Steve E. Ealick

Observation of an unexpected third receptor molecule in the crystal structure of human interferon-γ receptor complex

BACKGROUND Molecular interactions among cytokines and cytokine receptors form the basis of many cell-signaling pathways relevant to immune function. Interferon-gamma (IFN-gamma) signals through a multimeric receptor complex consisting of two different but structurally related transmembrane chains: the high-affinity receptor-binding subunit (IFN-gammaRalpha) and a species-specific accessory factor (AF-1 or IFN-gammaRbeta). In the signaling complex, the two receptors probably interact with one another through their extracellular domains. Understanding the atomic interactions of signaling complexes enhances the ability to control and alter cell signaling and also provides a greater understanding of basic biochemical processes. RESULTS The crystal structure of the complex of human IFN-gamma with the soluble, glycosylated extracellular part of IFN-gammaRalpha has been determined at 2.9 A resolution using multiwavelength anomalous diffraction methods. In addition to the expected 2:1 complex, the crystal structure reveals the presence of a third receptor molecule not directly associated with the IFN-gamma dimer. Two distinct intermolecular contacts, involving the edge strands of the C-terminal domains, are observed between this extra receptor and the 2:1 receptor-ligand complex thereby forming a 3:1 complex. CONCLUSIONS The observed interactions in the 2:1 complex of the high-affinity cell-surface receptor with the IFN-gamma cytokine are similar to those seen in a previously reported structure where the receptor chains were not glycosylated. The formation of beta-sheet packing interactions between pairs of IFN-gammaRalpha receptors in these crystals suggests a possible model for receptor oligomerization of Ralpha and the structurally homologous Rbeta receptors in the fully active IFN-gamma signaling complex.

The crystal structure of ADP-L-glycero-D-mannoheptose 6-epimerase: catalysis with a twist.

BACKGROUND ADP-L-glycero--mannoheptose 6-epimerase (AGME) is required for lipopolysaccharide (LPS) biosynthesis in most genera of pathogenic and non-pathogenic Gram-negative bacteria. It catalyzes the interconversion of ADP-D-glycero-D-mannoheptose and ADP-L-glycero-D-mannoheptose, a precursor of the seven-carbon sugar L-glycero-mannoheptose (heptose). Heptose is an obligatory component of the LPS core domain; its absence results in a truncated LPS structure resulting in susceptibility to hydrophobic antibiotics. Heptose is not found in mammalian cells, thus its biosynthetic pathway in bacteria presents a unique target for the design of novel antimicrobial agents. RESULTS The structure of AGME, in complex with NADP and the catalytic inhibitor ADP-glucose, has been determined at 2.0 A resolution by multiwavelength anomalous diffraction (MAD) phasing methods. AGME is a homopentameric enzyme, which crystallizes with two pentamers in the asymmetric unit. The location of 70 crystallographically independent selenium sites was a key step in the structure determination process. Each monomer comprises two domains: a large N-terminal domain, consisting of a modified seven-stranded Rossmann fold that is associated with NADP binding; and a smaller alpha/beta C-terminal domain involved in substrate binding. CONCLUSIONS The first structure of an LPS core biosynthetic enzyme leads to an understanding of the mechanism of the conversion between ADP-D-glycero--mannoheptose and ADP-L-glycero-D-mannoheptose. On the basis of its high structural similarity to UDP-galactose epimerase and the three-dimensional positions of the conserved residues Ser116, Tyr140 and Lys144, AGME was classified as a member of the short-chain dehydrogenase/reductase (SDR) superfamily. This study should prove useful in the design of mechanistic and structure-based inhibitors of the AGME catalyzed reaction.

Selenium-based MAD phasing: setting the sites on larger structures

We would like to acknowledge the generous support of grant GM-46733 from the National Institutes of Health. Research at CHESS is supported by grant DMR-9311772 from the National Science Foundation and the Mac-CHESS research resource is funded by grant RR-01646 from the National Institutes of Health.

A System for Integrated Collection and Analysis of Crystallographic Diffraction Data

A set of linked software modules has been installed at the A1 station of CHESS, the Cornell High Energy Synchrotron Source, which, with the underlying hardware, allows crystallographic users of the facility to evaluate crystals, collect diffraction images and process the images rapidly and with assurance of the quality of the resultant data, thereby making most efficient use of their beam time. The system includes a CCD detector and its controlling software, with a graphical user interface, a convenient oscillation camera featuring automated alignment with the X-ray beam, a program for optimizing crystal rotation range, and the HKL data-reduction package. Principles embodied in this system are applicable to other facilities where crystallographic data are routinely collected.

Charge coupled device X-ray detectors for macromolecular crystallography

We gratefully acknowledge CHESS and the MacCHESS staff for beam time and experimental support. We thank Daniel J Thiel and Richard L Walter for help with many aspects of the data collection, and Shelly R Armstrong, Andrew Karplus, Chen Mao, Wladek Minor, Michael G Rossmann, and Corey Strickland for providing crystals and/or data. We also thank our detector development team at Princeton University (Martin Novak, Fred Osterberg, John Shepherd, Mike Wall, Mark Tate), the Robert Wood Johnson Medical School (Eric Eikenberry) and Princeton Scientific Instruments (John Lowrance). Work at Cornell is supported by NIH grants RR01646 and GM48874 and the Keck Laboratory for Molecular Structure. The detector was developed primarily via support from DoE grant DE-FG02-87ER60522 to SMG.

O5′,6-Methanocytidine — Synthesis, Conformational Properties and Deamination by Cytidine Deaminase

Abstract The synthesis of O5′,6-methanocytidine (4), a pyrimidine nucleoside restricted to the anti conformation, is described. Molecular modeling studies suggest that 4 is more flexible than conventional cyclonucleosides because of its larger-than-usual bridging system and that it can exist in a number of low energy conformations where the glycosyl rotation angles (X) cover an ∼80° segment of the anti range. However, while both N-type (C2′-exo) and S-type (C3′-exo) sugar puckerings are possible, none of the low energy conformers adopt the C3′-endo or C2′-endo puckering modes generally seen for unconstrained nucleosides. The lowest energy conformer predicted for 4 (X = −152°, y = 73°, P = 206°) is similar to the X-ray structure of a related compound, namely 5-hydroxy-O5′,6 -methanouridine (12, X = −138°. Y = 63°, P = 200°). In solution, NMR evidence suggests an equilibrium between C2′-exo and C3′-exo puckerings for 4, and CD evidence suggests an average glycosyl rotation angle (X) of around −160°. O5′,6 -...

User-friendly interfaces for control of crystallographic experiments at CHESS

In designing a system to collect high quality diffraction data in an efficient manner, both hardware and software must be considered. This work focuses on the data collection software used at CHESS, the Cornell High Energy Synchrotron source, with emphasis on the interface between the user and the experimental components. For each type of detector used at CHESS, there is a graphical user interface (GUI) enabling the user to easily set up and run an experiment. For the CCD detector from Area Detector Systems Corp., this is a commercial product from ADSC, customized for CHESS. For the Princeton CCD detectors, a GUI has recently been developed to streamline communication between the user and the TV6 program which controls the detector. For Fuji imaging plates, a new GUI controls operation of the oscillation camera, including the imaging plate carousel; scanning of plates is done using the software provided by Fuji. Although these GUI’s are not identical, they have numerous similarities, making it easier for ...