Mapping the cofilin binding site on yeast G-actin by chemical cross-linking

Cofilin is a major cytoskeletal protein that binds to both monomeric actin (G-actin) and polymeric actin (F-actin) and is involved in microfilament dynamics. Although an atomic structure of the G-actin-cofilin complex does not exist, models of the complex have been built using molecular dynamics simulations, structural homology considerations, and synchrotron radiolytic footprinting data. The hydrophobic cleft between actin subdomains 1 and 3 and, alternatively, the cleft between actin subdomains 1 and 2 have been proposed as possible high-affinity cofilin binding sites. In this study, the proposed binding of cofilin to the subdomain 1/subdomain 3 region on G-actin has been probed using site-directed mutagenesis, fluorescence labeling, and chemical cross-linking, with yeast actin mutants containing single reactive cysteines in the actin hydrophobic cleft and with cofilin mutants carrying reactive cysteines in the regions predicted to bind to G-actin. Mass spectrometry analysis of the cross-linked complex revealed that cysteine 345 in subdomain 1 of mutant G-actin was cross-linked to native cysteine 62 on cofilin. A cofilin mutant that carried a cysteine substitution in the α3-helix (residue 95) formed a cross-link with residue 144 in actin subdomain 3. Distance constraints imposed by these cross-links provide experimental evidence for cofilin binding between actin subdomains 1 and 3 and fit a corresponding docking-based structure of the complex. The cross-linking of the N-terminal region of recombinant yeast cofilin to actin residues 346 and 374 with dithio-bis-maleimidoethane (12.4 Å) and via disulfide bond formation was also documented. This set of cross-linking data confirms the important role of the N-terminal segment of cofilin in interactions with G-actin.     

Cofilin, actin and their complex observed in vivo using fluorescence resonance energy transfer

Actin is the principal component of microfilaments. Its assembly/disassembly is essential for cell motility, cytokinesis, and a range of other functions. Recent evidence suggests that actin is present in the nucleus where it may be involved in the regulation of gene expression and that cofilin binds actin and can translocate into the nucleus during times of stress. In this report, we combine fluorescence resonance energy transfer and confocal microscopy to analyze the interactions of cofilin and G-actin within the nucleus and cytoplasm. By measuring the rate of photobleaching of fluorescein-labeled actin in the presence and absence of Cy5-labeled cofilin, we determined that almost all G-actin in the nucleus is bound to cofilin, whereas approximately (1/2) is bound in the cytoplasm. Using fluorescence resonance energy transfer imaging techniques we observed that a significant proportion of fluorescein-labeled cofilin in both the nucleus and cytoplasm binds added tetramethylrhodamine-labeled G-actin. Our data suggest there is significantly more cofilin-G-actin complex and less free cofilin in the nucleus than in the cytoplasm.     

Structural reorganization of the transferrin C-lobe and transferrin receptor upon complex formation: the C-lobe binds to the receptor helical domain

Human transferrin, a bilobal protein, with each lobe bearing a single iron-binding site, functions to transport iron into cells. While the N-terminal lobe alone does not measurably bind cellular transferrin receptors or serve as an iron donor for cells, the C-lobe is capable of both functions. We used hydroxyl radical-mediated protein footprinting and mass spectrometry to reveal the conformational changes that occur upon complex formation for the human transferrin C-lobe (residues 334-679) bound to the ectodomain of human transferrin receptor 1 (residues 121-760). Oxidation rates for proteolytic peptides in the C-lobe, the receptor, and their complex have been measured by mass spectrometry; upon formation of the complex, a dramatic decrease in modification rates, indicating protection of specific side chain groups, can be seen in C-lobe sequences corresponding to residues 381-401, 415-433, and 457-470. Peptide sequences experiencing modification rate decreases in the transferrin receptor upon C-lobe binding include residues 232-240, 365-371, 496-508, 580 and 581, 614-623, 634-646, 647-681, and 733-760. In addition, several peptides in the receptor exhibit enhancements in the rate of modification consistent with allosteric effects of complex formation. Using tandem mass spectrometry, the sites of modification with altered reactivity in the complex include Met382, Met389, Trp460, Met464, and Phe427 in the C-lobe and Tyr503, Pro581, Tyr611, Leu619, Met635, Phe650, Trp740, Trp754, and Phe760 within the transferrin receptor. Using available genetic, biochemical, and structural data, we confirm that the conserved RGD sequence (residues 646-648) in the helical domain of the transferrin receptor, including residues from Leu619 to Phe650, is a primary binding site for the transferrin C-lobe.     

Mapping the ADF/cofilin binding site on monomeric actin by competitive cross-linking and peptide array: evidence for a second binding site on monomeric actin

The binding sites for actin depolymerising factor (ADF) and cofilin on G-actin have been mapped by competitive chemical cross-linking using deoxyribonuclease I (DNase I), gelsolin segment 1 (G1), thymosin beta4 (Tbeta4), and vitamin D-binding protein (DbP). To reduce ADF/cofilin induced actin oligomerisation we used ADP-ribosylated actin. Both vitamin D-binding protein and thymosin beta4 inhibit binding by ADF or cofilin, while cofilin or ADF and DNase I bind simultaneously. Competition was observed between ADF or cofilin and G1, supporting the hypothesis that cofilin preferentially binds in the cleft between sub-domains 1 and 3, similar to or overlapping the binding site of G1. Because the affinity of G1 is much higher than that of ADF or cofilin, even at a 20-fold excess of the latter, the complexes contained predominantly G1. Nevertheless, cross-linking studies using actin:G1 complexes and ADF or cofilin showed the presence of low concentrations of ternary complexes containing both ADF or cofilin and G1. Thus, even with monomeric actin, it is shown for the first time that binding sites for both G1 and ADF or cofilin can be occupied simultaneously, confirming the existence of two separate binding sites. Employing a peptide array with overlapping sequences of actin overlaid by cofilin, we have identified five sequence stretches of actin able to bind cofilin. These sequences are located within the regions of F-actin predicted to bind cofilin in the model derived from image reconstructions of electron microscopical images of cofilin-decorated filaments. Three of the peptides map to the cleft region between sub-domains 1 and 3 of the upper actin along the two-start long-pitch helix, while the other two are in the DNase I loop corresponding to the site of the lower actin in the helix. In the absence of any crystal structures of ADF or cofilin in complex with actin, these studies provide further information about the binding sites on F-actin for these important actin regulatory proteins.     

The reactivity and function of thiol groups in trout actin

1. Considerable differences were found between the rates and degrees of modification of native trout actin with iodo[2-(14)C]acetate and iodo[1-(14)C]acetamide. 2. With iodoacetate, G- and F-actin were both labelled in the N-terminal peptide only. This modification had little effect on the ability of the actin to polymerize. 3. Iodoacetamide labelled three cysteine residues in both G- and F-actin. The modified cysteine residues were identified from the position of the corresponding tryptic peptides on peptide ;maps'. 4. The modification had little effect on the ability of G-actin to polymerize, to bind ATP or to bind Ca(2+), or on the ability of F-actin to depolymerize. 5. It is concluded that the three cysteine residues present on the ;surface' of the native trout actin molecule have no direct role in the polymerization processes, the binding of ATP, or the binding of Ca(2+).     

Antibody and peptide probes of interactions between the SH1-SH2 region of myosin subfragment 1 and actin's N-terminus.

The negatively charged residues in the N-terminus of actin and the 697-707 region on myosin subfragment 1 (S-1), containing the reactive cysteines SH1 and SH2, are known to be important for actin-activated myosin ATPase activity. The relationship between these two sites was first examined by monitoring the rates of SH1 and SH2 modification with N-ethylmaleimide in the presence of actin and, secondly, by testing for direct binding of SH1 peptides to the N-terminal segment on actin. While actin alone protected SH1 from N-ethylmaleimide modification, this effect was abolished by an antibody against the seven N-terminal amino acids on actin, F(ab)(1-7), and was greatly reduced when the charge of acidic residues at actin's N-terminus was altered by carbodiimide coupling of ethylenediamine. Neither F(ab)(1-7) nor ethylenediamine treatment reversed the effect of F-actin on SH2 reactivity in SH1-modified S-1. These results show a communication between the SH1 region on S-1 and actin's N-terminus in the acto-S-1 complex. To test whether such a communication involves the binding of the SH1 site on S-1 to the N-terminal segment of actin, the SH1 peptide IRICRKG-NH2(4+) was used. Cosedimentation experiments revealed the binding of three to six peptides per actin monomer. Peptide binding to actin was affected slightly, if at all, by F(ab)(1-7). The antibody also did not change the polymerization of G-actin by the peptides. The peptides caused a small reduction in the binding of S-1 to actin and did not change the binding of F(ab)(1-7).(ABSTRACT TRUNCATED AT 250 WORDS)     

The second ADF/cofilin actin-binding site exists in F-actin, the cofilin-G-actin complex, but not in G-actin.

ADF/cofilins are actin binding proteins that bind actin close to both the N- and C-termini (site 1), and we have found a second cofilin binding site (site 2) centered around helix 112-125 [Renoult, C., Ternent, D., Maciver, S.K., Fattoum, A., Astier, C., Benyamin, Y. & Roustan, C. (1999) J. Biol. Chem. 274, 28893-28899]. We proposed a model in which ADF/cofilin intercalated between subdomains 1 and 2 of two longitudinally associated actin monomers within the actin:cofilin cofilament, explaining the change in twist that ADF/cofilins induce in the filament [McGough, A. Pope, B., Chiu, W. & Weeds, A. (1998) J. Cell Biol. 138, 771-781]. Here, we have determined the fuller extent of the cofilin footprint on site 1 of actin. Site 1 is primarily the G-actin binding site. Experiments with both peptide mimetics and fluorescently labeled cofilin suggest that site 2 only becomes available for cofilin binding within the filament, possibly due to motion between subdomains 1 and 2 within an actin monomer. We have detected motion between subdomains 1 and 2 of G-actin by FRET induced by cofilin, to reveal the second cofilin-binding site. This motion may also explain how cofilins inhibit the nucleotide exchange of actin, and why the actin:cofilin complex is polymerizable without dissociation.     

Structural effects of cofilin on longitudinal contacts in F-actin

Structural effects of yeast cofilin on skeletal muscle and yeast actin were examined in solution. Cofilin binding to native actin was non-cooperative and saturated at a 1:1 molar ratio, with K(d)相似文献   

A structural basis for the pH-dependence of cofilin. F-actin interactions.

A marked pH-dependent interaction with F-actin is an important property of typical members of the actin depolymerizing factor (ADF)/cofilin family of abundant actin-binding proteins. ADF/cofilins tend to bind to F-actin with a ratio of 1 : 1 at pH values around 6.5, and to G-actin at pH 8.0. We have investigated the mechanism for the pH-sensitivity. We found no evidence for pH-dependent changes in the structure of cofilin itself, nor for the interaction of cofilin with G-actin. None of the actin-derived, cofilin-binding peptides that we had previously identified [Renoult, C., Ternent, D., Maciver, S.K., Fattoum, A., Astier, C., Benyamin, Y. & Roustan, C. (1999) J. Biol. Chem. 274, 28893-28899] bound cofilin in a pH-sensitive manner. However, we have detected a conformational change in region 75-105 in the actin subdomain 1 by the use of a peptide-directed antibody. A pH-dependent conformational change has also been detected spectroscopically in a similar peptide (84-103) on binding to cofilin. These results are consistent with a model in which pH-dependent motion of subdomain 1 relative to subdomain 2 (through region 75-105) of actin reveals a second cofilin binding site on actin (centered around region 112-125) that allows ADF/cofilin association with the actin filament. This motion requires salt in addition to low pH.     

Rapid formation and high diffusibility of actin-cofilin cofilaments at low pH.

Cofilin is a small actin-binding protein that is known to bind both F-actin and G-actin, severing the former. The interaction of cofilin with actin is pH-sensitive, F-actin being preferentially bound at low pH and G-actin at higher pH, within the physiological range. Diffusion coefficients of F-actin with cofilin were measured by the fluorescence recovery after photobleaching (FRAP) technique. This has the potential for simultaneous and direct measurement of average polymer length via the average diffusion coefficient of the polymers (DLM) as well as the fraction of polymerized actin, fLM, present in solution. In the range of cofilin-actin ratios up to 1 : 1 and at both pH 6.5 and pH 8.0, the diffusion coefficients of the polymers increased with the amount of cofilin present in the complex, in a co-operative manner to a plateau. We interpret this as indicating co-operative binding/severing and that filaments less than a certain length cannot be severed further. Under the conditions used here, filaments were found to be more motile at pH 6.5 than at pH 8.0. At pH 8.0, some actin is expected to be sequestered as ADP-actin-cofilin complexes, with the remaining actin being present as long slowly diffusing filaments. At pH 6.5, however, cofilin binds to F-actin to form short rapidly diffusing cofilaments. These filaments form very rapidly from cofilin-actin monomeric complexes, possibly indicating that this complex is able to polymerize without dissociation. These findings may be relevant to the nuclear import of actin-cofilin complexes.     

Mapping the interaction of cofilin with subdomain 2 on actin

Cofilin, a member of the actin-depolymerizing factor (ADF)/cofilin family of proteins, is a key regulator of actin dynamics. Cofilin binds to monomer (G-) and filamentous (F-) actin, severs the filaments, and increases their turnover rate. Electron microscopy studies suggested cofilin interactions with subdomains 2 and 1/3 on adjacent actin protomers in F-actin. To probe for the presence of a cryptic cofilin binding site in subdomain 2 in G-actin, we used transglutaminase-mediated cross-linking, which targets Gln41 in subdomain 2. The cross-linking proceeded with up to 85% efficiency with skeletal alpha-actin and WT yeast actin, yielding a single product corresponding to a 1:1 actin-cofilin complex but was strongly inhibited in Q41C yeast actin (in which Q41 was substituted with cysteine). LC-MS/MS analysis of the proteolytic fragments of this complex mapped the cross-linking to Gln41 on actin and Gly1 on recombinant yeast cofilin. The actin-cofilin (AC) heterodimer was purified on FPLC for analytical ultracentrifugation and electron microscopy analysis. Sedimentation equilibrium and velocity runs revealed oligomers of AC in G-actin buffer. In the presence of excess cofilin, the covalent AC heterodimer bound a second cofilin, forming a 2:1 cofilin/actin complex, as revealed by sedimentation results. Under polymerizing conditions the cross-linked AC formed mostly short filaments, which according to image reconstruction were similar to uncross-linked actin-cofilin filaments. Although a majority of the cross-linking occurs at Gln41, a small fraction of the AC cross-linked complex forms in the Q41C yeast actin mutant. This secondary cross-linking site was sequenced by MALDI-MS/MS as linking Gln360 in actin to Lys98 on cofilin. Overall, these results demonstrate that the region around Gln41 (subdomain 2) is involved in a weak binding of cofilin to G-actin.     

The two ADF-H domains of twinfilin play functionally distinct roles in interactions with actin monomers

Twinfilin is a ubiquitous and abundant actin monomer-binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (K(D) = 0.05 microM) than ATP-G-actin (K(D) = 0.47 microM) under physiological ionic conditions and forms a relatively stable (k(off) = 1.8 s(-1)) complex with ADP-G-actin. Data from native PAGE and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations, twinfilin, cofilin, and actin may also form a ternary complex. By systematic deletion analysis, we show that the actin-binding activity is located entirely in the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.     

An actin-interacting heptapeptide in the cofilin sequence

Cofilin, a 21-kDa actin-binding protein, has a hexapeptide sequence DAIKKK which is identical to the N-terminal portion (residues 2-7) of tropomyosin. The synthetic heptapeptide, DAIKKKL, corresponding to residues 122-128 of cofilin, inhibited the binding of cofilin to F-actin in a dose-dependent manner. The heptapeptide cosedimented with F-actin, decreased the fluorescence intensity of pyrene-labeled F-actin, and increased the rate of polymerization of G-actin. The hexapeptides, DIKKKL and DAIKKL, also inhibited the binding of cofilin to F-actin and affected the fluorescence intensity of pyrene-labeled F-actin and the rate of actin polymerization, like the heptapeptide. However, their effects were weaker than those of the heptapeptide. Moreover, the pentapeptide, DIKKL, had little or no effect. These results suggest that the heptapeptide sequence is specific for the interaction with actin and, therefore, may constitute part of the actin-binding domain of cofilin.     

Cofilin weakly interacts with 14-3-3 and therefore can only indirectly participate in regulation of cell motility by small heat shock protein HspB6 (Hsp20)

It has been previously reported that phosphorylated cofilin interacted with 14-3-3ζ protein to generate a sub-micromolar K(d) binary complex. Here we challenge this hypothesis by analyzing the direct association of recombinant cofilin with 14-3-3ζ using different in vitro biochemical methods. Phosphorylated cofilin at high concentration binds to 14-3-3 immobilized on nitrocellulose, however no complex formation was detected by means of native gel electrophoresis or chemical crosslinking. Intact dimeric or mutant monomeric 14-3-3 was unable to form stable complexes with phosphorylated or unphosphorylated cofilin detected by size-exclusion chromatography. In co-sedimentation assay 14-3-3 did not affect interaction of cofilin with F-actin. The data of native gel electrophoresis indicate that 14-3-3 did not affect interaction of cofilin with G-actin. Thus, cofilin only weakly interacts with 14-3-3 and therefore cannot directly compete with phosphorylated small heat shock protein HspB6 for its binding to 14-3-3. It is hypothesized that phosphorylated HspB6 might affect interaction of 14-3-3 with protein phosphatases (and/or protein kinases) involved in dephosphorylation (or phosphorylation) of cofilin and by this means regulate cofilin-dependent reorganization of cytoskeleton.     

The C-terminal tail of UNC-60B (actin depolymerizing factor/cofilin) is critical for maintaining its stable association with F-actin and is implicated in the second actin-binding site

Actin depolymerizing factor (ADF)/cofilin changes the twist of actin filaments by binding two longitudinally associated actin subunits. In the absence of an atomic model of the ADF/cofilin-F-actin complex, we have identified residues in ADF/cofilin that are essential for filament binding. Here, we have characterized the C-terminal tail of UNC-60B (a nematode ADF/cofilin isoform) as a novel determinant for its association with F-actin. Removal of the C-terminal isoleucine (Ile152) by carboxypeptidase A or truncation by mutagenesis eliminated F-actin binding activity but strongly enhanced actin depolymerizing activity. Replacement of Ile152 by Ala had a similar but less marked effect; F-actin binding was weakened and depolymerizing activity slightly enhanced. Truncation of both Arg151 and Ile152 or replacement of Arg151 with Ala also abolished F-actin binding and enhanced depolymerizing activity. Loss of F-actin binding in these mutants was accompanied by loss or greatly decreased severing activity. All of the variants of UNC-60B interacted with G-actin in an indistinguishable manner from wild type. Cryoelectron microscopy showed that UNC-60B changed the twist of F-actin to a similar extent to vertebrate ADF/cofilins. Helical reconstruction and structural modeling of UNC-60B-F-actin complex reveal how the C terminus of UNC-60B might be involved in one of the two actin-binding sites.     

Differences in the ionic interaction of actin with the motor domains of nonmuscle and muscle myosin II.

Changes in the actin-myosin interface are thought to play an important role in microfilament-linked cellular movements. In this study, we compared the actin binding properties of the motor domain of Dictyostelium discoideum (M765) and rabbit skeletal muscle myosin subfragment-1 (S1). The Dictyostelium motor domain resembles S1(A2) (S1 carrying the A2 light chain) in its interaction with G-actin. Similar to S1(A2), none of the Dictyostelium motor domain constructs induced G-actin polymerization. The affinity of monomeric actin (G-actin) was 20-fold lower for M765 than for S1(A2) but increasing the number of positive charges in the loop 2 region of the D. discoideum motor domain (residues 613-623) resulted in equivalent affinities of G-actin for M765 and for S1. Proteolytic cleavage and cross-linking approaches were used to show that M765, like S1, interacts via the loop 2 region with filamentous actin (F-actin). For both types of myosin, F-actin prevents trypsin cleavage in the loop 2 region and F-actin segment 1-28 can be cross-linked to loop 2 residues by a carbodiimide-induced reaction. In contrast with the S1, loop residues 559-565 of D. discoideum myosin was not cross-linked to F-actin, probably due to the lower number of positive charges. These results confirm the importance of the loop 2 region of myosin for the interaction with both G-actin and F-actin, regardless of the source of myosin. The differences observed in the way in which M765 and S1 interact with actin may be linked to more general differences in the structure of the actomyosin interface of muscle and nonmuscle myosins.     

Solution structure of human cofilin: actin binding, pH sensitivity, and relationship to actin-depolymerizing factor

Human actin-depolymerizing factor (ADF) and cofilin are pH-sensitive, actin-depolymerizing proteins. Although 72% identical in sequence, ADF has a much higher depolymerizing activity than cofilin at pH 8. To understand this, we solved the structure of human cofilin using nuclear magnetic resonance and compared it with human ADF. Important sequence differences between vertebrate ADF/cofilins were correlated with unique structural determinants in the F-actin-binding site to account for differences in biochemical activities of the two proteins. Cofilin has a short beta-strand at the C terminus, not found in ADF, which packs against strands beta3/beta4, changing the environment around Lys96, a residue essential for F-actin binding. A salt bridge involving His133 and Asp98 (Glu98 in ADF) may explain the pH sensitivity of human cofilin and ADF; these two residues are fully conserved in vertebrate ADF/cofilins. Chemical shift perturbations identified residues that (i) differ in their chemical environments between wild type cofilin and mutants S3D, which has greatly reduced G-actin binding, and K96Q, which does not bind F-actin; (ii) are affected when G-actin binds cofilin; and (iii) are affected by pH change from 6 to 8. Many residues affected by G-actin binding also show perturbation in the mutants or in response to pH. Our evidence suggests the involvement of residues 133-138 of strand beta5 in all of the activities examined. Because residues in beta5 are perturbed by mutations that affect both G-actin and F-actin binding, this strand forms a "boundary" or "bridge" between the proposed F- and G-actin-binding sites.     

Interactions between G-actin and myosin subfragment 1: immunochemical probing of the NH2-terminal segment on actin

The role of the N-terminal segment of actin in myosin-induced polymerization of G-actin was studied by using peptide antibodies directed against the first seven N-terminal residues of alpha-skeletal actin. Light scattering, fluorescence, and analytical ultracentrifugation experiments showed that the Fab fragments of these antibodies inhibited the polymerization of G-actin by myosin subfragment 1 (S-1) by inhibiting the binding of these proteins to each other. Fluorescence measurements using actin labeled with pyrenyliodoacetamide revealed that Fab inhibited the initial step in the binding of S-1 to G-actin. It is deduced from these results and from other literature data that the initial contact between G-actin and S-1 involves residues 1-7 on actin and residues 633-642 on the S-1 heavy chain. This interaction appears to be of major importance for the binding of S-1 and G-actin. The presence of additional myosin contact sites on G-actin was indicated by concentration-dependent recovery of S-1 binding to G-actin without displacement of Fab. The reduced Fab inhibition of S-1 binding to polymerizing and polymerized actin is consistent with the tightening of acto-S-1 binding at these sites or the creation of new sites upon formation of F-actin.     

Gln-41 is intermolecularly cross-linked to Lys-113 in F-actin by N-(4-azidobenzoyl)-putrescine.

The bifunctional reagent N-(4-azidobenzoyl)-putrescine was synthesized and covalently bound to rabbit skeletal muscle actin. The incorporation was mediated by guinea pig liver transglutaminase under conditions similar to those described by Takashi (1988, Biochemistry 27, 938-943); up to 0.5 M/M were incorporated into G-actin, whereas F-actin was refractory to incorporation. Peptide fractionation showed that at least 90% of the label was bound to Gln-41. The labeled G-actin was polymerized, and irradiation of the F-actin led to covalent intermolecular cross-linking. A cross-linked peptide complex was isolated from a tryptic digest of the cross-linked actin in which digestion was limited to arginine; sequence analysis as well as mass spectrometry indicated that the linked peptides contained residues 40-62 and residues 96-116, and that the actual cross-link was between Gln-41 and Lys-113. Thus the gamma-carboxyl group of Gln-41 must be within 10.7 A of the side chain (probably the amino group) of Lys-113 in an adjacent actin monomer. In the atomic model for F-actin proposed by Holmes et al. (1990, Nature 347, 44-49), the alpha-carbons of these residues in adjacent monomers along the two-start helices are sufficiently close to permit cross-linking of their side chains, and, pending atomic resolution of the side chains, the results presented here seem to support the proposed model.     

Identification of a cofilin-like actin-binding site on translationally controlled tumor protein (TCTP)

Translationally controlled tumor protein (TCTP) expression is suppressed during cancer cell reversion to a non-malignant phenotype. We identified a primary sequence of TCTP with homology to ADF/cofilin. We confirm that a synthetic peptide corresponding to this sequence binds specifically to actin and is displaced from actin by cofilin. TCTP peptide has higher affinity for G-actin than F-actin and does not block actin-filament depolymerization by cofilin. These results suggest that TCTP may channel active cofilin to F-actin, enhancing the cofilin-activity cycle in invasive tumor cells. Loss of TCTP may result in sequestration of active cofilin by a monomeric pool of actin.