Viperin is an IFN-inducible radical S-adenosylmethionine (SAM) enzyme that inhibits viral replication. We determined crystal structures of an anaerobically prepared fragment of mouse viperin (residues 45-362) complexed with S-adenosylhomocysteine (SAH) or 5′-deoxyadenosine (5′-dAdo) and L-methionine (L-Met). Viperin contains a partial (βα) 6 -barrel fold with a disordered N-terminal extension (residues 45-74) and a partially ordered C-terminal extension (residues 285-362) that bridges the partial barrel to form an overall closed barrel structure. Cys84, Cys88, and Cys91 located after the first β-strand bind a [4Fe-4S] cluster. The active site architecture of viperin with bound SAH (a SAM analog) or 5′-dAdo and L-Met (SAM cleavage products) is consistent with the canonical mechanism of 5′-deoxyadenosyl radical generation. The viperin structure, together with sequence alignments, suggests that vertebrate viperins are highly conserved and that fungi contain a viperin-like ortholog. Many bacteria and archaebacteria also express viperin-like enzymes with conserved active site residues. Structural alignments show that viperin is similar to several other radical SAM enzymes, including the molybdenum cofactor biosynthetic enzyme MoaA and the RNA methyltransferase RlmN, which methylates specific nucleotides in rRNA and tRNA. The viperin putative active site contains several conserved positively charged residues, and a portion of the active site shows structural similarity to the GTPbinding site of MoaA, suggesting that the viperin substrate may be a nucleoside triphosphate of some type.radical SAM | IFN-stimulated gene | antiviral cellular factor | free radical | S-adenosyl methionine V iruses exploit the metabolic machinery of host cells to replicate and spread to other cells. Although cytotoxic T cells and antibody-producing B cells can ultimately be produced in an adaptive response to the virus, innate immune mechanisms are used to respond to infection rapidly. Upon infection, cells can sense the presence of virus via pattern recognition receptors (1, 2) and produce IFNs that limit the spread of infection to other cells (3). IFNs induce the expression of hundreds of IFN-stimulated genes (ISGs), many of which are involved in various antiviral processes, including antigen presentation, apoptosis, and inhibition of viral replication (4-7).Viperin, the product of rsad-2, was first identified as a protein induced by exposure of human macrophages to IFN-γ and by infection of primary human fibroblasts with human cytomegalovirus (8,9). Early studies showed that viperin is induced in various cell types by IFN-α and IFN-β, associates with the cytosolic face of the endoplasmic reticulum (ER), and inhibits human cytomegalovirus replication when preexpressed in human fibroblasts (8). Since then, viperin has been shown to be induced by several factors, including lipopolysaccharide (10-12), and to inhibit a broad range of viruses, including HIV-1 (13), West Nile virus (14), hepatitis C virus (15, 16), dengue virus type 2 (17), influ...

The quinolinate synthase of prokaryotes and photosynthetic eukaryotes, NadA, contains a [4Fe-4S] cluster with unknown function. We report crystal structures of Pyrococcus horikoshii NadA in complex with dihydroxyacetone phosphate (DHAP), iminoaspartate analogues, and quinolinate. DHAP adopts a nearly planar conformation and chelates the [4Fe-4S] cluster via its keto and hydroxyl groups. The active site architecture suggests that the cluster acts as a Lewis acid in enediolate formation, like zinc in class II aldolases. The DHAP and putative iminoaspartate structures suggest a model for a condensed intermediate. The ensemble of structures suggests a two-state system, which may be exploited in early steps.

Diphthamide biosynthesis involves a carbon-carbon bond-forming reaction catalyzed by a radical S-adenosylmethionine (SAM) enzyme that cleaves a carbon-sulfur (C-S) bond in SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. Using rapid freezing, we have captured an organometallic intermediate with an iron-carbon (Fe-C) bond between ACP and the enzyme's [4Fe-4S] cluster. In the presence of the substrate protein, elongation factor 2, this intermediate converts to an organic radical, formed by addition of the ACP radical to a histidine side chain. Crystal structures of archaeal diphthamide biosynthetic radical SAM enzymes reveal that the carbon of the SAM C-S bond being cleaved is positioned near the unique cluster Fe, able to react with the cluster. Our results explain how selective C-S bond cleavage is achieved in this radical SAM enzyme.

The mechanism by which the binding of a neurotransmitter to a receptor leads to channel opening is a central issue in molecular neurobiology. The structure of the agonist binding domain of ionotropic glutamate receptors has led to a greater understanding of the changes in structure that accompany agonist binding and have provided important clues as to the link between these structural changes and channel activation and desensitization. However, because the binding domain has exhibited different structures in different crystallization conditions, understanding the structure in the absence of crystal packing is of considerable importance. The orientation of the two lobes of the binding domain in the presence of a full agonist, an antagonist, and several partial agonists was measured using NMR spectroscopy by employing residual dipolar couplings. For some partial agonists the solution conformation differs from that observed in the crystal. A model of channel activation based on the results is discussed.The majority of excitatory synaptic transmission in the vertebrate central nervous system is mediated by ionotropic glutamate receptors (iGluRs; 3). These receptors also play important roles in neuronal development and the formation of synaptic plasticity underlying higher order processes such as learning and memory (4). In addition, iGluRs are also associated with neurologic disorders including epilepsy and ischemic brain damage and neurodegenerative disorders such as Huntington's chorea, Parkinson's and Alzheimer's diseases. iGluRs are membrane-bound receptor ion channels composed of four subunits surrounding a central ion channel in which each subunit contributes to pore formation. Subunits are categorized by pharmacological properties, sequence, functionality, and biological role into those that are sensitive: (1) to the synthetic agonist α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA 1 ; GluR1-4); (2) to the naturally occurring neurotoxin kainate (GluR5-7, KA1, 2); and (3) to the synthetic agonist N-methyl-D-aspartic acid (NMDA; NR1, NR2A-D, NR3A-B).The three dimensional structures of the binding domain (S1S2) of a number of glutamate receptors are known from X-ray crystallography. In particular, the structures of GluR2 bound to a wide variety of agonists, partial agonists and antagonists have provided compelling clues *Corresponding author; telephone: 1-607-253-3877; fax: 1-607-253-3659; reo1@cornell.edu. Supporting Information Available This material is available free of charge via the Internet at http://pubs.acs.org. 1 Abbreviations: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid; BrW, (S)-5-bromowillardiine; ClW, (S)-5-chlorowillardiine; FW, (S)-5-fluorowillardiine; HW, (S)-willardiine ((S)-2-Amino-3-(3,4-dihydro-2,4-dioxopyrimidin-1(2H)-yl) propanoic acid); iGluR, ionotropic glutamate receptor; IW, (S)-5-iodowillardiine; IPTG, isopropyl-β-D-thiogalactoside; NW, (S)-5-nitrowillardiine; NMDA, N-methyl-D-aspartic acid; S1S2, extracellular ligand-binding domain of GluR2; RDC, r...