PubMed · 7686830
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M H Schreier. Immunophilins and immunosuppression.. https://pubmed.ncbi.nlm.nih.gov/7686830/
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Pin1 is an essential and conserved mitotic peptidyl-prolyl isomerase (PPIase) that is distinct from members of two other families of conventional PPIases, cyclophilins and FKBPs (FK-506 binding proteins). In response to their phosphorylation during mitosis, Pin1 binds and regulates members of a highly conserved set of proteins that overlaps with antigens recognized by the mitosis-specific monoclonal antibody MPM-2. Pin1 is here shown to be a phosphorylation-dependent PPIase that specifically recognizes the phosphoserine-proline or phosphothreonine-proline bonds present in mitotic phosphoproteins. Both Pin1 and MPM-2 selected similar phosphorylated serine-proline-containing peptides, providing the basis for the specific interaction between Pin1 and MPM-2 antigens. Pin1 preferentially isomerized proline residues preceded by phosphorylated serine or threonine with up to 1300-fold selectivity compared with unphosphorylated peptides. Pin1 may thus regulate mitotic progression by catalyzing sequence-specific and phosphorylation-dependent proline isomerization.
The nuclear magnetic resonance (NMR) solution structure of free, unligated cyclophilin A (CypA), which is an 18 kDa protein from human T-lymphocytes that was expressed in Escherichia coli for the present study, was determined using multidimensional heteronuclear NMR techniques. Sequence-specific resonance assignments for 99.5% of all backbone amide protons and non-labile hydrogen atoms provided the basis for collection of an input of 4101 nuclear Overhauser enhancement (NOE) upper distance constraints and 371 dihedral angle constraints for distance geometry calculations and energy minimization with the programs DIANA and OPAL. The average RMSD values of the 20 best energy-refined NMR conformers relative to the mean coordinates are 0.49 A for the backbone atoms and 0.88 A for all heavy atoms of residues 2 to 165. The molecular architecture includes an eight-stranded antiparallel beta-barrel that is closed by two amphipathic alpha-helices. Detailed comparisons with the crystal structure of free CypA revealed subtle but significant conformational differences that can in most cases be related to lattice contacts in the crystal structure. 15N spin relaxation times and NMR lineshape analyses for CypA in the free form and complexed with cyclosporin A (CsA) revealed transitions of polypeptide loops surrounding the ligand-binding site from locally flexible conformations in the free protein, some of which include well-defined conformational equilibria, to well-defined spatial arrangements in the CypA-CsA complex. Compared to the crystal structure of free CypA, where the ligand-binding area is extensively involved in lattice contacts, the NMR structure presents a highly relevant reference for studies of changes in structure and internal mobility of the binding pocket upon ligand binding, and possible consequences of this conformational variability for calcineurin recognition by the CypA-CsA complex.
The Escherichia coli trigger factor is a peptidyl-prolyl cis/trans isomerase (PPIase) which catalyzes proline-limited protein folding extremely well. It has been found associated with nascent protein chains as well as with the chaperone GroEL. The trigger factor utilizes protein regions outside the central catalytic domain for catalyzing refolding of unfolded proteins efficiently. Here we produced several fragments which encompass individual domains or combinations of the middle FKBP-like domain (M) with the N-terminal (N) and C-terminal (C) regions, respectively. These fragments appear to be stably folded. They show ordered structure and cooperative urea-induced unfolding transitions, and the far-UV CD spectrum of the intact trigger factor is well represented by the sum of the spectra of the fragments. This suggests that the native trigger factor shows a modular structure, which is composed of three fairly independent folding units. In the intact protein there is a slight mutual stabilization of these units. The high enzymatic activity in protein folding could not be restored by fusing alternatively the N or the C-terminal regions to the catalytic domain (in NM and MC constructs, respectively). Surprisingly, the high folding activity of the intact trigger factor has been regained partially by functional complementation of the overlapping NM and MC constructs.