Mapping of interaction between cytochrome P450 2B4 and cytochrome b5: the first evidence of two mutual orientations.

OBJECTIVES: The cytochrome P450 (P450) and cytochrome b5 are membrane hemoproteins composing together with flavoprotein NADPH:P450 reductase a mixed function oxidase (MFO) system. The knowledge of the interaction between P450 and its redox partners within a MFO system is fundamental to understand P450 reaction mechanism, an electron transport from its redox partner and also detoxification of xenobiotics and/or metabolism of endogenous substrates with all positive or negative aspects for organisms. METHODS: The chemical cross-linking by soluble carbodiimide (EDC) in combination with the liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) has been employed to characterize the contact surface regions involved in the transient interaction between two catalytic domains of P450 2B4 and cytochrome b5. RESULTS: The cross-linking reaction was accomplished in an equimolar catalytic complex of P450 2B4:cytochrome b5 and the covalent hetero-dimers detected on SDS-PAGE electrophoresis were analyzed (after in gel trypsin digestion) using LC-HRMS to identify cross-linked amino-acid residues. The computed in silico models of P450 2B4:cytochrome b5 complex using amino-acids participating in cross-links (Asp134, Lys139, Glu424 and Glu439 located on a proximal surface of P450 2B4) suggest interpretation that two different types of cytochrome b5 orientations are present in the studied interaction within a MFO system: the first allowing potential cytochrome b5 electron donation to P450, the second one inducing cytochrome b5 modulation of P450 structural changes. CONCLUSIONS: The results demonstrated the capability of the used experimental approach to map the interaction between P450 and cytochrome b5 suggesting the formation of multi-meric structures within a MFO system as interpretation of the two observed mutual orientations.

Carboxylated calixarenes bind strongly to CD69 and protect CD69(+) killer cells from suicidal cell death induced by tumor cell surface ligands.

We have recently identified a new class of high affinity ligands for CD69 leukocyte membrane receptor, carboxylated calixarenes. Of the three compounds investigated here, thiacalix[4]arene had the highest affinity for CD69 in direct binding assays, and proved to be the most specific inhibitor of CD69 identified so far in receptor precipitation and cellular activation experiments. Carboxylated calixarenes also proved effective at protection of CD69(high) lymphocytes from apoptosis triggered by a multivalent ligand or antibody. Thus, carboxylated calixarenes set a new paradigm for noncarbohydrate ligands for CD69 making them attractive for protection of killer cells in combined animal tumor therapies.