The NMR structure of dematin headpiece reveals a dynamic loop that is conformationally altered upon phosphorylation at a distal site.
Dematin (band 4.9) is found in the junctional complex of the spectrin cytoskeleton that supports the erythrocyte cell membrane. Dematin is a member of the larger class of cytoskeleton-associated proteins that contain a modular "headpiece" domain at their extreme C termini. The dematin headpiece domain provides the second F-actin-binding site required for in vitro F-actin bundling. The dematin headpiece is found in two forms in the cell, one of 68 residues (DHP) and one containing a 22-amino acid insert near its N terminus (DHP+22). In addition, dematin contains the only headpiece domain that is phosphorylated, in vivo. The 22-amino acid insert in DHP+22 appeared unstructured in NMR spectra; therefore, we have determined the three-dimensional structure of DHP by multidimensional NMR methods. Although the overall three-dimensional structure of DHP is similar to that of the villin headpiece, there are two novel characteristics revealed by this structure. First, unlike villin headpiece that contains a single buried salt bridge, DHP contains a buried charged cluster comprising residues Glu(39), Arg(66), Lys(70), and the C-terminal carboxylate of Phe(76). Second, (15)N relaxation experiments indicate that the longer "variable loop" region near the N terminus of DHP (residues 20-29) is dynamic, undergoing significantly greater motions that the rest of the structure. Furthermore, NMR chemical shift changes indicate that the conformation of the dynamic variable loop is altered by phosphorylation of serine 74, which is far in the sequence from the variable loop region. Our results suggest that phosphorylation of the dematin headpiece acts as a conformational switch within this headpiece domain.
Frank
Benjamin S
BS
Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118.
Vardar
Didem
D
Chishti
Athar H
AH
McKnight
C James
CJ
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry; D004910:Erythrocyte Membrane / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008840:Microfilament Proteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins; D010766:Phosphorylation; D011487:Protein Conformation; D012694:Serine / Q000737:chemistry
D000199:Actins; D001798:Blood Proteins; D002352:Carrier Proteins; C044805:DMTN protein, human; D008840:Microfilament Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; C026615:villin; D012694:Serine; D006639:Histidine