Structure of dimerized assimilatory NADPH-dependent sulfite reductase reveals the minimal interface for diflavin reductase binding.

NADPH-dependent assimilatory sulfite reductase (SiR) reduces sulfite by six electrons to make sulfide for incorporation into sulfur-containing biomolecules. SiR has two subunits: an NADPH, FMN, and FAD-binding diflavin flavoprotein and a siroheme/FeS cluster-containing hemoprotein. The molecular interactions that govern subunit binding have been unknown since the discovery of SiR over 50 years ago because SiR is flexible, thus has been intransigent for traditional high-resolution structural analysis.

Oligomer-to-monomer transition underlies the chaperone function of AAGAB in AP1/AP2 assembly.

Assembly of protein complexes is facilitated by assembly chaperones. Alpha and gamma adaptin-binding protein (AAGAB) is a chaperone governing the assembly of the heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) involved in clathrin-mediated membrane trafficking. Here, we found that before AP1/2 binding, AAGAB exists as a homodimer.

The characterization of novel monomeric creatine kinases in the early branching Alveolata species, Perkinsus marinus: Implications for phosphagen kinase evolution.

The genome of the unicellular molluscan parasite Perkinsus marinus contains at least five genes coding for putative creatine kinases (CK), a phosphoryl transfer enzyme which plays a key role in cellular energy transactions. Expression and kinetic analyses of three of the P. marinus CKs revealed them to be true CKs with catalytic properties in the range of typical metazoan CKs.

Neutron scattering maps the higher-order assembly of NADPH-dependent assimilatory sulfite reductase.

Precursor molecules for biomass incorporation must be imported into cells and made available to the molecular machines that build the cell. Sulfur-containing macromolecules require that sulfur be in its S oxidation state before assimilation into amino acids, cofactors, and vitamins that are essential to organisms throughout the biosphere. In α-proteobacteria, NADPH-dependent assimilatory sulfite reductase (SiR) performs the final six-electron reduction of sulfur.

Small-angle neutron scattering solution structures of NADPH-dependent sulfite reductase.

Sulfite reductase (SiR), a dodecameric complex of flavoprotein reductase subunits (SiRFP) and hemoprotein oxidase subunits (SiRHP), reduces sulfur for biomass incorporation. Electron transfer within SiR requires intra- and inter-subunit interactions that are mediated by the relative position of each protein, governed by flexible domain movements. Using small-angle neutron scattering, we report the first solution structures of SiR heterodimers containing a single copy of each subunit.

Siroheme synthase orients substrates for dehydrogenase and chelatase activities in a common active site.

Siroheme is the central cofactor in a conserved class of sulfite and nitrite reductases that catalyze the six-electron reduction of sulfite to sulfide and nitrite to ammonia. In Salmonella enterica serovar Typhimurium, siroheme is produced by a trifunctional enzyme, siroheme synthase (CysG). A bifunctional active site that is distinct from its methyltransferase activity catalyzes the final two steps, NAD-dependent dehydrogenation and iron chelation.

Bulged and Canonical G-Quadruplex Conformations Determine NDPK Binding Specificity.

Guanine-rich DNA strands can adopt tertiary structures known as G-quadruplexes (G4s) that form when Hoogsteen base-paired guanines assemble as planar stacks, stabilized by a central cation like K. In this study, we investigated the conformational heterogeneity of a G-rich sequence from the 5' untranslated region of the gene. This sequence adopted an extensively polymorphic G-quadruplex, including non-canonical bulged G-quadruplex folds that co-existed in solution.

NADPH-dependent sulfite reductase flavoprotein adopts an extended conformation unique to this diflavin reductase.

This is the first X-ray crystal structure of the monomeric form of sulfite reductase (SiR) flavoprotein (SiRFP-60) that shows the relationship between its major domains in an extended position not seen before in any homologous diflavin reductases. Small angle neutron scattering confirms this novel domain orientation also occurs in solution. Activity measurements of SiR and SiRFP variants allow us to propose a novel mechanism for electron transfer from the SiRFP reductase subunit to its oxidase metalloenzyme partner that, together, make up the SiR holoenzyme.

AIM2 inflammasome activation and regulation: A structural perspective.

Absent in melanoma 2 (AIM2) inflammasome is a multi-protein platform that recognizes aberrant cytoplasmic dsDNA and induces cytokine maturation, release and pyroptosis. It is composed of AIM2, apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1. Recent X-ray crystallographic and high resolution cryo-electron microscopic (cryo-EM) studies have revealed a series of structures in AIM2 inflammasome activation and regulation.

Quantification of Protein-Induced Membrane Remodeling Kinetics In Vitro with Lipid Multilayer Gratings.

The dynamic self-organization of lipids in biological systems is a highly regulated process that enables the compartmentalization of living systems at micro- and nanoscopic scales. Consequently, quantitative methods for assaying the kinetics of supramolecular remodeling such as vesicle formation from planar lipid bilayers or multilayers are needed to understand cellular self-organization. Here, a new nanotechnology-based method for quantitative measurements of lipid-protein interactions is presented and its suitability for quantifying the membrane binding, inflation, and budding activity of the membrane-remodeling protein Sar1 is demonstrated.

The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly.

Assimilatory NADPH-sulfite reductase (SiR) from Escherichia coli is a structurally complex oxidoreductase that catalyzes the six-electron reduction of sulfite to sulfide. Two subunits, one a flavin-binding flavoprotein (SiRFP, the α subunit) and the other an iron-containing hemoprotein (SiRHP, the β subunit), assemble to make a holoenzyme of about 800 kDa. How the two subunits assemble is not known.

Hrr25/CK1δ-directed release of Ltv1 from pre-40S ribosomes is necessary for ribosome assembly and cell growth.

Casein kinase 1δ/ε (CK1δ/ε) and their yeast homologue Hrr25 are essential for cell growth. Further, CK1δ is overexpressed in several malignancies, and CK1δ inhibitors have shown promise in several preclinical animal studies. However, the substrates of Hrr25 and CK1δ/ε that are necessary for cell growth and survival are unknown.

The maize (Zea mays L.) nucleoside diphosphate kinase1 (ZmNDPK1) gene encodes a human NM23-H2 homologue that binds and stabilizes G-quadruplex DNA.

Noncanonical forms of DNA like the guanine quadruplex (G4) play important roles in regulating transcription and translation through interactions with their protein partners. Although potential G4 elements have been identified in or near genes from species diverse as bacteria, mammals, and plants, little is known about how they might function as cis-regulatory elements or as binding sites for trans-acting protein partners. In fact, until now no G4 binding partners have been identified in the plant kingdom.

Visualization of retroviral envelope spikes in complex with the V3 loop antibody 447-52D on intact viruses by cryo-electron tomography.

Unlabelled: The gp120 portion of the envelope spike on human immunodeficiency virus type 1 (HIV-1) plays a critical role in viral entry into host cells and is a key target for the humoral immune response, and yet many structural details remain elusive. We have used cryoelectron tomography to visualize the binding of the broadly neutralizing monoclonal antibody (MAb) 447-52D to intact envelope spikes on virions of HIV-1 MN strain. Antibody 447-52D has previously been shown to bind to the tip of the V3 loop.

Cryo-EM structures of the actin:tropomyosin filament reveal the mechanism for the transition from C- to M-state.

Tropomyosin (Tm) is a key factor in the molecular mechanisms that regulate the binding of myosin motors to actin filaments (F-Actins) in most eukaryotic cells. This regulation is achieved by the azimuthal repositioning of Tm along the actin (Ac):Tm:troponin (Tn) thin filament to block or expose myosin binding sites on Ac. In striated muscle, including involuntary cardiac muscle, Tm regulates muscle contraction by coupling Ca(2+) binding to Tn with myosin binding to the thin filament.

Mutational analysis of sulfite reductase hemoprotein reveals the mechanism for coordinated electron and proton transfer.

Sulfite reductase catalyzes the six-electron reduction of sulfite to sulfide. The active site, found in the hemoprotein subunit (SiRHP), sits on the distal face of a negatively charged porphyrinoid called siroheme whose central iron atom is coupled to a proximal Fe(4)S(4) cluster. Four positively charged amino acids are positioned around the active site cavity.

Binding of anti-membrane-proximal gp41 monoclonal antibodies to CD4-liganded and -unliganded human immunodeficiency virus type 1 and simian immunodeficiency virus virions.

The broadly neutralizing monoclonal antibodies (MAbs) 4E10, 2F5, and Z13e1 target membrane-proximal external region (MPER) epitopes of HIV-1 gp41 in a manner that remains controversial. The requirements for initial lipid bilayer binding and/or CD4 ligation have been proposed. To further investigate these issues, we probed for binding of these MAbs to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) virions with protein A-conjugated gold (PAG) nanoparticles using negative-stain electron microscopy.

Linear IgE-epitope mapping and comparative structural homology modeling of hazelnut and English walnut 11S globulins.

Allergic reactions to walnuts and hazelnuts can be serious. The 11S globulins (legumins) have been identified as important allergens in these and other nuts and seeds. Here we identify the linear IgE-binding epitopes of walnut and hazelnut 11S globulins, and generate 3D 11S globulin models to map the locations of the epitopes for comparison to other allergenic homologues.

Cryoelectron tomography of HIV-1 envelope spikes: further evidence for tripod-like legs.

A detailed understanding of the morphology of the HIV-1 envelope (Env) spike is key to understanding viral pathogenesis and for informed vaccine design. We have previously presented a cryoelectron microscopic tomogram (cryoET) of the Env spikes on SIV virions. Several structural features were noted in the gp120 head and gp41 stalk regions.

The role of filament-packing dynamics in powering amoeboid cell motility.

Although several models have been proposed to account for how cytoskeleton polymerization drives protrusion in cell motility, the precise mechanism remains controversial. Here, we show that, in addition to force exerted directly against the membrane by growing filaments, the way elongating filaments pack also contributes to protrusion by generating an expansion of the cytoskeleton gel. Tomography shows that filament packing in the major sperm protein (MSP) -based nematode sperm-motility machinery resembles that observed with rigid rods.

The early evolution of the phosphagen kinases--insights from choanoflagellate and poriferan arginine kinases.

Arginine kinase (AK) is a member of a large family of phosphoryl transfer enzymes called phosphagen (guanidino) kinases. AKs are present in certain protozoans, sponges, cnidarians, and both lophotrochozoan and ecdysozoan protostomes. Another phosphagen kinase, creatine kinase (CK), is found in sponges, cnidarians, and both deuterostome and protostome groups but does not appear to be present in protozoans.

AIDS virus envelope spike structure.

The envelope (Env) spikes on HIV-1 and closely related SIV define the viral tropism, mediate the fusion process and are the prime target of the humoral response. Despite intensive efforts, Env has been slow to reveal its structural and functional secrets. Three gp120 subunits comprise the 'head' of Env and three gp41 subunits comprise the 'stalk' and other membrane-associated elements.

The capacity for the de novo biosynthesis of creatine is present in the tunicate Ciona intestinalis and is likely widespread in other protochordate and invertebrate groups.

Creatine kinase (CK) catalyzes the reversible transfer of thegamma-terminal phosphate of MgATP to the guanidine creatine (Cr) forming MgADP and phosphocreatine (PCr). The CK reaction plays a central role in both temporal and spatial ATP buffering in cells displaying high and variable rates of ATP turnover. There is a constant non-enzymatic conversion of Cr and PCr to creatinine that must be compensated for by biosynthesis and/or dietary uptake.