Vladimir V. Lunin ^1,9, Elena Dobrovetsky ^2,9 , Galina Khutoreskaya 2 , Rongguang Zhang ⁴ , Andrzej Joachimiak ⁴, Declan A. Doyle ⁵ , Alexey Bochkarev ^{2, 3, 6}, Michael E. Maguire ⁷, Aled M. Edwards ^{1, 2 , 3, 4, 6} and Christopher M. Koth ^2,8

The magnesium ion, Mg2+, is essential for myriad biochemical processes and remains the only major biological ion whose transport mechanisms remain unknown. The CorA family of magnesium transporters is the primary Mg2+ uptake system of most prokaryotes and a functional homologue of the eukaryotic mitochondrial magnesium transporter. Here we determine crystal structures of the full-length Thermotoga maritima CorA in an apparent closed state and its isolated cytoplasmic domain at 3.9 Å and 1.85 Å resolution, respectively. The transporter is a funnel-shaped homopentamer with two transmembrane helices per monomer.

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Structure of tracheal cytotoxin in complex with a heterodimeric pattern-recognition receptor.

Chung-I Chang^1,2, Yogarany Chelliah ^1,2, Dominika Borek ², Dominique Mengin-Lecreulx ³ and Johann Deisenhofer ^1,2

Tracheal cytotoxin (TCT), a naturally occurring fragment of Gram-negative peptidoglycan, is a potent elicitor of innate immune responses
in Drosophila. It induces the heterodimerization of its recognition
receptors, the peptidoglycan recognition proteins (PGRPs) LCa and LCx,
which activates the immune deficiency pathway...

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Structures of human insulin-degrading enzyme reveal a new substrate recognition mechanism.
Nature 443, 870-874 (19 October 2006)

Yuequan Shen ¹, Andrzej Joachimiak ², Marsha Rich Rosner ¹ and Wei-Jen Tang¹

Insulin-degrading enzyme (IDE), a Zn2+-metalloprotease, is involved in the clearance of insulin and amyloid-beta (refs 1–3). Loss-of-function mutations of IDE
in rodents cause glucose intolerance and cerebral accumulation
of amyloid-beta, whereas enhanced IDE activity effectively reduces
brain amyloid-beta (refs 4–7). Here we report structures of human
IDE in complex with four substrates (insulin B chain, amyloid-beta
peptide (1–40), amylin and glucagon). The amino- and carboxy-terminal
domains of IDE (IDE-N and IDE-C, respectively) form an enclosed cage
just large enough to encapsulate insulin.

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A Bird Flu Protein Link to Virulence
Smart viruses infect without killing the host, at least in the short term. In such low-level infections, the virus gains time to persist, reproduce, infect other individuals, and spread through the population. Some viruses persist in certain host populations without killing them, but in other hosts they have a rapid and deadly effect. The H5N1 virus strains responsible for recent lethal outbreaks of bird flu apparently existed for quite some time in wild goose populations without doing much harm.

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Ultrahigh-resolution study of protein atomic displacement parameters
at cryotemperatures obtained with a helium cryostat

Tatiana Petrova,^a,b,c Stephan Ginell,^aAndre Mitschler,^c Isabelle Hazemann,^c Thomas Schneider,^d Alexandra Cousido,^c Vladimir Y. Lunin,^b Andrzej Joachimiak ^a* and Alberto Podjarny^c
Two X-ray data sets for a complex of human aldose reductase (h-AR) with the inhibitor IDD 594 and the cofactor NADP+ were collected from two different parts of the same crystal to a resolution of 0.81 Å at 15 and 60 K using cold helium gas as cryogen. The contribution of temperature to the atomic B values was estimated by comparison of the independently refined models. It was found that although being slightly different for different kinds of atoms, the differences (B) in the isotropic equivalents B of atomic displacement parameters (ADPs) were approximately constant (about 1.7 Å2) for well ordered atoms as the temperature was increased from 15 to 60 K ...>> see related article

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The transition state for formation of the peptide bond in the ribosome

Asta Gindulyte*, Anat Bashan†, Ilana Agmon†, Lou Massa*, Ada Yonath†‡, and Jerome Karle§ >> Proc Natl Acad Sci U S A. 2006 Sep 5;103(36):13327-32
Using quantum mechanics and exploiting known crystallographic coordinates of tRNA substrate located in the ribosome peptidyl transferase center around the 2-fold axis, we have investigated the mechanism for peptide-bond formation. The calculation is based on a choice of 50 atoms assumed to be important in the mechanism. We used density functional theory to optimize the geometry and energy of the transition state (TS) for peptide-bond formation. The TS is formed simultaneously with the rotatory motion enabling the translocation of the A-site tRNA 3' end into the P site, and we estimated the magnitude of rotation angle between the A-site starting position and the place at which the TS occurs >> see related article

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The many faces of radiation-induced changes

Dominika Borek,^a Stephan L. Ginell,^b Marcin Cymborowski,^c Wladek Minor^c and Zbyszek Otwinowski ^a* >> J. Synchrotron Rad. (2007) 14, 24-33
During diffraction experiments even cryo-cooled protein crystals can be significantly damaged due to chemical and physical changes induced by absorbed X-ray photons. The character and scale of the observed effects depend strongly on the temperature and the composition of crystals. The absorption of radiation energy results in incremental regular changes to the crystal structure, making its impact on the process of solving the structure strongly correlated with other experimental variables. An understanding of all the dependencies is still limited and does not allow for a precise prediction of the outcome of a particular diffraction experiment. Results are presented of diffraction experiments performed under different experimental conditions. The influence of temperature and crystal composition on different characteristics of radiation damage is analyzed. The observed effects are discussed in terms of their impact on data processing and phasing procedures. >> see related article

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Crystal structure of poliovirus 3CD protein: virally encoded protease and precursor to the RNA-dependent RNA polymerase

Laura L. Marcotte,¹ Amanda B. Wass,¹ David W. Gohara,¹,Harsh B. Pathak,², Jamie J. Arnold,² David J. Filman,² Craig E. Cameron,² and James M. Hogle¹
J. Virol. 81 (7), April, 3583-3596 (2007)

Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3C ^(pro) and the viral polymerase 3D^(pol) and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4-A resolution and the G64S fidelity mutant of 3D^(pol)at a 3.0-A resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. >> see related article

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Structural conservation of RecF and Rad50: implications for DNA recognition and RecF function
Olga Koroleva¹, Nodar Makharashvili¹, Charmain T Courcelle², Justin Courcelle² and Sergey Korolev¹ The EMBO Journal (2007) 26, 867–877

RecF, together with RecO and RecR, belongs to a ubiquitous group of recombination mediators (RMs) that includes eukaryotic proteins such as Rad52 and BRCA2. RMs help maintain genome stability in the presence of DNA damage by loading RecA-like recombinases and displacing single-stranded DNA-binding proteins. Here, we present the crystal structure of RecF from Deinococcus radiodurans. RecF exhibits a high degree of structural similarity with the head domain of Rad50, but lacks its long coiled-coil region. The structural homology between RecF and Rad50 is extensive, encompassing the ATPase subdomain and the so-called 'Lobe II' subdomain of Rad50...
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Domain Architecture of Pyruvate Carboxylase, a Biotin-Dependent Multifunctional Enzyme Martin St. Maurice,¹ Laurie Reinhardt,¹ Kathy H. Surinya,² Paul V. Attwood,³ John C. Wallace,² W. Wallace Cleland,¹ Ivan Rayment¹ Science 24 August 2007: Vol. 317. no. 5841, pp. 1076 - 1079

Biotin-dependent multifunctional enzymes carry out metabolically important carboxyl group transfer reactions and are potential targets for the treatment of obesity and type 2 diabetes. These enzymes use a tethered biotin cofactor to carry an activated carboxyl group between distantly spaced active sites. The mechanism of this transfer has remained poorly understood. Here we report the complete structure of pyruvate carboxylase at 2.0 angstroms resolution, which shows its domain arrangement. The structure, when combined with mutagenic analysis, shows that intermediate transfer occurs between active sites on separate polypeptide chains. >> see related article

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Structure and Function of an Essential Component of the Outer Membrane Protein Assembly Machine Seokhee Kim,¹ Juliana C. Malinverni,² Piotr Sliz,^3,4 Thomas J. Silhavy,² Stephen C. Harrison,^3,4 Daniel Kahne^1,3 Science 17 August 2007: Vol. 317. no. 5840, pp. 961 - 964


Integral ß-barrel proteins are found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. The machine that assembles these proteins contains an integral membrane protein, called YaeT in Escherichia coli, which has one or more polypeptide transport–associated (POTRA) domains. The crystal structure of a periplasmic fragment of YaeT reveals the POTRA domain fold and suggests a model for how POTRA domains can bind different peptide sequences, as required for a machine that handles numerous ß-barrel protein precursors. Analysis of POTRA domain deletions shows which are essential and provides a view of the spatial organization of this assembly machine.>> see related article

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Synthetic antibodies for specific recognition and crystallization of structured RNA. Ye JD, Tereshko V, Frederiksen JK, Koide A, Fellouse FA, Sidhu SS, Koide S, Kossiakoff AA, Piccirilli JA. Proc Natl Acad Sci U S A. 2008 Jan 8;105(1):82-7

Antibodies that bind protein antigens are indispensable in biochemical research and modern medicine. However, knowledge of RNA-binding antibodies and their application in the ever-growing RNA field is lacking. Here we have developed a robust approach using a synthetic phage-display library to select specific antigen-binding fragments (Fabs) targeting a large functional RNA. We have solved the crystal structure of the first Fab-RNA complex at 1.95 Å. Capability in phasing and crystal contact formation suggests that the Fab provides a potentially valuable crystal chaperone for RNA. The crystal structure reveals that the Fab achieves specific RNA binding on a shallow surface with complementarity-determining region (CDR) sequence diversity, length variability, and main-chain conformational plasticity. The Fab-RNA interface also differs significantly from Fab-protein interfaces in amino acid composition and light-chain participation. These findings yield valuable insights for engineering of Fabs as RNA-binding modules and facilitate further development of Fabs as possible therapeutic drugs and biochemical tools to explore RNA biology. >> see related article

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Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase Matthew P. Blakeley, Federico Ruiz, Raul Cachau, Isabelle Hazemann, Flora Meilleur, Andre Mitschler, Stephan Ginell, Pavel Afonine, Oscar N. Ventura, Alexandra Cousido-Siah, Michael Haertlein, Andrzej Joachimiak, Dean Myles, and Alberto Podjarny PNAS | February 12, 2008 vol. 105 | no. 6 | 1844-1848

Enzymatic catalysis frequently involves proton translocation (PT). PT can occur on very short time scales (nanoseconds) and over long distances (tens of Angstroms), often involving multiple proton relay sites and proton wires (Grotthus-like mechanisms) (1). The nature of PT processes combined with the weak x-ray scattering signal from hydrogen atoms, which excludes their observation at resolutions <1.2 Å, make the structural characterization of PT processes very difficult. Recent advances in synchrotron and neutron sources, detectors, cryocooling, and software, coupled with the ability to obtain high-quality crystals, have led to an improved level of detail in protein structures, as exemplified in our recent structure of human aldose reductase (h-AR) determined at subatomic resolution (2) >> see related article

NADP Regulates the Yeast GAL Induction System P. Rajesh Kumar, Yao Yu, Rolf Sternglanz, Stephen Albert Johnston, Leemor Joshua-Tor Science 22 February 2008: Vol. 319. no. 5866, pp. 1090 - 1092

Transcriptional regulation of the galactose-metabolizing genes in Saccharomyces cerevisiae depends on three core proteins: Gal4p, the transcriptional activator that binds to upstream activating DNA sequences (UASGAL); Gal80p, a repressor that binds to the carboxyl terminus of Gal4p and inhibits transcription; and Gal3p, a cytoplasmic transducer that, upon binding galactose and adenosine 5'-triphosphate, relieves Gal80p repression. The current model of induction relies on Gal3p sequestering Gal80p in the cytoplasm. However, the rapid induction of this system implies that there is a missing factor. Our structure of Gal80p in complex with a peptide from the carboxyl-terminal activation domain of Gal4p reveals the existence of a dinucleotide that mediates the interaction between the two. Biochemical and in vivo experiments suggests that nicotinamide adenine dinucleotide phosphate (NADP) plays a key role in the initial induction event.>> see related article