Calbindin D28k EF-hand ligand binding and oligomerization: four high-affinity sites--three modes of action.
Calbindin D28k, a highly conserved protein with Ca2+-sensing and Ca2+-buffering capabilities, is abundant in brain and sensory neurons. This protein contains six EF-hand subdomains, four of which bind Ca2+ with high affinity. Calbindin D28k can be reconstituted from six synthetic peptides corresponding to the six EF-hands, indicating a single-domain structure with multiple interactions between the EF-hand subdomains. In this study, we have undertaken a detailed characterization of the Ca2+-binding and oligomerization properties of each individual EF-hand peptide using CD spectroscopy and analytical ultracentrifugation. Under the conditions tested, EF2 is monomeric and does not bind Ca2+, whereas EF6, which binds Ca2+ weakly, aggregates severely. We have therefore focused this study on the high-affinity binding sites, EF-hands 1, 3, 4, and 5. Our sedimentation equilibrium data show that, in the presence of Ca2+, EF-hands 1, 3, 4, and 5 all form dimers in solution in which the distribution between the monomer, dimer, and higher order oligomers differs. The processes of Ca2+ binding and oligomerization are linked to different degrees, and three main mechanisms emerge. For EF-hands 1 and 5, the dimer binds Ca2+ more strongly than the monomer and Ca2+ binding drives dimerization. For EF-hand 4, dimer formation requires only one of the monomers to be Ca2+-bound. In this case, the Ca2+ affinity is independent of dimerization. For EF-hand 3, dimerization occurs both in the absence and presence of Ca2+, while oligomerization increases in the presence of Ca2+.
Cedervall
Tommy
T
Department of Chemistry, Haverford College, Haverford, Pennsylvania 19041, USA.
André
Ingemar
I
Selah
Cheryl
C
Robblee
James P
JP
Krecioch
Peter C
PC
Fairman
Robert
R
Linse
Sara
S
Akerfeldt
Karin S
KS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D018257:Helix-Loop-Helix Motifs; D008024:Ligands; D008956:Models, Chemical; D008961:Models, Structural; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D064030:S100 Calcium Binding Protein G / Q000737:chemistry* / Q000378:metabolism; D014461:Ultracentrifugation
D064026:Calbindins; D008024:Ligands; D046912:Multiprotein Complexes; D064030:S100 Calcium Binding Protein G; D002118:Calcium