THE NMR SOLUTION CONFORMATION OF UNLIGATED HUMAN CYCLOPHILIN-A

The nuclear magnetic resonance (NMR) solution structure of free, unlig ated cyclophilin A (CypA), which is an 18 kDa protein from human T-lym phocytes that was expressed in Escherichia coli for the present study, was determined using multidimensional heteronuclear NMR techniques. S equence-specific resonance assignments for 99.5% of all backbone amide protons and non-labile hydrogen atoms provided the basis for collecti on of an input of 4101 nuclear Overhauser enhancement (NOE) upper dist ance constraints and 371 dihedral angle constraints for distance geome try calculations and energy minimization with the programs DIANA and O PAL. The average RMSD values of the 20 best energy-refined NMR conform ers relative to the mean coordinates are 0.49 Angstrom for the backbon e atoms and 0.88 Angstrom for all heavy atoms of residues 2 to 165. Th e molecular architecture includes an eight-stranded antiparallel beta- barrel that is closed by two amphipathic alpha-helices. Detailed compa risons with the crystal structure of free CypA revealed subtle but sig nificant conformational differences that can in most cases be related to lattice contacts in the crystal structure. N-15 spin relaxation tim es and NMR lineshape analyses for CypA in the free form and complexed with cyclosporin A (CsA) revealed transitions of polypeptide loops sur rounding the ligand-binding site from locally flexible conformations i n the free protein, some of which include well-defined conformational equilibria, to well-defined spatial arrangements in the CypA-CsA compl ex. Compared to the crystal structure of free CypA, where the ligand-b inding area is extensively involved in lattice contacts, the NMR struc ture presents a highly relevant reference for studies of changes in st ructure and internal mobility of the binding pocket upon ligand bindin g, and possible consequences of this conformational variability for ca lcineurin recognition by the CypA-CsA complex. (C) 1997 Academic Press Limited.