Domain analysis of the actin-binding and actin-remodeling activities of drebrin

Drebrin is an actin-binding protein which is expressed at highly levels in neurons. When introduced into fibroblasts, it has been known to bind to F-actin and to cause remodeling of F-actin. Here, we performed a domain analysis of the actin-binding and actin-remodeling activities of drebrin. Various fragments of drebrin cDNA were fused with green fluorescent protein cDNA and introduced into Chinese hamster ovary cells. Association of the fusion protein with F-actin and remodeling of the F-actin were examined. We found that the central 85-amino-acid sequence (residues 233-317) was sufficient for the binding to and remodeling of F-actin. The binding activity of this fragment was relatively low compared with that of full-length drebrin, but all the types of abnormalities of F-actin that are observed with full-length drebrin were also observed with this fragment. When this sequence was further fragmented, the actin-binding activity was greatly reduced and the actin-remodeling activity disappeared. The actin-binding activity of the central region of drebrin was confirmed by a cosedimentation assay of chymotryptic fragments of drebrin with purified actin. These data indicate that the actin-binding domain and actin-remodeling domain are identical and that this domain is located at the central region of drebrin.     

Biochemical characterisation of the actin-binding properties of utrophin

Utrophin is a large ubiquitously expressed cytoskeletal protein that is important for maturation of vertebrate neuromuscular junctions. It is highly homologous to dystrophin, the protein defective in Duchenne and Becker muscular dystrophies. Utrophin binds to the actin cytoskeleton via an N-terminal actin-binding domain, which is related to the actin-binding domains of members of the spectrin superfamily of proteins. We have determined the actin-binding properties of this utrophin domain and investigated its binding site on F-actin. An F-actin cosedimentation assay confirmed that the domain binds more tightly to beta-F-actin than to alpha-F-actin and that the full-length utrophin domain binds more tightly to both actin isoforms than a truncated construct, lacking a characteristic utrophin N-terminal extension. Both domain constructs exist in solution as compact monomers and bind to actin as 1:1 complexes. Analysis of the products of partial proteolysis of the domain in the presence of F-actin showed that the N-terminal extension was protected by binding to actin. The actin isoform dependence of utrophin binding could reflect differences at the N-termini of the actin isoforms, thus localising the utrophin-binding site on actin. The involvement of the actin N-terminus in utrophin binding was also supported by competition binding assays using myosin subfragment S1, which also binds F-actin near its N-terminus. Cross-linking studies suggested that utrophin contacts two actin monomers in the actin filament as does myosin S1. These biochemical approaches complement our structural studies and facilitate characterisation of the actin-binding properties of the utrophin actin-binding domain.     

Evidence that a 27-residue sequence is the actin-binding site of ABP-120

Proteolysis experiments of ABP-120 from Dictyostelium discoideum have previously demonstrated that removal of residues 89-115 from a tryptic peptide which retains actin binding activity, abolishes actin binding (Bresnick, A. R., Warren, V., and Condeelis, J. (1990) J. Biol. Chem. 265, 9236-9240). Antibodies made against a synthetic peptide of this 27-amino acid sequence (27-mer) specifically immunoprecipitate native ABP-120 from Dictyostelium high speed supernatants, demonstrating that the 27-mer sequence is on the surface of the molecule as expected for an active site. ABP-120 is inhibited in its binding to F-actin by Fab' fragments of the anti-27-mer IgG. Half-maximal inhibition occurs at an approximate molar ratio of 7 Fab' fragments/ABP-120 monomer. Viscoelastic measurements indicate that ABP-120 forms fewer cross-links with F-actin in the presence of the 27-mer synthetic peptide than in its absence. In F-actin cosedimentation assays, the binding of ABP-120 to actin is inhibited by the 27-mer synthetic peptide. Furthermore, the 27-mer synthetic peptide cosediments with F-actin, whereas a control hydrophobic peptide and a synthetic peptide of residues 69-88 of ABP-120 do not cosediment with F-actin. These observations suggest a direct involvement of the 27-mer sequence in the actin binding activity of ABP-120.     

Actin surface structure revealed by antibody imprints: evaluation of phage-display analysis of anti-actin antibodies

Phage-display peptide library analysis of an anti-F actin polyclonal antibody identified 12 amino acid residues of actin that appear, in its X-ray crystal structure, to be grouped together in a surface accessible conformational epitope. Phage epitope mapping was carried out by isolating immune complexes containing members of the J404 nonapeptide phage-display library formed in diluted antiserum and isolated on a protein A affinity matrix. Immunoreactive clones were grown as plaques, replica plated onto nitrocellulose, and labeled with anti-actin immune serum. One hundred and forty-four positively staining clones identified in this way were sequenced. Of these, 54 displayed peptides with sequence similarities. When the most abundantly selected sequence, KQTWQQLWD, was produced as a synthetic peptide and derivatized to ovalbumin, the complex was strongly recognized by the antiserum on Western blots and inhibited the binding of the antibody to immobilized F-actin by 60%. A scrambled version of this sequence WQDK WLQTQ, when coupled to ovalbumin, was not recognized by the antiserum and minimally inhibited binding of antiserum to immobilized F-actin by 10%. KQTWQQLWD contained four residues that corresponded, in frame, to a highly conserved six residue region of the chicken beta-actin sequence 351TFQQMW356 (identical residues are shown in bold). Examination of the rabbit skeletal muscle X-ray crystal structure suggested that within a 15 A radius of W356, nine additional residues were arranged on the actin surface in such a way that they could be mimicked by several of the selected phage sequences with root-mean-square deviation fits of 2.1-2.5 A. We conclude that phage-display analysis can provide information about the relative location of amino acids on the surfaces of proteins using antibody imprints of the protein surface structure.     

Two domains of the epidermal growth factor receptor are involved in cytoskeletal interactions

Epidermal growth factor receptor can interact directly with F-actin through an actin-binding domain. In the present study, a mutant EGFR, lacking a previously identified actin-binding domain (ABD 1), was still able to bind elements of the cytoskeleton. A second EGFR actin-binding domain (ABD 2) was identified in the region of the receptor that includes Tyr-1148 by a yeast two-hybrid assay. GST fusion proteins comprising ABD 1 or ABD 2 bound actin in vitro and competed for actin-binding with the full-length EGFR. EGFR binding to actin was also studied in intact cells using fluorescence resonance energy transfer (FRET). The localization of the EGFR/actin-binding complex changed after EGF stimulation. Fusion proteins containing mutations in ABD1 or ABD2 did not display a FRET signal. The results lead to the conclusion that the interaction between ABD1 and ABD2 and actin during EGF-induced signal transduction, and thus between EGFR and actin, are important in cell activation.     

Localization and synthesis of an antigenic determinant of herpes simplex virus glycoprotein D that stimulates the production of neutralizing antibody.

An antigenic determinant capable of inducing type-common herpes simplex virus (HSV)-neutralizing antibodies has been located on glycoprotein D (gD) of HSV type 1 (HSV-1). A peptide of 16 amino acids corresponding to residues 8 to 23 of the mature glycoprotein (residues 33 to 48 of the predicted gD-1 sequence) was synthesized. This peptide reacted with an anti-gD monoclonal antibody (group VII) previously shown to neutralize the infectivity of HSV-1 and HSV-2. The peptide was also recognized by polyclonal antibodies prepared against purified gD-1 but was less reactive with anti-gD-2 sera. Sera from animals immunized with the synthetic peptide reacted with native gD and neutralized both HSV-1 and HSV-2.     

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.     

Tetrahymena fimbrin localized in the division furrow bundles actin filaments in a calcium-independent manner

In cytokinesis, the contractile ring constricts the cleavage furrow. However, the formation and properties of the contractile ring are poorly understood. Fimbrin has two actin-binding domains and two EF-hand Ca(2+)-binding motifs. Ca(2+) binding to the EF-hand motifs inhibits actin-binding activity. In Tetrahymena, fimbrin is localized in the cleavage furrow during cytokinesis. In a previous study, Tetrahymena fimbrin was purified with an F-actin affinity column. However, the purified Tetrahymena fimbrin was broken in to a 60 kDa fragment of a 70 kDa full length fimbrin. In this study, we investigated the properties of recombinant Tetrahymena fimbrin. In an F-actin cosedimentation assay, Tetrahymena fimbrin bound to F-actin and bundled it in a Ca(2+)-independent manner, with a K(d) of 0.3 micro M and a stoichiometry at saturation of 1:1.4 (Tetrahymena fimbrin: actin). In the presence of 1 molecule of Tetrahymena fimbrin to 7 molecules of actin, F-actin was bundled. Immunofluorecence microscopy showed that a dotted line of Tetrahymena fimbrin along the cleavage furrow formed a ring structure. The properties and localization of Tetrahymena fimbrin suggest that it bundles actin filaments in the cleavage furrow and plays an important role in contractile ring formation during cytokinesis.     

The COOH terminus of the c-Abl tyrosine kinase contains distinct F- and G-actin binding domains with bundling activity [published erratum appears in J Cell Biol 1994 Mar;124(5):865]

The myristoylated form of c-Abl protein, as well as the P210bcr/abl protein, have been shown by indirect immunofluorescence to associate with F-actin stress fibers in fibroblasts. Analysis of deletion mutants of c-Abl stably expressed in fibroblasts maps the domain responsible for this interaction to the extreme COOH-terminus of Abl. This domain mediates the association of a heterologous protein with F-actin filaments after microinjection into NIH 3T3 cells, and directly binds to F-actin in a cosedimentation assay. Microinjection and cosedimentation assays localize the actin-binding domain to a 58 amino acid region, including a charged motif at the extreme COOH-terminus that is important for efficient binding. F-actin binding by Abl is calcium independent, and Abl competes with gelsolin for binding to F- actin. In addition to the F-actin binding domain, the COOH-terminus of Abl contains a proline-rich region that mediates binding and sequestration of G-actin, and the Abl F- and G-actin binding domains cooperate to bundle F-actin filaments in vitro. The COOH terminus of Abl thus confers several novel localizing functions upon the protein, including actin binding, nuclear localization, and DNA binding. Abl may modify and receive signals from the F-actin cytoskeleton in vivo, and is an ideal candidate to mediate signal transduction from the cell surface and cytoskeleton to the nucleus.     

Pleckstrin homology domains interact with filamentous actin.

A fraction of Bruton's tyrosine kinase (Btk) co-localizes with actin fibers upon stimulation of mast cells via the high affinity IgE receptor (FcepsilonRI). In this study, a molecular basis of the Btk co-localization with actin fibers is presented. Btk and other Tec family tyrosine kinases have a pleckstrin homology (PH) domain at their N termini. The PH domain is a short peptide module frequently found in signal-transducing proteins and cytoskeletal proteins. Filamentous actin (F-actin) is shown to be a novel ligand for a subset of PH domains, including that of Btk. The actin-binding site was mapped to a 10-residue region of the N-terminal region of Btk. Basic residues in this short stretch are demonstrated to be involved in actin binding. Isolated PH domains induced actin filament bundle formation. Consistent with these observations, Btk binds F-actin in vitro and in vivo. Wild-type Btk protein is in part translocated to the cytoskeleton upon FcepsilonRI cross-linking, whereas Btk containing a mutated PH domain is not. Phosphatidylinositol 3,4, 5-trisphosphate-mediated membrane translocation of Btk was enhanced in cytochalasin D-pretreated, FcepsilonRI-stimulated mast cells. These data indicate that PH domain-mediated F-actin binding plays a role in Btk co-localization with actin filaments.     

A new model for the interaction of dystrophin with F-actin

The F-actin binding and cross-linking properties of skeletal muscle dystrophin-glycoprotein complex were examined using high and low speed cosedimentation assays, microcapillary falling ball viscometry, and electron microscopy. Dystrophin-glycoprotein complex binding to F-actin saturated near 0.042 +/- 0.005 mol/ mol, which corresponds to one dystrophin per 24 actin monomers. Dystrophin-glycoprotein complex bound to F-actin with an average apparent Kd for dystrophin of 0.5 microM. These results demonstrate that native, full-length dystrophin in the glycoprotein complex binds F-actin with some properties similar to those measured for several members of the actin cross-linking super- family of proteins. However, we failed to observe dystrophin- glycoprotein complex-induced cross-linking of F-actin by three different methods, each positively controlled with alpha-actinin. Furthermore, high speed cosedimentation analysis of dystrophin- glycoprotein complex digested with calpain revealed a novel F-actin binding site located near the middle of the dystrophin rod domain. Recombinant dystrophin fragments corresponding to the novel actin binding site and the first 246 amino acids of dystrophin both bound F- actin but with significantly lower affinity and higher capacity than was observed with purified dystrophin-glycoprotein complex. Finally, dystrophin-glycoprotein complex was observed to significantly slow the depolymerization of F-actin, Suggesting that dystrophin may lie along side an actin filament through interaction with multiple actin monomers. These data suggest that although dystrophin is most closely related to the actin cross-linking superfamily based on sequence homology, dystrophin binds F-actin in a manner more analogous to actin side-binding proteins.     

Evidence of intramolecular regulation of the Dictyostelium discoideum 34 000 Da F-actin-bundling protein

Intramolecular interaction within the Ca(2+)-regulated 34 kDa actin-bundling protein from Dictyostelium discoideum was found to contribute to the regulation of its actin-binding activity. Recombinant N-terminally truncated proteins aa77-295, 124-295, and 139-295 bound actin at > or = 2:1 stoichiometry, which is 5-fold greater than the intact protein aa1-295 as assessed by cosedimentation with F-actin. These proteins also have enhanced cross-linking activity as assessed by viscometry and electron microscopy. All truncated 34 kDa proteins failed to bind (45)Ca(2+) on blots and displayed Ca(2+)-insensitive binding with actin, although most proteins possessed intact putative EF-hand Ca(2+)-binding motifs. An intramolecular interaction within the 34 kDa protein was inferred from direct demonstrations of domain-domain interaction among the truncated 34 kDa proteins both in the presence and absence of actin. The intramolecular interaction between interaction zone 1 (aa71-123) and interaction zone 2 (aa193-254) is proposed to maintain the N-terminal inhibitory region (aa1-76) in close proximity with the strong actin-binding site (aa193-254) in order to modulate the interaction of the intact protein with actin filaments.     

Sorting of ligand-activated epidermal growth factor receptor to lysosomes requires its actin-binding domain

Ligand-induced down-regulation of the epidermal growth factor receptor (EGFR) comprises activation of two sequential transport steps. The first involves endocytic uptake by clathrin-coated vesicles, the second transfer of endocytosed EGFR from endosomes to lysosomes. Here we demonstrate that the second transport step requires a domain of the EGFR that encompasses residues 985-996 and was previously found to interact with actin. Deletion of domain 989-994 (Delta989-994 EGFR) did not interfere with EGFR uptake but completely abrogated its degradation. In contrast, both uptake and degradation were affected for K721A EGFR, a kinase-deficient EGFR mutant. To measure intracellular EGFR sorting, we developed a novel cell fractionation assay toward which cells were co-transfected for chicken hepatic lectin, a receptor for agialoglycoproteins. These cells were incubated with agialofetuin-coupled colloidal gold, which was targeted to lysosomes after receptor-mediated endocytosis. Compartments within the lysosomal pathway gained buoyant density because of the presence of colloidal gold and could be isolated from cell homogenates by ultracentrifugation through a high-density sucrose cushion. In contrast to endocytosed wild type EGFR, both Delta989-994 EGFR and K721A EGFR were largely not retrieved in gold-containing endocytic compartments. These results are supported with morphological data. We conclude that sorting of endocytosed EGFR into the degradation pathway requires both its kinase activity and actin-binding domain.     

Antibodies directed against N-terminal residues on actin do not block acto-myosin binding

Several studies using a variety of approaches have suggested a possible role for the amino-terminal residues of skeletal muscle actin in acto-myosin interaction. In order to assess the significance of acto-S-1 contacts involving the N-terminal segment of actin, we have prepared polyclonal antisera against a synthetic peptide corresponding to the seven amino-terminal residues of rabbit skeletal muscle actin (alpha-N-terminal peptide). Affinity-purified immunoglobulin (Ig) G (and Fab) prepared from these antisera reacts strongly and specifically with the amino-terminal segment of both G- and F-actin but not with myosin subfragment 1 (S-1). This specificity was determined by Western blot analysis of actin and its proteolytic fragments and the inhibition of the above reactivity by the alpha-N-terminal peptide. The alpha-N-terminal peptide did not interact with S-1 in solution, affect S-1 and actin-activated S-1 MgATPase, or cause dissociation of the acto-S-1 complex. In separate experiments F-actin could be cosedimented with S-1 and affinity-purified IgG or Fab by using an air-driven ultracentrifuge. Densitometric analysis of sodium dodecyl sulfate/polyacrylamide gels of pellet and supernatant fractions from such experiments demonstrated the binding of both S-1 and IgG or Fab to the same F-actin protomer. Our results suggest that, while the acidic N-terminal amino acids of actin may contact the myosin head, these residues cannot be the main determinants of acto-S-1 interaction.     

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.     

Polyphosphoinositides inhibit the interaction of vinculin with actin filaments.

Binding of vinculin to adhesion plaque proteins is restricted by an intramolecular association of vinculin's head and tail regions. Results of previous work suggest that polyphosphoinositides disrupt this interaction and thereby promote binding of vinculin to both talin and actin. However, data presented here show that phosphatidylinositol 4,5-bisphosphate (PI4,5P2) inhibits the interaction of purified tail domain with F-actin. Upon re-examining the effect of PI4,5P2 on the actin and talin-binding activities of intact vinculin, we find that when the experimental design controls for the effect of magnesium on aggregation of PI4,5P2 micelles, polyphosphoinositides promote interactions with the talin-binding domain, but block interactions of the actin-binding domain. In contrast, if vinculin is trapped in an open confirmation by a peptide specific for the talin-binding domain of vinculin, actin binding is allowed. These results demonstrate that activation of the actin-binding activity of vinculin requires steps other than or in addition to the binding of PI4,5P2.     

Structure of the utrophin actin-binding domain bound to F-actin reveals binding by an induced fit mechanism

Utrophin is a large ubiquitously expressed cytoskeletal protein, homologous to dystrophin, the protein disrupted in Duchenne muscular dystrophy. The association of both proteins with the actin cytoskeleton is functionally important and is mediated by a domain at their N termini, conserved in members of the spectrin superfamily, including alpha-actinin, beta-spectrin and fimbrin. We present the structure of the actin-binding domain of utrophin in complex with F-actin, determined by cryo-electron microscopy and helical reconstruction, and a pseudo-atomic model of the complex, generated by docking the crystal structures of the utrophin domain and F-actin into the reconstruction. In contrast to the model of actin binding proposed for fimbrin, the utrophin actin-binding domain appears to associate with actin in an extended conformation. This conformation places residues that are highly conserved in utrophin and other members of the spectrin superfamily at the utrophin interface with actin, confirming the likelihood of this binding orientation. This model emphasises the importance of protein flexibility in modeling interactions and presents the fascinating possibility of a diversity of actin-binding mechanisms among related proteins.     

Interactions of elongation factor 1alpha with F-actin and beta-actin mRNA: implications for anchoring mRNA in cell protrusions

The targeting of mRNA and local protein synthesis is important for the generation and maintenance of cell polarity. As part of the translational machinery as well as an actin/microtubule-binding protein, elongation factor 1alpha (EF1alpha) is a candidate linker between the protein translation apparatus and the cytoskeleton. We demonstrate in this work that EF1alpha colocalizes with beta-actin mRNA and F-actin in protrusions of chicken embryo fibroblasts and binds directly to F-actin and beta-actin mRNA simultaneously in vitro in actin cosedimentation and enzyme-linked immunosorbent assays. To investigate the role of EF1alpha in mRNA targeting, we mapped the two actin-binding sites on EF1alpha at high resolution and defined one site at the N-terminal 49 residues of domain I and the other at the C-terminal 54 residues of domain III. In vitro actin-binding assays and localization in vivo of recombinant full-length EF1alpha and its various truncates demonstrated that the C terminus of domain III was the dominant actin-binding site both in vitro and in vivo. We propose that the EF1alpha-F-actin complex is the scaffold that is important for beta-actin mRNA anchoring. Disruption of this complex would lead to delocalization of the mRNA. This hypothesis was tested by using two dominant negative polypeptides: the actin-binding domain III of EF1alpha and the EF1alpha-binding site of yeast Bni1p, a protein that inhibits EF1alpha binding to F-actin and also is required for yeast mRNA localization. We demonstrate that either domain III of EF1alpha or the EF1alpha-binding site of Bni1p inhibits EF1alpha binding to beta-actin mRNA in vitro and causes delocalization of beta-actin mRNA in chicken embryo fibroblasts. Taken together, these results implicate EF1alpha in the anchoring of beta-actin mRNA to the protrusion in crawling cells.     

Antibody directed against the 142-148 sequence of the myosin heavy chain interferes with myosin-actin interaction.

It has been reported recently that the isolated and renatured 23-kDa N-terminal fragment of rabbit skeletal muscle myosin binds tightly to F-actin in an ATP-dependent manner [Muhlrad, A. (1989) Biochemistry 28, 4002-4010]. The binding to actin is of electrostatic nature and may involve a positively charged cluster of residues on the 23-kDa fragment stretching from Arg-143 to Arg-147. An octapeptide containing this positive cluster was synthesized and coupled to BSA through a cysteine residue added to the N-terminus of the peptide. Polyclonal antibody was raised against the BSA-coupled peptide in rabbits which recognized the N-terminal 23-kDa fragment of rabbit skeletal myosin subfragment 1, and a peptide comprised of residues 122-204 of the 23K fragment in Western blots. The purified antibody [IgG and F(ab)] inhibited the actin-activated ATPase activity of S1 without affecting its Mg2(+)- and K+(EDTA)-modulated ATPase activity. Both IgG and F(ab) decreased the binding of S1 to F-actin in a sedimentation assay, and actin inhibited the binding of both IgG and F(ab) to S1 in a competitive binding assay. The cysteine thiol of the synthetic octapeptide was labeled by the fluorescent thiol reagent monobromobimane, and the labeled peptide was found to bind to actin in a sedimentation assay. The results support the possibility that the positively charged Arg-143 to Arg-147 stretch of residues on the 23-kDa fragment participates in actin binding of myosin and may represent an essential constituent of the actin-S1 interface.     

The interfaces of actin and Acanthamoeba actobindin. Identification of a new actin-binding motif

Actobindin is an 88-amino acid polypeptide, containing two almost identical repeated domains of 33 and 34 residues. Depending on the molar ratios in which they are mixed, actobindin binds either one or two actin molecules. We cross-linked actobindin and actin in the 1:1 complex, using the zero-length cross-linker 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. The cross-linked peptides were purified after consecutive CNBr cleavage and trypsin and Staphylococcus protease V8 digestions, and the cross-linked side chains were identified by amino acid sequencing. Isopeptide linkages were formed between residues Glu-100 of actin and Lys-16 of actobindin. In addition, we found a connection between one or more of the acidic residues 1,2, or 3 of actin and Lys-16 and Lys-52 of actobindin. The cross-linked regions in actobindin contain Leu-Lys-His-Ala-Glu-Thr motifs, similar to sequences observed in several other actin-binding proteins.