Deswapping bovine odorant binding protein.
The X-ray structure of bovine Odorant Binding Protein (bOBP) revealed its association as a domain swapped dimer. bOBP, devoid of any cysteines, contrasts with other mammalian OBPs, which are monomeric and possess at least one disulfide bridge. We have produced a mutant of bOBP in which a glycine residue was inserted after position 121. This mutation yielded a monomeric bOBP-121Gly+ in which domain swapping has been reverted. Here, we have subsequently introduced two mutations, Trp64Cys and His155Cys, in view to stabilize the putative monomer with a disulfide bridge. We have determined the crystal structure of this triple mutant at 1.65 A resolution. The mutant protein is monomeric, stabilized by a disulfide bridge between Trp64Cys and His155Cys, with a backbone superimposable to that of native bOBP, with the exception of the hinge and of the 10 residues at the C-terminus. bOBP triple mutant binds 1-amino-anthracene, 1-octen-3-ol (bOBP co-purified ligand) and other ligands with microM Kd values comparable to those of the swapped dimer.
Ramoni
Roberto
R
Dipartimento di Produzioni Animali, Biotecnologie Veterinarie, Qualità e Sicurezza degli Alimenti, Università degli Studi di Parma, Via del Taglio 8, 43100 Parma, Italy.
Spinelli
Silvia
S
Grolli
Stefano
S
Conti
Virna
V
Merli
Elisa
E
Cambillau
Christian
C
Tegoni
Mariella
M
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D002850:Chromatography, Gel; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D019281:Dimerization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D000442:Octanols / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018035:Receptors, Odorant / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D000442:Octanols; D018035:Receptors, Odorant; C045648:odorant-binding protein; C006497:1-aminoadenosine; D000241:Adenosine; C038844:1-octen-3-ol