Villin‐type headpiece domains show a wide range of F‐actin‐binding affinities

Abstract

The villin-type "headpiece" domain is a modular motif found at the extreme C-terminus of larger "core" domains in over 25 cytoskeletal proteins in plants and animals. Although headpiece is classified as an F-actin-binding domain, it has been suggested that some expressed fusion-proteins containing headpiece may lack F-actin-binding in vivo. To determine the intrinsic F-actin affinity of headpiece domains, we quantified the F-actin affinity of seven headpiece domains and three N-terminal truncations, under identical in vitro conditions. The constructs are folded and adopt the native headpiece structure. However, they show a wide range of affinities that can be grouped into high, low, and nonspecific-binding categories. Computer models of the structure and charged surface potential of these headpiece domains suggest features important for high F-actin affinity. We conclude that not all headpiece domains are intrinsically F-actin-binding motifs, and suggest that the surface charge distribution may be an important element for F-actin recognition.

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

“…The consensus phosphorylation site specificity of the catalytic subunit of cyclic AMP dependent kinase is RRXSX but varies substantially and is quite similar to the KKASF site that is phosphorylated in dematin headpiece (12). Whereas the role of this phosphorylation in vivo is unknown, phosphorylation of dematin inhibits in vitro Factin bundling but not F-actin binding (1,13).…”

“…Most, but not all, headpiece domains are modular F-actinbinding motifs that retain binding activity when cleaved from the core domain (1). Headpiece domains were originally identified in the protein villin, an F-actin cross-linking protein found in the actin bundles that support the brush border membrane of the absorptive epithelium (2,3).…”

“…The NMR structures of villin headpiece (HP67) 1 and a small subdomain within villin headpiece (HP35) reveal a unique two-domain structure (4,17). The N-terminal half of villin headpiece has little regular secondary structure and its folding is pH-dependent due to a buried histidine residue.…”

“…The putative 10 residue dematin headpiece V-loop is the longest within any headpiece sequence. Although the NMR structure of HP67, in conjunction with mutagenic data, suggests the features that are critical for F-actin binding (1,4) no other headpiece domain structure has been determined to test the validity of the generalization made from villin headpiece on this class of sequences.…”

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.…”