Structure (v.13, #1)

A Response to Reimportation by Marcia Newcomer (1-2).
In this issue of Structure, describe elements of an innate defense mechanism that provides mammals a means to restrict bacterial growth. The host protein siderocalin scavenges struc turally dissimilar bacterial siderophores and prevents the uptake of Fe3+ already earmarked for bacterial import.

Oxygen, Metabolism, and Gene Expression: The T-Rex Connection by Matthew J. Wood; Gisela Storz (2-4).
In this issue of Structure, report the structure of the redox-sensing repressor from the gram-positive bacterium Thermus aquaticus (T-Rex), a protein that links gene expression to oxygen limitation and the metabolic state of the cell.

Resolving Protein Structure Dynamically by Sergey Yeremenko; Klaas J. Hellingwerf (4-6).
In this issue of Structure, report further innovations in the X-ray diffraction analysis of the dynamical changes in protein conformation; they use these methods to resolve the light-activated changes in the conformation of Photoactive Yellow Protein. This approach allows a straightforward reinforcement of X-ray diffraction data with spectroscopic data.

Oligomerization is important for the structure and function of many proteins, but frequently complicates their characterization. It is often desirable to obtain the protein in monomeric form. Here, we report a strategy that allows the generation of monomers from weakly associated oligomers but does not require knowledge of the three-dimensional structure of the protein. The dynamics of protein association are used in solution NMR spectroscopy to identify regions of the polypeptide chain that are likely to be responsible for the interaction. Protein sequence analysis further refines the selection, as conserved sites with moderate hydrophobicity are targeted for modification. Gel filtration and activity assays straightforwardly reveal the consequences of the change and are used to screen for the desired mutants. The strategy is demonstrated for the Rac1 binding domain of plexin-B1. A monomeric variant is generated which preserves the Rac1 binding activity and the wild-type protein structure.

The Structure and Function of the Outer Coat Protein VP9 of Banna Virus by Fauziah Mohd Jaafar; Houssam Attoui; Mohammad W. Bahar; Christian Siebold; Geoffrey Sutton; Peter P.C. Mertens; Philippe De Micco; David I. Stuart; Jonathan M. Grimes; Xavier De Lamballerie (17-28).
Banna virus (BAV: genus Seadornavirus, family Reoviridae) has a double-shelled morphology similar to rotavirus and bluetongue virus. The structure of BAV outer-capsid protein VP9 was determined by X-ray crystallography at 2.6 Å resolution, revealing a trimeric molecule, held together by an N-terminal helical bundle, reminiscent of coiled-coil structures found in fusion-active proteins such as HIV gp41. The major domain of VP9 contains stacked β sheets with marked structural similarities to the receptor binding protein VP8 of rotavirus. Anti-VP9 antibodies neutralize viral infectivity, and, remarkably, pretreatment of cells with trimeric VP9 increased viral infectivity, indicating that VP9 is involved in virus attachment to cell surface and subsequent internalization. Sequence similarities were also detected between BAV VP10 and VP5 portion of rotavirus VP4, suggesting that the receptor binding and internalization apparatus, which is a single gene product activated by proteoloysis in rotavirus, is the product of two separate genome segments in BAV.

Siderocalin (Lcn 2) Also Binds Carboxymycobactins, Potentially Defending against Mycobacterial Infections through Iron Sequestration by Margaret A. Holmes; Wendy Paulsene; Xu Jide; Colin Ratledge; Roland K. Strong (29-41).
Siderocalin, a member of the lipocalin family of binding proteins, is found in neutrophil granules, uterine secretions, and at markedly elevated levels in serum and synovium during bacterial infection; it is also secreted from epithelial cells in response to inflammation or tumorigenesis. Identification of high-affinity ligands, bacterial catecholate-type siderophores (such as enterochelin), suggested a possible function for siderocalin: an antibacterial agent, complementing the general antimicrobial innate immune system iron-depletion strategy, sequestering iron as ferric siderophore complexes. Supporting this hypothesis, siderocalin is a potent bacteriostatic agent in vitro under iron-limiting conditions and, when knocked out, renders mice remarkably susceptible to bacterial infection. Here we show that siderocalin also binds soluble siderophores of mycobacteria, including M. tubercu losis: carboxymycobactins. Siderocalin employs a degenerate recognition mechanism to cross react with these dissimilar types of siderophores, broadening the potential utility of this innate immune defense.

X-Ray Structure of a Rex-Family Repressor/NADH Complex Insights into the Mechanism of Redox Sensing by E. Allen Sickmier; Dimitris Brekasis; Shanthi Paranawithana; Jeffrey B. Bonanno; Mark S.B. Paget; Stephen K. Burley; Clara L. Kielkopf (43-54).
The redox-sensing repressor Rex regulates transcription of respiratory genes in response to the intra cellular NADH/NAD+ redox poise. As a step toward elucidating the molecular mechanism of NADH/NAD+ sensing, the X-ray structure of Thermus aquaticus Rex (T-Rex) bound to effector NADH has been determined at 2.9 Å resolution. The fold of the C-terminal domain of T-Rex is characteristic of NAD(H)-dependent enzymes, whereas the N-terminal domain is similar to a winged helix DNA binding motif. T-Rex dimerization is primarily mediated by “domain-swapped” α helices. Each NADH molecule binds to the C-terminal domain near the dimer interface. In contrast to NAD(H)-dependent enzymes, the nicotinamide is deeply buried within a hydrophobic pocket that appears to preclude substrate entry. We show that T-Rex binds to the Rex operator, and NADH but not NAD+ inhibits T-Rex/DNA binding activity. A mechanism for redox sensing by Rex family members is proposed by analogy with domain closure of NAD(H)-dependent enzymes.

A Structural Pathway for Signaling in the E46Q Mutant of Photoactive Yellow Protein by Sudarshan Rajagopal; Spencer Anderson; Vukica Srajer; Marius Schmidt; Reinhard Pahl; Keith Moffat (55-63).
In the bacterial photoreceptor photoactive yellow protein (PYP), absorption of blue light by its chromophore leads to a conformational change in the protein associated with differential signaling activity, as it executes a reversible photocycle. Time-resolved Laue crystallography allows structural snapshots (as short as 150 ps) of high crystallographic resolution (∼1.6 Å) to be taken of a protein as it functions. Here, we analyze by singular value decomposition a comprehensive time-resolved crystallographic data set of the E46Q mutant of PYP throughout the photocycle spanning 10 ns–100 ms. We identify and refine the structures of five distinct intermediates and provide a plausible chemical kinetic mechanism for their inter conversion. A clear structural progression is visible in these intermediates, in which a signal generated at the chromophore propagates through a distinct structural pathway of conserved residues and results in structural changes near the N terminus, over 20 Å distant from the chromophore.

A Vinculin Binding Domain from the Talin Rod Unfolds to Form a Complex with the Vinculin Head by Ian Fillingham; Alexandre R. Gingras; Evangelos Papagrigoriou; Bipin Patel; Jonas Emsley; David R. Critchley; Gordon C.K. Roberts; Igor L. Barsukov (65-74).
The cytoskeletal protein talin plays a key role in activating integrins and in coupling them to the actin cytoskeleton. Its N-terminal globular head, which binds β integrins, is linked to an extended rod having a C-terminal actin binding site and several vinculin binding sites (VBSs). The NMR structure of residues 755–889 of the rod (containing a VBS) is shown to be an amphipathic four-helix bundle with a left-handed topology. A talin peptide corresponding to the VBS binds the vinculin head; the X-ray crystallographic structure of this complex shows that the residues which interact with vinculin are buried in the hydrophobic core of the talin fragment. NMR shows that the interaction involves a major structural change in the talin fragment, including unfolding of one of its helices, making the VBS accessible to vinculin. Interestingly, the talin 755–889 fragment binds more than one vinculin head molecule, suggesting that the talin rod may contain additional as yet unrecognized VBSs.

Structure and Intracellular Targeting of the SARS-Coronavirus Orf7a Accessory Protein by Christopher A. Nelson; Andrew Pekosz; Chung A. Lee; Michael S. Diamond; Daved H. Fremont (75-85).
The open reading frame (ORF) 7a of the SARS-associated coronavirus (SARS-CoV) encodes a unique type I transmembrane protein of unknown function. We have determined the 1.8 Å resolution crystal structure of the N-terminal ectodomain of orf7a, revealing a compact seven-stranded β sandwich unexpectedly similar in fold and topology to members of the Ig superfamily. We also demonstrate that, in SARS-CoV- infected cells, the orf7a protein is expressed and retained intracellularly. Confocal microscopy studies using orf7a and orf7a/CD4 chimeras implicate the short cytoplasmic tail and transmembrane domain in trafficking of the protein within the endoplasmic reticulum and Golgi network. Taken together, our findings provide a structural and cellular framework in which to explore the role of orf7a in SARS-CoV pathogenesis.

A DNA Glycosylase from Pyrobaculum aerophilum with an 8-Oxoguanine Binding Mode and a Noncanonical Helix-Hairpin-Helix Structure by Gondichatnahalli M. Lingaraju; Alessandro A. Sartori; Dirk Kostrewa; Andrea E. Prota; Josef Jiricny; Fritz K. Winkler (87-98).
Studies of DNA base excision repair (BER) pathways in the hyperthermophilic crenarchaeon Pyrobaculum aerophilum identified an 8-oxoguanine-DNA glyco syl ase, Pa-AGOG (archaeal GO glycosylase), with distinct functional characteristics. Here, we describe its crystal structure and that of its complex with 8-oxoguanosine at 1.0 and 1.7 Å resolution, respectively. Characteristic structural features are identified that confirm Pa-AGOG to be the founding member of a functional class within the helix-hairpin-helix (HhH) superfamily of DNA repair enzymes. Its hairpin structure differs substantially from that of other proteins containing an HhH motif, and we predict that it interacts with the DNA backbone in a distinct manner. Furthermore, the mode of 8-oxoguanine recognition, which involves several hydrogen-bonding and π-stacking interactions, is unlike that observed in human OGG1, the prototypic 8-oxoguanine HhH-type DNA glycosylase. Despite these differences, the predicted kinked conformation of bound DNA and the catalytic mechanism are likely to resemble those of human OGG1.

Structural Mechanism of Inhibition of the Rho Transcription Termination Factor by the Antibiotic Bicyclomycin by Emmanuel Skordalakes; Andrew P. Brogan; Boon Saeng Park; Harold Kohn; James M. Berger (99-109).
Rho is a hexameric RNA/DNA helicase/translocase that terminates transcription of select genes in bacteria. The naturally occurring antibiotic, bicyclomycin (BCM), acts as a noncompetitive inhibitor of ATP turnover to disrupt this process. We have determined three independent X-ray crystal structures of Rho complexed with BCM and two semisynthetic derivatives, 5a-(3-formylphenylsulfanyl)-dihydrobicyclomycin (FPDB) and 5a-formylbicyclomycin (FB) to 3.15, 3.05, and 3.15 Å resolution, respectively. The structures show that BCM and its derivatives are nonnucleotide inhibitors that interact with Rho at a pocket adjacent to the ATP and RNA binding sites in the C-terminal half of the protein. BCM association prevents ATP turnover by an unexpected mechanism, occluding the binding of the nucleophilic water molecule required for ATP hydrolysis. Our data explain why only certain elements of BCM have been amenable to modification and serve as a template for the design of new inhibitors.

Structural Basis for Vertebrate Filamin Dimerization by Regina Pudas; Tiila-Riikka Kiema; P. Jonathan G. Butler; Murray Stewart; Jari Ylänne (111-119).
Filamins are essential in cell motility and many developmental processes. They are large actin cross linking proteins that contain actin binding domains in their N termini and a long rod region constructed from 24 tandem Ig domains. Dimerization is crucial for the actin crosslinking function of filamins and requires the most C-terminal Ig domain. We describe here the crystal structure of this 24th Ig domain (Ig24) of human filamin C and show how it mediates dimerization. The dimer interface is novel and quite different to that seen in the Dictyostelium discoideum filamin analog. The sequence signature of the dimerization interface suggests that the C-terminal domains of all vertebrate filamins share the same dimerization mechanism. Furthermore, we show that point mutations in the dimerization interface disrupt the dimer and that the dissociation constant for recombinant Ig24 is in the micromolar range.

Inference of Protein Function from Protein Structure by Debnath Pal; David Eisenberg (121-130).

The “Roll and Lock” Mechanism of Force Generation in Muscle by Michael A. Ferenczi; Sergey Y. Bershitsky; Natalia Koubassova; Verl Siththanandan; William I. Helsby; Pierre Panine; Manfred Roessle; Theyencheri Narayanan; Andrey K. Tsaturyan (131-141).
Muscle force results from the interaction of the globular heads of myosin-II with actin filaments. We studied the structure-function relationship in the myosin motor in contracting muscle fibers by using temperature jumps or length steps combined with time-resolved, low-angle X-ray diffraction. Both perturbations induced simultaneous changes in the active muscle force and in the extent of labeling of the actin helix by stereo-specifically bound myosin heads at a constant total number of attached heads. The generally accepted hypothesis assumes that muscle force is generated solely by tilting of the lever arm, or the light chain domain of the myosin head, about its catalytic domain firmly bound to actin. Data obtained suggest an additional force-generating step: the “roll and lock” transition of catalytic domains of non-stereo-specifically attached heads to a stereo-specifically bound state. A model based on this scheme is described to quantitatively explain the data.

Crystal Structure and Functional Implications of Pyrococcus furiosus Hef Helicase Domain Involved in Branched DNA Processing by Tatsuya Nishino; Kayoko Komori; Daisuke Tsuchiya; Yoshizumi Ishino; Kosuke Morikawa (143-153).
DNA and RNA frequently form various branched intermediates that are important for the transmission of genetic information. Helicases play pivotal roles in the processing of these transient intermediates during nucleic acid metabolism. The archaeal Hef helicase/ nuclease is a representative protein that processes flap- or fork-DNA structures, and, intriguingly, its C-terminal half belongs to the XPF/Mus81 nuclease family. Here, we report the crystal structure of the helicase domain of the Hef protein from Pyrococcus furiosus. The structure reveals a novel helical insertion between the two conserved helicase core domains. This positively charged extra region, structurally similar to the “thumb” domain of DNA polymerase, plays critical roles in fork recognition. The Hef helicase/nuclease exhibits sequence similarity to the Mph1 helicase from Saccharomyces cerevisiae; XPF/Rad1, involved in DNA repair; and a putative Hef homolog identified in mammals. Hence, our findings provide a structural basis for the functional mechanisms of this helicase/nuclease family.

Structural Basis for the Regulation of Insulin-like Growth Factors by IGF Binding Proteins by Igor Siwanowicz; Grzegorz M. Popowicz; Magdalena Wisniewska; Robert Huber; Klaus-Peter Kuenkele; Kurt Lang; Richard A. Engh; Tad A. Holak (155-167).
Insulin-like growth factor binding proteins (IGFBPs) control the extracellular distribution, function, and activity of IGFs. Here, we report an X-ray structure of the binary complex of IGF-I and the N-terminal domain of IGFBP-4 (NBP-4, residues 3–82) and a model of the ternary complex of IGF-I, NBP-4, and the C-terminal domain (CBP-4, residues 151–232) derived from diffraction data with weak definition of the C-terminal domain. These structures show how the IGFBPs regulate IGF signaling. Key features of the structures include (1) a disulphide bond ladder that binds to IGF and partially masks the IGF residues responsible for type 1 IGF receptor (IGF-IR) binding, (2) the high-affinity IGF-I interaction site formed by residues 39–82 in a globular fold, and (3) CBP-4 interactions. Although CBP-4 does not bind individually to either IGF-I or NBP-4, in the ternary complex, CBP-4 contacts both and also blocks the IGF-IR binding region of IGF-I.