Plant Villin Headpiece Domain Demonstrates a Novel Surface Charge Pattern and High Affinity for F-Actin

Abstract

Plants utilize multiple isoforms of villin, an F-actin regulating protein with an N-terminal gelsolin-like core and a distinct C-terminal headpiece domain. Unlike their vertebrate homologues, plant villins have a much longer linker polypeptide connecting the core and headpiece. Moreover, the linker-headpiece connection region in plant villins lacks sequence homology to the vertebrate villin sequences. It is unknown to what extent the plant villin headpiece structure and function resemble those of the well-studied vertebrate counterparts. Here we present the first solution NMR structure and backbone dynamics characterization of a headpiece from plants, villin isoform 4 from Arabidopsis thaliana. The villin 4 headpiece is a 63-residue domain (V4HP63) that adopts a typical headpiece fold with an aromatics core and a tryptophan-centered hydrophobic cap within its C-terminal subdomain. However, V4HP63 has a distinct N-terminal subdomain fold as well as a novel, high mobility loop due to the insertion of serine residue in the canonical sequence that follows the variable length loop in headpiece sequences. The domain binds actin filaments with micromolar affinity, like the vertebrate analogues. However, the V4HP63 surface charge pattern is novel and lacks certain features previously thought necessary for high-affinity F-actin binding. Utilizing the updated criteria for strong F-actin binding, we predict that the headpiece domains of all other villin isoforms in A. thaliana have high affinity for F-actin.

Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins

“…Native villin 4 contains a 190-residue fragment (positions 721–911) that is predicted to be mostly disordered [ 54 ]. Further analysis of this IDR reveals that it consists of a basic stretch at its N-terminus (~145 residues), followed by a predicted regulatory PEST motif [ 55 , 56 ] and a C-terminal acidic portion (35 residues) ( Figs 2 and S4 ).…”

“…Further analysis of this IDR reveals that it consists of a basic stretch at its N-terminus (~145 residues), followed by a predicted regulatory PEST motif [ 55 , 56 ] and a C-terminal acidic portion (35 residues) ( Figs 2 and S4 ). As an extension of our previous work with the villin 4 headpiece domain in isolation [ 54 ], we elected to focus our segmental isotopic labeling studies on a construct consisting of the 35-residue acidic IDR fragment joined to the headpiece. To render this system compatible with SML, it was first necessary to construct an IDR derivative fused at its C-terminus to an LPXTG sortase recognition motif, along with a headpiece derivative displaying an accessible polyglycine unit at its N-terminus ( Fig 1 ).…”

“…For the production of the C-terminal ligation partner, we utilized a 63-residue headpiece fragment (HP63, residues 912–974 in villin 4) that was previously shown to adopt a stable fold in solution [ 54 ]. For use in SML, we further augmented the HP63 polypeptide with an N-terminal diglycine that could be revealed upon TEV protease cleavage of a His 6 affinity tag.…”

“…The construct consisting of unlabeled FH8-IDR and 15 N-labeled headpiece reported a 15 N-HSQC signature nearly identical to that of the isolated headpiece domain ( Figs 4D and S8 ) [ 54 ]. The same headpiece signature was also present in the 15 N-HSQC spectrum of a fully 15 N-labeled analog ( Fig 4C ), however as expected several of the resonances were obscured by overlap with signals from the IDR.…”

“…In regards to plant villin 4, the N-terminal subdomain of the headpiece (residues 912–940, S4 Fig ) was previously shown to be temperature sensitive and exhibited significant tertiary structure alterations over the range of 10–40°C [ 54 ]. In addition, replacement of the native E 910 DLPA 914 residues on the surface of the N-terminal subdomain with GGGGG resulted in major destabilization of the headpiece when this fragment (G 5 -HP60, S2 Fig ) was characterized in isolation.…”