1.9 A crystal structure of interleukin 6: implications for a novel mode of receptor dimerization and signaling.

Interleukin 6 (IL-6) has many biological activities in vivo, and deregulation has been implicated in many disease processes. IL-6, a 185 amino acid polypeptide was refolded, purified and crystallized. The crystals diffracted to beyond 1.9 A and the structure was solved using single isomorphous replacement. The X-ray structure of IL-6 is composed of a four helix bundle linked by loops and an additional mini-helix. 157 out of 185 residues are well defined in the final structure, with 18 N-terminal and 8 A-B loop amino acids displaying no interpretable electron density. The three-dimensional structure has been used to construct a model of IL-6 interacting with the IL-6 receptor (alpha-chain) and gp130 (beta-chain) that gives new insight into the process of molecular recognition and signaling. Based on this model, we predict a fourth binding site on IL-6, a low affinity IL-6-IL-6 interaction, which may be necessary for the sequential assembly of a functional hexameric IL-6 receptor complex.

Human immunodeficiency virus 1 reverse transcriptase. Template binding, processivity, strand displacement synthesis, and template switching.

We have analyzed the kinetics of DNA synthesis catalyzed by reverse transcriptase from human immunodeficiency virus 1 (HIV-1). Reverse transcriptase, overproduced in Escherichia coli and purified to homogeneity, has polymerase and RNase H activity. Reverse transcriptase forms a stable complex with poly(rA).oligo(dT) primer-templates in the absence of Mg2+ and dTTP with an equilibrium dissociation constant of 3 nM. Synthesis from these preformed complexes can be initiated, and restricted to a single processive cycle, by the simultaneous addition of Mg2+, dTTP, and excess competitor RNA. Preformed complexes decay with a maximal half-life of 2-3 min. Synthesis on poly(rA) templates is processive with an incorporation rate of 10-15 nucleotides/s at 37 degrees C. Processivity varies widely with the template used, increasing from a few to greater than 300 nucleotides in the order: poly(dA) less than double-stranded DNA less than single-stranded DNA less than single-stranded RNA less than poly(rA). On double-stranded DNA reverse transcriptase catalyzes limited strand-displacement synthesis of up to 50 nucleotides. On RNA-DNA hybrids significant DNA synthesis is observed only after degradation of the RNA strand by the RNase H activity of reverse transcriptase. Intermolecular strand switching occurs with poly(rA) templates. At low ionic strength reverse transcriptase can use multiple templates with a single primer, leading to products of greater than template length. Reverse transcriptase and primer do not have to dissociate during the exchange of template strands, thus allowing processive DNA synthesis across template borders.

Bacteriorhodopsin precursor. Characterization and its integration into the purple membrane.

Halobacterium halobium spheroplasts synthesize and accumulate a bacteriorhodopsin precursor. By labeling of the precursor with [35S]Met and [3H]Leu followed by Edman degradation, we have confirmed the previous conclusion from the DNA sequencing that the precursor contains 13 additional amino acids at the NH2 terminus of bacteriorhodopsin. Although not processed in the spheroplasts, it integrates into the purple membrane in the correctly folded conformation. This was shown by the mode of cleavage by a number of proteolytic enzymes, the site of attachment of retinal, and the formation of oligomers on reaction with bifunctional cross-linking reagents. In all these respects, the behavior of the precursor was identical with that of native mature bacteriorhodopsin in the purple membrane. Finally, the precursor was not processed to bacteriorhodopsin even when the spheroplasts were subsequently allowed to revert to rod-shaped cells. This suggests that either the processing of the precursor is cotranslational or that the NH2 terminus of the precursor becomes inaccessible to the processing enzyme in the spheroplasts following integration into the membrane.

Anaerobic and aerobic coproporphyrinogen III oxidases of Rhodopseudomonas spheroides. Mechanism and stereochemistry of vinyl group formation.

An improved method for the preparation of various species of porphobilinogen stereospecifically labelled with 3H in the side chains (at C-6, C-7 and C-8) is described. These labelled samples were used to study the mechanism and stereochemistry of anaerobic as well as aerobic coproporphyrinogen III oxidase of light-grown Rhodopseudomonas spheroides. It was shown that both the oxidases catalyse the conversion of the propionate side chains of coproporphyrinogen III into the vinyl groups of protoporphyrinogen IX, (formula; see text) with the labilization of the pro-S-hydrogen atom at the beta-position. These results are similar to those previously recorded for such conversions in animal and plant systems. In the light of the cumulative information available to date, mechanisms for the conversion, (formula; see text) are discussed and doubt is cast on the participation of hydroxylated intermediates in the process.

5-Aminolevulinic acid dehydratase. The role of sulphydryl groups in 5-aminolevulinic acid dehydratase from bovine liver.

The thiophilic reagent 5,5'-dithiobis(2-nitrobenzoic acid) Nbs2) reacts with four sulphydryl groups in native 5-aminolevulinic acid dehydratase from bovine liver (groups I, II, III and IV). All four of these groups exhibit various degrees of half-site reactivity. Groups I and II are highly reactive and their rates of reaction with Nbs2 have been investigated using stopped-flow analysis. The reaction of these groups with Nbs2 results in the formation of an intramolecular disulphide bond which may be reduced with dithioerythritol to regenerate the free sulphydryl groups. Groups I and II appear to be at, or near, the catalytic site whereas group III is involved in the maintenance of conformation in the native enzyme.

5-Aminolevulinic acid dehydratase: alkylation of an essential thiol in the bovine-liver enzyme by active-site-directed reagents.

1. 5-Aminolevulinic acid dehydratase from bovine liver has been shown to be inactivated by 5-halolevulinic acids and 3-halolevulinic acids. 2. The substrate, 5-aminolevulinic acid, protects the enzyme from modification by 5-halolevulinic acids. 3. Using tritiated chlorolevulinic acids, it was shown that four of the subunits in the octameric enzyme are preferentially modified. 4. The susceptible enzyme group modified is an --SH group of a reactive cysteine at or near the active site. 5. Oxidized enzyme is not affected by either 5-chlorolevulinic acid or 3-chlorolevulinic acid. 6. Evidence is presented which suggests that 5-chlorolevulinic acid is acting as an active-site-directed reagent.