplasmic Component of a Novel Exopolysaccharide Secretin by Carrie-Lynn Keiski; Michael Harwich; Sumita Jain; Ana Mirela Neculai; Patrick Yip; Howard Robinson; John C. Whitney; Laura Riley; Lori L. Burrows; Dennis E. Ohman; P. Lynne Howell (pp. 265-273).
The opportunistic pathogen Pseudomonas aeruginosa causes chronic biofilm infections in cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by overproduction of the exopolysaccharide alginate. Here we show that AlgK, a protein essential for production of high molecular weight alginate, is an outer membrane lipoprotein that contributes to the correct localization of the porin AlgE. Our 2.5 Å structure shows AlgK is composed of 9.5 tetratricopeptide-like repeats, and three putative sites of protein-protein interaction have been identified. Bioinformatics analysis suggests that BcsA, PgaA, and PelB, involved in the production and export of cellulose, poly-β-1,6- N-Acetyl-d-glucosamine, and Pel exopolysaccharide, respectively, share the same topology as AlgK/E. Together, our data suggest that AlgK plays a role in the assembly of the alginate biosynthetic complex and represents the periplasmic component of a new type of outer membrane secretin that differs from canonical bacterial capsular polysaccharide secretion systems.Display Omitted► AlgK is an outer membrane lipoprotein ► AlgK contributes to the correct localization of the porin AlgE ► AlgK is composed of 9.5 tetratricopeptide-like repeats ► AlgE/K, BcsA, PgaA and PelB represent a new family of outer membrane secretins.


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