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Structure (v.17, #5)

In This Issue (pp. ix-x).

Flexibility Promotes Fidelity by J. Jefferson P. Perry; Kenichi Hitomi; John A. Tainer (pp. 633-634).
Y-family lesion bypass polymerases can process 8-oxoguanine, a potentially mutagenic form of oxidative DNA damage, error-free. In this issue of Structure, Rechkoblit et al. reveal that Y-family polymerases overcome 8-oxoguanine:adenine mismatches using a mechanism that screens for conformational heterogeneity.

One More Piece in the Fibrillin Puzzle by Dirk Hubmacher; Dieter P. Reinhardt (pp. 635-636).
Jensen et al. report the crystal structure of a human fibrillin-1 hybrid domain in this issue of Structure. This domain is found exclusively in the fibrillin/latent transforming growth factor-β binding protein superfamily and shares structural features with two other domains in these proteins, the TB/8-Cys and the cbEGF domains.

Plant Photosystem I Design in the Light of Evolution by Alexey Amunts; Nathan Nelson (pp. 637-650).
Photosystem I (PSI) is a membrane protein complex that catalyzes sunlight-driven transmembrane electron transfer as part of the photosynthetic machinery. Photosynthetic organisms appeared on the Earth about 3.5 billion years ago and provided an essential source of potential energy for the development of life. During the course of evolution, these primordial organisms were phagocytosed by more sophisticated eukaryotic cells, resulting in the evolvement of algae and plants. Despite the extended time interval between primordial cyanobacteria and plants, PSI has retained its fundamental mechanism of sunlight conversion. Being probably the most efficient photoelectric apparatus in nature, PSI operates with a quantum efficiency close to 100%. However, adapting to different ecological niches necessitated structural changes in the PSI design. Based on the recently solved structure of plant PSI, which revealed a complex of 17 protein subunits and 178 prosthetic groups, we analyze the evolutionary development of PSI. In addition, some aspects of PSI structure determination are discussed.

Structure of the Noncatalytic Domains and Global Fold of the Protein Disulfide Isomerase ERp72 by Guennadi Kozlov; Pekka Määttänen; Joseph D. Schrag; Greg L. Hura; Lisa Gabrielli; Miroslaw Cygler; David Y. Thomas; Kalle Gehring (pp. 651-659).
Protein disulfide isomerases are a family of proteins that catalyze the oxidation and isomerization of disulfide bonds in newly synthesized proteins in the endoplasmic reticulum. The family includes general enzymes such as PDI that recognize unfolded proteins, and others that are selective for specific classes of proteins. Here, we report the X-ray crystal structure of central non-catalytic domains of a specific isomerase, ERp72 (also called CaBP2 and protein disulfide-isomerase A4) from Rattus norvegicus. The structure reveals strong similarity to ERp57, a PDI-family member that interacts with the lectin-like chaperones calnexin and calreticulin but, unexpectedly, ERp72 does not interact with calnexin as shown by isothermal titration calorimetry and nuclear magnetic resonance (NMR) spectroscopy. Small-angle X-ray scattering (SAXS) of ERp72 was used to develop models of the full-length protein using both rigid body refinement and ab initio simulated annealing of dummy atoms. The two methods show excellent agreement and define the relative positions of the five thioredoxin-like domains of ERp72 and potential substrate or chaperone binding sites.

Keywords: PROTEINS

Autoinhibitory Interactions between the PDZ2 and C-terminal Domains in the Scaffolding Protein NHERF1 by Hong Cheng; Jianquan Li; Ruzaliya Fazlieva; Zhongping Dai; Zimei Bu; Heinrich Roder (pp. 660-669).
Na+/H+ exchanger regulatory factor (NHERF1) is a signaling adaptor protein comprising two PDZ domains and a C-terminal ezrin-binding (EB) motif. To understand the role of intramolecular interactions in regulating its binding properties, we characterized the complex between the second PDZ domain PDZ2 and the C-terminal 242–358 fragment of NHERF1 using NMR and fluorescence methods. NMR chemical shift and relaxation data implicate 11 C-terminal residues in binding and, together with a thermodynamic analysis of mutant proteins, indicate that the EB region becomes helical when bound to PDZ2. Both specific contacts between PDZ2 and EB as well as nonspecific interactions involving a 100-residue flexible linker contribute to stabilizing two structurally distinct closed conformations of NHERF1. The affinity of mutant proteins for an extrinsic ligand is inversely related to the helix-forming propensity of the EB motif. The findings provide a structural framework for understanding how autoinhibitory interactions modulated the binding properties of NHERF1.


Structure and Site-Specific Recognition of Histone H3 by the PHD Finger of Human Autoimmune Regulator by Suvobrata Chakravarty; Lei Zeng; Ming-Ming Zhou (pp. 670-679).
Human autoimmune regulator (AIRE) functions to control thymic expression of tissue-specific antigens via sequence-specific histone H3 recognition by its plant homeodomain (PHD) finger. Mutations in the AIRE PHD finger have been linked to autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Here we report the three-dimensional solution structure of the first PHD finger of human AIRE bound to a histone H3 peptide. The structure reveals a detailed network of interactions between the protein and the amino-terminal residues of histone H3, and particularly key electrostatic interactions of a conserved aspartic acid 297 in AIRE with the unmodified lysine 4 of histone H3 (H3K4). NMR binding study with H3 peptides carrying known posttranslational modifications flanking H3K4 confirms that transcriptional regulation by AIRE through its interactions with histone H3 is confined to the first N-terminal eight residues in H3. Our study offers a molecular explanation for the APECED mutations and helps define a subclass of the PHD finger family proteins that recognize histone H3 in a sequence-specific manner.

Keywords: PROTEIN; DNA

Polymerase Translocation with Respect to Single-Stranded Nucleic Acid: Looping or Wrapping of Primer around a Poly(A) Polymerase by ChangZheng Li; Huiying Li; SuFeng Zhou; Eric Sun; Janice Yoshizawa; Thomas L. Poulos; Paul D. Gershon (pp. 680-689).
Vaccinia virus protein VP55 translocates continuously with respect to single-stranded nucleic acid while extending its 3′end. Here, all key sites of polymerase-primer interaction were identified, demonstrating the wrapping or looping of polyadenylation primer around the polymerase during translocation. Side-chain substitutions at one of the sites indicated its requirement for tail extension beyond ∼12 nucleotides in length, and conformational changes observed upon oligonucleotide binding suggested allosteric connectivity during translocation. Conformational changes in VP39 upon VP55 binding suggested that, within the VP55-VP39 complex, VP39's mRNA 5′ cap binding site closes. The crystallographic structure showed a PAPase catalytic center without side-chain substitutions, possessing two metal ions and with all known reactive and catalytic groups represented, fitting a classical two-metal ion mechanism for phosphoryl transfer.


Crystal Structure of E. coli RecE Protein Reveals a Toroidal Tetramer for Processing Double-Stranded DNA Breaks by Jinjin Zhang; Xu Xing; Andrew B. Herr; Charles E. Bell (pp. 690-702).
Escherichia coli RecE protein is part of the classical RecET recombination system that has recently been used in powerful new methods for genetic engineering. RecE binds to free double-stranded DNA (dsDNA) ends and processively digests the 5′-ended strand to form 5′-mononucleotides and a 3′-overhang that is a substrate for single strand annealing promoted by RecT. Here, we report the crystal structure of the C-terminal nuclease domain of RecE at 2.8 Å resolution. RecE forms a toroidal tetramer with a central tapered channel that is wide enough to bind dsDNA at one end, but is partially plugged at the other end by the C-terminal segment of the protein. Four narrow tunnels, one within each subunit of the tetramer, lead from the central channel to the four active sites, which lie about 15 Å from the channel. The structure, combined with mutational studies, suggests a mechanism in which dsDNA enters through the open end of the central channel, the 5′-ended strand passes through a tunnel to access one of the four active sites, and the 3′-ended strand passes through the plugged end of the channel at the back of the tetramer.

Keywords: DNA

Crystal Structures of Two Archaeal 8-Oxoguanine DNA Glycosylases Provide Structural Insight into Guanine/8-Oxoguanine Distinction by Frédérick Faucher; Stéphanie Duclos; Viswanath Bandaru; Susan S. Wallace; Sylvie Doublié (pp. 703-712).
Among the four DNA bases, guanine is particularly vulnerable to oxidative damage and the most common oxidative product, 7,8-dihydro-8-oxoguanine (8-oxoG), is the most prevalent lesion observed in DNA molecules. Fortunately, 8-oxoG is recognized and excised by the 8-oxoguanine DNA glycosylase (Ogg) of the base excision repair pathway. Ogg enzymes are divided into three separate families, namely, Ogg1, Ogg2, and archaeal GO glycosylase (AGOG). To date, structures of members of both Ogg1 and AGOG families are known but no structural information is available for members of Ogg2. Here we describe the first crystal structures of two archaeal Ogg2: Methanocaldococcus janischii Ogg and Sulfolobus solfataricus Ogg. A structural comparison with OGG1 and AGOG suggested that the C-terminal lysine of Ogg2 may play a key role in discriminating between guanine and 8-oxoG. This prediction was substantiated by measuring the glycosylase/lyase activity of a C-terminal deletion mutant of MjaOgg.


Conserved Cysteine Residues of GidA Are Essential for Biogenesis of 5-Carboxymethylaminomethyluridine at tRNA Anticodon by Takuo Osawa; Koichi Ito; Hideko Inanaga; Osamu Nureki; Kozo Tomita; Tomoyuki Numata (pp. 713-724).
The 5-carboxymethylaminomethyl modification of uridine (cmnm5U) at the anticodon first position occurs in tRNAs that read split codon boxes ending with purine. This modification is crucial for correct translation, by restricting codon-anticodon wobbling. Two conserved enzymes, GidA and MnmE, participate in the cmnm5U modification process. Here we determined the crystal structure of Aquifex aeolicus GidA at 2.3 Å resolution. The structure revealed the tight interaction of GidA with FAD. Structure-based mutation analyses allowed us to identify two conserved Cys residues in the vicinity of the FAD-binding site that are essential for the cmnm5U modification in vivo. Together with mutational analysis of MnmE, we propose a mechanism for the cmnm5U modification process where GidA, but not MnmE, attacks the C6 atom of uridine by a mechanism analogous to that of thymidylate synthase. We also present a tRNA-docking model that provides structural insights into the tRNA recognition mechanism for efficient modification.


Impact of Conformational Heterogeneity of OxoG Lesions and Their Pairing Partners on Bypass Fidelity by Y Family Polymerases by Olga Rechkoblit; Lucy Malinina; Yuan Cheng; Nicholas E. Geacintov; Suse Broyde; Dinshaw J. Patel (pp. 725-736).
7,8-Dihydro-8-oxoguanine (oxoG), the predominant oxidative DNA damage lesion, is processed differently by high-fidelity and Y-family lesion bypass polymerases. Although high-fidelity polymerases extend predominantly from an A base opposite an oxoG, the Y-family polymerases Dpo4 and human Pol η preferentially extend from the oxoG•C base pair. We have determined crystal structures of extension Dpo4 ternary complexes with oxoG opposite C, A, G, or T and the next nascent base pair. We demonstrate that neither template backbone nor the architecture of the active site is perturbed by the oxoG (anti)•C and oxoG•A pairs. However, the latter manifest conformational heterogeneity, adopting both oxoG (syn)•A (anti) and oxoG (anti)•A (syn) alignment. Hence, the observed reduced primer extension from the dynamically flexible 3′-terminal primer base A is explained. Because of homology between Dpo4 and Pol η, such a dynamic screening mechanism might be utilized by Dpo4 and Pol η to regulate error-free versus error-prone bypass of oxoG and other lesions.


Proton-Linked Dimerization of a Retroviral Capsid Protein Initiates Capsid Assembly by Graham D. Bailey; Jae K. Hyun; Alok K. Mitra; Richard L. Kingston (pp. 737-748).
In mature retroviral particles, the capsid protein (CA) forms a shell encasing the viral replication complex. Human immunodeficiency virus (HIV) CA dimerizes in solution, through its C-terminal domain (CTD), and this interaction is important for capsid assembly. In contrast, other retroviral capsid proteins, including that of Rous sarcoma virus (RSV), do not dimerize with measurable affinity. Here we show, using X-ray crystallography and other biophysical methods, that acidification causes RSV CA to dimerize in a fashion analogous to HIV CA, and that this drives capsid assembly in vitro. A pair of aspartic acid residues, located within the CTD dimer interface, explains why dimerization is linked to proton binding. Our results show that despite overarching structural similarities, the intermolecular forces responsible for forming and stabilizing the retroviral capsid differ markedly across retroviral genera. Our data further suggest that proton binding may regulate RSV capsid assembly, or modulate stability of the assembled capsid.


Structural Mechanism of SDS-Induced Enzyme Activity of Scorpion Hemocyanin Revealed by Electron Cryomicroscopy by Yao Cong; Qinfen Zhang; David Woolford; Thorsten Schweikardt; Htet Khant; Matthew Dougherty; Steven J. Ludtke; Wah Chiu; Heinz Decker (pp. 749-758).
Phenoloxidases (POs) occur in all organisms and are involved in skin and hair coloring in mammals, and initiating melanization in wound healing. Mutation or overexpression of PO can cause albinism or melanoma, respectively. SDS can convert inactive PO and the oxygen carrier hemocyanin (Hc) into enzymatically active PO. Here we present single-particle cryo-EM maps at subnanometer resolution and pseudoatomic models of the 24-oligomeric Hc from scorpion Pandinus imperator in resting and SDS-activated states. Our structural analyses led to a plausible mechanism of Hc enzyme PO activation: upon SDS activation, the intrinsically flexible Hc domain I twists away from domains II and III in each subunit, exposing the entrance to the active site; this movement is stabilized by enhanced interhexamer and interdodecamer interactions, particularly in the central linker subunits. This mechanism could be applicable to other type 3 copper proteins, as the active site is highly conserved.

Keywords: PROTEINS

Structure and Interdomain Interactions of a Hybrid Domain: A Disulphide-Rich Module of the Fibrillin/LTBP Superfamily of Matrix Proteins by Sacha A. Jensen; Sarah Iqbal; Edward D. Lowe; Christina Redfield; Penny A. Handford (pp. 759-768).
The fibrillins and latent transforming growth factor-β binding proteins (LTBPs) form a superfamily of structurally-related proteins consisting of calcium-binding epidermal growth factor-like (cbEGF) domains interspersed with 8-cysteine-containing transforming growth factor β-binding protein-like (TB) and hybrid (hyb) domains. Fibrillins are the major components of the extracellular 10–12 nm diameter microfibrils, which mediate a variety of cell-matrix interactions. Here we present the crystal structure of a fibrillin-1 cbEGF9-hyb2-cbEGF10 fragment, solved to 1.8 Å resolution. The hybrid domain fold is similar, but not identical, to the TB domain fold seen in previous fibrillin-1 and LTBP-1 fragments. Pairwise interactions with neighboring cbEGF domains demonstrate extensive interfaces, with the hyb2-cbEGF10 interface dependent on Ca2+ binding. These observations provide accurate constraints for models of fibrillin organization within the 10–12 nm microfibrils and provide further molecular insights into how Ca2+ binding influences the intermolecular interactions and biomechanical properties of fibrillin-1.


Recognition of AT-Rich DNA Binding Sites by the MogR Repressor by Aimee Shen; Darren E. Higgins; Daniel Panne (pp. 769-777).
The MogR transcriptional repressor of the intracellular pathogen Listeria monocytogenes recognizes AT-rich binding sites in promoters of flagellar genes to downregulate flagellar gene expression during infection. We describe here the 1.8 Å resolution crystal structure of MogR bound to the recognition sequence 5′ ATTTTTTAAAAAAAT 3′ present within the flaA promoter region. Our structure shows that MogR binds as a dimer. Each half-site is recognized in the major groove by a helix-turn-helix motif and in the minor groove by a loop from the symmetry-related molecule, resulting in a “crossover” binding mode. This oversampling through minor groove interactions is important for specificity. The MogR binding site has structural features of A-tract DNA and is bent by ∼52° away from the dimer. The structure explains how MogR achieves binding specificity in the AT-rich genome of L. monocytogenes and explains the evolutionary conservation of A-tract sequence elements within promoter regions of MogR-regulated flagellar genes.

Keywords: DNA

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