Insights into a single rod-like helix in activated radixin required for membrane-cytoskeletal cross-linking.
The members of the ezrin-radixin-moesin (ERM) family of proteins function as membrane-cytoskeletal cross-linkers in actin-rich cell surface structures. ERM proteins are thereby thought to be essential for cortical cytoskeleton organization, cell motility, adhesion, and proliferation. These modular polypeptides consist of a central helix-rich region, termed the alpha-domain, that connects an N-terminal FERM domain required for membrane binding and a C-terminal region which contains a major actin-binding motif. Conformational regulation of ERM protein function occurs by association of the FERM and C-terminal domains, whereby the membrane- and actin-binding activities are mutually suppressed and the protein is thought to take an inactive "closed" form. Here we report in vitro and in vivo studies of radixin to address the role of the alpha-domain in conformational activation of ERM proteins. Remarkably, an isolated alpha-domain comprised of radixin(311-469) forms a monomeric, stable helical rod that spans 240 A in length from the N-terminus to the C-terminus, most likely stabilized by extensive salt bridge interactions. By fusing green fluorescent protein variants to the FERM and C-terminal domains, we probed in vitroconformational changes impacted by the presence of the alpha-domain using fluorescence resonance energy transfer (FRET). Furthermore, deletion of this unusually long alpha-helical structure (radixin residues 314-411) prevents ERM membrane targeting in vivo.
Hoeflich
Klaus P
KP
Division of Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada.
Tsukita
Sachiko
S
Hicks
Leslie
L
Kay
Cyril M
CM
Tsukita
Shoichiro
S
Ikura
Mitsuhiko
M
D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D031541:Fluorescence Resonance Energy Transfer; D007739:L Cells; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D025581:Neurofibromin 2 / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011487:Protein Conformation; D017433:Protein Structure, Secondary / Q000235:genetics; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
D001798:Blood Proteins; D003432:Cross-Linking Reagents; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D025581:Neurofibromin 2; D010446:Peptide Fragments; C071776:radixin