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FKBP59, a 59-kDa FK506 binding protein, was discovered in heterooligomeric complexes containing nontransformed, non-DNA binding, steroid receptors. Sequence similarity search and secondary structure prediction suggested that the protein has a multi-domain organization, the N-terminal domain having a great similarity to human FKBP12 (12-kDa FK506-binding protein). FKBP59 binds immunosuppressant FK506 and has peptidylprolyl cis-trans-isomerase activity, both properties being localized in the N-terminal domain (FKBP59-I). In order to characterize its conformational features and to better understand its biological significance, we overexpressed and 15N-labeled this domain (149 amino acids) in Escherichia coli and initiated an NMR structural study in solution. Almost complete sequence-specific assignment of the 1H and 15N resonances was achieved using two-dimensional and three-dimensional homonuclear and heteronuclear experiments. Localization of the secondary structure elements was derived essentially from C alpha H chemical shift distribution along the sequence, the short-range and medium-range NOE connectivities and exchange kinetics of amide protons. The domain has a structured part comprising six beta-strands and a three-turn alpha-helix between K87 and M96. The first 17 residues are highly flexible and show no regular secondary structure. The beta-sheet structure, derived from long-range connectivities between backbone protons, consists of six beta-strands defined as follows: B1, V22-I24; B2, V32-K37; B3, D50-L61; B4, T64-S68 and F76-L80; B5, E100-K107; B6, L127-F137. They are organized in an antiparallel beta-sheet with the connecting topology +1, +3, +1, -3, +1. The alpha-helix connects strand B4 to strand B5. Globally, the structure of FKBP59-I, derived from the present work, is similar to the NMR-derived structures of uncomplexed FKBP12. However, several conformational differences were noted at this level of structural analysis. The beta-sheet of the FKBP59 domain has an additional strand at the N-terminal and the alpha-helix is longer by about one helical turn. In addition, strand B4 has two components, separated by a large bulge (seven residues); the first component was observed in the X-ray or NMR structures of complexed FKBP12 but not in the NMR-derived, uncomplexed structure.

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The effect of recombinant FKBP-59/HBI or of its first N-terminal domain FKBP-59/HBI-I on the phosphatase activity of calcineurin (a Ca(+2)-calmodulin dependent phosphatase) was tested in vitro in the presence or absence of the immunosuppressant drug FK506. Contrarily to the inhibition observed with the immunosuppressant complex FKBP-12-FK506, no significant inhibition was observed with FKBP-59/HBI or FKBP-59/HBI-I in the presence of FK506, even though FKBP-59/HBI-1 is nearly 55% homologous to the immunophilin FKBP-12. Inhibition was tested both with native calcineurin (calcineurin A: Mr 58-59 kDa) and with protease activated, calmodulin independent calcineurin (calcineurin A: Mr 45 kDa). There was no competitive effect of FKBP-59 on the inhibitory activity of the FKBP-12-FK506 complex, even when the molar concentration of FKBP-59/HBI was 100 times higher than that of FKBP-12. Clearly, although the first domain of FKBP-59/HBI displays several structural and functional features of FKBP-12, it does not interact with calcineurin.

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Flavocytochrome b2 catalyzes the oxidation of lactate at the expense of cytochrome c. After flavin (FMN) reduction by the substrate, reducing equivalents are transferred one by one to heme b2, and from there on to cytochrome c. The crystal structure of the enzyme is known at 2.4-A resolution, and specific roles in catalysis have been assigned to active side chains. Tyr143 in particular, located at the interface between the flavodehydrogenase moiety and the heme-binding domain, was thought to take part in substrate binding, as well as to orient the heme-binding domain for efficient electron transfer. A first study of the properties of a Tyr143Phe mutant showed that the major effect of the mutation was to decrease the rate of electron transfer from flavin to heme [Miles, C.S., Rouvière-Fourmy, N., Lederer, F., Mathews, F.S., Reid, G.A., Black, M.T., & Chapman, S.K. (1992) Biochem. J. 285, 187-192]. In the present paper, we focus on the effect of the mutation on catalysis of lactate dehydrogenation. We report the deuterium kinetic isotope effects on flavin reduction as measured with stopped-flow methods and on cytochrome c reduction in the steady-state using L-[2-2H]lactate. For the wild-type enzyme, isotope effects on FMN reduction, D(kredF) and D(kredF)/Km), were 7.2 +/- 0.9 and 4.2 +/- 1.3, respectively, and for the Y143F mutant values of 4.4 +/- 0.5 and 3.9 +/- 1.1 were obtained. Calculations, from deuterium isotope effects, of substrate Kd values, combined with knowledge of kcat/Km values, lead to the conclusion that Tyr143 does stabilize the Michaelis complex by hydrogen bonding to a substrate carboxylate, as was postulated; but the mutation does not destabilize the transition state more than the Michaelis complex.(ABSTRACT TRUNCATED AT 250 WORDS)

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It has been previously proposed that the rabbit p59-HBI (Heat shock protein Binding Immunophilin) or rFKBP59 (FK506 Binding Protein), found associated with the 90 kDa heat shock protein in nontransformed steroid receptor complexes, has three domains structurally related to hFKBP12 (Callebaut, I., Renoir, J.M., Lebeau, M.C., Massol, N., Burny, A., Baulieu, E.E. and Mornon, J.P. (1992) Proc. Natl. Acad. Sci., USA 89, 6270-6274). Here we report the overexpression, as fusion proteins in E. coli, of the full length p59-HBI and a series of p59-HBI mutants delimiting these domains and their respective peptidyl prolyl cis trans isomerase (PPlase) activity. The PPlase activity of p59-HBI is comparable to that of hFKBP12 and is due to domain p59-HBI I which displays the highest homology with this immunophilin. The residual enzymatic activity found in domain p59-HBI II is discussed.

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The role of Tyr-143 in the catalytic cycle of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) has been examined by replacement of this residue with phenylalanine. The electron-transfer steps in wild-type and mutant flavocytochromes b2 have been investigated by using steady-state and stopped-flow kinetic methods. The most significant effect of the Tyr-143----Phe mutation is a change in the rate-determining step in the reduction of the enzyme. For wild-type enzyme the main rate-determining step is proton abstraction at the C-2 position of lactate, as shown by the 2H kinetic-isotope effect. However, for the mutant enzyme it is clear that the slowest step is interdomain electron transfer between the FMN and haem prosthetic groups. In fact, the rate of haem reduction by lactate, as determined by the stopped-flow method, is decreased by more than 20-fold, from 445 +/- 50 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 21 +/- 2 s-1 in the mutant enzyme. Decreases in kinetic-isotope effects seen with [2-2H]lactate for mutant enzyme compared with wild-type, both for flavin reduction (from 8.1 +/- 1.4 to 4.3 +/- 0.8) and for haem reduction (from 6.3 +/- 1.2 to 1.6 +/- 0.5) also provide support for a change in the nature of the rate-determining step. Other kinetic parameters determined by stopped-flow methods and with two external electron acceptors (cytochrome c and ferricyanide) under steady-state conditions are all consistent with this mutation having a dramatic effect on interdomain electron transfer. We conclude that Tyr-143, an active-site residue which lies between the flavodehydrogenase and cytochrome domains of flavocytochrome b2, plays a key role in facilitating electron transfer between FMN and haem groups.

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