Structure (v.14, #5)

Editorial by Chris Lima; Joseph D. Puglisi; Andrej Sali; Lara Szewczak (801).

Molecular Glue to Cement a Phage by Carolyn M. Teschke (803-804).
The structure of the protein Dec that “decorates” phage L capsids is described in this issue of Structure by . Intriguingly, the Dec protein trimers bind to only a subset of quasi-equivalent sites on the capsids.

Broken Symmetry in Homing Endonucleases by Frederick S. Gimble (804-806).
Homing DNA endonucleases are highly site-specific enzymes that initiate the transfer of mobile DNA elements. In this issue of Structure, Spiegel et al. report the structure of the I-CeuI homing enzyme and describe how a symmetric homodimeric enzyme acquired specificity for an asymmetric substrate.

Turning up the HEAT on Translation by Simon E.V. Phillips (806-807).
Eukaryotic Initiation Factor 4G forms the core of the translation initiation complex. Bellsolell et al. report the structure of its C-terminal region as two HEAT domains that define a new class and map binding sites for its regulatory factors ().

Deposition Diseases and 3D Domain Swapping by Melanie J. Bennett; Michael R. Sawaya; David Eisenberg (811-824).
Protein aggregation is a feature of both normal cellular assemblies and pathological protein depositions. Although the limited order of aggregates has often impeded their structural characterization, 3D domain swapping has been implicated in the formation of several protein aggregates. Here, we review known structures displaying 3D domain swapping in the context of amyloid and related fibrils, prion proteins, and macroscopic aggregates, and we discuss the possible involvement of domain swapping in protein deposition diseases.

RNA Kink-Turns as Molecular Elbows: Hydration, Cation Binding, and Large-Scale Dynamics by Filip Rázga; Martin Zacharias; Kamila Réblová; Jaroslav Koča; Jiří Šponer (825-835).
The presence of Kink-turns (Kt) at key functional sites in the ribosome (e.g., A-site finger and L7/L12 stalk) suggests that some Kink-turns can confer flexibility on RNA protuberances that regulate the traversal of tRNAs during translocation. Explicit solvent molecular dynamics demonstrates that Kink-turns can act as flexible molecular elbows. Kink-turns are associated with a unique network of long-residency static and dynamical hydration sites that is intimately involved in modulating their conformational dynamics. An implicit solvent conformational search confirms the flexibility of Kink-turns around their X-ray geometries and identifies a second low-energy region with open structures that could correspond to Kink-turn geometries seen in solution experiments. An extended simulation of Kt-42 with the factor binding site (helices 43 and 44) shows that the local Kt-42 elbow-like motion fully propagates beyond the Kink-turn, and that there is no other comparably flexible site in this rRNA region. Kink-turns could mediate large-scale adjustments of distant RNA segments.

Highly Discriminatory Binding of Capsid-Cementing Proteins in Bacteriophage L by Liang Tang; Eddie B. Gilcrease; Sherwood R. Casjens; John E. Johnson (837-845).
Cementing proteins that bind to the virion surface have been described in double-stranded DNA viruses such as herpesvirus, adenovirus, and numerous bacteriophages. The three-dimensional structure of bacteriophage L determined by electron cryo-microscopy reveals binding modes of two cementing proteins—one, called Dec, encoded by phage gene orf134 and the other by an as yet unidentified gene. These two proteins form homotrimers and bind at the quasi 3-fold axes nearest the icosahedral 2-fold axes and at the icosahedral 3-fold vertices, respectively. They do not bind at the quasi 3-fold axes near the icosahedral 5-fold vertices. These observations indicate precise recognition of the two cementing proteins at a subset of the quasi equivalent sites on the phage capsid. Sequence analysis shows striking similarity between the C-terminal portion of phage L Dec protein and five regions in the long tail fiber of a T4-like phage, suggesting functional parallelism between them.
Keywords: MICROBIO;

High-Resolution Structural Validation of the Computational Redesign of Human U1A Protein by Neil Dobson; Gautam Dantas; David Baker; Gabriele Varani (847-856).
Achieving atomic-level resolution in the computational design of a protein structure remains a challenging problem despite recent progress. Rigorous experimental tests are needed to improve protein design algorithms, yet studies of the structure and dynamics of computationally designed proteins are very few. The NMR structure and backbone dynamics of a redesigned protein of 96 amino acids are compared here with the design target, human U1A protein. We demonstrate that the redesigned protein reproduces the target structure to within the uncertainty of the NMR coordinates, even as 65 out of 96 amino acids were simultaneously changed by purely computational methods. The dynamics of the backbone of the redesigned protein also mirror those of human U1A, suggesting that the protein design algorithm captures the shape of the potential energy landscape in addition to the local energy minimum.

YfhJ, a Molecular Adaptor in Iron-Sulfur Cluster Formation or a Frataxin-like Protein? by Chiara Pastore; Salvatore Adinolfi; Martjin A. Huynen; Vladimir Rybin; Stephen Martin; Mathias Mayer; Bernd Bukau; Annalisa Pastore (857-867).
The yfhJ gene is part of the isc operon, which encodes the machinery devoted to assemble iron-sulfur clusters in prokaryotes. Its transcript is a small acidic protein that binds the desulfurase IscS, which is essential in iron-specific metabolic pathways. To understand its cellular role, we have characterized the structure of YfhJ in solution and its interactions with potential cellular partners. It contains a modified winged helix motif, usually present in DNA binding proteins, and is able to bind iron cations. The IscS interaction surface is the same as that involved in iron binding. This observation and the pattern of conservation through species strongly suggest that YfhJ is a molecular adaptor that is able to modulate the function of IscS in iron-sulfur cluster formation. The remarkable similarity between the in vitro behavior of YfhJ and that of the protein frataxin also suggests new hypotheses regarding the functional role of both proteins.

The Structure of I-CeuI Homing Endonuclease: Evolving Asymmetric DNA Recognition from a Symmetric Protein Scaffold by P. Clint Spiegel; Brett Chevalier; Django Sussman; Monique Turmel; Claude Lemieux; Barry L. Stoddard (869-880).
Homing endonucleases are highly specific catalysts of DNA strand breaks, leading to the transfer of mobile intervening sequences containing the endonuclease ORF. We have determined the structure and DNA recognition behavior of I-CeuI, a homodimeric LAGLIDADG endonuclease from Chlamydomonas eugametos. This symmetric endonuclease displays unique structural elaborations on its core enzyme fold, and it preferentially cleaves a highly asymmetric target site. This latter property represents an early step, prior to gene fusion, in the generation of asymmetric DNA binding platforms from homodimeric ancestors. The divergence of the sequence, structure, and target recognition behavior of homing endonucleases, as illustrated by this study, leads to the invasion of novel genomic sites by mobile introns during evolution.

GTP-Ras Disrupts the Intramolecular Complex of C1 and RA Domains of Nore1 by Elena Harjes; Stefan Harjes; Sabine Wohlgemuth; Karl-Heinz Müller; Elmar Krieger; Christian Herrmann; Peter Bayer (881-888).
The novel Ras effector mNore1, capable of inducing apoptosis, is a multidomain protein. It comprises a C1 domain homologous to PKC and an RA domain similar to the Ras effectors AF-6 and RalGDS. Here, we determine the affinity of these two domains to the active forms of Ras and Rap1 using isothermal calorimetric titration. The interaction of Ras/Rap1-GTP with the RA domain of mNore1 is weakened significantly by direct binding of the C1 domain to the RA domain. In order to analyze this observation in atomic detail, we solved the C1 solution structure by NMR. By determining chemical shifts and relaxation rates, we can show an intramolecular complex of C1-RA. GTP-Ras titration and binding to RA disrupts this complex and displaces the C1 domain. Once the C1 domain tumbles freely in solution, a lipid binding interface becomes accessible. Furthermore, we provide evidence of phosphatidylinositol 3-phosphate binding of the free C1 domain.

Entry of SARS coronavirus into its target cell requires large-scale structural transitions in the viral spike (S) glycoprotein in order to induce fusion of the virus and cell membranes. Here we describe the identification and crystal structures of four distinct α-helical domains derived from the highly conserved heptad-repeat (HR) regions of the S2 fusion subunit. The four domains are an antiparallel four-stranded coiled coil, a parallel trimeric coiled coil, a four-helix bundle, and a six-helix bundle that is likely the final fusogenic form of the protein. When considered together, the structural and thermodynamic features of the four domains suggest a possible mechanism whereby the HR regions, initially sequestered in the native S glycoprotein spike, are released and refold sequentially to promote membrane fusion. Our results provide a structural framework for understanding the control of membrane fusion and should guide efforts to intervene in the SARS coronavirus entry process.

Structure of the Mammalian NOS Regulator Dimethylarginine Dimethylaminohydrolase: A Basis for the Design of Specific Inhibitors by Daniel Frey; Oliver Braun; Christophe Briand; Milan Vašák; Markus G. Grütter (901-911).
Dimethylarginine dimethylaminohydrolase (DDAH) is involved in the regulation of nitric oxide synthase (NOS) by metabolizing the free endogenous arginine derivatives Nω -methyl-L-arginine (MMA) and Nω,Nω -dimethyl-L-arginine (ADMA), which are competitive inhibitors of NOS. Here, we present high-resolution crystal structures of DDAH isoform 1 (DDAH-1) isolated from bovine brain in complex with different inhibitors, including S-nitroso-L-homocysteine and Zn2+, a regulator of this mammalian enzyme. The structure of DDAH-1 consists of a propeller-like fold similar to other arginine-modifying enzymes and a flexible loop, which adopts different conformations and acts as a lid at the entrance of the active site. The orientation and interaction mode of inhibitors in the active site give insight into the regulation and the molecular mechanism of the enzyme. The presented structures provide a basis for the structure-based development of specific DDAH-1 inhibitors that might be useful in the therapeutic treatment of NOS dysfunction-related diseases.

Two Structurally Atypical HEAT Domains in the C-Terminal Portion of Human eIF4G Support Binding to eIF4A and Mnk1 by Lluís Bellsolell; Park F. Cho-Park; Francis Poulin; Nahum Sonenberg; Stephen K. Burley (913-923).
The X-ray structure of the C-terminal region of human eukaryotic translation initiation factor 4G (eIF4G) has been determined at 2.2 Å resolution, revealing two atypical HEAT-repeat domains. eIF4G recruits various translation factors and the 40S ribosomal subunit to the mRNA 5′ end. In higher eukaryotes, the C terminus of eIF4G (4G/C) supports translational regulation by recruiting eIF4A, an RNA helicase, and Mnk1, the kinase responsible for phosphorylating eIF4E. Structure-guided surface mutagenesis and protein-protein interaction assays were used to identify binding sites for eIF4A and Mnk1 within the HEAT-repeats of 4G/C. p97/DAP5, a translational modulator homologous to eIF4G, lacks an eIF4A binding site in the corresponding region. The second atypical HEAT domain of the 4G/C binds Mnk1 using two conserved aromatic/acidic-box (AA-box) motifs. Within the first AA-box, the aromatic residues contribute to the hydrophobic core of the domain, while the acidic residues form a negatively charged surface feature suitable for electrostatic interactions with basic residues in Mnk1.
Keywords: RNA;

Rounding up: Engineering 12-Membered Rings from the Cyclic 11-Mer TRAP by Jonathan G. Heddle; Takeshi Yokoyama; Ichiro Yamashita; Sam-Yong Park; Jeremy R.H. Tame (925-933).
The protein TRAP (trp RNA binding attenuation protein) forms a highly thermostable ring-shaped 11-mer. By linking in tandem two, three, or four DNA sequences encoding TRAP monomers, we have engineered new rings that consist of 12 TRAP subunits and bind 12 ligand molecules. The hydrogen bonding pattern and buried surface area within and between subunits are essentially identical between the 11-mer and 12-mer crystal structures. Why do the artificial proteins choose to make single 12-mer rings? The 12-mer rings are highly sterically strained by their peptide linkers and far from thermostable. That proteins choose to adopt a strained conformation of few subunits rather than an unstrained one with 11 subunits demonstrates the importance of entropic factors in controlling protein-protein interactions in general.

Divergence of Interdomain Geometry in Two-Domain Proteins by Jung-Hoon Han; Nicola Kerrison; Cyrus Chothia; Sarah A. Teichmann (935-945).
For homologous protein chains composed of two domains, we have determined the extent to which they conserve (1) their interdomain geometry and (2) the molecular structure of the domain interface. This work was carried out on 128 unique two-domain architectures. Of the 128, we find 75 conserve their interdomain geometry and the structure of their domain interface; 5 conserve their interdomain geometry but not the structure of their interface; and 48 have variable geometries and divergent interface structure. We describe how different types of interface changes or the absence of an interface is responsible for these differences in geometry. Variable interdomain geometries can be found in homologous structures with high sequence identities (70%).