Actin and tubulin binding domains of synapsins Ia and Ib

Synapsins Ia and Ib are neuronal phosphoproteins involved with the regulated clustering of small synaptic vesicles at the presynaptic terminus. In vitro they bind and bundle filaments of both actin and tubulin. Previously, we identified an actin binding domain in the NH2-terminal 25-kDa fragment (N25) generated by 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage of synapsin I and found that a complementary COOH-terminal 52-kDa portion of the molecule (N52) contained either a second actin binding site or a site of self-association [Petrucci, T. P., & Morrow, J. S. (1987) J. Cell. Biol. 105, 1355]. Using direct binding assays between actin, tubulin, and specific synapsin NTCB-derived peptides, we confirm the ability of purified N25 to bind but not bundle actin and demonstrate that the complementary N52 (or N50) fragments from synapsins Ia and Ib and a 14-kDa fragment derived from the middle of the molecule also associate directly with actin. An antibody specific for N25 inhibits the actin binding activity of N25 and the actin bundling but not the actin binding activity of intact synapsin I. Similar studies conducted with purified tubulin and tubulin immobilized on Sepharose demonstrate that both tubulin and actin bind at approximately the same sites in the NH2-terminal half of synapsin I. Although the fragments derived from the COOH terminus of both synapsin Ia and synapsin Ib (N40b/N34) were devoid of measurable actin binding activity after NTCB cleavage, they were specifically labeled in the intact molecule by a photoactivated cross-linker bound to F-actin. Collectively, these results indicate that synapsins Ia and Ib possess two actin and tubulin binding domains located in the NH2-terminal half of the molecule and suggest that a third actin binding domain is located in the COOH-terminal region. The NH2-terminal sites are found in NTCB peptides N25 and N14, while the third site, apparently of lower affinity, resides in N40b/N34. It is hypothesized that, in the intact molecule, the two NH2-terminal domains contribute to a single high-affinity actin and/or tubulin binding site in the "globular" head region of synapsin I, while the third actin binding domain constitutes the topographically distinct site required for the actin bundling activity of the native molecule. The 45-residue COOH extension that distinguishes synapsin Ia from synapsin Ib appears not to be involved with actin binding, since no differences were found in the ability of N40b and N34 to be photo-cross-linked to actin.(ABSTRACT TRUNCATED AT 400 WORDS)     

The domain organization and properties of individual domains of DNA topoisomerase V, a type 1B topoisomerase with DNA repair activities.

Topoisomerase V (Topo V) is a type IB (eukaryotic-like) DNA topoisomerase. It was discovered in the hyperthermophilic prokaryote Methanopyrus kandleri and is the only topoisomerase with associated apurinic/apyrimidinic (AP) site-processing activities. The structure of Topo V in the free and DNA-bound states was probed by limited proteolysis at 37 degrees C and 80 degrees C. The Topo V protein is comprised of (i) a 44-kDa NH(2)-terminal core subdomain, which contains the active site tyrosine residue for topoisomerase activity, (ii) an immediately adjacent 16-kDa subdomain that contains degenerate helix-hairpin-helix (HhH) motifs, (iii) a protease-sensitive 18-kDa HhH "hinge" region, and (iv) a 34-kDa COOH-terminal HhH domain. Three truncated Topo V polypeptides comprising the NH(2)-terminal 44-kDa and 16-kDa domains (Topo61), the 44-, 16-, and 18-kDa domains (Topo78), and the COOH-terminal 34-kDa domain (Topo34) were cloned, purified, and characterized. Both Topo61 and Topo78 are active topoisomerases, but in contrast to Topo V these enzymes are inhibited by high salt concentrations. Topo34 has strong DNA-binding ability but shows no topoisomerase activity. Finally, we demonstrate that Topo78 and Topo34 possess AP lyase activities that are important in base excision DNA repair. Thus, Topo V has at least two active sites capable of processing AP DNA. The significance of multiple HhH motifs for the Topo V processivity is discussed.     

High and low molecular weight rabbit secretory components. Evidence for the deletion of the second and third domains in the smaller polypeptide

Rabbit secretory components exist in two forms which differ in apparent mass by about 25 kDa. Each of these two forms were reduced, carboxymethylated, and extensively digested with trypsin. The resulting peptides were purified by reverse-phase high performance liquid chromatography and characterized by NH2- and COOH-terminal sequence determination and/or amino acid analysis. They were aligned with the protein sequence predicted from the cDNA nucleotide sequence encoding the rabbit poly(Ig) receptor (Mostov, K. E., Friedlander, M., and Blobel, G. (1984) Nature 308, 37-43). All peptides belonging to the fourth and fifth domains except one (positions 488-496) were accounted for in both forms. In addition, limited tryptic proteolysis of the native low Mr secretory components produced the intact 18-kDa NH2-terminal domain (positions 1-117) and the 30-kDa fragment encompassing the fourth and fifth domains. These results suggest that the smaller polypeptide derives from the larger secretory component form by the deletion of the second and third domains.     

Isolation and molecular and functional properties of the amino-terminal domain of colicin A

A plasmid was constructed which allowed easy and efficient production and purification of the NH2-terminal domain of colicin A. In only three steps, an homogenous 18-kDa polypeptide was obtained. The NH2- and COOH-terminal sequences of the protein were determined and showed that it corresponded to the NH2-terminal 171 amino acid residues of the 63-kDa colicin A. Although colicin A is a highly asymmetric protein, hydrodynamic studies indicated that the NH2-terminal domain (designated AT) has a globular structure. This fragment is not the receptor-binding domain of colicin A but is required for the transfer of colicin A across the outer membrane of sensitive cells. However, it has a low affinity for phospholipid films and this affinity is not pH-dependent, in contrast to that of colicin A.     

Characterization of a 48-kDa nucleic-acid-binding fragment of nucleolin

Nucleolin (C23 or 100 kDa) is an abundant single-stranded-nucleic-acid-binding nucleolar protein proposed to be involved in the early stages of ribosome assembly. A stable 48-kDa fragment of the protein was produced either by proteolytic activity present in nucleolar extracts or by added trypsin. The hydrodynamic and DNA-binding properties of the 48-kDa fragment were compared with the parent molecule. Protein sequencing indicated that the fragment begins at residue 282; amino acid composition of the fragment including 10-12 methylated arginine residues suggested that the fragment contains the entire COOH-terminal two-thirds of the protein. The 48-kDa fragment was more globular than nucleolin, as indicated by a lower frictional coefficient (1.3 vs. 2.0 for nucleolin) and a similar sedimentation coefficient (4.1-4.3S) in spite of the reduction in molecular mass. Although the 48-kDa fragment retained single-stranded-DNA-binding activity, the binding capacity and the ability to reassociate DNA were about fivefold and sixfold lower, respectively, than nucleolin. Similarly, tenfold higher concentrations of the 48-kDa fragment were required to form nucleoprotein aggregates. These results suggest that nucleolin contains a globular COOH-terminal domain for nucleic-acid binding and a NH2-terminal region which is involved in protein-protein interactions and modulating nucleic-acid-binding activity.     

Two soluble forms of bovine carboxypeptidase H have different NH2-terminal sequences

Carboxypeptidase H is an important enzyme in the biosynthesis of many peptide hormones. Development of a rapid isolation procedure led to the purification of two soluble forms from acidic extracts of bovine pituitary glands. These two forms differed in apparent molecular size (56 and 53 kDa). Both forms were found in the anterior lobe while only the 53-kDa form was found in posterior lobe. Digestion with N-glycosidase F demonstrated that these two forms are not due to alternative glycosylation of a common polypeptide core. Both forms bind antibodies raised against a COOH-terminal peptide of the full-length protein showing that the difference between them is not due to proteolysis at the COOH terminus. These results also argue against the idea that proteolysis of COOH-terminal domains converts the membrane-associated form of this protein into a soluble form. NH2-terminal sequence analysis demonstrated different NH2 termini. The NH2-terminal sequence of the 56-kDa form begins at the site predicted for signal peptide cleavage. Ion-exchange chromatography resolved the 56-kDa form from the 53-kDa form. The two forms were catalytically active with very similar properties. These results show that bovine carboxypeptidase H can be posttranslationally processed at alternative sites and provide evidence against the idea of a prosequence that must be removed before enzyme activity can be expressed.     

A functional linker in human topoisomerase I is required for maximum sensitivity to camptothecin in a DNA relaxation assay.

Human topoisomerase I is composed of four major domains: the highly charged NH(2)-terminal region, the conserved core domain, the positively charged linker domain, and the highly conserved COOH-terminal domain. Near complete enzyme activity can be reconstituted by combining recombinant polypeptides that approximate the core and COOH-terminal domains, although DNA binding is reduced somewhat for the reconstituted enzyme (Stewart, L., Ireton, G. C., and Champoux, J. J. (1997) J. Mol. Biol. 269, 355-372). A reconstituted enzyme comprising the core domain plus a COOH-terminal fragment containing the complete linker region exhibits the same biochemical properties as a reconstituted enzyme lacking the linker altogether, and thus detachment of the linker from the core domain renders the linker non-functional. The rate of religation by the reconstituted enzyme is increased relative to the forms of the enzyme containing the linker indicating that in the intact enzyme the linker slows religation. Relaxation of plasmid DNA by full-length human topoisomerase I or a 70-kDa form of the enzyme that is missing only the non-essential NH(2)-terminal domain (topo70) is inhibited approximately 16-fold by the anticancer compound, camptothecin, whereas the reconstituted enzyme is nearly resistant to the inhibitory effects of the drug despite similar affinities for the drug by the two forms of the enzyme. Based on these results and in light of the crystal structure of human topoisomerase I, we propose that the linker plays a role in hindering supercoil relaxation during the normal relaxation reaction and that camptothecin inhibition of DNA relaxation depends on a direct effect of the drug on DNA rotation that is also dependent on the linker.     

Domain structure of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a glycoprotein of the endoplasmic reticulum

We present and evaluate a model for the secondary structure and membrane orientation of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the glycoprotein of the endoplasmic reticulum that controls the rate of cholesterol biosynthesis. This model is derived from proteolysis experiments that separate the 97-kilodalton enzyme into two domains, an NH2-terminal membrane-bound domain of 339 residues and a COOH-terminal water-soluble domain of 548 residues that projects into the cytoplasm and contains the catalytic site. These domains were identified by reaction with antibodies against synthetic peptides corresponding to specific regions in the molecule. Computer modeling of the reductase structure, based on the amino acid sequence as determined by molecular cloning, predicts that the NH2-terminal domain contains 7 membrane-spanning regions. Analysis of the gene structure reveals that each proposed membrane-spanning region is encoded in a separate exon and is separated from the adjacent membrane-spanning region by an intron. The COOH-terminal domain of the reductase is predicted to contain two beta-structures flanked by a series of amphipathic helices, which together may constitute the active site. The NH2-terminal membrane-bound domain of the reductase bears some resemblance to rhodopsin, the photoreceptor protein of retinal rod disks and the only other intracellular glycoprotein whose amino acid sequence is known.     

Mammalian heterogeneous nuclear ribonucleoprotein complex protein A1. Large-scale overproduction in Escherichia coli and cooperative binding to single-stranded nucleic acids

Characterization of mammalian heterogeneous nuclear ribonucleoprotein complex protein A1 is reported after large-scale overproduction of the protein in Escherichia coli and purification to homogeneity. A1 is a single-stranded nucleic acid binding protein of 320 amino acids and 34,214 Da. The protein has two domains. The NH2-terminal domain is globular, whereas the COOH-terminal domain of about 120 amino acids has low probability of alpha-helix structure and is glycinerich. Nucleic acid binding properties of recombinant A1 were compared with those of recombinant and natural proteins corresponding to the NH2-terminal domain. A1 bound to single-stranded DNA-cellulose with higher affinity than the NH2-terminal domain peptides. Protein-induced fluorescence enhancement was used to measure equilibrium binding properties of the proteins. A1 binding to poly (ethenoadenylate) was cooperative with the intrinsic association constant of 1.5 X 10(5) M-1 at 0.4 M NaCl and a cooperativity parameter of 30. The NH2-terminal domain peptides bound noncooperatively and with a much lower association constant. With these peptides and with intact A1, binding was fully reversed by increasing [NaCl]; yet. A1 binding was much less salt-sensitive than binding by the NH2-terminal domain peptides. A synthetic polypeptide analog of the COOH-terminal domain was prepared and was found to bind tightly to poly-(ethenoadenylate). The results are consistent with the idea that the COOH-terminal domain contributes to A1 binding through both cooperative protein-protein interaction and direct interaction with the nucleic acid.     

Interchain disulfide bonding in the regulatory subunit of cAMP-dependent protein kinase I

The two protomers of the purified regulatory subunit from porcine cAMP-dependent protein kinase I have been shown to be covalently cross-linked by interchain disulfide bonding. Limited proteolysis which cleaves the polypeptide chain into two fragments demonstrated that the disulfide bonding was associated exclusively with the fragment that corresponded to the NH2-terminal region of the polypeptide chain. This NH2-terminal fragment accounted for approximately 15 to 20% of the molecule. The disulfide bonding was further characterized by alkylating the cysteines in the native regulatory subunit. Following oxidation with performic acid, each regulatory subunit contained 7 cysteic acid residues; however, under denaturing conditions, but without prior reduction, only 5 cysteine residues could be alkylated with iodoacetic acid. Following limited proteolysis, all five of these cysteines were associated with the larger COOH-terminal, cAMP binding domain. In contrast, if the denatured subunit was first reduced prior to alkylation, all 7 cysteine residues were alkylated. The 2 cysteines that were only accessible to alkylation after prior reduction were both associated with the NH2-terminal end of the polypeptide chain ultimately with a 5,400 peptide. Alkylation of the isolated, denatured NH2-terminal domain with iodoacetic acid resulted in no covalent modification unless the fragment was first reduced with dithiothreitol. The NH2-terminal and COOH-terminal domains were shown to be linked by a region of the polypeptide chain that is rich in both proline and arginine. It is the arginine-rich site that is readily prone to proteolytic cleavage.     

Quaternary structure of the HSC70 cochaperone HIP

HSC70 interacting protein (HIP) is an essential cytoplasmic cochaperone involved in the regulation of HSC70 chaperone activity and the maturation of progesterone receptor. To determine the quaternary structure and the gross conformation of the protein in solution, a wide array of biochemical and biophysical techniques has been used. Size-exclusion chromatography and sedimentation velocity indicate the presence of a single species with a Stokes radius, R(s), of 55 A and a sedimentation coefficient, s degrees (20,w), of 4.34 S. The combination of these data gives a molecular mass of 101 000 Da, a value close to that of the theoretical molecular mass of a dimer (87 090 Da). Sedimentation equilibrium, performed at various protein concentrations and rotor speeds, gives a molecular mass of 88 284 Da, almost in exact agreement with the molecular mass of a dimer. On the basis of these data, a frictional ratio f/f(0) of 1.6 is obtained, suggesting an elongated shape for the HIP dimer. Secondary structure predictions, supported by circular dichroism experiments, indicate that HIP is an almost all alpha-protein, able to form extended coiled coils. Using threading and comparative model building methods, a structural model of a segment of HIP involved in HSC70 binding has been constructed and potential sites of interaction between HIP and HSC70 are proposed on the basis of electrostatic as well as shape complementarity. Altogether, these results indicate that HIP is an elongated dimer, able to bind two HSC70 molecules through its TPR regions, and suggest the existence of a versatile binding site on HSC70 that may be involved in the interaction of the chaperone with the cochaperones or other interacting proteins.     

Roles of H1 domains in determining higher order chromatin structure and H1 location

Peptides derived from calf thymus H1 and rat liver H1, comprising only the globular and COOH-terminal domains of the intact molecule and therefore lacking NH2-terminal domains, have been shown by reconstitution to be as effective as the complete H1 molecule in inducing higher-order-chromatin structure. As the globular domain of H1 alone cannot induce chromatin folding, our results demonstrate that this function is primarily controlled by the COOH-terminal domain of the molecule. Surprisingly, these peptides do not locate correctly with respect to the nucleosome. This is demonstrated by their failure to confer upon reconstitutes the ability to protect DNA fragments of chromatosome length when digested with micrococcal nuclease. The precise placement of the H1 molecule (globular domain) with respect to the nucleosome is shown to be influenced by the "tail" domains of both H1 and the core histones.     

A characteristic property of vitamin K-dependent plasma protein Z

Evidence is presented for rapid, limited proteolysis of protein Z by alpha-thrombin. This alpha-thrombin-catalyzed proteolysis of protein Z occurred at a single peptide linkage, between Arg-365 and Gly-366, located in the COOH-terminal portion. The resulting NH2-terminal large fragment (PZt) and the COOH-terminal peptide (C-peptide) were isolated and chemically characterized. The C-peptide consisted of 31 amino acid residues including one galactosamine-type Thr residue and was assigned to the position from Gly-366 to the COOH-terminal residue of Val-396 in protein Z. The NH2-terminal large fragment, PZt, constituted the remainder of protein Z. The abilities to bind calcium of intact protein Z, PZt, and the derivative of protein Z devoid of the NH2-terminal gamma-carboxyglutamic acid (Gla) domain (Gla-domainless), prepared with the known chymotrypsin treatment, were examined by equilibrium dialysis. The results indicated that intact protein Z and PZt contain four calcium binding sites with dissociation constants of 0.1 mM. Moreover, the Scatchard plot analysis showed positive cooperativity, suggesting the presence of at least two initial sites for calcium binding. In contrast, the Gla-domainless protein Z had no calcium binding site, indicating that the domain of protein Z functional for calcium binding occurs within the NH2-terminal Gla domain. This differed from factor X, factor IX, protein S, and protein C, all of which contain one or two calcium binding site(s) independent on their Gla-domains.     

Domain structure of the large subunit of Escherichia coli carbamoyl phosphate synthetase. Location of the binding site for the allosteric inhibitor UMP in the COOH-terminal domain

The large subunit of Escherichia coli carbamoyl phosphate synthetase (a polypeptide of 117.7 kDa that consists of two homologous halves) is responsible for carbamoyl phosphate synthesis from NH3 and for the binding of the allosteric activators ornithine and IMP and of the inhibitor UMP. Elastase, trypsin, and chymotrypsin inactivate the enzyme and cleave the large subunit at a site approximately 15 kDa from the COOH terminus (demonstrated by NH2-terminal sequencing). UMP, IMP, and ornithine prevent this cleavage and the inactivation. Upon irradiation with ultraviolet light in the presence of [14C]UMP, the large subunit is labeled selectively and specifically. The labeling is inhibited by ornithine and IMP. Cleavage of the 15-kDa COOH-terminal region by prior treatment of the enzyme with trypsin prevents the labeling on subsequent irradiation with [14C]UMP. The [14C]UMP-labeled large subunit is resistant to proteolytic cleavage, but if it is treated with SDS the resistance is lost, indicating that UMP is cross-linked to its binding site and that the protection is due to conformational factors. In the presence of SDS, the labeled large subunit is cleaved by trypsin or by V8 staphylococcal protease at a site located 15 or 25 kDa, respectively, from the COOH terminus (shown by NH2-terminal sequencing), and only the 15- or 25-kDa fragments are labeled. Similarly, upon cleavage of the aspartyl-prolyl bonds of the [14C]UMP-labeled enzyme with 70% formic acid, labeling was found only in the 18.5-kDa fragment that contains the COOH terminus of the subunit. Thus, UMP binds to the COOH-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection and localization of a cytoplasmic domain on the beta-subunit of Na+,K+-ATPase. A monoclonal antibody study

A monoclonal antibody directed against the beta-subunit of dog kidney Na+,K+-ATPase was generated. Immunoblots demonstrate that monoclonal antibody III 18A binds exclusively to the denaturated beta-subunit. Binding experiments with membranes and whole cells reveal that III 18A binds to membranes but not to whole cells, indicating that the antibody binds to a cytoplasmic domain on the native beta-subunit. To localize the antibody-binding epitope, purified membrane-bound enzyme was fragmented by protease treatment. Tryptic digestion yields a 30-kDa fragment of the beta-subunit, which still retains the binding capacity for the antibody. Thus III 18A probably does not bind to the NH2-terminal segment of the protein. On the other hand, fragmentation of the beta-subunit with low concentrations of papain, which is known to yield a 40-kDa NH2-terminal and a 16-kDa COOH-terminal fragment, results in a complete loss of III 18A binding. These results suggest that the antibody-binding epitope is localized at or near a papain cleavage site on the COOH-terminal part of the beta-subunit. This is inconsistent with a structure model of the beta-subunit containing only a single transmembrane hydrophobic segment with a cytoplasmic NH2-terminal portion, but agrees quite well with a hypothetical structure with four intramembrane segments.     

Expression of plectin mutant cDNA in cultured cells indicates a role of COOH-terminal domain in intermediate filament association

Plectin is an intermediate filament (IF) binding protein of exceptionally large size. Its molecular structure, revealed by EM and predicted by its sequence, indicates an NH2-terminal globular domain, a long rodlike central domain, and a globular COOH-terminal domain containing six highly homologous repeat regions. To examine the role of the various domains in mediating plectin's interaction with IFs, we have constructed rat cDNAs encoding truncated plectin mutants under the control of the SV-40 promoter. Mutant proteins expressed in mammalian COS and PtK2 cells could be distinguished from endogenous wild type plectin by virtue of a short carboxy-terminal antigenic peptide (P tag). As shown by conventional and confocal immunofluorescence microscopy, the transient expression of plectin mutants containing all six or the last four of the repeat regions of the COOH-terminus, or the COOH-terminus and the rod, associated with IF networks of both the vimentin and the cytokeratin type and eventually caused their collapse into perinuclear aggregates. Similar effects were observed upon expression of a protein encoded by a full length cDNA construct. Microtubules and microfilaments were unaffected. Unexpectedly, mutants containing the rod without any of the COOH-terminal repeats, accumulated almost exclusively within the nuclei of cells. When the rod was extended by the first one and a half of the COOH-terminal repeats, mutant proteins showed a partial cytoplasmic distribution, although association with intermediate filaments was not observed. Nuclear and diffuse cytoplasmic distribution was also observed upon expression of the NH2-terminal domain without rod. These results indicate that sequences located roughly within the last two thirds of the globular COOH-terminus are indispensable for association of plectin with intermediate filaments in living cells.     

Localization of the actin-binding sites of Acanthamoeba myosin IB and effect of limited proteolysis on its actin-activated Mg2+-ATPase activity

Acanthamoeba myosin IB contains a 125-kDa heavy chain that has high actin-activated Mg2+-ATPase activity when 1 serine residue is phosphorylated. The heavy chain contains two F-actin-binding sites, one associated with the catalytic site and a second which allows myosin IB to cross-link actin filaments but has no direct effect on catalytic activity. Tryptic digestion of the heavy chain initially produces an NH2-terminal 62-kDa peptide that contains the ATP-binding site and the regulatory phosphorylation site, and a COOH-terminal 68-kDa peptide. F-actin, in the absence of ATP, protects this site and tryptic cleavage then produces an NH2-terminal 80-kDa peptide. Both the 62- and the 80-kDa peptides retain the (NH+4,EDTA)-ATPase activity of native myosin IB and both bind to F-actin in an ATP-sensitive manner. However, only the 80-kDa peptide retains a major portion of the actin-activated Mg2+-ATPase activity. This activity requires phosphorylation of the 80-kDa peptide by myosin I heavy chain kinase but, in contrast to the activity of intact myosin IB, it has a simple, hyperbolic dependence on the concentration of F-actin. Also unlike myosin IB, the 80-kDa peptide cannot cross-link F-actin filaments indicating the presence of only a single actin-binding site. These results allow the assignment of the actin-binding site involved in catalytic activity to the region near, and possibly on both sides of, the tryptic cleavage site 62 kDa from the NH2 terminus, and the second actin-binding site to the COOH-terminal 45-kDa domain. Thus, the NH2-terminal 80 kDa of the myosin IB heavy chain is functionally similar to the 93-kDa subfragment 1 of muscle myosin and most likely has a similar organization of functional domains.     

Novel alternative splicings of BPAG1 (bullous pemphigoid antigen 1) including the domain structure closely related to MACF (microtubule actin cross-linking factor).

BPAG1 (bullous pemphigoid antigen 1) was originally identified as a 230-kDa hemidesmosomal protein and belongs to the plakin family, because it consists of a plakin domain, a coiled-coil rod domain and a COOH-terminal intermediate filament binding domain. To date, alternatively spliced products of BPAG1, BPAG1e, and BPAG1n are known. BPAG1e is expressed in epithelial tissues and localized to hemidesmosomes, on the other hand, BPAG1n is expressed in neural tissues and muscles and has an actin binding domain at the NH(2)-terminal of BPAG1e. BPAG1 is also known as a gene responsible for Dystonia musculorum (dt) neurodegeneration syndrome of the mouse. Another plakin family protein MACF (microtubule actin cross-linking factor) has also an actin binding domain and the plakin domain at the NH(2)-terminal. However, in contrast to its high homology with BPAG1 at the NH(2)-terminal, the COOH-terminal structure of MACF, including a microtubule binding domain, resembles dystrophin rather than plakins. Here, we investigated RNAs and proteins expressed from the BPAG1 locus and suggest novel alternative splicing variants, which include one consisting of the COOH-terminal domain structure homologous to MACF. The results indicate that BPAG1 has three kinds of cytoskeletal binding domains and seems to play an important role in linking the different types of cytoskeletons.     

Substructure and higher structure of chicken smooth muscle alpha-actinin molecule

Substructure of chicken gizzard smooth muscle alpha-actinin molecule was deduced by domainal mapping of the proteolytic fragments with alpha-chymotrypsin. There were three chymotryptic cleavage sites (Sites I, II, and III, from the amino terminus). Cleavage at Site I generated two fragments, i.e. an NH2-terminal 36-kDa fragment and a COOH-terminal 70-kDa fragment. The 70-kDa fragment generated either a 55-kDa fragment by cleavage at Site II or a 65-kDa fragment by cleavage at Site III. Purified NH2-terminal 36-kDa fragment bound to F-actin, whereas the 55-kDa fragment formed a dimeric molecule. Circular dichroism and electron microscopic experiments demonstrated that the alpha-helical content of the 55-kDa fragment was 14% higher than that of native gizzard alpha-actinin, coinciding with the apparently rod-shaped configuration of this fragment. A 110-kDa product was generated from two 55-kDa fragments in a cross-linking study with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Two cross-linkable sites in the 55-kDa, A- and B-site, were shown to be involved in this reaction. Further, it was demonstrated by using N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide labeling and immunoblotting analyses that the A-site on one 55-kDa fragment was cross-linked to the B-site on the other. These results suggest that smooth muscle alpha-actinin formed an antiparallel dimeric molecule in which the 55-kDa fragments connected the two actin-binding domains composed of the 36-kDa fragments.     

Mapping of the functional domains in the amino-terminal region of calponin.

Three chymotryptic fragments accounting for almost the entire amino acid sequence of gizzard calponin (Takahashi, K., and Nadal-Ginard, B. (1991) J. Biol. Chem. 266, 13284-13288) were isolated and characterized. They encompass the segments of residues 7-144 (NH2-terminal 13-kDa peptide), 7-182 (NH2-terminal 22-kDa peptide), and 183-292 (COOH-terminal 13-kDa peptide). They arise from the sequential hydrolysis of the peptide bonds at Tyr182-Gly183 and Tyr144-Ala145 which were protected by the binding of F-actin to calponin. Only the NH2-terminal 13- and 22-kDa fragments were retained by immobilized Ca(2+)-calmodulin, but only the larger 22 kDa entity cosedimented with F-actin and inhibited, in the absence of Ca(2+)-calmodulin, the skeletal actomyosin subfragment-1 ATPase activity as the intact calponin. Since the latter peptide differs from the NH2-terminal 13-kDa fragment by a COOH-terminal 38-residue extension, this difference segment appears to contain the actin-binding domain of calponin. Zero-length cross-linked complexes of F-actin and either calponin or its 22-kDa peptide were produced. The total CNBr digest of the F-actin-calponin conjugate was fractionated over immobilized calmodulin. The EGTA-eluted pair of cross-linked actin-calponin peptides was composed of the COOH-terminal actin segment of residues 326-355 joined to the NH2-terminal calponin region of residues 52-168 which seems to contain the major determinants for F-actin and Ca(2+)-calmodulin binding.