The NMR Structure of Dematin Headpiece Reveals a Dynamic Loop That Is Conformationally Altered upon Phosphorylation at a Distal Site

Abstract

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 Factin-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.

A global role for EKLF in definitive and primitive erythropoiesis

“…Labeled samples were hybridized to 22K Compugen mouse long-oligonucleotide microarrays (Compugen, San Jose, CA) and analyzed using previously described methods. 28,29 All data and rankings used to define statistically robust differential expression were exported into GeneSpring 7.2 (Agilent Technologies, PaloAlto, CA) for visualization. Full documentation of experimental parameters and raw data are available for download in accordance with MIAME guidelines 30 via a Signet interface (Agilent Technologies).…”

On the unyielding hydrophobic core of villin headpiece

“…1). [14][15][16] In addition, this region is considerably more dynamic than the remainder of the protein as demonstrated by 15 N-relaxation analysis. 15,17,18 Prior analysis of several different crystal structures of villin headpiece have provided strong evidence that the different conformations in the V-Loop represent distinct substates of a highly dynamic ensemble.…”

“…[14][15][16] In addition, this region is considerably more dynamic than the remainder of the protein as demonstrated by 15 N-relaxation analysis. 15,17,18 Prior analysis of several different crystal structures of villin headpiece have provided strong evidence that the different conformations in the V-Loop represent distinct substates of a highly dynamic ensemble. 5 Despite crystallizing in the P2 1 2 1 2 1 space group, the conformation of the V-Loop region in HP67 L61G is structurally more similar to chain B of wild-type villin headpiece within the P6 1 crystal lattice than it is to wild-type headpiece crystallized in the P2 1 2 1 2 1 space group (PDB Accession IDs: 2RJX, 2RJY; Supporting Information Fig.…”

“…Unlike the dynamic V-Loop, the C-terminal subdomain has been shown by 15 N relaxation analysis to be comparatively rigid. 15,17,18 As a result, the structural perturbation in the C-terminal subdomain of L61[GL] is significant.…”

“…15,17,18 As a result, the structural perturbation in the C-terminal subdomain of L61[GL] is significant. Relative to the wild-type structure, helix 4 of L61[GL] is unwound (Figs.…”