PubMed Health⌕ Search

Biomedical subjects

J Kallen

Publications and source records attributed to J Kallen.

31 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↗
PubMed1993

X-ray structure of a decameric cyclophilin-cyclosporin crystal complex.

Human cyclophilin A (CypA), a ubiquitous intracellular protein of 165 amino acids, is the major receptor for the cyclic undecapeptide immunosuppressant drug cyclosporin A (CsA), which prevents allograft rejection after transplant surgery and is efficacious in the field of autoimmune diseases. CsA prevents T-cell proliferation by blocking the calcium-activated pathway leading to interleukin-2 transcription. Besides their ability to bind CsA, the cyclophilin isoforms also have peptidyl-prolyl isomerase activity and enhance the rate of protein folding. The macrolide FK506 acts similarly to CsA and its cognate receptor FKBP also has peptidyl-prolyl isomerase activity. Inhibition of this enzymatic activity alone is not sufficient to achieve immunosuppression. A direct molecular interaction between the drug-immunophilin complex (CsA-CypA, or FK506-FKBP) and the phosphatase calcineurin, is responsible for modulating the T-cell receptor signal transduction pathway. Here we describe the crystal structure of a decameric CypA-CsA complex. The crystallographic asymmetric unit is composed of a pentamer of 1:1 cyclophilin-cyclosporin complexes of rather exact non-crystallographic fivefold symmetry. The 2.8 A electron density map is of high quality. The five independent cyclosporin molecules are clearly identifiable, providing an unambiguous picture of the detailed interactions between a peptide drug and its receptor. It broadly confirms the results of previous NMR, X-ray and modelling studies, but provides further important structural details which will be of use in the design of drugs that are analogues of CsA.

Amino Acid Isomerases↗
PubMed1992

The X-ray structure of a tetrapeptide bound to the active site of human cyclophilin A.

Human cyclophilin A (165 residues) has peptidyl-prolyl cis-trans isomerase activity. Here we report a high-resolution three-dimensional X-ray structure of a substrate, ac-Ala-Ala-Pro-Ala-amc (ac, acetyl; amc, amidomethylcoumarin) bound to the active-site of cyclophilin. The structure consisting of a dimer of complexes and 135 water molecules was refined to a crystallographic R-factor of 17.7% for all data in the range 8 A-2.3 A.

Amino Acid Isomerases↗
PubMed1992

Cyclosporin A-cyclophilin complex formation. A model based on X-ray and NMR data.

The previously determined 3D NMR solution structure of cyclophilin-bound cyclosporin A (CsA) was docked onto the X-ray crystal structure of cyclophilin. Intermolecular nuclear Overhauser effects (NOE) between CsA and cyclophilin were used as constraints in a restrained energy minimization to generate a model of the complex which satisfied all the NOE distance constraints. The model shows that the residues 9 to 11 and 1 to 5 of the cyclic CsA molecule are in contact with cyclophilin. Comparing the model of the CsA-cyclophilin complex to the X-ray crystal structure of a complex of cyclophilin with a substrate for peptidyl-proline cis-trans isomerase activity, i.e. the linear tetrapeptide substrate ac-Ala-Ala-Pro-Ala-amc (ac, acetyl; amc, amidomethylcoumarin), one notices that the contacting peptide segments in the two ligands are oriented in opposite directions, and that the side chain of MeVal-11 of CsA superposes rather precisely with the position of the prolyl residue in ac-Ala-Ala-Pro-Ala-amc.

Amino Acid Isomerases↗
PubMed1991

Structure of human cyclophilin and its binding site for cyclosporin A determined by X-ray crystallography and NMR spectroscopy.

The protein cyclophilin is the major intracellular receptor for the immunosuppressive drug cyclosporin A. Cyclosporin A acts as an inhibitor of T-cell activation and can prevent graft rejection in organ and bone marrow transplantation. Cyclophilin may be responsible for mediating this immunosuppressive response. Cyclophilin also catalyses the interconversion of the cis and trans isomers of the peptidyl-prolyl amide bonds of peptide and protein substrates. Here we report the X-ray crystal structure of human recombinant cyclophilin complexed with a tetrapeptide and the identification, by nuclear magnetic resonance spectroscopy, of the specific binding site for cyclosporin A. Cyclophilin has an eight-stranded antiparallel beta-barrel structure. The prolyl isomerase substrate-binding site is coincident with the cyclosporine-binding site. These results may help to provide a structural basis for rationalizing the immunosuppressive function of the cyclosporin-cyclophilin system and will also be important in the design of improved immunosuppressant drugs.

Amino Acid Isomerases↗
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

Crystallization and preliminary X-ray crystallographic study of interleukin-8.

Interleukin-8 (neutrophil-activating factor; NAP-1) has been crystallized by the vapour diffusion technique to give single crystals suitable for three-dimensional structural study at a resolution higher than 2.4 A. The crystals belong to the space group P3(1)21 or P3(2)21 and have unit cell dimensions a = b = 40.9 A, c = 90.3 A.

Chemotactic Factors↗
PubMed1989

Complexes of aspartate aminotransferase with hydroxylamine derivatives: spectral studies in solution and in the crystalline state.

Hydroxylamine and its derivatives of general formula H2NOR react with aldehydes and aldimines to produce oximes. If R corresponds to the side chain of a natural amino acid, such compounds can be thought of as analogs of the corresponding amino acids, lacking the alpha-carboxylate group. Oximes formed between such compounds and pyridoxal phosphate in the active site of aspartate amino-transferase mimic external aldimine intermediates that occur during catalysis by this enzyme. The properties of oxime derivatives of mitochondrial aspartate aminotransferase with hydroxylamine and 6 compounds H2NOR were studied by absorption spectroscopy and circular dichroism in solution and by linear dichroism in crystals. Stable oximes, absorbing at lambda max congruent to 380 nm and exhibiting a negative Cotton effect, were obtained with the carboxylate-containing compounds. The oximes formed with carboxylate-free compounds showed somewhat different properties and stability. With H-Tyr a stable complex absorbing at lambda max congruent to 370 nm rather than at 380 nm, was obtained, H-Ala and H-Phe produced unstable oximes with the initial absorption band at lambda max congruent to 380 nm that was gradually replaced by a band at lambda max congruent to 340 nm. The species absorbing at 340 nm were shown to be coenzyme-inhibitor complexes which were gradually released from the enzyme. A similar 330-340 nm absorption band was observed upon reaction of the free coenzyme with all hydroxylamine inhibitors at neutral pH-values. The results of the circular dichroism experiments in solution and the linear dichroism studies in microcrystals of mAspAT indicate that the coenzyme conformation in these inhibitor/enzyme complexes is similar to that occurring in an external aldimine analogue, the 2-MeAsp/mAspAT complex. Co-crystallizations of the enzyme with the H2NOR compounds were also carried out. Triclinic crystals were obtained in all cases, suggesting that the "closed" structure cannot be stabilized by a single carboxylate group.

Animals↗