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Hepatitis B virus core protein has 183 amino acids divided into an assembly domain and an arginine-rich C-terminal domain (CTD) that regulates essential functions including genome packaging, reverse transcription, and intracellular trafficking. Here, we investigated the CTD in empty hepatitis B virus (HBV) T=4 capsids. We examined wild-type core protein (Cp183-WT) and a mutant core protein (Cp183-EEE), in which three CTD serines are replaced with glutamate to mimic phosphorylated protein. We found that Cp183-WT capsids were less stable than Cp183-EEE capsids. When we tested CTD sensitivity to trypsin, we detected two different populations of CTDs differentiated by their rate of trypsin cleavage. Interestingly, CTDs from Cp183-EEE capsids exhibited a much slower rate of proteolytic cleavage when compared with CTDs of Cp183-WT capsids. Cryo-electron microscopy studies of trypsin-digested capsids show that CTDs at five-fold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at five-fold, increase capsid stability and reduce CTD exposure. Our studies show that quasi-equivalent CTDs exhibit different rates of exposure and thus might perform distinct functions during the hepatitis B virus lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle.

LP2086 is a family of outer membrane lipoproteins from Neisseria meningitidis, which elicits bactericidal antibodies and are currently undergoing human clinical trials in a bivalent formulation where each antigen represents one of the two known LP2086 subfamilies. Here we report the NMR structure of the recombinant LP2086 variant B01, a representative of the LP2086 subfamily B. The structure reveals a novel fold composed of two domains: a “taco-shaped” N-terminal β-sheet and a C-terminal β-barrel connected by a linker. The structure in micellar solution is consistent with a model of LP2086 anchored to the outer membrane bilayer through its lipidated N terminus. A long flexible chain connects the folded part of the protein to the lipid anchor and acts as spacer, making both domains accessible to the host immune system. Antibodies broadly reactive against members from both subfamilies have been mapped to the N terminus. A surface of subfamily-defining residues was identified on one face of the protein, offering an explanation for the induction of subfamily-specific bactericidal antibodies.

Maturation of noninfectious nodavirus provirions occurs by autoproteolytic cleavage of most of the 180 copies of the alpha-protein that make up the icosahedral capsid. This maturation, which is much slower than viral assembly, produces an infectious particle that is more stable than the provirion and makes viral uncoating thermodynamically distinct from assembly, allowing assembly and (a time-delayed) uncoating to occur under similar conditions. The results of structural, computational, and molecular genetic studies suggest that maturation depends both on intrasubunit strain, produced during assembly, and on a critical aspartic acid residue. This residue lies in a hydrophobic pocket that is stabilized by intersubunit contacts. It is close to the scissile bond and exhibits an environmentally elevated pKa. The apparent involvement of a single acidic residue in the hydrolytic cleavage of a peptide bond contrasts with the involvement of 2 such residues in acid proteases.

Calcium transport into everted membrane vesicles of Escherichia coli was found to have two components, one phosphate-dependent and the other phosphate-independent. In vesicles prepared in a glycerol buffer, calcium/proton exchange was phosphate independent but net uptake of 45Ca2+ required phosphate. In vesicles prepared in a sucrose buffer both phosphate-independent and phosphate-dependent accumulation of 45Ca2+ occurred. Both calcium/proton exchange and phosphate-independent uptake of 45Ca2+ were inactivated by treatment with trypsin, chymotrypsin, or N,N'-dicyclohexylcarbodiimide but not by N-ethylmaleimide. Phosphate-dependent uptake of 45Ca2+ was inhibited by N-ethylmaleimide but not by trypsin, chymotrypsin, or N,N'-dicyclohexylcarbodiimide. Under conditions permitting only phosphate-dependent uptake of 45Ca2+, concomitant uptake of 32Pi was also observed. Both uptake and efflux of the two ions occurred with a 1:1 ratio. These results imply the existence of two calcium transport systems in everted membrane vesicles, one of which catalyzes exchange of calcium ions and protons, the other of which catalyzes cotransport of calcium and phosphate.

NAD+ had a biphasic effect on the NADH oxidase activity in electron transport particles from Mycobacterium phlei. The oxidase was inhibited competitively by NAD+ at concentrations above 0.05 mM. NAD+ in concentrations from 0.02 to 0.05 mM resulted in maximum stimulation of both NADH oxidation and oxygen uptake with concentrations of substrate both above and below the apparent K-M. Oxygen uptake and cyanide sensitivity indicated that the NAD+ stimulatory effect was linked to the terminal respiratory chain. The stimulatory effect was specific for NAD+. NAD+ was also specific in protecting the oxidase during heating at 50 degrees and against inactivation during storage at 0 degrees. NAD+ glycohydrolase did not affect stimulation nor heat protection of the NADH oxidase activity if the particles were previously preincubated with NAD+. Binding studies revealed that the particles bound approximately 3.6 pmol of [14C1NAD+ per mg of electron transport particle protein. Although bound NAD+ represented only a small fraction of the total added NAD+ necessary for maximal stimulation, removal of the apparently unbound NAD+ by Sephadex chromatography revealed that particles retained the stimulated state for at least 48 hours. Further addition of NAD+ to stimulated washed particles resulted in competitive inhibition of oxidase activity. Desensitization of the oxidase to the stimulatory effect of NAD+ was achieved by heating the particles at 50 degrees for 2 min without appreciable loss of enzymatic activity. Kinetic studies indicated that addition of NADH to electron transport particles prior to preincubation with NAD+ inhibited stimulation. In addition, NADH inhibited binding of [14C]NAD+. The utilization of artificial electron acceptors, which act as a shunt of the respiratory chain at or near the flavoprotein component, indicated that NAD+ acts as at the level of the NADH dehydrogenase at a site other than the catalytic one resulting in a conformational change which causes restoration as well as protection of oxidase activity.

Several fluorescence techniques have been used to estimate the depth, in the membrane, of the endogenous tryptophans of membrane-bound proteins. We reported recently the use of phosphatidylcholines specifically brominated at different positions of the sn-2 acyl chain for this purpose (Markello, T., Zlotnick, A., Everett, J., Tennyson, J., and Holloway, P. W. (1985) Biochemistry 24, 2895-2901). The membranes made from these brominated lipids will have the brominated lipid in both monolayers, and so the estimated depth of the fluorophore will be relative to either the inner or outer surface of the membrane, but will not distinguish between these two extremes. To differentiate between these two models vesicles have now been made with an asymmetric distribution of brominated lipid, by use of phosphatidylcholine exchange protein. The asymmetric vesicles were isolated by virtue of their density, and their asymmetry was established by addition of an amphipathic fluorescent carbazole compound. With these vesicles it was shown that the tryptophan in the membrane-binding domain of cytochrome b5 which is quenched by bromolipid is located 0.7 nm below the outer surface of the membrane vesicles, rather than 0.7 nm from the inner surface.

We have purified to homogeneity a peptidoglycan-associated protein from Haemophilus influenzae. Our purification process used differential extraction of cell envelopes with nondenaturing detergents. Solubilization of this protein was accomplished by heating a peptidoglycan-enriched subcellular fraction in the presence of one of several nondenaturing detergents at 55-60 degrees C. The purified protein migrated as a single band, with a Mr approximately 15,000, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein contains covalently linked fatty acids, is rich in tyrosine, but lacks methionine and tryptophan. Amino acid analysis also revealed the presence of glycerylcysteine, which has been shown to be the site of fatty acylation in other bacterial lipoproteins. Over 87% of the primary structure has been determined by sequencing high pressure liquid chromatography purified fragments derived from several endoproteinase digests. This protein belongs to a family of proteins, known as peptidoglycan associated lipoproteins, which appear to be components of the outer membranes of most Gram-negative bacteria.