Biochem. J. (2007) 405
(473–480) (Printed in Great Britain)
The mammalian oxysterol-binding protein-related proteins (ORPs) bind 25-hydroxycholesterol in an evolutionarily conserved pocket
Monika SUCHANEK*, Riikka HYNYNEN†, Gerd WOHLFAHRT‡, Markku LEHTO†, Marie JOHANSSON†, Hannu SAARINEN†, Anna RADZIKOWSKA*, Christoph THIELE*1 and Vesa M. OLKKONEN†1
*Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany, †Department of Molecular Medicine, National Public Health Institute, Biomedicum, P.O. Box 104, FIN-00251 Helsinki, Finland, and ‡Orion Pharma, Computer-Aided Drug Design, P.O. Box 65, FIN-02101 Espoo, Finland
OSBP (oxysterol-binding protein) homologues, ORPs (OSBP-related proteins), constitute a 12-member family in mammals. We employed an in vitro [3H]25OH (25-hydroxycholesterol)-binding assay with purified recombinant proteins as well as live cell photo-cross-linking with [3H]photo-25OH and [3H]photoCH (photo-cholesterol), to investigate sterol binding by the mammalian ORPs. ORP1 and ORP2 [a short ORP consisting of an ORD (OSBP-related ligand-binding domain) only] were in vitro shown to bind 25OH. GST (glutathione S-transferase) fusions of the ORP1L [long variant with an N-terminal extension that carries ankyrin repeats and a PH domain (pleckstrin homology domain)] and ORP1S (short variant consisting of an ORD only) variants bound 25OH with similar affinity (ORP1L, Kd=9.7×10-8 M; ORP1S, Kd=8.4 ×10-8 M), while the affinity of GST–ORP2 for 25OH was lower (Kd=3.9×10-6 M). Molecular modelling suggested that ORP2 has a sterol-binding pocket similar to that of Saccharomyces cerevisiae Osh4p. This was confirmed by site-directed mutagenesis of residues in proximity of the bound sterol in the structural model. Substitution of Ile249 by tryptophan or Lys150 by alanine markedly inhibited 25OH binding by ORP2. In agreement with the in vitro data, ORP1L, ORP1S, and ORP2 were cross-linked with photo-25OH in live COS7 cells. Furthermore, in experiments with either truncated cDNAs encoding the OSBP-related ligand-binding domains of the ORPs or the full-length proteins, photo-25OH was bound to OSBP, ORP3, ORP4, ORP5, ORP6, ORP7, ORP8, ORP10 and ORP11. In addition, the ORP1L variant and ORP3, ORP5, and ORP8 were cross-linked with photoCH. The present study identifies ORP1 and ORP2 as OSBPs and suggests that most of the mammalian ORPs are able to bind sterols.
Key words: cholesterol, 25-hydroxycholesterol, molecular homology modelling, OSBP-related protein (ORP), oxysterol-binding protein (OSBP), photo-cross-linking.
Abbreviations used: DMEM, Dulbecco's modified Eagle's medium; EMEM, Eagle's minimal essential medium; FCS, fetal calf serum; GST, glutathione S-transferase; MBP, maltose-binding protein; 25OH, 25-hydroxycholesterol; OSBP, oxysterol-binding protein; photoCH, photo-cholesterol; ORD, OSBP-related ligand-binding domain; ORP, OSBP-related protein; PH domain, pleckstrin homology domain; PIP, phosphoinositide; wt, wild-type.
1Correspondence may be addressed to either of the authors (email thiele@mpi-cbg.de or vesa.olkkonen@ktl.fi).
Received 1 February 2007/4 April 2007; accepted 11 April 2007
Published as BJ Immediate Publication 11 April 2007, doi:10.1042/BJ20070176
© The Authors Journal compilation © 2007 Biochemical Society
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