How an assembly factor enhances covalent FAD attachment to the flavoprotein subunit of complex II.

The membrane-bound complex II family of proteins is composed of enzymes that catalyze succinate and fumarate interconversion coupled with reduction or oxidation of quinones within the membrane domain. The majority of complex II enzymes are protein heterotetramers with the different subunits harboring a variety of redox centers. These redox centers are used to transfer electrons between the site of succinate-fumarate oxidation/reduction and the membrane domain harboring the quinone.

Short Arrestin-3-Derived Peptides Activate JNK3 in Cells.

Arrestins were first discovered as suppressors of G protein-mediated signaling by G protein-coupled receptors. It was later demonstrated that arrestins also initiate several signaling branches, including mitogen-activated protein kinase cascades. Arrestin-3-dependent activation of the JNK family can be recapitulated with peptide fragments, which are monofunctional elements distilled from this multi-functional arrestin protein.

Display of the human mucinome with defined O-glycans by gene engineered cells.

Mucins are a large family of heavily O-glycosylated proteins that cover all mucosal surfaces and constitute the major macromolecules in most body fluids. Mucins are primarily defined by their variable tandem repeat (TR) domains that are densely decorated with different O-glycan structures in distinct patterns, and these arguably convey much of the informational content of mucins. Here, we develop a cell-based platform for the display and production of human TR O-glycodomains (~200 amino acids) with tunable structures and patterns of O-glycans using membrane-bound and secreted reporters expressed in glycoengineered HEK293 cells.

Structure based virtual screening identifies small molecule effectors for the sialoglycan binding protein Hsa.

Infective endocarditis (IE) is a cardiovascular disease often caused by bacteria of the viridans group of streptococci, which includes Streptococcus gordonii and Streptococcus sanguinis. Previous research has found that serine-rich repeat (SRR) proteins on the S. gordonii bacterial surface play a critical role in pathogenesis by facilitating bacterial attachment to sialylated glycans displayed on human platelets.

A Novel Class of Common Docking Domain Inhibitors That Prevent ERK2 Activation and Substrate Phosphorylation.

Extracellular signal-regulated kinases (ERK1/2) are mitogen-activated protein kinases (MAPKs) that play a pro-tumorigenic role in numerous cancers. ERK1/2 possess two protein-docking sites that are distinct from the active site: the D-recruitment site (DRS) and the F-recruitment site. These docking sites facilitate substrate recognition, intracellular localization, signaling specificity, and protein complex assembly.

Structural Basis of Arrestin-Dependent Signal Transduction.

Arrestins are a small family of proteins with four isoforms in humans. Remarkably, two arrestins regulate signaling from >800 G protein-coupled receptors (GPCRs) or nonreceptor activators by simultaneously binding an activator and one out of hundreds of other signaling proteins. When arrestins are bound to GPCRs or other activators, the affinity for these signaling partners changes.

Differential manipulation of arrestin-3 binding to basal and agonist-activated G protein-coupled receptors.

Non-visual arrestins interact with hundreds of different G protein-coupled receptors (GPCRs). Here we show that by introducing mutations into elements that directly bind receptors, the specificity of arrestin-3 can be altered. Several mutations in the two parts of the central "crest" of the arrestin molecule, middle-loop and C-loop, enhanced or reduced arrestin-3 interactions with several GPCRs in receptor subtype and functional state-specific manner.

Novel aspects of sialoglycan recognition by the Siglec-like domains of streptococcal SRR glycoproteins.

Serine-rich repeat glycoproteins are adhesins expressed by commensal and pathogenic Gram-positive bacteria. A subset of these adhesins, expressed by oral streptococci, binds sialylated glycans decorating human salivary mucin MG2/MUC7, and platelet glycoprotein GPIb. Specific sialoglycan targets were previously identified for the ligand-binding regions (BRs) of GspB and Hsa, two serine-rich repeat glycoproteins expressed by Streptococcus gordonii While GspB selectively binds sialyl-T antigen, Hsa displays broader specificity.

Peptide mini-scaffold facilitates JNK3 activation in cells.

Three-kinase mitogen-activated protein kinase (MAPK) signaling cascades are present in virtually all eukaryotic cells. MAPK cascades are organized by scaffold proteins, which assemble cognate kinases into productive signaling complexes. Arrestin-3 facilitates JNK activation in cells, and a short 25-residue arrestin-3 peptide was identified as the critical JNK3-binding element.

Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II.

Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate:ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol:fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate.

Arrestin expression in E. coli and purification.

Purified arrestin proteins are necessary for biochemical, biophysical, and crystallographic studies of these versatile regulators of cell signaling. Described herein is a basic protocol for arrestin expression in E. coli and purification of the tag-free wild-type and mutant arrestins.

A conserved phenylalanine as a relay between the α5 helix and the GDP binding region of heterotrimeric Gi protein α subunit.

G protein activation by G protein-coupled receptors is one of the critical steps for many cellular signal transduction pathways. Previously, we and other groups reported that the α5 helix in the G protein α subunit plays a major role during this activation process. However, the precise signaling pathway between the α5 helix and the guanosine diphosphate (GDP) binding pocket remains elusive.

Characterization of fibrinogen binding by glycoproteins Srr1 and Srr2 of Streptococcus agalactiae.

The serine-rich repeat glycoproteins of Gram-positive bacteria comprise a large family of cell wall proteins. Streptococcus agalactiae (group B streptococcus, GBS) expresses either Srr1 or Srr2 on its surface, depending on the strain. Srr1 has recently been shown to bind fibrinogen, and this interaction contributes to the pathogenesis of GBS meningitis.

Plasticity of the quinone-binding site of the complex II homolog quinol:fumarate reductase.

Respiratory processes often use quinone oxidoreduction to generate a transmembrane proton gradient, making the 2H(+)/2e(-) quinone chemistry important for ATP synthesis. There are a variety of quinones used as electron carriers between bioenergetic proteins, and some respiratory proteins can functionally interact with more than one quinone type. In the case of complex II homologs, which couple quinone chemistry to the interconversion of succinate and fumarate, the redox potentials of the biologically available ubiquinone and menaquinone aid in driving the chemical reaction in one direction.

Involvement of distinct arrestin-1 elements in binding to different functional forms of rhodopsin.

Solution NMR spectroscopy of labeled arrestin-1 was used to explore its interactions with dark-state phosphorylated rhodopsin (P-Rh), phosphorylated opsin (P-opsin), unphosphorylated light-activated rhodopsin (Rh*), and phosphorylated light-activated rhodopsin (P-Rh*). Distinct sets of arrestin-1 elements were seen to be engaged by Rh* and inactive P-Rh, which induced conformational changes that differed from those triggered by binding of P-Rh*. Although arrestin-1 affinity for Rh* was seen to be low (K(D) > 150 μM), its affinity for P-Rh (K(D) ~80 μM) was comparable to the concentration of active monomeric arrestin-1 in the outer segment, suggesting that P-Rh generated by high-gain phosphorylation is occupied by arrestin-1 under physiological conditions and will not signal upon photo-activation.

Engineering visual arrestin-1 with special functional characteristics.

Arrestin-1 preferentially binds active phosphorylated rhodopsin. Previously, a mutant with enhanced binding to unphosphorylated active rhodopsin (Rh*) was shown to partially compensate for lack of rhodopsin phosphorylation in vivo. Here we showed that reengineering of the receptor binding surface of arrestin-1 further improves the binding to Rh* while preserving protein stability.

Structural basis for malfunction in complex II.

Complex II couples oxidoreduction of succinate and fumarate at one active site with that of quinol/quinone at a second distinct active site over 40 Å away. This process links the Krebs cycle to oxidative phosphorylation and ATP synthesis. The pathogenic mutation or inhibition of human complex II or its assembly factors is often associated with neurodegeneration or tumor formation in tissues derived from the neural crest.

Bacillus cereus phosphopentomutase is an alkaline phosphatase family member that exhibits an altered entry point into the catalytic cycle.

Bacterial phosphopentomutases (PPMs) are alkaline phosphatase superfamily members that interconvert α-D-ribose 5-phosphate (ribose 5-phosphate) and α-D-ribose 1-phosphate (ribose 1-phosphate). We investigated the reaction mechanism of Bacillus cereus PPM using a combination of structural and biochemical studies. Four high resolution crystal structures of B.

Geometric restraint drives on- and off-pathway catalysis by the Escherichia coli menaquinol:fumarate reductase.

Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate.

Mechanism of potassium-channel selectivity revealed by Na(+) and Li(+) binding sites within the KcsA pore.

Potassium channels allow K(+) ions to diffuse through their pores while preventing smaller Na(+) ions from permeating. Discrimination between these similar, abundant ions enables these proteins to control electrical and chemical activity in all organisms. Selection occurs at the narrow selectivity filter containing structurally identified K(+) binding sites.

Expression, purification and preliminary X-ray analysis of the Neisseria meningitidis outer membrane protein PorB.

The Neisseria meningitidis outer membrane protein PorB was expressed in Escherichia coli and purified from inclusion bodies by denaturation in urea followed by refolding in buffered LDAO on a size-exclusion column. PorB has been crystallized in three different crystal forms: C222, R32 and P6(3). The C222 crystal form may contain either one or two PorB monomers in the asymmetric unit, while both the R32 and P6(3) crystal forms contained one PorB monomer in the asymmetric unit.

The enteropathy of prostaglandin deficiency.

Background: Small intestinal ulcers are frequent complications of therapy with nonsteroidal anti-inflammatory drugs (NSAIDs). We present here a genetic deficiency of eicosanoid biosynthesis that illuminates the mechanism of NSAID-induced ulcers of the small intestine.

Methods: Eicosanoids and metabolites were measured by isotope dilution with mass spectrometry.

Helix dipole movement and conformational variability contribute to allosteric GDP release in Galphai subunits.

Heterotrimeric G proteins (Galphabetagamma) transmit signals from activated G protein-coupled receptors (GPCRs) to downstream effectors through a guanine nucleotide signaling cycle. Numerous studies indicate that the carboxy-terminal alpha5 helix of Galpha subunits participates in Galpha-receptor binding, and previous EPR studies suggest this receptor-mediated interaction induces a rotation and translation of the alpha5 helix of the Galpha subunit [Oldham, W. M.

A threonine on the active site loop controls transition state formation in Escherichia coli respiratory complex II.

In Escherichia coli, the complex II superfamily members succinate:ubiquinone oxidoreductase (SQR) and quinol:fumarate reductase (QFR) participate in aerobic and anaerobic respiration, respectively. Complex II enzymes catalyze succinate and fumarate interconversion at the interface of two domains of the soluble flavoprotein subunit, the FAD binding domain and the capping domain. An 11-amino acid loop in the capping domain (Thr-A234 to Thr-A244 in quinol:fumarate reductase) begins at the interdomain hinge and covers the active site.