Structural basis for methyl transfer by a radical SAM enzyme

The radical S-adenosyl-L-methionine (SAM) enzymes RlmN and Cfr methylate 23S ribosomal RNA, modifying the C2 or C8 position of adenosine 2503. The methyl groups are installed by a two-step sequence involving initial methylation of a conserved Cys residue (RlmN Cys(355)) by SAM. Methyl transfer to the substrate requires reductive cleavage of a second equivalent of SAM. Crystal structures of RlmN and RlmN with SAM show that a single molecule of SAM coordinates the [4Fe-4S] cluster. Residue Cys(355) is S-methylated and located proximal to the SAM methyl group, suggesting the SAM that is involved in the initial methyl transfer binds at the same site. Thus, RlmN accomplishes its complex reaction with structural economy, harnessing the two most important reactivities of SAM within a single site.     

Structural basis of trans-inhibition in a molybdate/tungstate ABC transporter

Transport across cellular membranes is an essential process that is catalyzed by diverse membrane transport proteins. The turnover rates of certain transporters are inhibited by their substrates in a process termed trans-inhibition, whose structural basis is poorly understood. We present the crystal structure of a molybdate/tungstate ABC transporter (ModBC) from Methanosarcina acetivorans in a trans-inhibited state. The regulatory domains of the nucleotide-binding subunits are in close contact and provide two oxyanion binding pockets at the shared interface. By specifically binding to these pockets, molybdate or tungstate prevent adenosine triphosphatase activity and lock the transporter in an inward-facing conformation, with the catalytic motifs of the nucleotide-binding domains separated. This allosteric effect prevents the transporter from switching between the inward-facing and the outward-facing states, thus interfering with the alternating access and release mechanism.     

Structural basis of Wnt recognition by Frizzled

Wnts are lipid-modified morphogens that play critical roles in development principally through engagement of Frizzled receptors. The 3.25 angstrom structure of Xenopus Wnt8 (XWnt8) in complex with mouse Frizzled-8 (Fz8) cysteine-rich domain (CRD) reveals an unusual two-domain Wnt structure, not obviously related to known protein folds, resembling a "hand" with "thumb" and "index" fingers extended to grasp the Fz8-CRD at two distinct binding sites. One site is dominated by a palmitoleic acid lipid group projecting from serine 187 at the tip of Wnt's thumb into a deep groove in the Fz8-CRD. In the second binding site, the conserved tip of Wnt's "index finger" forms hydrophobic amino acid contacts with a depression on the opposite side of the Fz8-CRD. The conservation of amino acids in both interfaces appears to facilitate ligand-receptor cross-reactivity, which has important implications for understanding Wnt's functional pleiotropy and for developing Wnt-based drugs for cancer and regenerative medicine.     

Structural basis of mitochondrial tethering by mitofusin complexes

Vesicle fusion involves vesicle tethering, docking, and membrane merger. We show that mitofusin, an integral mitochondrial membrane protein, is required on adjacent mitochondria to mediate fusion, which indicates that mitofusin complexes act in trans (that is, between adjacent mitochondria). A heptad repeat region (HR2) mediates mitofusin oligomerization by assembling a dimeric, antiparallel coiled coil. The transmembrane segments are located at opposite ends of the 95 angstrom coiled coil and provide a mechanism for organelle tethering. Consistent with this proposal, truncated mitofusin, in an HR2-dependent manner, causes mitochondria to become apposed with a uniform gap. Our results suggest that HR2 functions as a mitochondrial tether before fusion.     

Structural basis for ribosome recruitment and manipulation by a viral IRES RNA

Canonical cap-dependent translation initiation requires a large number of protein factors that act in a stepwise assembly process. In contrast, internal ribosomal entry sites (IRESs) are cis-acting RNAs that in some cases completely supplant these factors by recruiting and activating the ribosome using a single structured RNA. Here we present the crystal structures of the ribosome-binding domain from a Dicistroviridae intergenic region IRES at 3.1 angstrom resolution, providing a view of the prefolded architecture of an all-RNA translation initiation apparatus. Docking of the structure into cryo-electron microscopy reconstructions of an IRES-ribosome complex suggests a model for ribosome manipulation by a dynamic IRES RNA.     

Structural basis for double-stranded RNA processing by Dicer

The specialized ribonuclease Dicer initiates RNA interference by cleaving double-stranded RNA (dsRNA) substrates into small fragments about 25 nucleotides in length. In the crystal structure of an intact Dicer enzyme, the PAZ domain, a module that binds the end of dsRNA, is separated from the two catalytic ribonuclease III (RNase III) domains by a flat, positively charged surface. The 65 angstrom distance between the PAZ and RNase III domains matches the length spanned by 25 base pairs of RNA. Thus, Dicer itself is a molecular ruler that recognizes dsRNA and cleaves a specified distance from the helical end.     

Structural basis for gluten intolerance in celiac sprue

Celiac Sprue, a widely prevalent autoimmune disease of the small intestine, is induced in genetically susceptible individuals by exposure to dietary gluten. A 33-mer peptide was identified that has several characteristics suggesting it is the primary initiator of the inflammatory response to gluten in Celiac Sprue patients. In vitro and in vivo studies in rats and humans demonstrated that it is stable toward breakdown by all gastric, pancreatic, and intestinal brush-border membrane proteases. The peptide reacted with tissue transglutaminase, the major autoantigen in Celiac Sprue, with substantially greater selectivity than known natural substrates of this extracellular enzyme. It was a potent inducer of gut-derived human T cell lines from 14 of 14 Celiac Sprue patients. Homologs of this peptide were found in all food grains that are toxic to Celiac Sprue patients but are absent from all nontoxic food grains. The peptide could be detoxified in in vitro and in vivo assays by exposure to a bacterial prolyl endopeptidase, suggesting a strategy for oral peptidase supplement therapy for Celiac Sprue.     

Arabidopsis NPL1: a phototropin homolog controlling the chloroplast high-light avoidance response

Chloroplasts relocate their positions in a cell in response to the intensity of incident light, moving to the side wall of the cell to avoid strong light, but gathering at the front face under weak light to maximize light interception. Here, Arabidopsis thaliana mutants defective in the avoidance response were isolated, and the mutated gene was identified as NPL1 (NPH-like 1), a homolog of NPH1 (nonphototropic hypocotyl 1), a blue light receptor used in phototropism. Hence, NPL1 is likely a blue light receptor regulating the avoidance response under strong light.     

Structural basis of toll-like receptor 3 signaling with double-stranded RNA

Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA), a molecular signature of most viruses, and triggers inflammatory responses that prevent viral spread. TLR3 ectodomains (ECDs) dimerize on oligonucleotides of at least 40 to 50 base pairs in length, the minimal length required for signal transduction. To establish the molecular basis for ligand binding and signaling, we determined the crystal structure of a complex between two mouse TLR3-ECDs and dsRNA at 3.4 angstrom resolution. Each TLR3-ECD binds dsRNA at two sites located at opposite ends of the TLR3 horseshoe, and an intermolecular contact between the two TLR3-ECD C-terminal domains coordinates and stabilizes the dimer. This juxtaposition could mediate downstream signaling by dimerizing the cytoplasmic Toll interleukin-1 receptor (TIR) domains. The overall shape of the TLR3-ECD does not change upon binding to dsRNA.     

Structural basis for allosteric regulation of GPCRs by sodium ions

Pharmacological responses of G protein-coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR structures. We reengineered the human A(2A) adenosine receptor by replacing its third intracellular loop with apocytochrome b(562)RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure allowed us to identify 57 ordered water molecules inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved aspartate residue Asp(2.50). Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured water molecules, sodium ions, and lipids/cholesterol in GPCR stabilization and function.     

Structural basis of glmS ribozyme activation by glucosamine-6-phosphate

The glmS ribozyme is the only natural catalytic RNA known to require a small-molecule activator for catalysis. This catalytic RNA functions as a riboswitch, with activator-dependent RNA cleavage regulating glmS messenger RNA expression. We report crystal structures of the glmS ribozyme in precleavage states that are unliganded or bound to the competitive inhibitor glucose-6-phosphate and in the postcleavage state. All structures superimpose closely, revealing a remarkably rigid RNA that contains a preformed active and coenzyme-binding site. Unlike other riboswitches, the glmS ribozyme binds its activator in an open, solvent-accessible pocket. Our structures suggest that the amine group of the glmS ribozyme-bound coenzyme performs general acid-base and electrostatic catalysis.     

Structural basis of gating by the outer membrane transporter FecA

Siderophore-mediated acquisition systems facilitate iron uptake. We present the crystallographic structure of the integral outer membrane receptor FecA from Escherichia coli with and without ferric citrate at 2.5 and 2.0 angstrom resolution. FecA is composed of three distinct domains: the barrel, plug, and NH2-terminal extension. Binding of ferric citrate triggers a conformational change of the extracellular loops that close the external pocket of FecA. Ligand-induced allosteric transitions are propagated through the outer membrane by the plug domain, signaling the occupancy of the receptor in the periplasm. These data establish the structural basis of gating for receptors dependent on the cytoplasmic membrane protein TonB. By compiling available data for this family of receptors, we propose a mechanism for the energy-dependent transport of siderophores.