Flipping a genetic switch by subunit exchange.

The bacteriophage T4 AsiA protein is a multifunctional protein that simultaneously acts as both a repressor and activator of gene expression during the phage life cycle. These dual roles with opposing transcriptional consequences are achieved by modification of the host RNA polymerase in which AsiA binds to conserved region 4 (SR4) of sigma(70), altering the pathway of promoter selection by the holoenzyme. The mechanism by which AsiA flips this genetic switch has now been revealed, in part, from the three-dimensional structure of AsiA and the elucidation of its interaction with SR4. The structure of AsiA is that of a novel homodimer in which each monomer is constructed as a seven-helix bundle arranged in four overlapping helix-loop-helix elements. Identification of the protein interfaces for both the AsiA homodimer and the AsiA-sigma(70) complex reveals that these interfaces are coincident. Thus, the AsiA interaction with sigma(70) necessitates that the AsiA homodimer dissociate to form an AsiA-SR4 heterodimer, exchanging one protein subunit for another to alter promoter choice by RNA polymerase.

Structure of the RCK domain from the E. coli K+ channel and demonstration of its presence in the human BK channel.

The intracellular C-terminal domain structure of a six-transmembrane K+ channel from Escherichia coli has been solved by X-ray crystallography at 2.4 A resolution. The structure is representative of a broad class of domains/proteins that regulate the conductance of K+ (here referred to as RCK domains) in prokaryotic K+ transporters and K+ channels. The RCK domain has a Rossmann-fold topology with unique positions, not commonly conserved among Rossmann-fold proteins, composing a well-conserved salt bridge and a hydrophobic dimer interface. Structure-based amino acid sequence alignments and mutational analysis are used to demonstrate that an RCK domain is also present and is an important component of the gating machinery in eukaryotic large-conductance Ca2+ activated K+ channels.

Biophysical analysis of the endoplasmic reticulum-resident chaperone/heat shock protein gp96/GRP94 and its complex with peptide antigen.

Animals vaccinated with heat shock protein (HSP)--peptide complexes develop specific protective immunity against cancers from which the HSPs were originally isolated. This autologous specific immunity has been demonstrated using a number of HSP--peptide antigen complexes. A prototypical HSP-based cancer vaccine is the gp96--peptide antigen complex, which is currently undergoing human clinical trials. Here, we analyzed the structure of a recombinant wild-type and a mutant gp96 protein and their peptide complexes using a number of biophysical techniques. Gel filtration chromatography, dynamic light scattering, and equilibrium analytical ultracentrifugation demonstrated that both a wild-type gp96 and a gp96 mutant lacking a dimerization domain formed higher order structures. More detailed analysis using scanning transmission electron microscopy indicated that both the wild-type and dimerization deletion mutant gp96 protein were organized, unexpectedly, into large aggregates. Size distributions ranged from dimers to octamers and higher. Circular dichroism and intrinsic Trp fluorescence suggested that the gp96 dimerization domain deletion mutant protein was more compact than the wild-type gp96. A fluorescent peptide antigen was synthesized, and the peptide-binding properties of wild-type and the dimerization domain deletion mutant gp96 were studied. Fluorescence lifetime and anisotropy decay showed that the bound antigenic peptide was located in a hydrophobic pocket, with considerable free space for the rotation of the probe. Deletion of the dimerization domain affected the peptide-binding microenvironment, although peptide-binding affinity was reduced by only a small extent. Peptide--gp96 complexes were extremely stable, persisting for many days in the cold. The extraordinary stability of peptide--gp96 complexes and the plasticity of the peptide-binding pocket support the proposed relay of diverse peptides to MHC and/or other molecules via molecular recognition.

A robust, detergent-friendly method for mass spectrometric analysis of integral membrane proteins.

Recent breakthroughs in the high-resolution structural elucidation of ion channels and transporters are prompting a growing interest in methods for characterizing integral membrane proteins. These methods are proving extremely valuable in facilitating the production of X-ray diffraction-grade crystals. Here we present a robust and straightforward mass spectrometric procedure that utilizes matrix-assisted laser desorption/ionization to analyze integral membrane proteins in the presence of detergents. The utility of this method is illustrated with examples of high-quality mass spectral data obtained from membrane proteins for which atomic resolution structural studies are ongoing.

Hybrid and complex glycans are linked to the conserved N-glycosylation site of the third eight-cysteine domain of LTBP-1 in insect cells.

Covalent association of LTBP-1 (latent TGF-beta binding protein-1) to latent TGF-beta is mediated by the third eight-cysteine (also referred to as TB) module of LTBP-1, a domain designated as CR3. Spodoptera frugiperda (Sf9) cells have proved a suitable cell system in which to study this association and to produce recombinant CR3, and we show here that another lepidopteran cell line, Trichoplusia niTN-5B1-4 (High-Five) cells, allows the recovery of large amounts of functional recombinant CR3. CR3 contains an N-glycosylation site, which is conserved in all forms of LTBP known to date. When we examined the status of this N-glycosylation using MALDI-TOF mass spectrometry and enzymatic analysis, we found that CR3 is one of the rare recombinant peptides modified with complex glycans in insect cells. Sf9 cells mainly processed the fucosylated paucomannosidic structure (GlcNAc)(2)(Mannose)(3)Fucose, although hybrid and complex N-glycosylations were also detected. In High-Five cells, the peptide was found to be modified with a wide variety of hybrid and complex sugars in addition to paucomanosidic oligosaccharides. Most glycans had one or two fucose residues bound through alpha1,3 and alpha1,6 linkages to the innermost GlcNAc. On the basis of these results and on the structure of an eight-cysteine domain from fibrillin-1, we present a model of glycosylated CR3 and discuss the role of glycosylation in eight-cysteine domain protein-protein interactions.

Inhibition of neutrophil cathepsin G by oxidized mucus proteinase inhibitor. Effect of heparin.

Oxidation of mucus proteinase inhibitor (MPI) transforms Met73, the P'1 residue of its active center into methionine sulfoxide and lowers its affinity for neutrophil elastase [Boudier, C., and Bieth, J. G. (1994) Biochem. J. 303, 61-68]. Here, we show that the oxidized inhibitor has also a decreased affinity for neutrophil cathepsin G and pancreatic chymotrypsin. The Ki of the oxidized MPI-cathepsin G complex (1.2 microM) is probably too high to be compatible with significant inhibition of cathepsin G in inflammatory lung secretions. Stopped-flow kinetics shows that, within the inhibitor concentration range used, the mechanism of inhibition of cathepsin G and chymotrypsin by oxidized MPI is consistent with a one-step reaction, [equation in text] whereas the inhibition of elastase takes place in two steps, [equation in text]. Heparin, which accelerates the inhibition of the three proteinases by native MPI, also favors their interaction with oxidized MPI. Flow calorimetry shows that heparin binds oxidized MPI with Kd, Delta H degrees, and Delta S degrees values close to those reported for native MPI. In the presence of heparin, oxidized MPI inhibits cathepsin G via a two-step reaction characterized by Ki = 0.22 microM, k2 = 0.1 s-1, k-2 = 0.023 s-1, and Ki = 42 nM. Under these conditions, in vivo inhibition of cathepsin G is again possible. Heparin also improves the inhibition of chymotrypsin and elastase by oxidized MPI by increasing their kass or k2/Ki and decreasing their Ki. Our data suggest that oxidation of MPI during chronic bronchitis may lead to cathepsin G-mediated lung tissue degradation and that heparin may be a useful adjuvant of MPI-based therapy of acute lung inflammation in cystic fibrosis.

Instability of the amyloidogenic cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type.

A cystatin C variant with L68Q substitution and a truncation of 10 NH2-terminal residues is the major constituent of the amyloid deposited in the cerebral vasculature of patients with the Icelandic form of hereditary cerebral hemorrhage with amyloidosis (HCHWA-I). Variant and wild type cystatin C production, processing, secretion, and clearance were studied in human cell lines stably overexpressing the cystatin C genes. Immunoblot and mass spectrometry analyses demonstrated monomeric cystatin C in cell homogenates and culture media. While cystatin C formed concentration-dependent dimers, the HCHWA-I variant dimerized at lower concentrations than the wild type protein. Amino-terminal sequence analysis revealed that the variant and normal proteins produced and secreted are the full-length cystatin C. Pulse-chase experiments demonstrated similar levels of normal and variant cystatin C production and secretion. However, the secreted variant cystatin C exhibited an increased susceptibility to a serine protease in conditioned media and in human cerebrospinal fluid, explaining its depletion from the cerebrospinal fluid of HCHWA-I patients. Thus, the amino acid substitution may induce unstable cystatin C with intact inhibitory activity and predisposition to self-aggregation and amyloid fibril formation.

Inhibition of neutrophil serine proteinases by suramin.

Suramin, a hexasulfonated naphtylurea recently used as an anti-tumor drug, is a potent inhibitor of human neutrophil elastase, cathepsin G, and proteinase 3. The complexes it forms with these enzymes are partially active on synthetic substrates, but full inhibition takes place when elastase activity is measured with fibrous elastin or when cathepsin G activity is measured using platelet aggregation. One molecule of elastase binds four molecules of suramin with a Ki of 2 x 10(-7) M as determined by enzyme inhibition or intrinsic fluorescence enhancement of suramin. The binding curves show no sign of cooperativity or anticooperativity. The Ki for the complexes with cathepsin G and proteinase 3 are 8 x 10(-8) and 5 x 10(-7) M, respectively. Ionic strength increases the Ki of the elastase-suramin complex in a way that suggests that four of the six sulfonate groups of suramin form ionic interactions with basic residues of the enzyme and that at saturation almost all arginines of elastase form salt bridges with suramin. The neutrophil proteinase-inhibitory activity of suramin might be used to prevent tissue destruction and thrombus formation in diseases where massive infiltration and activation of neutrophils take place.

Mapping the suramin-binding sites of human neutrophil elastase: investigation by fluorescence resonance energy transfer and molecular modeling.

Neutrophil elastase (NE), a mediator of inflammation, binds with high affinity numerous anionic molecules including suramin, a polysulfated naphthylurea, which inhibits it with a Ki of 0.2 microM and a 4:1 suramin:NE stoichiometry and thus constitutes a potential therapeutic agent. In an attempt to locate the suramin molecules on NE, we investigated the NE-suramin interaction using steady-state and time-resolved fluorescence spectroscopy. The time-resolved intensity decay of NE, a protein with three Trp residues, in positions 27, 141, and 237 (chymotrypsin numbering system) was best described by a three-exponential function with lifetimes ranging from 0.22 to 2.28 ns. Comparison of the accessibility of the three lifetime classes to the fluorescence quenchers acrylamide and iodide with the computed solvent accessibility of the three Trp residues in the crystal structure of NE indicates that the main, if not the sole, contribution to the 2.28 ns lifetime class is brought about by the fully buried Trp 141 residue. The addition of suramin to NE induces a sharp decrease in NE fluorescence and a corresponding increase in suramin fluorescence due to an efficient fluorescence resonance energy transfer (FRET) between the Trp residues of NE, acting as donors, and the naphthalene rings of suramin, behaving as acceptors. From the fate of the longest lifetime class in the presence of variable suramin concentrations, we deduce that two suramins are bound at less than 17 A from Trp 141, whereas the two others are located at least 29 A from Trp 141. Moreover, neither the binding of suramin to NE nor the FRET process was modified when NE was complexed with a peptide chloromethylketone inhibitor, suggesting that suramin does not directly interfere with the substrate binding site of NE. These data were used as constraints to model the NE-suramin complex.

Influence of low molecular mass heparin on the kinetics of neutrophil elastase inhibition by mucus proteinase inhibitor.

Commercial low molecular mass heparin accelerates the inhibition of neutrophil elastase by mucus proteinase inhibitor, the predominant antielastase of lung secretions (Faller, B., Mély, Y., Gérard, D., and Bieth, J.G. (1992) Biochemistry 31, 8285-8290). To study the kinetic mechanism of this rate enhancement, we have isolated a 4.5-kDa heparin fragment from commercial heparin. This compound is fairly monodisperse as shown by analytical ultracentrifugation. It binds elastase and inhibitor with a 1:1 stoichiometry and an equilibrium dissociation constant of 3 and 210 nM, respectively. It also forms a tight complex with EI. Flow calorimetry shows that the inhibitor-heparin interaction is characterized by a large negative enthalpy change (delta H0 = -45.2 kJ mol-1) and a small entropy change (delta S = -23.7 J K-1 mol-1). Stopped-flow kinetics run under pseudo-first-order conditions ([Io] >> [Eo]) show that in the absence of heparin the inhibition conforms to a simple bimolecular reaction, [formula: see text] where, ka = 3.1 x 10(6) M-1 s-1, kd = 10(-4) s-1, and Ki = 33 pM, whereas in the presence of heparin, E and I react via a two-step mechanism, [formula: see text] where Ki* = 86 nM, k2 = 2.2 s-1, k-2 = 10(-3) s-1, and Ki = 37 pM. Thus, heparin increases both the rate of inhibition by promoting the formation of a high affinity EI* intermediate and the rate of EI dissociation. Since the dissociation is negligible in bronchial secretions where the inhibitor concentration is much higher than Ki, it may be concluded that heparin significantly potentiates the inhibitor's antielastase potential in vivo.

Demonstration of a two-step reaction mechanism for the inhibition of heparin-bound neutrophil elastase by alpha 1-proteinase inhibitor.

Heparin decreases the rate of inhibition of neutrophil elastase by alpha 1-proteinase inhibitor as a result of its strong binding to the enzyme. Here, we used the slow-binding kinetic approach to decide whether the enzyme-inhibitor interaction proceeds via a two-step mechanism and to identify the step that is affected by heparin. The inhibition kinetics was assessed under pseudo-first-order conditions using conventional or stopped-flow spectrophotometry. In the absence of heparin, the pseudo-first-order rate constant of inhibition increased linearly with the inhibitor concentration indicating that within the experimental concentration range (< or = 6 microM) the enzyme-inhibitor association conforms either to a simple bimolecular reaction (E+I kass-->EI with kass = 10(7) M-1 s-1) or to a two-step reaction (E+I Ki*<==>EI* k2-->EI with Ki* > 0.4 microM and k2 > 4 s-1). In the presence of heparin, the rate constant of inhibition varied hyperbolically with the inhibitor concentration, indicating that the inhibition is a two-step process with Ki* = 80 nM and K2 = 0.15 s-1. Thus, heparin has two opposite effects on the elastase + alpha 1-proteinase inhibitor interaction: it favors the association by decreasing Ki* but impairs it by decreasing k2. This rationalizes the previously demonstrated rate-depressing effect of the sulfated polymer. Heparin does not significantly alter the stability of the irreversible elastase-alpha 1-proteinase inhibitor complex.