Novel mechanism of regulation of protein 4.1G binding properties through Ca2+/calmodulin-mediated structural changes.
Protein 4.1G (4.1G) is a widely expressed member of the protein 4.1 family of membrane skeletal proteins. We have previously reported that Ca(2+)-saturated calmodulin (Ca(2+)/CaM) modulates 4.1G interactions with transmembrane and membrane-associated proteins through binding to Four.one-ezrin-radixin-moesin (4.1G FERM) domain and N-terminal headpiece region (GHP). Here we identify a novel mechanism of Ca(2+)/CaM-mediated regulation of 4.1G interactions using a combination of small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy analyses. We document that GHP intrinsically disordered coiled structure switches to a stable compact structure upon binding of Ca(2+)/CaM. This dramatic conformational change of GHP inhibits in turn 4.1G FERM domain interactions due to steric hindrance. Based upon sequence homologies with the Ca(2+)/CaM-binding motif in protein 4.1R headpiece region, we establish that the 4.1G S(71)RGISRFIPPWLKKQKS peptide (pepG) mediates Ca(2+)/CaM binding. As observed for GHP, the random coiled structure of pepG changes to a relaxed globular shape upon complex formation with Ca(2+)/CaM. The resilient coiled structure of pepG, maintained even in the presence of trifluoroethanol, singles it out from any previously published CaM-binding peptide. Taken together, these results show that Ca(2+)/CaM binding to GHP, and more specifically to pepG, has profound effects on other functional domains of 4.1G.
Center for Geo-Environmental Science, Graduate School of Engineering and Resource Science, Akita University, Akita, Japan. nunomura@gipc.akita-u.ac.jp
Wataru
W
Nunomura
Yuji
Y
Jinbo
Noriyoshi
N
Isozumi
Shinya
S
Ohki
Yoshinobu
Y
Izumi
Norio
N
Matsushima
Yuichi
Y
Takakuwa
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D002147:Calmodulin; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; C028841:erythrocyte membrane band 4.1 protein; D002118:Calcium