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V Mikol

Publications and source records attributed to V Mikol.

27 records · Page 2Linked to original sources
PubMed1994

X-ray structure of a cyclophilin B/cyclosporin complex: comparison with cyclophilin A and delineation of its calcineurin-binding domain.

The crystal structure of a complex between recombinant human cyclophilin B (CypB) and a cyclosporin A (CsA) analog has been determined and refined at 1.85-A resolution to a crystallographic R factor of 16.0%. The overall structures of CypB and of cyclophilin A (CypA) are similar; however, significant differences occur in two loops and at the N and C termini. The CsA-binding pocket in CypB has the same structure as in CypA and cyclosporin shows a similar bound conformation and network of interactions in both CypB and CypA complexes. The network of the water-mediated contacts is also essentially conserved. The higher potency of the CypB/CsA complex versus CypA/CsA in inhibiting the Ca(2+)- and calmodulin-dependent protein phosphatase calcineurin is discussed in terms of the structural differences between the two complexes. The three residues Arg90, Lys113, and Ala128 and the loop containing Arg158 on the surface of CypB are likely to modulate the differences in calcineurin inhibition between CypA and CypB.

Amino Acid Isomerases↗
PubMed1994

The X-ray structure of (MeBm2t)1-cyclosporin complexed with cyclophilin A provides an explanation for its anomalously high immunosuppressive activity.

For most of the cyclosporin A (CsA) analogs, there is generally a good correlation between cyclophilin binding and immunosuppression. However, this relationship does not seem to hold for 4-[(E)-2-butenyl]-4,4,N-trimethyl-L-threonine1 (MeBm2t)1-CsA. Its affinity for cyclophilin was reported to be approximately 1% that of CsA and its immunosuppressive activity in vitro was shown to be approximately 30% that of CsA. We report here the crystal structure of a complex between recombinant human cyclophilin A (CypA) and (MeBm2t)1-CsA which has been determined by X-ray crystallography at 2.2 A resolution and refined to an R-factor of 16.3%. (MeBm2t)1-CsA shows a similar bound conformation and network of interactions to CypA as CsA. The measured lower affinity for CypA cannot therefore be explained by a different mode of binding. We propose that the poor affinity to CypA could be accounted for by the existence of an equilibrium in aqueous solution between a 'cyclophilin bound conformation' and a 'non-binding conformation' of (MeBm2t)1-CsA. The relatively high immunosuppressive activity is suggested to result from slight conformational differences observed in the effector domain.

Amino Acid Isomerases↗
PubMed1993

X-ray structure of a monomeric cyclophilin A-cyclosporin A crystal complex at 2.1 A resolution.

The crystal structure of a complex between recombinant human cyclophilin A (Cyp) and cyclosporin A (CsA) has been determined from a novel orthorhombic crystal form that contains only one monomer of complex per asymmetric unit rather than five in the previously determined tetragonal structure. The structure has been refined at 2.1 A resolution to a crystallographic R-factor of 16.7%. The conformation of Cyp is practically unchanged with respect to the tetragonal form. A certain number of previously undefined side-chains have been located in the electron density and a very detailed picture of the ordered solvent structure has been obtained. The interactions between CsA and Cyp are conserved. A network of the possibly conserved, water-mediated contacts is described. The structure of CsA in the monomeric complex is similar to that of the decameric complex, but shows a few small differences in the so-called effector domain of CsA, probably due to differences in crystal environment. The fact that this monomeric crystal form can be obtained shows that the formation of pentamer or decamer complexes is not a generally observed phenomenon and is not a prerequisite for biological activity.

Amino Acid Isomerases↗
PubMed1993

Cyclosporins. Structure-activity relationships.

Cyclosporin A (Sandimmun) achieves immunosuppressive activity by complex formation with cyclophilin and subsequent binding of the binary complex to and inhibiting protein phosphatase 2B (calcineurin). Complexes of nonimmunosuppressive cyclophilin binding cyclosporin analogues do not inhibit protein phosphatase 2B, suggesting a crucial role for this enzyme in T cell activation. Binding of cyclosporin A to cyclophilins A, B, and C, respectively, results in complexes of significantly different inhibitory potency. The cyclosporin molecule thus has two functional domains, one mediating cyclophilin binding and a second one endowing affinity of the complex to calcineurin, thereby inhibiting its enzyme activity. Structure-activity studies and x-ray crystallography of cyclosporin-cyclophilin complexes indicate a crucial role of leucine side chains in positions 4 and 6 of the cyclosporin macrocycle for the calcineurin interaction.

Amino Acid Sequence↗
PubMed1990

Crystallisation and preliminary X-ray diffraction studies of cyclophilin-tetrapeptide and cyclophilin-cyclosporin complexes.

Recombinant human cyclophilin has been co-crystallised with a number of peptides to give crystals suitable for X-ray analysis. The crystal complexes for which heavy-atom derivatives have been prepared and X-ray data collected are: cyclophilin with N-acetyl-Ala-Ala-Pro-Ala-amidomethyl-coumarin (I) which crystallises in space group P2(1)2(1)2(1) with a = 108.2, b = 123.0, c = 35.8 A, and cyclophilin with cyclosporin (II) which crystallises as tetragonal plates in space group P4(1)2(1)2 or P4(3)2(1)2 with a = b = 94.98, c = 278.55 A.

Amino Acid Isomerases↗
PubMed1990

Diagnostic of precipitant for biomacromolecule crystallization by quasi-elastic light-scattering.

The translational diffusion coefficient D25,w of hen egg-white lysozyme and concanavalin A from the jack bean is measured in various precipitating agent solutions as a function of salt and protein concentration using quasi-elastic light-scattering. With some precipitants, in undersaturated protein solutions, a protein or salt concentration dependence of the diffusion coefficient of the scatters is observed. It can be correlated with the inability of the protein to crystallize in this precipitant once the solution is supersaturated. These variations of D25,w are interpreted in terms of non-specific interactions and/or aggregation that prevent the protein from making appropriate contacts to form a crystal. With other precipitants known to lead to crystallization, no significant variation of the diffusion coefficient with increasing concentration was observed, indicating that under such conditions up to saturation the proteins remain essentially monodisperse. Application of this technique to find crystallization conditions of other proteins is discussed.

Ammonium Sulfate↗
PubMed1990

Experimental determination of water equilibration rates in the hanging drop method of protein crystallization.

The hanging drop method for protein crystallization consists of equilibrating a water droplet containing the protein and a precipitant against a reservoir solution at a higher precipitant concentration. The time for water equilibration--which affects the kinetics of crystallization--to reach 90% of completion is shown to vary between about 25 h and more than 25 days, depending on experimental conditions. Experiments were performed with three of the most widely used precipitants (ammonium sulfate, polyethylene glycol, 2-methyl-2,4-pentanediol), combining various parameters expected to affect the rate of water evaporation. The most dramatic effects were obtained when varying temperature and initial drop volume. A simple empirical equation for estimating the kinetics of water equilibration under given crystallization conditions is proposed.

Chemical Phenomena↗
PubMed1988

A high resolution diffracting crystal form of the complex between yeast tRNAAsp and aspartyl-tRNA synthetase.

Three new crystal forms of the complex between yeast tRNAAsp and aspartyl-tRNA synthetase have been produced. The best crystals, obtained after modifying both purification and crystallization conditions, belong to space group P2(1)2(1)2(1) and diffract to 2.7 A. Unit cell parameters are a = 210.4 A, b = 145.3 A and c = 86.0 A (1 A = 0.1 nm), with one dimeric enzyme and two tRNA molecules in the asymmetric unit.

Amino Acyl-tRNA Synthetases↗