Application of electrospray ionization mass spectrometry for studying human immunodeficiency virus protein complexes

Mass spectrometry (MS) with electrospray ionization (ESI) has shown utility for studying noncovalent protein complexes, as it offers advantages in sensitivity, speed, and mass accuracy. The stoichiometry of the binding partners can be easily deduced from the molecular weight measurement. In many examples of protein complexes, the gas phase-based measurement is consistent with the expected solution phase binding characteristics. This quality suggests the utility of ESI-MS for investigating solution phase molecular interactions. Complexes composed of proteins from the human immunodeficiency virus (HIV) have been studied using ESI-MS. Multiply charged protein dimers from HIV integrase catalytic core (F185K) and HIV protease have been observed. Furthermore, the ternary complex between HIV protease dimer and inhibitor pepstatin A was studied as a function of solution pH. Zinc binding to zinc finger-containing nucleocapsid protein (NCp7) and the NCp7-psi RNA 1:1 stoichiometry complex was also studied by ESI-MS. No protein-RNA complex was observed in the absence of zinc, consistent with the role of the zinc finger motifs for RNA binding. Proteins Suppl. 2:28–37, 1998. © 1998 Wiley-Liss, Inc.     

Interaction of the poliovirus receptor CD155 with the dynein light chain Tctex-1 and its implication for poliovirus pathogenesis.

The cellular receptor for poliovirus CD155 (or PVR) is the founding member of a new class of membrane-associated immunoglobulin-like proteins, which include the mouse tumor-associated antigen E4 (Tage4) and three proteins termed "nectins." Using a yeast two-hybrid screen we have discovered that the cytoplasmic domain of CD155 associates strongly and specifically with Tctex-1, a light chain of the dynein motor complex, the latter representing the major driving force for retrograde transport of endocytic vesicles and membranous organelles. We confirmed the interaction biochemically and by co-immunoprecipitation, and we mapped the Tctex-1 binding site to a SKCSR motif within the juxtamembrane region of CD155. Tctex-1 immunoreactivity was detected in mouse sciatic nerve and spinal cord (two tissues of central importance for poliovirus pathogenesis) in punctate, possibly vesicular, patterns. We propose that the cytoplasmic domain may target CD155-containing endocytic vesicles to the microtubular network. Neurotropic viruses like poliovirus, herpesvirus, rabies virus, and pseudorabies virus all utilize neuronal retrograde transport to invade the central nervous system. Association with Tctex-1 and, hence, with the dynein motor complex may offer an explanation for how poliovirus hijacks the cellular transport machinery to retrogradely ascend along the axon to the neuronal cell body.     

Reconstitution of acetylcholine receptor from Torpedo californica with highly purified phospholipids: Effect of α-tocopherol,phylloquinone, and other terpenoid quinones

Acetylcholine receptor from $\text{Torpedo californica}$ can be incorporated by the cholate dialysis procedure into liposomes prepared with crude soybean phospholipids (asolectin). Vesicles reconstituted with asolectin depleted of neutral lipids or with a mixture of pure phospholipids, are less active in catalyzing carbamylcholine-sensitive Na⁺ flux. Inclusion of α-tocopherol or certain quinones such as coenzyme Q₁₀ or vitamin K₁ during reconstitution yields vesicles with carbamylcholine-sensitive Na⁺ flux which, under optimal conditions, was considerably higher than that observed with vesicles reconstituted with crude phospholipid mixtures.     

NMR identification of protein surfaces using paramagnetic probes

Paramagnetic agents produce line broadening and thus cancellation of anti phase cross-peak components in two-dimensional correlated nuclear magnetic resonance spectra. The specificity of this effect was examined to determine its utility for identifying surface residues of proteins. Ubiquitin and hen egg white lysozyme, for which X-ray crystal structures and proton NMR assignments are available, served as test cases. Two relaxation reagents were employed, 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy and the gadolinium (III) diethylenetriaminepentaacetate complex ion. Correlations were sought between reagent-produced decreases of side-chain cross-peak volumes in double-quantum-filtered proton correlation (DQF-COSY) spectra and the solvent-exposed side-chain surface area of the corresponding residues. The lanthanide complex produced strong effects ascribable to association with carboxylate groups but was not otherwise useful in delineating surface residues. The nitroxyl, on the other hand, produced clear distinctions among the Val, Leu, and Ile residues that generally paralleled side-chain exposure in the crystal, although consistent correlations were not observed with residues of other types. Although an instance of possible specific protein-nitroxyl association was noted, the nitroxyl appears to be a tool for identifying hydrophobic surface residues.     

Regioselective enzymatic aminoacylation of lobucavir to give an intermediate for lobucavir prodrug

Synthesis of lobucavir prodrug, L-valine, [(1S,2R,3R)-3-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (BMS 233866), requires regioselective coupling of one of the two hydroxyl groups of lobucavir (BMS 180194) with valine. Either hydroxyl group of lobucavir could be selectively aminoacylated with valine by using enzymatic reactions. N-[(Phenylmethoxy)carbonyl]-L-valine, [(1R,2R,4S)-2-(2-amino-6-oxo-1H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester (3, 82.5% yield), was obtained by selective hydrolysis of N,N′-bis[(phenylmethoxy)carbonyl]bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester (1) with lipase M, and L-valine, [(1R,2R,4S)-2-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (4, 87% yield) was obtained by hydrolysis of bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester, dihydrochloride (2), with lipase from Candida cylindracea. The final intermediate for lobucavir prodrug, N-[(phenylmethoxy)carbonyl]-L-valine, [(1S,2R,4R)-3-(2-amino-6-oxo-1H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester (5), could be obtained by transesterification of lobucavir using ChiroCLEC™ BL (61% yield), or more selectively by using immobilized lipase from Pseudomonas cepacia (84% yield).     

Progressive changes in adherens junction structure during intestinal adenoma formation in Apc mutant mice

The C57BL/6J-Min/+ (Min/+) mouse bears a mutant Apc gene and therefore is an important in vivo model of intestinal tumorigenesis. Min/+ mice develop adenomas that exhibit loss of the wild-type Apc allele (Apc(Min/-)). Previously, we found that histologically normal enterocytes bearing a truncated Apc protein (Apc(Min/+)) migrated more slowly in vivo than enterocytes with either wild-type Apc (Apc(+/+)) or with heterozygous loss of Apc protein (Apc(1638N)). To study this phenotype further, we determined the effect of the Apc(Min) mutation upon cell-cell adhesion by examining the components of the adherens junction (AJ). We observed a reduced association between E-cadherin and beta-catenin in Apc(Min/+) enterocytes. Subcellular fractionation of proteins from Apc(+/+), Apc(Min/+), and Apc(Min/-) intestinal tissues revealed a cytoplasmic localization of intact E-cadherin only in Apc(Min/+), suggesting E-cadherin internalization in these enterocytes. beta-Catenin tyrosine phosphorylation was also increased in Apc(Min/+) enterocytes, consistent with its dissociation from E-cadherin. Furthermore, Apc(Min/+) enterocytes showed a decreased association between beta-catenin and receptor protein-tyrosine phosphatase beta/zeta (RPTPbeta/zeta), and Apc(Min/-) cells demonstrated an association between beta-catenin and receptor protein-tyrosine phosphatase gamma. In contrast to the Apc(Min/+) enterocytes, Apc(Min/-) adenomas displayed increased expression and association of E-cadherin, beta-catenin, and alpha-catenin relative to Apc(+/+) controls. These data show that Apc plays a role in regulating adherens junction structure and function in the intestine. In addition, discovery of these effects in initiated but histologically normal tissue (Apc(Min/+)) defines a pre-adenoma stage of tumorigenesis in the intestinal mucosa.     

Rapid purification and crystal structure analysis of a small protein carrying two terminal affinity tags

Small peptide tags are often fused to proteins to allow their affinity purification in high-throughput structure analysis schemes. To assess the compatibility of small peptide tags with protein crystallization and to examine if the tags alter the three-dimensional structure, the N-terminus of the chicken alpha-spectrin SH3 domain was labeled with a His6 tag and the C-terminus with a StrepII tag. The resulting protein, His6-SH3-StrepII, consists of 83 amino-acid residues, 23 of which originate from the tags. His6-SH3-StrepII is readily purified by dual affinity chromatography, has very similar biophysical characteristics as the untagged protein domain and crystallizes readily from a number of sparse-matrix screen conditions. The crystal structure analysis at 2.3 A resolution proves native-like structure of His6-SH3-StrepII and shows the entire His6 tag and part of the StrepII tag to be disordered in the crystal. Obviously, the fused affinity tags did not interfere with crystallization and structure analysis and did not change the protein structure. From the extreme case of His6-SH3-StrepII, where affinity tags represent 27% of the total fusion protein mass, we extrapolate that protein constructs with N- and C-terminal peptide tags may lend themselves to biophysical and structural investigations in high-throughput regimes.     

Phosphopantetheine adenylyltransferase from Escherichia coli: investigation of the kinetic mechanism and role in regulation of coenzyme A biosynthesis

Phosphopantetheine adenylyltransferase (PPAT) from Escherichia coli is an essential hexameric enzyme that catalyzes the penultimate step in coenzyme A (CoA) biosynthesis and is a target for antibacterial drug discovery. The enzyme utilizes Mg-ATP and phosphopantetheine (PhP) to generate dephospho-CoA (dPCoA) and pyrophosphate. When overexpressed in E. coli, PPAT copurifies with tightly bound CoA, suggesting a feedback inhibitory role for this cofactor. Using an enzyme-coupled assay for the forward-direction reaction (dPCoA-generating) and isothermal titration calorimetry, we investigated the steady-state kinetics and ligand binding properties of PPAT. All substrates and products bind the free enzyme, and product inhibition studies are consistent with a random bi-bi kinetic mechanism. CoA inhibits PPAT and is competitive with ATP, PhP, and dPCoA. Previously published structures of PPAT crystallized at pH 5.0 show half-the-sites reactivity for PhP and dPCoA and full occupancy by ATP and CoA. Ligand-binding studies at pH 8.0 show that ATP, PhP, dPCoA, and CoA occupy all six monomers of the PPAT hexamer, although CoA exhibits two thermodynamically distinct binding modes. These results suggest that the half-the-sites reactivity observed in PPAT crystal structures may be pH dependent. In light of previous studies on the regulation of CoA biosynthesis, the PPAT kinetic and ligand binding data suggest that intracellular PhP concentrations modulate the distribution of PPAT monomers between high- and low-affinity CoA binding modes. This model is consistent with PPAT serving as a “backup” regulator of pathway flux relative to pantothenate kinase.     

A model invalidation-based approach for elucidating biological signalling pathways,applied to the chemotaxis pathway in R. sphaeroides

Background  

Developing methods for understanding the connectivity of signalling pathways is a major challenge in biological research. For this purpose, mathematical models are routinely developed based on experimental observations, which also allow the prediction of the system behaviour under different experimental conditions. Often, however, the same experimental data can be represented by several competing network models.