Journal of Molecular Biology
Regular articleThe NMR solution conformation of unligated human cyclophilin A1☆,
Introduction
The protein cyclophilin A (CypA) from human T lymphocytes consists of 165 amino acid residues and possesses peptidyl-prolyl cis-trans isomerase activity Handschumacher et al 1984, Fischer et al 1989. CypA has attracted keen interest in biomedical research, since it functions as an intracellular receptor of the immunosuppressive cyclic undecapeptide cyclosporin A (CsA), which acts as an inhibitor of specific signal transduction pathways leading to T lymphocyte activation Borel 1986, Schreiber 1991. Because of its high affinity for this drug, CypA has an important role in the prevention of organ transplant rejection (Borel, 1989). Renewed interest in research about cyclophilin has recently been sparked by the findings that CypA binds to the HIV-1 Gag protein (Luban et al., 1993), and that it is specifically incorporated in HIV-1 virions and required for their infectious activity Franke et al 1994, Thali et al 1994.
Interest in the molecular basis of immunosuppressive action spurred vigorous research on the CypA/CsA system, including three-dimensional structure determinations by nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography (for a review, see Braun et al., 1995). Structural studies of the CypA/CsA system yielded a first important result with the NMR determination of the conformation of CypA-bound CsA Fesik et al 1991, Weber et al 1991, Wuthrich et al 1991a, which was found to be very different from the structure of free CsA in crystals or in non-polar solvents (Loosli et al., 1985). Subsequently, the secondary structure of CypA in solution was determined (Wüthrich et al., 1991b), which was then used to support the tracing of a low-resolution electron density map to yield the crystal structure of CypA complexed with a linear tetrapeptide substrate (Kallen et al., 1991). On the basis of the crystal structure, the NMR structure of CypA-bound CsA and measurements of intermolecular nuclear Overhauser enhancement (NOE) distance constraints identifying CypA-CsA contacts, a molecular model of the CypA-CsA complex was generated (Spitzfaden et al., 1992). Subsequently, a crystal structure of free CypA and more than ten structure determinations of ligated cyclophilins in crystals and in solution have been reported (see Discussion and Table 4 below). We now add the NMR solution structure of free recombinant CypA expressed in Escherichia coli, and report comparative studies of internal mobility of CypA in the free form and in the CypA-CsA complex through measurements of 15N spin relaxation times. These data are analyzed with regard to mechanistic aspects of ligand binding by CypA and recognition of the CypA-CsA complex by calcineurin (CaN).
Section snippets
Assignments of the polypeptide backbone and aliphatic side-chain resonances
Complete sequence-specific NMR assignments for free CypA (Table 1) were obtained based nearly entirely on scalar coupling connectivities. Assignments of backbone resonances were established using 3D CBCA(CO)NH (Grzesiek & Bax, 1992) and ct-HNCA (Madsen & Sørensen, 1992) spectra. The backbone assignments are in good agreement with those obtained previously (Spitzfaden et al., 1992), using amino acid identification with residue-specific 15N labeling (Wüthrich et al., 1991a) and 3D 15N-resolved [1
Discussion
The ligand-binding area of CypA has been characterized at variable degrees of precision in the different structure determinations listed in Table 4. As a basis for the following discussion on implications of the present, new results for mechanistic aspects of ligand binding by CypA and of intermolecular recognition between the CypA-CsA complex and CaN, we use a review of the data up to 1994 by Braun et al. (1995) and the analysis of the high-resolution X-ray crystal structure of free human CypA
Sample preparation
Cyclophilin A labeled uniformly with 13C, with 15N, or with both 13C and 15N, was overexpressed in E. coli. Cells were grown on 1 or 2 l of Martek CELTONE rich medium containing M9 salts (Sambrook et al., 1989). Purification of CypA and the preparation of the CypA-CsA complex were as described (Weber et al., 1991). The yield of purified protein was about 35 mg/l. Samples were dialyzed extensively against an optimized NMR-buffer containing 10 mM Na+/K+-phosphate (pH 6.5), 100 mM NaNO3, 5 mM [2H10
Supplementary Files
The Supplementary material is, regrettably, no longer available.
Acknowledgements
We thank Dr K. Memmert (Sandoz) for assistance during the production of labeled CypA, and Drs U. Hommel, J. Kallen, H. Widmer, M. Zurini (Sandoz) and P. Luginbühl (ETHZ) for helpful discussions on various aspects of cyclophilin structure and function. Financial support was obtained from Sandoz Pharma AG, Basel, and the Schweizerischer Nationalfonds (project 31.32033.91). We thank Mrs R. Hug for the careful processing of the manuscript.
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2010, Journal of Molecular BiologyCitation Excerpt :Reference spectra were obtained with the respective CT delay and omission of the CPMG delay period.64 Chemical shifts for CypA alone have been published previously.38 Chemical shifts for CypA in complex with CAN were determined from both a 3D 15N-edited nuclear Overhauser enhancement spectroscopy-HSQC collected on a 600-MHz spectrometer at 25 °C with a mixing period of 80 ms and a titration experiment of 15N-labeled CypA R55A (0.5 mM) with increasing concentrations of CAN (0.13–2.0 mM), which allowed us to follow CypA resonance shifts as a function of [CAN].13
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Supplementary material comprising three Tables is available fromDOI:10.1006/jmbi.1997.1220
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Edited by P. E. Wright
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Present addresses: M. Ottiger, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0510, USA; O. Zerbe, Departement für Pharmazie, ETH, CH-8057 Zürich, Switzerland.