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Characterization of the Actin Cross-linking Properties of the Scruin-Calmodulin Complex from the Acrosomal Process of Limulus Sperm

1996; Elsevier BV; Volume: 271; Issue: 5 Linguagem: Inglês

10.1074/jbc.271.5.2651

1083-351X

Mitchell C. Sanders, Michael Way, Jun Sakai, Paul Matsudaira,

Silk-based biomaterials and applications

During activation of the Limulus sperm acrosomal process, actin filaments undergo a change in twist that is linked with the conversion from a coiled to a straight scruin-actin bundle. Since scruin had not been purified, its identity as an actin-binding protein has not been demonstrated. Using HECAMEG (methyl-6-O-(N-heptylcarbamoyl)-α-D-glucopyranoside) detergent extraction in concert with high calcium, we purified native scruin and identified it as an equimolar complex with calmodulin. 125I-Calmodulin overlays and calmodulin-Sepharose indicate that scruin binds calmodulin in calcium but not in EGTA. Overlay experiments also map the calmodulin binding site between the putative N- and C-terminal β-propeller domains within residues 425-446. Immunofluorescence microscopy reveals that calmodulin colocalizes with scruin and actin in the coiled bundle. Although scruin binds calmodulin, pelleting assays and electron microscopy show that the scruin cross-links F-actin into bundles independently of calcium. Based on our biochemical and structural studies, we suggest a model to explain how scruin controls a change in twist of actin filaments during the acrosome reaction. We predict that calcium subtly alters scruin conformation through its calmodulin subunit and the conformation change in scruin causes a shift in the relative positions of the scruin-bound actin subunits. During activation of the Limulus sperm acrosomal process, actin filaments undergo a change in twist that is linked with the conversion from a coiled to a straight scruin-actin bundle. Since scruin had not been purified, its identity as an actin-binding protein has not been demonstrated. Using HECAMEG (methyl-6-O-(N-heptylcarbamoyl)-α-D-glucopyranoside) detergent extraction in concert with high calcium, we purified native scruin and identified it as an equimolar complex with calmodulin. 125I-Calmodulin overlays and calmodulin-Sepharose indicate that scruin binds calmodulin in calcium but not in EGTA. Overlay experiments also map the calmodulin binding site between the putative N- and C-terminal β-propeller domains within residues 425-446. Immunofluorescence microscopy reveals that calmodulin colocalizes with scruin and actin in the coiled bundle. Although scruin binds calmodulin, pelleting assays and electron microscopy show that the scruin cross-links F-actin into bundles independently of calcium. Based on our biochemical and structural studies, we suggest a model to explain how scruin controls a change in twist of actin filaments during the acrosome reaction. We predict that calcium subtly alters scruin conformation through its calmodulin subunit and the conformation change in scruin causes a shift in the relative positions of the scruin-bound actin subunits. INTRODUCTIONIn many examples of cell motility including: cytokinesis, phagocytosis, exocytosis, chemotaxis, and extension of the lamella, movement or force is generated by either actin-myosin interactions or the reversible assembly of actin filaments(1.Stossel T.P. Science. 1993; 260: 1086-1094Crossref PubMed Scopus (903) Google Scholar). Contrary to these examples, extension of the acrosomal process in Limulus sperm may be a movement of an actin spring in which potential energy, stored as a coiled bundle at the base of sperm body, is unleashed at fertilization to uncoil and extrude the bundle through a channel in the nucleus(2.Tilney L.G. J. Cell Biol. 1975; 64: 289-310Crossref PubMed Scopus (150) Google Scholar, 3.Tilney L.G. Clain J.G. Tilney M.S. J. Cell Biol. 1979; 81: 229-253Crossref PubMed Scopus (31) Google Scholar). During the uncoiling process, the actin bundle untwists by an impressive 60° per 700 nm. This action is accompanied by slippage and a modest (−0.23° per subunit) untwisting of the actin filaments(4.De Rosier D. Tilney L. Flicker P. J. Mol. Biol. 1980; 137: 375-389Crossref PubMed Scopus (36) Google Scholar, 5.De Rosier D.J. Tilney L.G. Bonder E.M. Frankl P. J. Cell Biol. 1982; 93: 324-337Crossref PubMed Scopus (28) Google Scholar). As a result of these events, the bundle forms a 60-μm-long membrane extension, which bridges the egg jelly coat to fuse with the egg plasma membrane.The factors that maintain the coiled state of the bundle or signal its rotation and slippage are unknown, but the target of their action must be scruin, an actin-associated protein in the acrosomal process. Previous studies show that the acrosomal process consists of a 1:1 complex of actin and scruin (Mr 102,000)(6.Schmid M.F. Matsudaira P. Jeng T.W. Jakana J. Towns-Andrews E. Bordas J. Chiu W. J. Mol. Biol. 1991; 221: 711-725Crossref PubMed Scopus (30) Google Scholar). In EM ( 1The abbreviations used are: EMelectron microscopyHECAMEGmethyl-6-O-(N-heptylcarbamoyl)-α-D-glucopyranosideHPLChigh performance liquid chromatographyPAGEpolymacrylamide gel electrophoresisGSTglutathione S-transferase.) reconstructions, scruin decorates the outside of an actin filament, with each scruin molecule bound to a pair of actin subunits along the actin one-start helix(7.Schmid M.F. Agris J.M. Jakana J. Matsudaira P. Chiu W. J. Cell Biol. 1994; 124: 341-350Crossref PubMed Scopus (69) Google Scholar). Presumably, actin cross-links are maintained by interactions between scruin proteins on neighboring filaments.Based on sequence analysis, limited proteolysis, and EM image reconstructions, scruin is organized into two 40-kDa domains connected by a highly helical protease-sensitive neck(7.Schmid M.F. Agris J.M. Jakana J. Matsudaira P. Chiu W. J. Cell Biol. 1994; 124: 341-350Crossref PubMed Scopus (69) Google Scholar, 8.Owen C. De Rosier D. J. Cell Biol. 1993; 123: 337-344Crossref PubMed Scopus (79) Google Scholar, 9.Way M. Sanders M. Garcia C. Sakai J. Matsudaira P. J. Cell Biol. 1995; 128: 51-60Crossref PubMed Scopus (63) Google Scholar). Each domain consists of a six-fold repeat of ~50 amino acids that based on the work of Bork and Doolittle(10.Bork P. Doolittle R.F. J. Mol. Biol. 1994; 236: 1277-1282Crossref PubMed Scopus (161) Google Scholar), is predicted to fold into a four stranded β-sheet motif(9.Way M. Sanders M. Garcia C. Sakai J. Matsudaira P. J. Cell Biol. 1995; 128: 51-60Crossref PubMed Scopus (63) Google Scholar). This motif typifies a protein superfamily, which includes galactose oxidase(10.Bork P. Doolittle R.F. J. Mol. Biol. 1994; 236: 1277-1282Crossref PubMed Scopus (161) Google Scholar), several open reading frames in the genome of pox viruses(11.Massung R.F. Liu L.I. Qi J. Knight J.C. Yuran T.E. Kerlavage A.R. Parsons J.M. Venter J.C. Esposito J.J. Virology. 1994; 201: 215-240Crossref PubMed Scopus (211) Google Scholar), a mouse placental transcript, MIPP(12.Chang-Yeh A. Mold D.E. Huang R.C. Nucleic Acids Res. 1991; 19: 3667-3672Crossref PubMed Scopus (64) Google Scholar), and kelch, the Drosophila gene that is important for nutrient transport during oogenesis(13.Xue F. Cooley L. Cell. 1993; 72: 681-693Abstract Full Text PDF PubMed Scopus (373) Google Scholar). Although there is some understanding of the structural organization of scruin, its regulation and biochemical properties are not understood because the protein has not been purified in a native, soluble state.To identify the mechanism that causes the dynamic conformational changes in the acrosomal process during the acrosome reaction, we must first purify scruin and characterize its actin binding properties. In this report, we describe the isolation of scruin and report its association with calmodulin. Furthermore, we show that the scruin-calmodulin complex cross-links F-actin into bundles but, surprisingly, the cross-linking activity is independent of calcium. Our results suggests that scruin is always bound to actin filaments, and we hypothesize that during the acrosome reaction the conformational changes in the actin filament and acrosomal process may be caused by a subtle conformation change in scruin.EXPERIMENTAL PROCEDURESMaterialsPepstatin, leupeptin, aprotinin, benzamidine, and ATP were purchased from Sigma. HECAMEG and calcium ionophore A23187 were purchased from Calbiochem. 125I-Calmodulin was purchased from DuPont NEN. Artificial sea water was purchased from Tropic Marin. Protein assays were performed with the Bio-Rad assay reagent.Purification of the True DischargeThe true discharge was isolated using a protocol modified from Schmid et al.,(6.Schmid M.F. Matsudaira P. Jeng T.W. Jakana J. Towns-Andrews E. Bordas J. Chiu W. J. Mol. Biol. 1991; 221: 711-725Crossref PubMed Scopus (30) Google Scholar). Twenty ml of sperm, collected from 30-40 adult male horseshoe crabs, was divided into six aliquots, layered on 33 ml of a 1:1 mixture of artificial sea water and 50 mM calcium chloride, and then gently mixed. Sperm were activated by adding 100 μl of 19 mM A23187 (in dimethyl sulfoxide) to each tube and incubated in the dark for 30 min. To prevent proteolysis, a mixture of protease inhibitors were added to each step (final concentrations: 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, 0.1 mM pepstatin A, 1.0 KIU/ml aprotinin, and 0.2 μg/ml leupeptin). The activated sperm were sheared three times through a 21-gauge needle and centrifuged twice at 2420 × g for 10 min to remove the sperm heads. The supernatant was then centrifuged at 43,140 × g for 15 min to pellet the acrosomal bundles. Each pellet was resuspended in 5 ml of scruin buffer A (10 mM Tris, pH 8.0, 1 mM dithiothreitol, 1 mM CaCl2, 100 mM NaCl, 0.01% NaN3) with 19 mM HECAMEG for 15 min on ice. The volume was diluted to 30 ml with scruin buffer A and centrifuged at 43,140 × g for 15 min. The pellets were pooled into 1 ml of buffer A and pelleted at 16,000 × g in a microcentrifuge for 15 min. To quantify the stoichiometry of scruin and calmodulin by HPLC, this washing step was repeated 5 times to reduce the protein contaminants. In a typical preparation, 5.2 mg of true discharge were obtained as determined by the Bio-Rad assay. The purity of the true discharge preparations was assessed by SDS-PAGE.Purification of Soluble ScruinScruin was dissociated from actin by gently shaking the isolated bundles in 1 ml of 1 M calcium for 1 h at room temperature, and then centrifuging the extract for 15 min in a microcentrifuge (16,000 × g) to remove most of the actin filaments. The low speed supernatant containing primarily scruin, calmodulin, actin, and some minor contaminants (3.7 mg of total protein) was successively filtered through 0.8-, 0.45-, and 0.2-μm filters. In the later stages of the project, nucleic acids in the extract were removed by filtration through a 0.8-μm filter and then a Qiagen Tip 5. The filtrate was loaded onto a Superdex 200 HR FPLC gel filtration column pre-equilibrated and run in scruin buffer A supplemented with inhibitors. The fractions that contained scruin were pooled (1.1 mg of total protein), loaded onto a Q-HiTrap (Pharmacia Biotech Inc.) ion exchange column with scruin buffer A, and eluted with a 0-1 M NaCl gradient in buffer A. Scruin protein eluted in 0.4 M NaCl. The scruin-containing fractions (300 μg of total protein) were either shell-frozen in liquid nitrogen and stored as a lyophilate or concentrated by Ultrafree-MC centrifugal membrane filters (Millipore) and used immediately.Identification of CalmodulinThe stoichiometry of the scruin-actin-calmodulin complex was determined by size exclusion chromatography through a TSK 300SWLX column (Toyo Soda) under denaturing conditions (6 M guanidine HCl). The absorbance was measured at 230 nm. The molar ratio was calculated from the integrated peak areas. The 17-kDa polypeptide was identified as calmodulin by protein sequence obtained by mass spectrometry or chemical sequencing. First, the scruin-calmodulin complex was dissociated in 6 M guanidine HCl and separated by size exclusion chromatography. The purified calmodulin was cleaved with cyanogen bromide in 70% formic acid for 12 h as described previously (Matsudaira, 1992). Following incubation the sample was diluted in water and dried under vacuum in a fume hood. The fragments were purified by reverse phase chromatography on the HPLC. The fractions that were found to contain a single polypeptide by mass spectrometry (LASERMAT, Finnigan MAT) were N-terminally sequenced by gas phase sequencing on a model 2090E sequencer (Beckman Instruments Inc.) equipped for the identification of phenythiohydantoin amino acids. In later experiments, Limulus sperm calmodulin was purified from sperm with a phenyl-Sepharose column on the FPLC using standard methods(14.Gopalakrishna R. Anderson W.B. Biochem. Biophys. Res. Commun. 1982; 104: 830-836Crossref PubMed Scopus (717) Google Scholar). The purified protein was confirmed to be calmodulin by calcium-dependent mobility shift on SDS-PAGE and sequence by mass spectrometry using a PerSeptive Biosystems Voyager Elite time of flight mass spectrometer.Identification of the Calmodulin Binding Site125I-Calmodulin gel overlays were performed as described previously (15.Glenney Jr., J. Weber K. Methods Enzymol. 1983; 102: 204-210Crossref PubMed Scopus (25) Google Scholar) with minor modifications(16.Matsudaira P. Carraway K.L. Carraway C.A.C. The Cytoskeleton: A Practical Approach. IRL Press, Oxford1992: 73-98Google Scholar). 125I-Calmodulin nitrocellulose blot overlays were performed as described (17.Flanagan S.D. Yost B. Anal. Biochem. 1984; 140: 510-519Crossref PubMed Scopus (48) Google Scholar) using 0.05% Tween 20 and 30 mg/ml bovine serum albumin as the blocking agents. For scruin, we found that either method worked, but the nitrocellulose blot overlays had a higher signal to noise ratio. In contrast, the myosin I positive control only worked with the gel overlay method. The percent binding was quantitated using NIH Image software and is represented as the ratio of the signal intensity and the total signal in calcium and EGTA × 100. A value of zero indicates the signal was not above the background levels. Competition assays with scruin and the peptide PSN1 (described below) were performed in calcium only.Protein Expression of Scruin FragmentsThe calmodulin binding site in scruin was mapped using either expressed scruin fragments or synthetic peptides to specific regions in scruin as described below. The 454C and 590C domains of scruin were expressed in Escherichia coli and purified as described previously(9.Way M. Sanders M. Garcia C. Sakai J. Matsudaira P. J. Cell Biol. 1995; 128: 51-60Crossref PubMed Scopus (63) Google Scholar). Additionally, three constructs, GST1, GST2, and GST3, consisting of residues 390-429, 430-469, and 470-509 of scruin fused to the GST domain, respectively, were engineered by polymerase chain reaction using standard methods. Briefly, an in-frame BamHI site was introduced 5′ to the first amino acid codon and a TAG stop/EcoRI site after the last codon. The resulting polymerase chain reaction products were cloned into the BamHI-EcoRI sites of pGEX-2T (Pharmacia) and the fidelity of the final expression constructs confirmed by sequencing. GST1 and GST3 were transformed into DH5α and expressed upon addition of isopropyl-1-thio-β-D-galactopyranoside. However, because the GST2 fusion construct did not express in E. coli under a number of expression conditions, a synthetic peptide, PSN1 (CGAAKKVQRRWRRYIEQKSITKRM), was synthesized. The N-terminal sequence CG was included to aid in generating a sequence-specific polyclonal antibody. Two other synthesized peptides to a sequence near the protease-sensitive neck region of scruin and a sequence in β-scruin (R34, CKAKPQPGSKPTSVK; R35, CTTRSGSRKTQKTLK, respectively) were also used as controls(18.Way M. Sanders M. Chafel M. Tu Y.-H. Knight A. Matsudaira P. J. Cell Sci. 1995; 108: 3155-3162PubMed Google Scholar).Actin Cosedimentation AssaysCosedimentation assays with purified scruin (0-4 μM), sperm calmodulin (15 μM), and rabbit skeletal actin (2 μM) were performed in the presence of 1 mM EGTA or 1 mM calcium as described previously(16.Matsudaira P. Carraway K.L. Carraway C.A.C. The Cytoskeleton: A Practical Approach. IRL Press, Oxford1992: 73-98Google Scholar). Briefly, samples were incubated for either 4°C overnight or for 1 h at room temperature and were centrifuged for 30 min in a bench-top ultracentrifuge (TL100, Beckman Instruments Inc., Fullerton, CA) at 75,000 rpm at 4°C. Pellets and supernatants from the centrifugation were adjusted to equal volumes and analyzed by SDS-PAGE, and electron microscopy. To determine whether the ability of scruin to bind and bundle F-actin was affected by calmodulin, exogenous sperm calmodulin was added to scruin at 3.75-fold molar excess.Reconstitution of Scruin-Actin BundlesScruin-actin bundles were formed by incubating scruin and actin, with or without exogenous sperm calmodulin at a molar ratio of at a 2:1:3.75, respectively, for 1 h at room temperature prior to absorbing the samples onto carbon films. The samples were negatively stained with 1.0% uranyl acetate and examined in a Phillips 410 electron microscope at an accelerating voltage of 80 kV. In some instances calmodulin was added to the samples at 0-5-fold molar excess of scruin.Immunofluorescence MicroscopyA rabbit polyclonal antiserum (R213.5) was generated against scruin; the specificity to crude sperm extracts has been described elsewhere(20.Friedberg F. Rhoads A.R. Bioessays. 1994; 16: 853-855Crossref PubMed Scopus (16) Google Scholar). The mouse monoclonal anti-calmodulin antiserum was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). To reduce the nonspecific background of the anti-scruin antibody for immunofluorescence, the antibody was affinity-purified against scruin blotted to nitrocellulose membrane (19.Sambrook J.E. Fritsch F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 18.16-18.18Google Scholar). Sperm were fixed and stained as described elsewhere(18.Way M. Sanders M. Chafel M. Tu Y.-H. Knight A. Matsudaira P. J. Cell Sci. 1995; 108: 3155-3162PubMed Google Scholar). Coverslips were examined in a Zeiss Axioskop microscope using differential interference contrast optics and a 100×/NA 1.3 Plan Neofluar objective configured with a Bio-Rad MRC600 Confocal ion laser. The ratio of the fluorescent intensity in the bundle and the entire sperm was quantified using the standard confocal image analysis software. For reproduction, the images were transferred to Adobe Photoshop and Illustrator (Adobe Systems Inc., Mountain View, CA) for cropping, contrast/brightness adjustment, placement of each image panel, and annotation.RESULTSTreatment of true discharges, isolated in Triton X-100, with 1 M calcium disassociated scruin from actin. However, after a few hours, the soluble scruin precipitated from solution (not shown). Subsequent experiments determined that long term solubility depended on the removal of the Triton X-100 by dialysis at high ionic strength (0.45 M NaCl). Other treatments such as denaturing agents (8 M urea) and low pH (0.2 M glycine, pH 4.2) were found to also disassociate the scruin actin bundles, but less than half of the scruin remained active, as judged by high speed sedimentation with rabbit skeletal actin (100,000 × g for 30 min: not shown). Based on these findings, we replaced Triton X-100 with the non ionic detergent HECAMEG. HECAMEG is easier to remove by gel filtration or dialysis (critical micelle concentration 19.5 mM) and does not absorb at 280 nm. After demembranation of the actin bundle with HECAMEG and extraction of scruin with 1 M CaCl2, one half of the actin pellets at low g-forces leaving scruin in the supernatant (Fig. 1A). The minor protein contaminants, DNA, and HECAMEG in the calcium extract were removed by gel filtration chromatography through Superdex HR 200 and ion exchange (Fig. 2). Based on relative molecular mass (Mr) standards, scruin fractionates as a monomer with an apparent molecular weight of 107,000.Figure 2:Copurification of calmodulin and scruin by gel filtration and quaternary amine ion exchange chromatography. A, the low speed supernatant was chromatographed through Superdex 200 HR (top panel). SDS-PAGE (bottom panel) of the fractions shows scruin and a 17-kDa polypeptide co-fractionate. B, a pool of scruin-containing fractions was loaded onto a Q-HiTrap column. The chromatograph (top panel) indicates a single peak elutes at at 40% buffer B. SDS-PAGE (bottom panel) shows that scruin co-purifies with a 17-kDa band that was determined by internal protein sequencing to be calmodulin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Identification of CalmodulinAt each stage of the purification, a 17-kDa polypeptide consistently cofractionated with scruin (Figure 1:, Figure 2:). Although the N terminus was blocked to chemical sequencing, we obtained sequence from two fragments generated by cyanogen bromide cleavage (MKDTDSEEEI and MIREADIDGDGQVNYEEFVTM). Data base searches with these two sequences showed an exact match with calmodulin from Drosophila. Additionally, this 17-kDa protein shows the characteristic calcium-dependent mobility shift on SDS-PAGE gels and is recognized by a monoclonal antibody to calmodulin (not shown).The presence of calmodulin was also confirmed by immunofluorescence microscopy (Fig. 3). In unactivated sperm cells, calmodulin is localized to the base of the nucleus as a ring of fluorescence, which colocalizes with the rhodamine phalloidin staining pattern of F-actin or immunostaining of scruin. Quantitation of the fluorescence showed that 71% (S.D. = ± 7.6, n = 7) of the calmodulin was found to localize to the bundle at the base with a less apparent staining in the nucleus and the perimeter of the acrosomal vesicle. No calmodulin staining was observed within the flagellar region of the sperm or the interior of the acrosomal vesicle.Figure 3:Distribution of scruin and calmodulin in Limulus sperm. a and b, DIC images of unactivated sperm show the presence of long flagella and an apical vesicle. c, phalloidin (green) and α-scruin (red) colocalize to the coiled actin bundle at the base of the nucleus. d, calmodulin (green) primarily colocalizes with phalloidin (red) in the bundle at the base of the sperm. Additionally, the calmodulin also is also located in the nuclear region and at the perimeter of the acrosomal vesicle. Bar = 5.0 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine the stoichiometry of the scruin, actin, and calmodulin in the acrosomal process, we quantified the peak areas of samples separated by gel exclusion chromatography in denaturing conditions. Based on the integrated peak areas and the known molecular masses for the proteins, the scruin:actin:calmodulin molar ratio was 1.00:1.15:0.97 (Fig. 1B). Although scruin and calmodulin were always seen to co-purify, the molar ratio of the two proteins was sometimes 1:0.5, depending on the individual preparation. This variability in the ratio of scruin to calmodulin after purification either is due to the extraction conditions or is merely a consequence of an equilibrium for binding and subsequent dissociation of calmodulin during purification.Calmodulin Binding SiteTo confirm that calmodulin is a scruin subunit, we tested binding of exogenous bovine calmodulin to scruin. By two criteria: 125I-calmodulin blot and gel overlays (Fig. 4) and calmodulin-Sepharose chromatography (not shown), calmodulin binds scruin in calcium but not in EGTA.Figure 4:Scruin binds calmodulin avidly in calcium based on blot overlays with 125I-calmodulin. Purified scruin was electrophoresed through SDS-PAGE gels and either stained with Coomassie Blue (A) or electroblotted to nitrocellulose membranes (B and C). The membranes were incubated with 125I-bovine calmodulin in the presence (B) or absence (C) of calcium. The calmodulin bound only to scruin in calcium; little or no binding was detected in EGTA. Similar results were observed with gel overlays (not shown). Myosin I bound calmodulin in calcium and EGTA as reported previously (data not shown; (24.Howe C.L. Mooseker M.S. J. Cell Biol. 1983; 97: 974-985Crossref PubMed Scopus (74) Google Scholar)). D, competition experiments with scruin and PSN1, the peptide containing the calmodulin binding site. Calmodulin binding to scruin was inhibited by nanomolar concentrations of PSN1. The estimated Ki is <50 nM. Each point is an average of two intensity values.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Although scruin is predicted to be mainly β-sheet, secondary structure analysis predicted that the neck region (Fig. 5a) between the N- and C-terminal domains of scruin is highly helical and amphipathic. A comparison of this region with the calmodulin binding IQ motif of MYO2 highlights the similar pattern of basic and hydrophobic residues. A helical wheel representation of a portion of the neck region clearly shows a basic and a hydrophobic face of the helix (Fig. 5b; Refs. 20 and 21). Because calmodulin binds to the basic face of an amphipathic helix, we examined calmodulin binding to various synthetic peptides, proteolytic fragments, and GST fusions that spanned the neck region. Two GST fusion constructs, GST1 and GST3, contain sequences that border outside of the predicted calmodulin binding site. These fusion constructs did not bind the radiolabeled calmodulin (Table 1). The third GST fusion (GST2) which contains this region did not express in E. coli; however, a synthetic peptide, PSN1, to the region 425-446 did bind calmodulin in EGTA and calcium. The peptide at a concentration of 50 nM also inhibited calmodulin binding to intact scruin by 61% (Fig. 4D). In other experiments, the C-terminal half of scruin (454C and 590C, produced by expression in E. coli; (9.Way M. Sanders M. Garcia C. Sakai J. Matsudaira P. J. Cell Biol. 1995; 128: 51-60Crossref PubMed Scopus (63) Google Scholar)), a tryptic digestion of scruin, or natural breakdown products of scruin did not bind to the radiolabeled calmodulin in the presence or absence of calcium (not shown). These proteolytic sites have been previously mapped to the protease-sensitive neck region of scruin(9.Way M. Sanders M. Garcia C. Sakai J. Matsudaira P. J. Cell Biol. 1995; 128: 51-60Crossref PubMed Scopus (63) Google Scholar), suggesting that the calmodulin binding site is not in the C- or N-terminal halves of scruin.Figure 5:The putative calmodulin binding site. a, the protease-sensitive neck region of scruin contains an amphipathic helix that is similar to the calmodulin binding IQ motif of MYO 2. Bold text delineates conserved residue matches. b, the helical wheel representation of the peptide PSN1 identifies basic and hydrophobic faces, which are characteristic of a calmodulin binding motif.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Tabled 1 Open table in a new tab Characterization of Actin Cross-linking ActivityBecause scruin is the only actin-associated protein in the acrosomal process, we tested it for actin binding activity using a cosedimentation assay and electron microscopy. Scruin binds actin independently of calcium. In calcium and EGTA, the majority of the scruin is found associated with actin in the pellet (Fig. 6). There is no difference between binding in EGTA and calcium. Furthermore, the amount of actin found in the pellet is invariant suggesting that scruin does not alter the F- to G-actin equilibrium. In the absence of actin, scruin remains in the supernatant in high g-force sedimentation assays (not shown). Since exogenously added calmodulin may affect the binding affinity of scruin for actin, cosedimentation assays were also performed with a 3.75-fold molar ratio of exogenously Limulus calmodulin (not shown). Examination of the samples by electron microscope before centrifugation shows that actin bundles form in the presence and absence of calcium (Fig. 7). The results from sedimentation assays and electron microscopy show that the presence or absence of excess calmodulin does not detectably affect actin binding activity of scruin.Figure 6:Cosedimentation of scruin with actin in EGTA and calcium. A, various concentrations of scruin were incubated with 2 μM actin in the presence of EGTA (□) and calcium (•). SDS-PAGE samples of supernatants and pellets show that in calcium and EGTA scruin binds avidly. B, quantitation of the pelleting assays shows the slightly higher affinity for actin in calcium than in EGTA. However, there is no apparent difference in actin binding in the presence of exogenous sperm calmodulin (data not shown). In the absence of actin, all scruin remained in the supernatant (data not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7:Reconstitution of scruin actin bundles. Based on electron microscopy, scruin is capable of forming bundles with rabbit skeletal F-actin in the presence of EGTA (a) and calcium (b). These bundles have a similar packing to the Limulus bundles (c) but are not crystalline. In the absence