Functional implication with the metal-binding properties of KChIP1.
To investigate the metal-binding properties of KChIP1, the interaction of KChIP1 and mutated KChIP1 with divalent cations (Mg(2+), Ca(2+), Sr(2+), and Ba(2+)) was explored by 8-anilinonaphthalene-1-sulfonate (ANS) fluorescence. It showed that KChIP1 possessed two types of Ca(2+)-binding sites, high-affinity and low-affinity Ca(2+)-binding sites. However, only low-affinity-binding site for Mg(2+), Sr(2+), and Ba(2+) was observed. The metal-binding properties of KChIP1 are not appreciably affected after removal of the N-terminal portion and EF-hand 1. Deleting the EF-hand 4 of KChIP1 abolishes its high-affinity Ca(2+)-binding site, but retains the intact low-affinity-binding site for metal ions. A decrease in the nonpolarity of ANS-binding site occurs with all mutants. However, the binding of ANS with KChIP1 is no longer observed after removal of EF-hands 3 and 4. Intermolecular interaction assessed by chemical cross-linking suggested that KChIP1 had a propensity to form dimer in the absence of metal ions, and a KChIP1 tetramer was pronouncedly produced in the presence of metal ions. Noticeably, the oligomerization state depends on the integrity of EF-hand 4. Taken together, our data suggest that EF-hand 4 is of structural importance as well as functional importance for fulfilling the physiological function of KChIP1.
Chang
Long-Sen
LS
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan. lschang@mail.nsysu.edu.tw
Chen
Chia-Yi
CY
Wu
Tony T
TT
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates; D001464:Barium / Q000737:chemistry*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D019281:Dimerization; D051605:Kv Channel-Interacting Proteins; D008274:Magnesium / Q000737:chemistry*; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013324:Strontium / Q000737:chemistry*; D013329:Structure-Activity Relationship
D000817:Anilino Naphthalenesulfonates; D002135:Calcium-Binding Proteins; D051605:Kv Channel-Interacting Proteins; D008670:Metals; D011994:Recombinant Proteins; D001464:Barium; C027132:1-anilino-8-naphthalenesulfonate; D008274:Magnesium; D002118:Calcium; D013324:Strontium