Crystal Structure of the RNA Lariat Debranching Enzyme Dbr1 with Hydrolyzed Phosphorothioate RNA Product.

The RNA lariat debranching enzyme is the sole enzyme responsible for hydrolyzing the 2'-5' phosphodiester bond in RNA lariats produced by the spliceosome. Here, we test the ability of Dbr1 to hydrolyze branched RNAs (bRNAs) that contain a 2'-5'-phosphorothioate linkage, a modification commonly used to resist degradation. We attempted to cocrystallize a phosphorothioate-branched RNA (PS-bRNA) with wild-type Dbr1 (EhDbr1) but observed in-crystal hydrolysis of the phosphorothioate bond.

Quaternary structure independent folding of voltage-gated ion channel pore domain subunits.

Every voltage-gated ion channel (VGIC) has a pore domain (PD) made from four subunits, each comprising an antiparallel transmembrane helix pair bridged by a loop. The extent to which PD subunit structure requires quaternary interactions is unclear. Here, we present crystal structures of a set of bacterial voltage-gated sodium channel (BacNa) 'pore only' proteins that reveal a surprising collection of non-canonical quaternary arrangements in which the PD tertiary structure is maintained.

Structure and function of a flavin-dependent S-monooxygenase from garlic ().

Allicin is a component of the characteristic smell and flavor of garlic (). A flavin-containing monooxygenase (FMO) produced by (AsFMO) was previously proposed to oxidize -allyl-l-cysteine (SAC) to alliin, an allicin precursor. Here, we present a kinetic and structural characterization of AsFMO that suggests a possible contradiction to this proposal.

Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1.

Intron lariats are circular, branched RNAs (bRNAs) produced during pre-mRNA splicing. Their unusual chemical and topological properties arise from branch-point nucleotides harboring vicinal 2',5'- and 3',5'-phosphodiester linkages. The 2',5'-bonds must be hydrolyzed by the RNA debranching enzyme Dbr1 before spliced introns can be degraded or processed into small nucleolar RNA and microRNA derived from intronic RNA.

Unexpected features and mechanism of heterodimer formation of a herpesvirus nuclear egress complex.

Herpesvirus nucleocapsids escape from the nucleus in a process orchestrated by a highly conserved, viral nuclear egress complex. In human cytomegalovirus, the complex consists of two proteins, UL50 and UL53. We solved structures of versions of UL53 and the complex by X-ray crystallography.

Structural basis of lariat RNA recognition by the intron debranching enzyme Dbr1.

The enzymatic processing of cellular RNA molecules requires selective recognition of unique chemical and topological features. The unusual 2',5'-phosphodiester linkages in RNA lariats produced by the spliceosome must be hydrolyzed by the intron debranching enzyme (Dbr1) before they can be metabolized or processed into essential cellular factors, such as snoRNA and miRNA. Dbr1 is also involved in the propagation of retrotransposons and retroviruses, although the precise role played by the enzyme in these processes is poorly understood.

Molecular mechanism and structure of the Saccharomyces cerevisiae iron regulator Aft2.

The paralogous iron-responsive transcription factors Aft1 and Aft2 (activators of ferrous transport) regulate iron homeostasis in Saccharomyces cerevisiae by activating expression of iron-uptake and -transport genes when intracellular iron is low. We present the previously unidentified crystal structure of Aft2 bound to DNA that reveals the mechanism of DNA recognition via specific interactions of the iron-responsive element with a Zn(2+)-containing WRKY-GCM1 domain in Aft2. We also show that two Aft2 monomers bind a [2Fe-2S] cluster (or Fe(2+)) through a Cys-Asp-Cys motif, leading to dimerization of Aft2 and decreased DNA-binding affinity.

Structure of HHARI, a RING-IBR-RING ubiquitin ligase: autoinhibition of an Ariadne-family E3 and insights into ligation mechanism.

A distinct mechanism for ubiquitin (Ub) ligation has recently been proposed for the RING1-IBR-RING2 (RBR) family of E3s: an N-terminal RING1 domain recruits a thioester-linked intermediate complex between Ub and the E2 UbcH7, and a structurally distinct C-terminal RING2 domain displays a catalytic cysteine required for Ub ligation. To obtain insights into RBR E3s, we determined the crystal structure of the human homolog of Ariadne (HHARI), which reveals the individual RING1, IBR, and RING2 domains embedded in superdomains involving sequences specific to the Ariadne RBR subfamily. The central IBR is flanked on one side by RING1, which is exposed and binds UbcH7.

Structure of the Chlamydia trachomatis immunodominant antigen Pgp3.

Chlamydia trachomatis infection is the most common sexually transmitted bacterial disease. Left untreated, it can lead to ectopic pregnancy, pelvic inflammatory disease, and infertility. Here we present the structure of the secreted C.

Recognition of nucleoside monophosphate substrates by Haemophilus influenzae class C acid phosphatase.

The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD(+) utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5',3'-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5'-AMP, 3'-AMP, and 2'-AMP.

Structure of avian thymic hormone, a high-affinity avian beta-parvalbumin, in the Ca2+-free and Ca2+-bound states.

Originally isolated on the basis of its capacity to stimulate T-cell maturation and proliferation, avian thymic hormone (ATH) is nevertheless a parvalbumin, one of two beta-lineage isoforms expressed in birds. We recently learned that addition of Ca(2+)-free ATH to a solution of 8-anilinonaphthalene-1-sulfonate (ANS) markedly increases ANS emission. This behavior, not observed in the presence of Ca(2+), suggests that apolar surface area buried in the Ca(2+)-bound state becomes solvent accessible upon Ca(2+) removal.

Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum.

The bifunctional proline catabolic flavoenzyme, proline utilization A (PutA), catalyzes the oxidation of proline to glutamate via the sequential activities of FAD-dependent proline dehydrogenase (PRODH) and NAD(+)-dependent Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Although structures for some of the domains of PutA are known, a structure for the full-length protein has not previously been solved. Here we report the 2.

Crystal Structures of the histidine acid phosphatase from Francisella tularensis provide insight into substrate recognition.

Histidine acid phosphatases catalyze the transfer of a phosphoryl group from phosphomonoesters to water at acidic pH using an active-site histidine. The histidine acid phosphatase from the category A pathogen Francisella tularensis (FtHAP) has been implicated in intramacrophage survival and virulence, motivating interest in understanding the structure and mechanism of this enzyme. Here, we report a structure-based study of ligand recognition by FtHAP.

Structural and biophysical properties of metal-free pathogenic SOD1 mutants A4V and G93A.

Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by the destruction of motor neurons in the spinal cord and brain. A subset of ALS cases are linked to dominant mutations in copper-zinc superoxide dismutase (SOD1). The pathogenic SOD1 variants A4V and G93A have been the foci of multiple studies aimed at understanding the molecular basis for SOD1-linked ALS.

Structural and biophysical properties of the pathogenic SOD1 variant H46R/H48Q.

Over 100 mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause an inherited form of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Two pathogenic SOD1 mutations, His46Arg (H46R) and His48Gln (H48Q), affect residues that act as copper ligands in the wild type enzyme. Transgenic mice expressing a human SOD1 variant containing both mutations develop paralytic disease akin to ALS.

A conserved active site tyrosine residue of proline dehydrogenase helps enforce the preference for proline over hydroxyproline as the substrate.

Proline dehydrogenase (PRODH) catalyzes the oxidation of l-proline to Delta-1-pyrroline-5-carboxylate. PRODHs exhibit a pronounced preference for proline over hydroxyproline (trans-4-hydroxy-l-proline) as the substrate, but the basis for specificity is unknown. The goal of this study, therefore, is to gain insight into the structural determinants of substrate specificity of this class of enzyme, with a focus on understanding how PRODHs discriminate between the two closely related molecules, proline and hydroxyproline.

Three crystal forms of the bifunctional enzyme proline utilization A (PutA) from Bradyrhizobium japonicum.

Proline utilization A proteins (PutAs) are large (1000-1300 residues) membrane-associated bifunctional flavoenzymes that catalyze the two-step oxidation of proline to glutamate by the sequential action of proline dehydrogenase and Delta(1)-pyrroline-5-carboxylate dehydrogenase domains. Here, the first successful crystallization efforts for a PutA protein are described. Three crystal forms of PutA from Bradyrhizobium japonicum are reported: apparent tetragonal, hexagonal and centered monoclinic.

Structure of the Hsp110:Hsc70 nucleotide exchange machine.

Hsp70s mediate protein folding, translocation, and macromolecular complex remodeling reactions. Their activities are regulated by proteins that exchange ADP for ATP from the nucleotide-binding domain (NBD) of the Hsp70. These nucleotide exchange factors (NEFs) include the Hsp110s, which are themselves members of the Hsp70 family.

Cooperative assembly of TGF-beta superfamily signaling complexes is mediated by two disparate mechanisms and distinct modes of receptor binding.

Dimeric ligands of the transforming growth factor-beta (TGF-beta) superfamily signal across cell membranes in a distinctive manner by assembling heterotetrameric complexes of structurally related serine/threonine-kinase receptor pairs. Unlike complexes of the bone morphogenetic protein (BMP) branch that apparently form due to avidity from membrane localization, TGF-beta complexes assemble cooperatively through recruitment of the low-affinity (type I) receptor by the ligand-bound high-affinity (type II) pair. Here we report the crystal structure of TGF-beta3 in complex with the extracellular domains of both pairs of receptors, revealing that the type I docks and becomes tethered via unique extensions at a composite ligand-type II interface.

Crystal structures of the DNA-binding domain of Escherichia coli proline utilization A flavoprotein and analysis of the role of Lys9 in DNA recognition.

PutA (proline utilization A) from Escherichia coli is a 1320-amino-acid residue protein that is both a bifunctional proline catabolic enzyme and an autogenous transcriptional repressor. Here, we report the first crystal structure of a PutA DNA-binding domain along with functional analysis of a mutant PutA defective in DNA binding. Crystals were grown using a polypeptide corresponding to residues 1-52 of E.

Evidence for structural plasticity of heavy chain complementarity-determining region 3 in antibody-ssDNA recognition.

Anti-DNA antibodies play important roles in the pathogenesis of autoimmune diseases. They also represent a unique and relatively unexplored class of DNA-binding protein. Here, we present a study of conformational changes induced by DNA binding to an anti-ssDNA Fab known as DNA-1.

Structures of the Escherichia coli PutA proline dehydrogenase domain in complex with competitive inhibitors.

Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the flavin-dependent oxidation of proline to Delta(1)-pyrroline-5-carboxylate. Here we present a structure-based study of the PRODH active site of the multifunctional Escherichia coli proline utilization A (PutA) protein using X-ray crystallography, enzyme kinetic measurements, and site-directed mutagenesis. Structures of the PutA PRODH domain complexed with competitive inhibitors acetate (K(i) = 30 mM), L-lactate (K(i) = 1 mM), and L-tetrahydro-2-furoic acid (L-THFA, K(i) = 0.

Structure of an anti-DNA fab complexed with a non-DNA ligand provides insights into cross-reactivity and molecular mimicry.

Antibodies that recognize DNA (anti-DNA) are part of the autoimmune response underlying systemic lupus erythematosus. To better understand molecular recognition by anti-DNA antibodies, crystallographic studies have been performed using an anti-ssDNA antigen-binding fragment (Fab) known as DNA-1. The previously determined structure of a DNA-1/dT5 complex revealed that thymine bases insert into a narrow groove, and that ligand recognition primarily involves the bases of DNA.

Crystal structure of rat alpha-parvalbumin at 1.05 Angstrom resolution.

The crystal structure of rat alpha-parvalbumin has been determined at 1.05 Angstrom resolution, using synchrotron data collected at Advanced Photon Source beamline 19-ID. After refinement with SHELX, employing anisotropic displacement parameters and riding hydrogen atoms, R = 0.

MRSAD: using anomalous dispersion from S atoms collected at Cu Kalpha wavelength in molecular-replacement structure determination.

The use of single-wavelength anomalous dispersion (SAD) from S atoms collected in-house to overcome model bias in molecular-replacement (MR) structure determination is demonstrated. The test case considered is a P6(5)22 anti-ssDNA Fab crystal with a theoretical anomalous signal of 0.8% and a diffraction limit of 2.