The isolated sixth gelsolin repeat and headpiece domain of villin bundle F-actin in the presence of calcium and are linked by a 40-residue unstructured sequence

Villin is an F-actin regulating, modular protein with a gelsolin-like core and a distinct C-terminal "headpiece" domain. Localized in the microvilli of the absorptive epithelium, villin can bundle F-actin and, at higher calcium concentrations, is capable of a gelsolin-like F-actin severing. The headpiece domain can, in isolation, bind F-actin and is crucial for F-actin bundling by villin. While the three-dimensional structure of the isolated headpiece is known, its conformation in the context of attachment to the villin core remains unexplored. Furthermore, the dynamics of the linkage of the headpiece to the core has not been determined. To address these issues, we employ a 208-residue modular fragment of villin, D6-HP, which consists of the sixth gelsolin-like domain of villin (D6) and the headpiece (HP). We demonstrate that this protein fragment requires calcium for structural stability and, surprisingly, is capable of Ca2+-dependent F-actin bundling, suggesting that D6 contains a cryptic F-actin binding site. NMR resonance assignments and 15N relaxation measurements of D6-HP in 5 mM Ca2+ demonstrate that D6-HP consists of two independent structural domains (D6 and HP) connected by an unfolded 40-residue linker sequence. The headpiece domain in D6-HP retains its structure and interacts with D6 only through the linker sequence without engaging in other interactions. Chemical shift values indicate essentially the same secondary structure elements for D6 in D6-HP as in the highly homologous gelsolin domain 6. Thus, the headpiece domain of villin is structurally and functionally independent of the core domain.     

Multistate folding of the villin headpiece domain

The villin headpiece (HP67) is a 67 residue, monomeric protein derived from the C-terminal domain of villin. Wild-type HP67 (WT HP67) is the smallest fragment of villin that retains strong in vitro actin-binding activity. WT HP67 is made up of two subdomains, which form a tightly packed interface. The C-terminal subdomain of WT HP67, denoted HP35, is rich in helical structure, folds in isolation, and has been widely used as a model system for folding studies. In contrast, very little is known about the folding of the intact villin headpiece domain. Here, NMR, CD and H/2H amide exchange measurements are used to follow the pH, thermal and urea-induced unfolding of WT HP67 and a mutant (HP67 H41Y) in which a buried conserved histidine in the N-terminal subdomain, His41, has been mutated to Tyr. Although most small proteins display two-state equilibrium unfolding, the results presented here demonstrate that unfolding of the villin headpiece is a multistate process. The presence of a folded N-terminal subdomain is shown to stabilize the C-terminal subdomain, increasing the midpoints of the thermal and urea-induced unfolding transitions and increasing protection factors for H/2H exchange. Histidine 41 has been shown to act as a pH-dependent switch in wild-type HP67: the N-terminal subdomain is unfolded when His41 is protonated, while the C-terminal subdomain remains folded irrespective of the protonation state of His41. Mutation of His41 to Tyr eliminates the segmental pH-dependent unfolding of the headpiece. The mutation stabilizes both domains, but folding is still multistate, indicating that His41 is not solely responsible for the unusual equilibrium unfolding behavior of villin headpiece domain.     

In vivo analysis of functional domains from villin and gelsolin

Transfected CV1 cells were used to compare the in vivo effects of various domains of villin and gelsolin. These two homologous actin modulating proteins both contain a duplicated severin-like sequence. Villin has in addition a carboxy-terminal domain, the headpiece, which accounts for its bundling activity. The effects of the villin-deleted mutants were compared with those of native villin. Our results show that essential domains of villin required to induce the growth of microvilli and F-actin redistribution are present in the first half of the core and in the headpiece. We also show that the second half of the villin core cannot be exchanged by its homolog in gelsolin. When expressed at high levels of CV1 cells, full length gelsolin completely disrupted stress fibers without change of the cell shape. Addition of the villin headpiece to gelsolin had no effect on the phenotype induced by gelsolin alone. Expression of the first half of gelsolin induced similar modifications as capping proteins and rapid cell mortality; this deleterious effect on the cell structure was also observed when the headpiece was linked to the first half of gelsolin. In cells expressing the second half of gelsolin, a dotted F-actin staining was often seen. Moreover elongated dorsal F-actin structures were observed when the headpiece was linked to the second gelsolin domain. These studies illustrate the patent in vivo severing activity of gelsolin as well as the distinct functional properties of villin core in contrast to gelsolin.     

Solution structures of the C-terminal headpiece subdomains of human villin and advillin, evaluation of headpiece F-actin-binding requirements

Headpiece (HP) is a 76-residue F-actin-binding module at the C terminus of many cytoskeletal proteins. Its 35-residue C-terminal subdomain is one of the smallest known motifs capable of autonomously adopting a stable, folded structure in the absence of any disulfide bridges, metal ligands, or unnatural amino acids. We report the three-dimensional solution structures of the C-terminal headpiece subdomains of human villin (HVcHP) and human advillin (HAcHP), determined by two-dimensional 1H-NMR. They represent the second and third structures of such C-terminal headpiece subdomains to be elucidated so far. A comparison with the structure of the chicken villin C-terminal subdomain reveals a high structural conservation. Both C-terminal subdomains bind specifically to F-actin. Mutagenesis is used to demonstrate the involvement of Trp 64 in the F-actin-binding surface. The latter residue is part of a conserved structural feature, in which the surface-exposed indole ring is stacked on the proline and lysine side chain embedded in a PXWK sequence motif. On the basis of the structural and mutational data concerning Trp 64 reported here, the results of a cysteine-scanning mutagenesis study of full headpiece, and a phage display mutational study of the 69-74 fragment, we propose a modification of the model, elaborated by Vardar and coworkers, for the binding of headpiece to F-actin.     

How to Arm a Supervillin: Designing F-Actin Binding Activity into Supervillin Headpiece

Villin-type headpiece domains are compact motifs that have been used extensively as model systems for protein folding. Although the majority of headpiece domains bind actin, there are some that lack this activity. Here, we present the first NMR solution structure and ¹⁵N-relaxation analysis of a villin-type headpiece domain natively devoid of F-actin binding activity, that of supervillin headpiece (SVHP). The structure was found to be similar to that of other headpiece domains that bind F-actin. Our NMR analysis demonstrates that SVHP lacks a conformationally flexible region (V-loop) present in all other villin-type headpiece domains and which is essential to the phosphoryl regulation of dematin headpiece. In comparing the electrostatic surface potential map of SVHP to that of other villin-type headpiece domains with significant affinity for F-actin, we identified a positive surface potential conserved among headpiece domains that bind F-actin but absent from SVHP. A single point mutation (L38K) in SVHP, which creates a similar positive surface potential, endowed SVHP with specific affinity for F-actin that is within an order of magnitude of the tightest binding headpiece domains. We propose that this effect is likely conferred by a specific buried salt bridge between headpiece and actin. As no high-resolution structural information exists for the villin-type headpiece F-actin complex, our results demonstrate that through positive mutagenesis, it is possible to design binding activity into homologous proteins without structural information of the counterpart's binding surface.     

Isolation of a domain of villin retaining calcium-dependent interaction with G-actin, but devoid of F-actin fragmenting activity

Villin is an F-actin binding protein located in the microfilament bundle of intestinal epithelial cell microvilli. Extensive in vitro proteolysis with Staphylococcus aureus V8 protease results in the production of a stable domain (apparent Mr 44000) which can be isolated due to its Ca2+-dependent interaction with G-actin bound to immobilized DNase-I, the standard procedure for the purification of villin. This 44-kDa fragment retains a single Ca2+ binding site with an apparent Kd = 2 X 10(-6) M, binds to G-actin, and inhibits the rate of actin polymerization. However, the 44-kDa domain does not shown any Ca2+-activated severing activity nor does it compete with villin for F-actin binding. These results suggest that villin contains three domains: headpiece containing an F-actin binding site, 44-kDa fragment containing a G-actin binding site, and an amino-terminal fragment responsible for the Ca2+-dependent severing activity.     

Human lymphocyte-specific protein 1, the protein overexpressed in neutrophil actin dysfunction with 47-kDa and 89-kDa protein abnormalities (NAD 47/89), has multiple F-actin binding domains

Human lymphocyte-specific protein 1 (LSP1) is an F-actin binding protein, which has an acidic N-terminal half and a basic C-terminal half. In the basic C-terminal half, there are amino acid sequences highly homologous to the actin-binding domains of two known F-actin binding proteins: caldesmon and the villin headpieces (CI, CII, VI, VII). However, the exact numbers and locations of the F-actin binding domains within LSP1 are not clearly defined. In this report, we utilized 125I-labeled F-actin ligand blotting and high-speed F-actin cosedimentation assays to analyze the F-actin binding properties of truncated LSP1 peptides and to define the F-actin binding domains. Results show that LSP1 has at least three and potentially a fourth F-actin binding domain. All F-actin binding domains are located in the basic C-terminal half and correspond to the caldesmon and villin headpiece homologous regions. LSP1 181-245 and LSP1 246-295, containing sequences homologous to caldesmon F-actin binding site I and II, respectively (CI, CII), binds F-actin; similarly, LSP1 306-339 can bind F-actin and contains two inseparable villin headpiece-like F-actin binding domains (VI, VII). Although LSP1 1-305, which does not contain VI and VII regions, retains F-actin binding activity, its binding affinity for F-actin is much weaker than that of full-length LSP1. Site-directed mutagenesis of the basic amino acids in the KRYK (VI) or KYEK (VII) sequences to acidic amino acids create mutants that bind F-actin with lower affinity than full-length wild-type LSP1. High KCl concentrations decrease full-length LSP1 binding to F-actin, suggesting the affinity between LSP1 and F-actin is mainly through electrostatic interaction.     

Mapping the functional surface of domain 2 in the gelsolin superfamily

The crystal structure of the F-actin binding domain 2 of severin, the gelsolin homologue from Dictyostelium discoideum, has been determined by multiple isomorphous replacement and refined to 1.75 A resolution. The structure reveals an alpha-helix-beta-sheet sandwich similar to the domains of gelsolin and villin, and contains two cation-binding sites, as observed in other domain 1 and domain 2 homologues. Comparison of the structures of several gelsolin family domains has identified residues that may mediate F-actin binding in gelsolin domain 2 homologues. To assess the involvement of these residues in F-actin binding, three mutants of human gelsolin domain 2 were assayed for F-actin binding activity and thermodynamic stability. Two of the mutants, RRV168AAA and RLK210AAA, demonstrated a lowered affinity for F-actin, indicating a role for those residues in filament binding. Using both structural and biochemical data, we have constructed a model of the gelsolin domain 1-domain 2-F-actin complex. This model highlights a number of interactions that may serve as positive and negative determinants of filament end- and side-binding.     

Competition between intradomain and interdomain interactions: a buried salt bridge is essential for villin headpiece folding and actin binding

Villin-type headpiece domains are ～70 residue motifs that reside at the C-terminus of a variety of actin-associated proteins. Villin headpiece (HP67) is a commonly used model system for both experimental and computational studies of protein folding. HP67 is made up of two subdomains that form a tightly packed interface. The isolated C-terminal subdomain of HP67 (HP35) is one of the smallest autonomously folding proteins known. The N-terminal subdomain requires the presence of the C-terminal subdomain to fold. In the structure of HP67, a conserved salt bridge connects N- and C-terminal subdomains. This buried salt bridge between residues E39 and K70 is unusual in a small protein domain. We used mutational analysis, monitored by CD and NMR, and functional assays to determine the role of this buried salt bridge. First, the two residues in the salt bridge were replaced with strictly hydrophobic amino acids, E39M/K70M. Second, the two residues in the salt bridge were swapped, E39K/K70E. Any change from the wild-type salt bridge residues results in unfolding of the N-terminal subdomain, even when the mutations were made in a stabilized variant of HP67. The C-terminal subdomain remains folded in all mutants and is stabilized by some of the mutations. Using actin sedimentation assays, we find that a folded N-terminal domain is essential for specific actin binding. Therefore, the buried salt bridge is required for the specific folding of the N-terminal domain which confers actin-binding activity to villin-type headpiece domains, even though the residues required for this specific interaction destabilize the C-terminal subdomain.     

Association of villin with phosphatidylinositol 4,5-bisphosphate regulates the actin cytoskeleton

Villin, an epithelial cell actin-binding protein, severs actin in vitro and in vivo. Previous studies report that phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulates actin severing by villin, presumably by interaction with villin. However, direct association of villin with PIP(2) has never been characterized. In this report, we presented mutational analysis to identify the PIP(2)-binding sites in villin. Villin (human) binds PIP(2) with a K(d) of 39.5 microm, a stoichiometry of 3.3, and a Hill coefficient of 1. We generated deletion mutants of villin lacking putative PIP(2)-binding sites and examined the impact of these mutations on PIP(2) binding and actin dynamics. Our analysis revealed the presence of three PIP(2)-binding sites, two in the amino-terminal core and one in the carboxyl-terminal headpiece of human villin. Synthetic peptides analogous with these sites confirmed the binding domains. Circular dichroism and quenching of intrinsic tryptophan fluorescence revealed a significant conformational change in these peptides ensuing in their association with PIP(2). By using site-directed mutagenesis (arginine 138 to alanine), we demonstrated the presence of an identical F-actin and PIP(2)-binding site in the capping and severing domain of villin. In contrast, the mutants lysine 822 and 824 to alanine demonstrated the presence of an overlapping F-actin and PIP(2)-binding site in the actin cross-linking domain of villin. Consistent with this observation, association of villin with PIP(2) inhibited the actin capping and severing functions of villin and enhanced the actin bundling function of villin. Our studies revealed that structural changes induced by association with PIP(2) could regulate the actin-modifying functions of villin. This study provided biochemical proof of the functional significance of villin association with PIP(2) and identified the molecular mechanisms involved in the regulation of actin dynamics by villin and PIP(2).     

The Allosteric Mechanism Induced by Protein Kinase A (PKA) Phosphorylation of Dematin (Band 4.9)

Dematin (band 4.9) is an F-actin binding and bundling protein best known for its role within red blood cells, where it both stabilizes as well as attaches the spectrin/actin cytoskeleton to the erythrocytic membrane. Here, we investigate the structural consequences of phosphorylating serine 381, a covalent modification that turns off F-actin bundling activity. In contrast to the canonical doctrine, in which phosphorylation of an intrinsically disordered region/protein confers affinity for another domain/protein, we found the converse to be true of dematin: phosphorylation of the well folded C-terminal villin-type headpiece confers affinity for its intrinsically disordered N-terminal core domain. We employed analytical ultracentrifugation to demonstrate that dematin is monomeric, in contrast to the prevailing view that it is trimeric. Next, using a series of truncation mutants, we verified that dematin has two F-actin binding sites, one in the core domain and the other in the headpiece domain. Although the phosphorylation-mimicking mutant, S381E, was incapable of bundling microfilaments, it retains the ability to bind F-actin. We found that a phosphorylation-mimicking mutant, S381E, eliminated the ability to bundle, but not bind F-actin filaments. Lastly, we show that the S381E point mutant caused the headpiece domain to associate with the core domain, leading us to the mechanism for cAMP-dependent kinase control of dematin''s F-actin bundling activity: when unphosphorylated, dematin''s two F-actin binding domains move independent of one another permitting them to bind different F-actin filaments. Phosphorylation causes these two domains to associate, forming a compact structure, and sterically eliminating one of these F-actin binding sites.     

Hair-forming activity of human lymphocyte specific protein 1 requires cooperation between its caldesmon-like domains and the villin headpiece-like domains.

LSP1 is an F-actin binding with multiple F-actin binding domains. Overexpression of LSP1 in NAD 47/89 patient's neutrophils created hair-like projections on the patient's neutrophil cell surfaces and inhibited neutrophil cell motility and transfection of LSP1 in serial cell lines recreate the NAD 47/89 phenotype and produce branching hair-like surface projections. Although LSP1 contains hair-forming ability and LSP1 F-actin binding domains have been defined, the LSP1 domains responsible for its hair-forming activity, the relationship to the F-actin binding domains, and the required domain interactions, if any, for hair formation are not well understood. To define the hair-forming domains of LSP1, the relationship to the known F-actin binding domains, and binding domain interactions, LSP1 truncates, which include or exclude the different F-actin binding domains, were created by PCR. LSP1 mutants were created by site-directed mutagenesis to define the amino acids important for hair formation. Sf9 cells were infected with recombinant baculovirus expressing the cDNA of LSP1 truncates and mutants, and the morphology of infected Sf9 cells was documented by DIC optics. Results show that (1) the hair-forming activity of LSP1 is localized to the basic C-terminal half of the molecule, which contains all of the F-actin binding domains; (2) both the caldesmon-like domains and the villin headpiece-like domains are required for the hair-forming activity of LSP1; (3) basic amino acids in the villin headpiece regions are crucial for the hair-forming activity of LSP1 molecule. The results suggest cooperation between the caldesmon-like domains and the villin headpiece-like domains are required for the hair-forming activity of human LSP1 in cells.     

A phage display-based method for determination of relative affinities of mutants. Application of the actin-binding motifs in thymosin beta 4 and the villin headpiece

We propose phage display combined with enzyme-linked immunosorbent assay as a tool for the systematic analysis of protein-protein interactions by investigating the binding behavior of variants to a partner protein. Via enzyme-linked immunosorbent assay we determine both the amount of fusion protein presented at the phage surface and the amount of complex formed, the ratio of which is proportional to the affinity. Hence this method enables us to calculate the relative affinities of a large number of mutants. As model systems, we investigated actin-binding motifs conserved in a number of proteins binding monomeric or filamentous actin. The hexapeptide motifs LKKTET, present in thymosin beta4, and LKKEKG, present in the villin headpiece, were mutated, and the variants were analyzed. Study of the positional tolerance allows postulating that the motifs, although similar in primary structures adopt different conformations when bound to actin. In addition, our data show that the second and the fourth amino acid of the thymosin beta4 motif and the first three residues of the villin headpiece motif are most important for actin binding. The latter result challenges the charged crown hypothesis for the villin headpiece filamentous actin interaction.     

Dimerization and actin-bundling properties of villin and its role in the assembly of epithelial cell brush borders

Villin is a major actin-bundling protein in the brush border of epithelial cells. In this study we demonstrate for the first time that villin can bundle actin filaments using a single F-actin binding site, because it has the ability to self-associate. Using fluorescence resonance energy transfer, we demonstrate villin self-association in living cells in microvilli and in growth factor-stimulated cells in membrane ruffles and lamellipodia. Using sucrose density gradient, size-exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight, the majority of villin was identified as a monomer or dimer. Villin dimers were also identified in Caco-2 cells, which endogenously express villin and Madin-Darby canine kidney cells that ectopically express villin. Using truncation mutants of villin, site-directed mutagenesis, and fluorescence resonance energy transfer, an amino-terminal dimerization site was identified that regulated villin self-association in parallel conformation as well as actin bundling by villin. This detailed analysis describes for the first time microvillus assembly by villin, redefines the actin-bundling function of villin, and provides a molecular mechanism for actin bundling by villin, which could have wider implications for other actin cross-linking proteins that share a villin-like headpiece domain. Our study also provides a molecular basis to separate the morphologically distinct actin-severing and actin-bundling properties of villin.     

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

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.     

Rational design, structural and thermodynamic characterization of a hyperstable variant of the villin headpiece helical subdomain

A hyperstable variant of the small independently folded helical subdomain (HP36) derived from the F-actin binding villin headpiece was designed by targeting surface electrostatic interactions and helical propensity. A double mutant N68A, K70M was significantly more stable than wild type. The Tm of wild type in aqueous buffer is 73.0 degrees C, whereas the double mutant did not display a complete unfolding transition. The double mutant could not be completely unfolded even by 10 M urea. In 3 M urea, the Tm of wild type is 54.8 degrees C while that of the N68AK70M double mutant is 73.9 degrees C. Amide H/2H exchange studies show that the pattern of exchange is very similar for wild type and the double mutant. The structures of a K70M single mutant and the double mutant were determined by X-ray crystallography and are identical to that of the wild type. Analytical ultracentrifugation demonstrates that the proteins are monomeric. The hyperstable mutant described here is expected to be useful for folding studies of HP36 because studies of the wild type domain have sometimes been limited by its marginal stability. The results provide direct evidence that naturally occurring miniature protein domains have not been evolutionarily optimized for global stability. The stabilizing effect of this double mutant could not be predicted by sequence analysis because K70 is conserved in the larger intact headpiece for functional reasons.     

Identification of a functional switch for actin severing by cytoskeletal proteins

Actin severing is vital for the organization of the actin cytoskeleton during cell motility. Severing of F-actin by the homologous proteins villin and gelsolin requires unphysiologically high calcium concentrations (20-200 microM). Here we demonstrate that high calcium releases an autoinhibited conformation in villin that is maintained by two low affinity calcium binding sites (aspartic acids 467 and 715) that interact with a cluster of basic residues in the S2 domain of villin. Mutation of either of these sites as well as tyrosine phosphorylation alters the conformation of villin resulting in a protein that can sever actin in nanomolar calcium. These results suggest that tyrosine phosphorylation rather than high calcium may be the mechanism by which villin and other related proteins sever actin in vivo.     

Arabidopsis VILLIN5, an Actin Filament Bundling and Severing Protein, Is Necessary for Normal Pollen Tube Growth

A dynamic actin cytoskeleton is essential for pollen germination and tube growth. However, the molecular mechanisms underlying the organization and turnover of the actin cytoskeleton in pollen remain poorly understood. Villin plays a key role in the formation of higher-order structures from actin filaments and in the regulation of actin dynamics in eukaryotic cells. It belongs to the villin/gelsolin/fragmin superfamily of actin binding proteins and is composed of six gelsolin-homology domains at its core and a villin headpiece domain at its C terminus. Recently, several villin family members from plants have been shown to sever, cap, and bundle actin filaments in vitro. Here, we characterized a villin isovariant, Arabidopsis thaliana VILLIN5 (VLN5), that is highly and preferentially expressed in pollen. VLN5 loss-of-function retarded pollen tube growth and sensitized actin filaments in pollen grains and tubes to latrunculin B. In vitro biochemical analyses revealed that VLN5 is a typical member of the villin family and retains a full suite of activities, including barbed-end capping, filament bundling, and calcium-dependent severing. The severing activity was confirmed with time-lapse evanescent wave microscopy of individual actin filaments in vitro. We propose that VLN5 is a major regulator of actin filament stability and turnover that functions in concert with oscillatory calcium gradients in pollen and therefore plays an integral role in pollen germination and tube growth.     

Crystal structure of a pH-stabilized mutant of villin headpiece

Villin-type headpiece domains are compact F-actin-binding motifs that have been used extensively as a model system to investigate protein folding by both experimental and computational methods. Villin headpiece (HP67) harbors a highly helical, thermostable, and autonomously folding subdomain in the C terminus (HP35), and because of this feature, HP67 is usually considered to be composed of a N- and C-terminal subdomain. Unlike the C-terminal subdomain, the N-terminal subdomain consists mainly of loops and turns, and the folding is dependent upon the presence of the C-terminal subdomain. The pH sensitivity of this subdomain is thought to arise from, at least partially, protonation of H41 buried in the hydrophobic core. Substitution of this histidine with tyrosine, another permissive residue at this position for naturally occurring sequences, increases not only the pH stability of HP67 but also the thermal stability and the cooperativity of thermal unfolding over a wide pH range (0.9-7.5). The crystal structures of wild-type HP67 and the H41Y mutant, determined under the same conditions, indicate that the H41Y substitution causes only localized rearrangement around the mutated residue. The F-actin-binding motif remains essentially the same after the mutation, accounting for the negligible effect of the mutation on F-actin affinity. The hydrogen bond formed between the imidazole ring of H41 and the backbone carbonyl of E14 of HP67 is eliminated by the H41Y mutation, which renders the extreme N terminus of H41Y more mobile; the hydrogen bond formed between the imidazole ring of H41 and the backbone nitrogen of D34 is replaced with that between the hydroxyl group of Y41 and the backbone nitrogen of D34 after the H41Y substitution. The increased hydrophobicity of tyrosine compensates for the loss of hydrogen bonds in the extreme N terminus and accounts for the increased stability and cooperativity of the H41Y mutant.     

Dematin exhibits a natively unfolded core domain and an independently folded headpiece domain

Dematin is an actin‐binding protein originally identified in the junctional complex of the erythrocyte plasma membrane, and is present in many nonerythroid cells. Dematin headpiece knockout mice display a spherical red cell phenotype and develop a compensated anemia. Dematin has two domains: a 315‐residue, proline‐rich “core” domain and a 68‐residue carboxyl‐terminal villin‐type “headpiece” domain. Expression of full‐length dematin in E. coli as a GST recombinant protein results in truncation within a proline, glutamic acid, serine, threonine rich region (PEST). Therefore, we designed a mutant construct that replaces the PEST sequence. The modified dematin has high actin binding activity as determined by actin sedimentation assays. Negative stain electron microscopy demonstrates that the modified dematin also exhibits actin bundling activity like that of native dematin. Circular dichroism (CD) and NMR spectral analysis, however, show little secondary structure in the modified dematin. The lack of secondary structure is also observed in native dematin purified from human red blood cells. ¹⁵N‐HSQC NMR spectra of modified dematin indicate that the headpiece domain is fully folded whereas the core region is primarily unfolded. Our finding suggests that the core is natively unfolded and may serve as a scaffold to organize the components of the junctional complex.