Peter J. Belshaw

Cyclosporin A inhibits CD40 ligand expression in T lymphocytes.

The ligand for CD40 is expressed on activated T lymphocytes and delivers contact-dependent activation signals to B lymphocytes. The mechanisms regulating CD40 ligand gene expression are largely unknown. Optimal expression of CD40 ligand required activation of protein kinase C and a rise in intracellular calcium concentration. CD40 ligand expression was inhibited by pretreatment of T cells with cyclosporin A. Cyclosporin A analogues inhibited CD40 ligand expression with a potency mirroring the ability of each compound to inhibit calcineurin activity, indicating that calcineurin plays a key role in CD40 ligand gene expression. Cyclosporin A inhibited IL-4-driven CD40 ligand-dependent IgE isotype switching in PBMC but did not inhibit IgE synthesis induced by CD40 mAb plus IL-4. PBMC derived from transplant patients receiving cyclosporin A failed to express CD40 ligand upon stimulation. These results suggest that patients receiving cyclosporin A may be deficient in CD40 ligand-dependent T cell help.

Crystal structures of cyclophilin A complexed with cyclosporin A and N-methyl-4-[(E)-2-butenyl]-4,4-dimethylthreonine cyclosporin A

BACKGROUND Cyclophilin (CyP) is a ubiquitious intracellular protein that binds the immunosuppressive drug cyclosporin A (CsA). CyP-CsA forms a ternary complex with calcineurin and thereby inhibits T-cell activation. CyP also has enzymatic activity, catalyzing the cis-trans isomerization of peptidyl-prolyl amide bonds. RESULTS We have determined the structure of human cyclophilin A (CyPA) complexed with CsA to 2.1 A resolution. We also report here the structure of CyPA complexed with an analog of CsA, CsA (MeBm2t1-CsA), which binds less well to CyPA, but has increased immunosuppressive activity. Comparison of these structures with previously determined structures of unligated CyPA and CyPA complexed with a candidate substrate for the isomerase activity, the dipeptide AlaPro, reveals that subtle conformational changes occur in both CsA and CyPA on complex formation. CONCLUSIONS MeBm2t1-CsA binds to CyPA in an essentially similar manner to CsA. The 100-fold weaker affinity of its binding may be attributable to the close contact between MeBmt1 and the active site residue Ala103 of CyPA, which causes small conformational changes in both protein and drug. One change, the slight movement of MeLeu6 in CsA relative to MeBm2t1-CsA, may be at least partially responsible for the higher affinity of the CyPA-MeBm2t1-CsA complex for calcineurin. Our comparison between CyPA-CsA and CyPA-AlaPro suggests that CsA is probably not an analog of the natural substrate, confirming that the catalytic activity of CyPA is not related to its role in immunosuppression either structurally or functionally.

Kinetics and regioselectivity of peptide-to-heterocycle conversions by microcin B17 synthetase

BACKGROUND The Escherichia coli peptide antibiotic microcin B17 (MccB17) contains four oxazole and four thiazole rings introduced post-translationally in the 69 amino acid McbA gene product, an MccB17 precursor, by the microcin B,C,D enzyme complex. Both monocyclic and 4,2-bis-heterocyclic moieties are generated. The enzymatic cyclization involves 14 of the last 43 amino acids of McbA and requires the presence of the first 26 amino acids that function as a specificity-conferring propeptide. RESULTS We have constructed maltose-binding protein (MBP)-McbA1-46 fusion proteins and have mutagenized the Gly39-Ser40-Cys41 (GSC) wild-type sequence to assess the regioselectivity and chemoselectivity of MccB17-synthetase-mediated heterocycle formation at the first two loci, residues 40 and 41 of McbA. Four single-site and four double-site substrates showed substantial differences in turnover as assessed by western assays, UV-visible spectroscopy and mass spectrometry. Cysteine-derived thiazoles form at a greater rate than serine-derived oxazoles. Formation of bis-heterocycles is sensitive both to composition and sequence context. CONCLUSIONS The E. coli McbB,C,D MccB17 synthetase is the first peptide heterocyclization enzyme to be characterized. This study reveals substantial regioselectivity and chemoselectivity (thiazole > oxazole) at the most amino-terminal bis-heterocyclization site of McbA. The heterocyclization of GSS and GCC mutants of McbA1-46 by MccB17 synthetase demonstrates that the complex can efficiently generate tandem bis-oxazoles and bis-thiazoles, moieties not found in MccB17 but present in natural products such as hennoxazole and bleomycin. The observations suggest a common enzymatic mechanism for the formation of peptide-derived heterocyclic natural products.

THE CONFORMATION OF CYCLOSPORIN A BOUND TO CYCLOPHILIN IS ALTERED (ONCE AGAIN) FOLLOWING BINDING TO CALCINEURIN: AN ANALYSIS OF RECEPTOR-LIGAND-RECEPTOR INTERACTIONS

Analyses of the complexation of cyclosporin A (CsA) by cyclophilin and the unusual properties of MeBmztl-CsA lead us to propose a conformational change upon binding of the cyclophilin-CsA complex to the protein phosphatase, calcineurin. Structural analyses of natural products bound to their protein receptors can provide a detailed understanding of phenomena ranging from enzymatic catalysis to signal transduction. This is especially true when such analy- ses are combined with experimental results from other disciplines such as synthetic chemistry and molecular bi- ology. In fact, it may be argued that the power of structure determination resides primarily in its ability to ex- plain experimental data, and in doing so to facilitate the formulation of hypotheses which then can direct further experiments. In this report we describe a proposed model for the interactions in a multimeric complex involving two proteins and a natural product ligand. The model is based on crystallographic and NMR spectroscopic anal- yses of one of the proteins and its complex with the natural product, and on biochemical and biological data on a synthetic analog of the natural product.