title: NMR studies of [U-13C]cyclosporin A bound to cyclophilin: bound conformation and portions of cyclosporin involved in binding.
abstract: Cyclosporin A (CsA), a potent immunosuppressant, is known to bind with high specificity to cyclophilin (CyP), a 17.7 kDa protein with peptidyl-prolyl isomerase activity. In order to investigate the three-dimensional structure of the CsA/CyP complex, we have applied a variety of multidimensional NMR methods in the study of uniformly 13C-labeled CsA bound to cyclophilin. The 1H and 13C NMR signals of cyclosporin A in the bound state have been assigned, and from a quantitative interpretation of the 3D NOE data, the bound conformation of CsA has been determined. Three-dimensional structures of CsA calculated from the NOE data by using a distance geometry/simulated appealing protocol were found to be very different from previously determined crystalline and solution conformations of uncomplexed CsA. In addition, from CsA/CyP NOEs, the portions of CsA that interact with cyclophilin were identified. For the most part, those CsA residues with NOEs to cyclophilin were the same residues important for cyclophilin binding and immunosuppressive activity as determined from structure/activity relationships. The structural information derived in this study together with the known structure/activity relationships for CsA analogues may prove useful in the design of improved immunosuppressants. Moreover, the approach that is described for obtaining the structural information is widely applicable to the study of small molecule/large molecule interactions.
authors.affiliation: Pharmaceutical Discovery Division, Abbott Laboratories, Abbott Park, Illinois 60064.
authors.forename: S W
authors.initials: SW
authors.lastname: Fesik
authors.forename: R T
authors.initials: RT
authors.lastname: Gampe
authors.forename: H L
authors.initials: HL
authors.lastname: Eaton
authors.forename: G
authors.initials: G
authors.lastname: Gemmecker
authors.forename: E T
authors.initials: ET
authors.lastname: Olejniczak
authors.forename: P
authors.initials: P
authors.lastname: Neri
authors.forename: T F
authors.initials: TF
authors.lastname: Holzman
authors.forename: D A
authors.initials: DA
authors.lastname: Egan
authors.forename: R
authors.initials: R
authors.lastname: Edalji
authors.forename: R
authors.initials: R
authors.lastname: Simmer
mesh_terms: D000591:Amino Acid Isomerases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002151:Calorimetry; D002247:Carbon Isotopes; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D003524:Cyclosporins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008968:Molecular Conformation; D019696:Peptidylprolyl Isomerase; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D002247:Carbon Isotopes; D002352:Carrier Proteins; D003524:Cyclosporins; D011994:Recombinant Proteins; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase