Structural basis for LFA-1 inhibition upon lovastatin binding to the CD11a I-domain.

The lymphocyte function-associated antigen (LFA-1) belongs to the family of beta2-integrins and plays an important role in T-cell activation and leukocyte migration to sites of inflammation. We report here that lovastatin, a drug clinically used for lowering cholesterol levels, inhibits the interaction of human LFA-1 with its counter-receptor intercellular adhesion molecule-1. Using nuclear magnetic resonance spectroscopy and X-ray crystallography we show that the inhibitor binds to a highly conserved domain of the LFA-1 alpha-chain called the I-domain. The first three-dimensional structure of an integrin inhibitor bound to its receptor reveals atomic details for a hitherto unknown mode of LFA-1 inhibition. It also sheds light into possible mechanisms of LFA-1 mediated signalling and will support the design of novel anti-adhesive and immunosuppressive drugs.

In vitro biosynthesis of [Thr2,Leu5,D-Hiv8,Leu10]-cyclosporin, a cyclosporin-related peptolide, with immunosuppressive activity by a multienzyme polypeptide.

A new cyclic peptolide (SDZ 214-103), which is produced by the fungus Cylindrotrichumoligospermum (Corda) BONORDEN (Dreyfuss, M. M., Schreier, M. H., Tscherter, H., and Wenger, R. (June 15, 1988) European Patent Application 0 296 123 A2) and is closely related to cyclosporin A (CyA), has as the main structural difference D-2-hydroxyisovaleric acid in ester linkage at position 8 instead of D-alanine in the cyclosporins. This peptolide exerts similar biological activities to CyA. We were able to prepare an enzyme fraction of crude extracts of the mycelium, which is capable of synthesizing the peptolide with consumption of the constitutive amino acids, D-2-hydroxyisovaleric acid, ATP, and S-adenosyl-L-methionine. The in vitro product co-chromatographs with authentic peptolide on thin layer chromatography and high performance liquid chromatography and shows similar immunosuppressive activity in vitro. The enzyme does not synthesize CyA, whereas cyclosporin synthetase does not synthesize the peptolide. Peptolide synthetase has a high molecular weight (in the same range as cyclosporin synthetase) and also does not appear to be glycosylated. The enzyme cross-reacts with antibodies directed specifically against cyclosporin synthetase.

In vitro biosynthesis of [Thr2, Leu5, D-Hiv8, Leu10]cyclosporin, a cyclosporin-related immunosuppressive peptolide.

We were able to prepare an enzyme fraction from crude extracts of the mycelium of the fungus Cylindrotrichum Bonorden, which is capable of synthesizing the new peptolide SDZ 214-103 under consumption of the constitutive amino acids, D-2-hydroxyisovaleric acid, ATP and S-adenosyl-L-methionine. The enzyme does not synthesize CyA, while cyclosporin synthetase does not synthesize the peptolide. Peptolide synthetase has a high molecular weight in the same range as cyclosporin synthetase (about 1.5 MDa).

Cell-free biosynthesis of new cyclosporins.

An enzyme preparation, isolated from extracts of the fungus Beauveria nivea (previously designated Tolypocladium inflatum), is able to synthesize cyclosporins (Cy's) in vitro. At suboptimal temperature it was possible to yield about 50 micrograms of CyA per ml. The enzyme also produces several of the naturally occurring congeners of CyA, such as the Cy's B, C, D, G, M, O, Q, U and V and some of the analogues known to be produced by the fungus via precursor directed biosynthesis, like dihydro-CyA, [N-methyl-L-beta-cyclohexylalanine]CyA, [L-allylglycine]CyA and [D-serine]CyA. Furthermore, Cy's not obtainable by the fungus could be prepared by the enzyme system in the presence of the appropriate precursor amino acids; the synthesis of [N-methyl-(+)-2-amino-3-hydroxy-4,4-dimethyloctanoic acid]CyA, [L-norvaline, N-methyl-L-norvaline]CyA, [L-norvaline, N-methyl-L-norvaline]CyA, [L-allo-isoleucine, N-methyl-L-allo-isoleucine]CyA, [L-allo-isoleucine]CyA, [D-2-aminobutyric acid]CyA and [beta-chloro-D-alanine]CyA could be established. The immunosuppressive effects of the new derivatives are discussed.

An improved method for two-dimensional gel-electrophoresis: analysis of mutationally altered ribosomal proteins of Escherichia coli.

An improved method for the two-dimensional electrophoretic analysis of ribosomal proteins on acrylamide gel slabs has been developed by combining the procedures for the first dimension of Mets and Bogorad (1974) and for the second dimension of Kaltschmidt and Wittmann (1970) and by introducing several modifications. Ribosomal proteins of various Escherichia coli mutants have been analyzed by the new method. Advantages are that (1) it requires only small amounts of protein (100-200 micrograms 70S ribosomal proteins), (2) reproducibility is very high, and (3) it makes it easier to identify mutational alterations in proteins S10, L4, L10, and L21 which hardly migrate out of the sample gel with our previous electrophoresis procedure. Furthermore, the new method can be nicely adapted to analysis of the ribosomal proteins from other organisms, such as Bacilli or yeast.

Synthesis of ribosomal proteins in merodiploid strains and in minicells of Escherichia coli.

Merodiploid strains of Escherichia coli containing episomes which carry one or several of the ribosomal protein (r-protein) transcriptional units were analysed to see whether the increase in the number of gene copies leads to an increased synthesis of the respective r-proteins. It was found that the amount of ribosomal proteins was (with the only exception of ribosomal protein S20) independent of the number of gene copies present. The comparison of the in vivo stability of r-proteins in haploid and merodiploid strains did not, within the time resolution of the experiment, provide any evidence for an increased rate of degradation of those proteins coded by more than one gene copy. These results indicate a tight coupling between the amount of ribosomal proteins synthesized and the level required irrespective of the number of gene copies present. With the aid of minicells from a strain containing the episome F'101 which carries the thr-leu segment of the chromosome it was demonstrated that (i) in vivo synthesis of r-protein S20 could proceed in the absence of the synthesis of ribosomal RNA and of other r-proteins, and (ii) r-protein S20 was degraded under conditions where it was not assembled into ribosomes.

Cold-sensitive growth of a mutant of Escherichia coli with an altered ribosomal protein S8: analysis of revertants.

26 cold-resistant revertants of a cold-sensitive Escherichia coli mutant with an altered ribosomal protein S8 were analyzed for their ribosomal protein pattern by two-dimensional polyacrylamide gel electrophoresis. It was found that 16 of them had acquired the apparent wild-type form of protein S8, one exhibits a more strongly altered SC than the original mutant and two revertants regained the wildtype form of S8 and, in addition, possess alterations in protein L30. The ribosomes of the residual revertants showed no detectable difference from those of the parental S8 mutant. The mutation leading to the more strongly altered S8 was genetically not separable from the primary S8 mutation; this indicates that both mutations are very close to each other or at the same site. The structural gene for ribosomal protein L30 was mapped relative to two other ribosomal protein genes (for proteins S5 and S8) by the aid of one of the L30 mutants: The relative order obtained is: aroE....rpmD(L30)....rpsE(S5)....rpsH(S8)....rpsL(S12). The L30 mutation impairs growth and ribosomal assembly at 20 degrees C and is therefore the first example of a mutant with defined 50S alteration that has (partial) cold-sensitive ribosome assembly. A double mutant was constructed which possesses both the S8 and the L30 mutations. It was found that the L30 mutation had a slight antagonistic effect on the growth inhibition caused by the S8 mutation. Thus the L30 mutants might have possibly arisen from the original S8 mutant first as S8/L30 double mutants which was followed by the loss of the original S8 lesion.

Localized mutagenesis of the aroE-strA section of the Escherichia coli chromosome coding for ribosomal proteins.

In order to obtain E. coli strains altered in ribosomal proteins the following isolation technique was used: Phage P1 grown in a streptomycin resistant E. coli strain, was mutagenized by hydroxylamine or nitrous acid, and was used to transduce into a strain auxotrophic for aroE. Transductants with streptomycin resistance and aroE prototrophy were selected and tested for their growth at various temperatures (20 degrees, 30 degrees and 42 degrees) and their response to different antibiotics. Ribosomes from seventeen transductants with an altered response to temperature or antibiotics were isolated. They were tested for alterations in their ribosomal subunit profiles by sucrose centrifugation and for altered ribosomal proteins by two dimensional gel electrophoresis. Two strains showed accumulation of 50S ribosomal precursors and three strains had an altered 50S protein L18. This protein belongs to the 5S RNA-protein complex having GTPase and ATPase activity.