How can Ca2+ selectively activate recoverin in the presence of Mg2+? Surface plasmon resonance and FT-IR spectroscopic studies.
We investigated the relationship between metal ion selective conformational changes of recoverin and its metal-bound coordination structures. Recoverin is a 23 kDa heterogeneously myristoylated Ca(2+)-binding protein that inhibits rhodopsin kinase. Upon accommodating two Ca(2+) ions, recoverin extrudes a myristoyl group and associates with the lipid bilayer membrane, which was monitored by the surface plasmon resonance (SPR) technique. Large changes in SPR signals were observed for Sr(2+), Ba(2+), Cd(2+), and Mn(2+) as well as Ca(2+), indicating that upon binding to these ions, recoverin underwent a large conformational change to extrude the myristoyl group, and thereby interacted with lipid membranes. In contrast, no SPR signal was induced by Mg(2+), confirming that even though it accommodates two Mg(2+) ions, recoverin does not induce the large conformational change. To investigate the coordination structures of metal-bound Ca(2+) binding sites, FT-IR studies were performed. The EF-hands, Ca(2+)-binding regions each comprising 12 residues, arrange to coordinate Ca(2+) with seven oxygen ligands, two of which are provided by a conserved bidentate Glu at the 12th relative position in the EF-hand. FT-IR analysis confirmed that Sr(2+), Ba(2+), Cd(2+), and Mn(2+) were coordinated to COO(-) of Glu by a bidentate state as well as Ca(2+), while coordination of COO(-) with Mg(2+) was a pseudobridging state with six-coordinate geometry. These SPR and FT-IR results taken together reveal that metal ions with seven-coordinate geometry in the EF-hands induce a large conformational change in recoverin so that it extrudes the myristoyl group, while metal ions with six-coordinate geometry in the EF-hands such as Mg(2+) remain the myristoyl group sequestered in recoverin.
Ozawa
T
T
Department of Chemistry, School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Fukuda
M
M
Nara
M
M
Nakamura
A
A
Komine
Y
Y
Kohama
K
K
Umezawa
Y
Y
D000818:Animals; D001464:Barium / Q000737:chemistry / Q000378:metabolism; D002104:Cadmium / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002413:Cations, Divalent; D002417:Cattle; D020832:EF Hand Motifs; D005136:Eye Proteins; D051597:Hippocalcin; D008074:Lipoproteins; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D019814:Myristic Acid / Q000378:metabolism; D009419:Nerve Tissue Proteins; D011485:Protein Binding; D011487:Protein Conformation; D051603:Recoverin; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013324:Strontium / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods
D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D005136:Eye Proteins; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D002104:Cadmium; D019814:Myristic Acid; D051603:Recoverin; D051597:Hippocalcin; D001464:Barium; D008345:Manganese; D008274:Magnesium; D002118:Calcium; D013324:Strontium