The complete amino acid sequence of a biologically active 197-residue fragment of M protein isolated from type 5 group A streptococci

The complete amino acid sequence of a peptic fragment (Pep M5) of the group A streptococcal type 5 M protein, the antiphagocytic cell surface molecule of the bacteria, is described. This fragment, comprising nearly half of the native M molecule, is biologically active in that it has the ability to interact with opsonic antibodies as well as to evoke such an antibody response in rabbits. The sequence of Pep M5 was determined by automated Edman degradations of the uncleaved molecule and its enzymatically derived peptides. The primary peptides for Edman degradation were the arginine peptides obtained by tryptic digestion. The tryptic cleavage of Pep M5 was limited to the arginyl peptide bonds by derivatizing the epsilon-amino groups of lysine residues by reductive dihydroxypropylation. The overlapping peptides were generated by digestion of the unmodified Pep M5 with chymotrypsin, V8 protease, and subtilisin. The sequence thus established for the Pep M5 molecule consists of a total of 197 residues (Mr = 22,705). The Pep M5 protein contains some identical, or nearly so, repeating sequences: four 7-residue segments and two 10-residue segments. However, extensive sequence repeats of the kind previously reported within the partial sequence of another M protein serotype, namely Pep M24, were absent. The Pep M5 sequence is distinct from, but exhibits some homology with, the partial sequences of two other M protein serotypes, namely, Pep M6 and Pep M24. Furthermore, the 7-residue periodicity of the nonpolar and charged residues, an alpha-helical coiled-coil structural characteristic that was previously observed within the partial sequences of M proteins, was found to extend over a significant part of the Pep M5 sequence. The implication of these results to the function and immunological diversity in M proteins is discussed.     

The tip of the coiled-coil rod determines the filament formation of smooth muscle and nonmuscle myosin

Myosin II self-assembles to form thick filaments that are attributed to its long coiled-coil tail domain. The present study has determined a region critical for filament formation of vertebrate smooth muscle and nonmuscle myosin II. A monoclonal antibody recognizing the 28 residues from the C-terminal end of the coiled-coil domain of smooth muscle myosin II completely inhibited filament formation, whereas other antibodies recognizing other parts of the coiled-coil did not. To determine the importance of this region in the filament assembly in vivo, green fluorescent protein (GFP)-tagged smooth muscle myosin was expressed in COS-7 cells, and the filamentous localization of the GFP signal was monitored by fluorescence microscopy. Wild type GFP-tagged smooth muscle myosin colocalized with F-actin during interphase and was also recruited into the contractile ring during cytokinesis. Myosin with the nonhelical tail piece deleted showed similar behavior, whereas deletion of the 28 residues at the C-terminal end of the coiled-coil domain abolished this localization. Deletion of the corresponding region of GFP-tagged nonmuscle myosin IIA also abolished this localization. We conclude that the C-terminal end of the coiled-coil domain, but not the nonhelical tail piece, of myosin II is critical for myosin filament formation both in vitro and in vivo.     

Structure and flexibility of the tropomyosin overlap junction

To be effective as a gatekeeper regulating the access of binding proteins to the actin filament, adjacent tropomyosin molecules associate head-to-tail to form a continuous super-helical cable running along the filament surface. Chimeric head-to-tail structures have been solved by NMR and X-ray crystallography for N- and C-terminal segments of smooth and striated muscle tropomyosin spliced onto non-native coiled-coil forming peptides. The resulting 4-helix complexes have a tight coiled-coil N-terminus inserted into a separated pair of C-terminal helices, with some helical unfolding of the terminal chains in the striated muscle peptides. These overlap complexes are distinctly curved, much more so than elsewhere along the superhelical tropomyosin cable. To verify whether the non-native protein adducts (needed to stabilize the coiled-coil chimeras) perturb the overlap, we carried out Molecular Dynamics simulations of head-to-tail structures having only native tropomyosin sequences. We observe that the splayed chains all refold and become helical. Significantly, the curvature of both the smooth and the striated muscle overlap domain is reduced and becomes comparable to that of the rest of the tropomyosin cable. Moreover, the measured flexibility across the junction is small. This and the reduced curvature ensure that the super-helical cable matches the contours of F-actin without manifesting localized kinking and excessive flexibility, thus enabling the high degree of cooperativity in the regulation of myosin accessibility to actin filaments.     

YspD: A Potential Therapeutic Target for Drug Design to Combat Yersinia enterocolitica Infection

YspD is an annotated hydrophilic translocator of Ysa–Ysp type III secretion system of Yersinia enterocolitica. YspD has sequence, secondary structure and three-dimensional structure similar to other hydrophilic translocators. All hydrophilic translocators lack transmembrane region and possess intramolecular coiled-coil region. Disordered regions are mostly clustered at the N-terminal. Large loops provide flexibility, allowing conformational changes during oligomerization and protein–protein interaction. LcrV and PcrV have globular N-terminal and C-terminal domains, connected by intramolecular coiled-coil region. YspD, IpaD, SipD and BipD lack globular N-terminal and C-terminal domains. Their N-terminal and C-terminal domain have a bundle like structure connected by the intramolecular coiled-coil. The intramolecular coiled-coil regions (helix-5&9) of YspD showed maximum conservation, followed by helices at N-terminal. Polar interactions are mainly involved during dimerization of YspD, involving polar residues from helix-9 of both the YspD molecules. A methionine forms the boundary of interaction between the two YspD molecules. The two YspD molecules are arranged in antiparallel fashion to form the dimer. N-terminal of YspB interacted with C-terminal of YspD molecule to form a pentameric complex, consisting four YspD molecules and one YspB molecule. Sequence, structural similarity and presence of specific motifs in YspD (like chaperone protein) indicate the ability of N-terminal domain to show self-chaperoning activity and regulate folding and conformational state of YspD during its journey from the bacterial cytoplasm to the needle tip. Structural analysis of YspD and its mechanism of interaction with other proteins would enable us to design drugs against this hydrophilic protein to combat Yersinia infection.

     

Push and pull of tropomyosin's opposite effects on myosin attachment to actin. A chimeric tropomyosin host-guest study

Tropomyosin is a ubiquitous actin-binding protein with an extended coiled-coil structure. Tropomyosin-actin interactions are weak and loosely specific, but they potently influence myosin. One such influence is inhibitory and is due to tropomyosin's statistically preferred positions on actin that sterically interfere with actin's strong attachment site for myosin. Contrastingly, tropomyosin's other influence is activating. It increases myosin's overall actin affinity ～4-fold. Stoichiometric considerations cause this activating effect to equate to an ～4(7)-fold effect of myosin on the actin affinity of tropomyosin. These positive, mutual, myosin-tropomyosin effects are absent if Saccharomyces cerevisiae tropomyosin replaces mammalian tropomyosin. To investigate these phenomena, chimeric tropomyosins were generated in which 38-residue muscle tropomyosin segments replaced a natural duplication within S. cerevisiae tropomyosin TPM1. Two such chimeric tropomyosins were sufficiently folded coiled coils to allow functional study. The two chimeras differed from TPM1 but in opposite ways. Consistent with steric interference, myosin greatly decreased the actin affinity of chimera 7, which contained muscle tropomyosin residues 228-265. On the other hand, myosin S1 increased by an order of magnitude the actin affinity of chimera 3, which contained muscle tropomyosin residues 74-111. Similarly, myosin S1-ADP binding to actin was strengthened 2-fold by substitution of chimera 3 tropomyosin for wild-type TPM1. Thus, a yeast tropomyosin was induced to mimic the activating behavior of mammalian tropomyosin by inserting a mammalian tropomyosin sequence. The data were not consistent with direct tropomyosin-myosin binding. Rather, they suggest an allosteric mechanism, in which myosin and tropomyosin share an effect on the actin filament.     

Type 1 M protein of Streptococcus pyogenes. N-terminal sequence and peptic fragments

Limited proteolysis of the surface of type 1 Streptococcus pyogenes by pepsin gives rise to fragment Pep M1 of Mr 20270 as the main product which covers the N-terminal part of the M protein. The amino acid sequence was determined of the N-terminal region of the M protein representing the most exposed part of the molecule on the surface fibrils of streptococcal cells, which seems to be very important for the differentiation of the individual serological types. The sequence differs from the homologous N-terminal sequences of types 5, 6 and 24, and shows a homology with sequences repeating in the chain of type 24. Fragment Pep M1 binds to fibrinogen; the absence of its 30 N-terminal amino acid residues, however, abolishes this interaction which is believed to play a role in the virulence of S. pyogenes.     

Unfolding domains of recombinant fusion alpha alpha-tropomyosin.

The thermal unfolding of the coiled-coil alpha-helix of recombinant alpha alpha-tropomyosin from rat striated muscle containing an additional 80-residue peptide of influenza virus NS1 protein at the N-terminus (fusion-tropomyosin) was studied with circular dichroism and fluorescence techniques. Fusion-tropomyosin unfolded in four cooperative transitions: (1) a pretransition starting at 35 degrees C involving the middle of the molecule; (2) a major transition at 46 degrees C involving no more than 36% of the helix from the C-terminus; (3) a major transition at 56 degrees C involving about 46% of the helix from the N-terminus; and (4) a transition from the nonhelical fusion domain at about 70 degrees C. Rabbit skeletal muscle tropomyosin, which lacks the fusion peptide but has the same tropomyosin sequence, does not exhibit the 56 degrees C or 70 degrees C transition. The very stable fusion unfolding domain of fusion-tropomyosin, which appears in electron micrographs as a globular structural domain at one end of the tropomyosin rod, acts as a cross-link to stabilize the adjacent N-terminal domain. The least stable middle of the molecule, when unfolded, acts as a boundary to allow the independent unfolding of the C-terminal domain at 46 degrees C from the stabilized N-terminal unfolding domain at 56 degrees C. Thus, strong localized interchain interactions in coiled-coil molecules can increase the stability of neighboring domains.     

Slac2-c (synaptotagmin-like protein homologue lacking C2 domains-c), a novel linker protein that interacts with Rab27, myosin Va/VIIa,and actin

Slac2-a (synaptotagmin-like protein (Slp) homologue lacking C2 domains-a)/melanophilin is a melanosome-associated protein that links Rab27A on melanosomes with myosin Va, an actin-based motor protein, and formation of the tripartite protein complex (Rab27A.Slac2-a.myosin Va) has been suggested to regulate melanosome transport (Fukuda, M., Kuroda, T. S., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 12432-12436). Here we report the structure of a novel form of Slac2, named Slac2-c, that is homologous to Slac2-a. Slac2-a and Slac2-c exhibit the same overall structure, consisting of a highly conserved N-terminal Slp homology domain (about 50% identity) and a less conserved C-terminal myosin Va-binding domain (about 20% identity). As with other Slac2 members and the Slp family, the Slp homology domain of Slac2-c was found to interact specifically with the GTP-bound form of Rab27A/B both in vitro and in intact cells, and the C-terminal domain of Slac2-c interacted with myosin Va and myosin VIIa. In addition, we discovered that the most C-terminal conserved region of Slac2-a (amino acids 400-590) and Slac2-c (amino acids 670-856), which is not essential for myosin Va binding, directly binds actin and that expression of these regions in PC12 cells and melanoma cells colocalized with actin filaments at the cell periphery, suggesting a novel role of Slac2-a/c in capture of Rab27-containing organelles in the actin-enriched cell periphery.     

Crosslinking of actin filaments and inhibition of actomyosin subfragment-1 ATPase activity by streptococcal M6 protein

M proteins are antiphagocytic molecules on the surface of group A streptococci having physical characteristics similar to those of mammalian tropomyosin. Both are alpha-helical coiled-coil fibrous structures with a similar seven-residue periodicity of nonpolar and charged amino acids. To determine if M protein is functionally similar to tropomyosin we studied the interaction of M protein with F-actin. At low ionic strength, M protein binds to actin weakly with a stoichiometry different from that of tropomyosin. M protein does not compete with tropomyosin for the binding to actin, indicating that it is functionally different from tropomyosin. M protein does compete with myosin subfragment-1 for binding to actin and induces the formation of bundles of actin filaments. The formation of actin aggregates is associated with a sharp reduction in the rate of ATP hydrolysis by subfragment-1. Intact streptococci having M protein on their surface are shown to bind to actin.     

Heptad motifs within the distal subdomain of the coiled-coil rod region of M protein from rheumatic fever and nephritis associated serotypes of group A streptococci are distinct from each other: Nucleotide sequence of the M57 gene and relation of the deduced amino acid sequence to other M proteins

Streptococcal M protein, a dimeric alpha helical coiled-coil molecule, is an antigenically variable virulence factor on the surface of the bacteria. Our recent conformational analysis of the complete sequence of the M6 protein led us to propose a basic model for the M protein consisting of an extended central coiled-coil rod domain flanked by a variable N-terminal and a conserved C-terminal end domains. The central coiled-coil rod domain of M protein, which constitutes the major part of the M molecule, is made up of repeating heptads of the generalized sequence a-b-c-d-e-f-g, wherein a and d are predominantly apolar residues. Based on the differences in the heptad pattern of apolar residues and internal sequence homology, the central coiled-coil rod domain of M protein could be further divided into three subdomains I, II, and III. The streptococcal sequelae rheumatic fever (RF) and acute glomerulonephritis (AGN) have been known to be associated with distinct serotypes. Consistent with this, we observed that the AGN associated M49 protein exhibits a heptad motif that is distinct from the RF associated M5 and M6 proteins. Asn and Leu predominated in the a and d positions, respectively, in subdomain I of the M5 and M6 proteins, whereas apolar residues predominated in both these positions in the M49 protein. To establish whether the heptad motif of M49 is unique to this protein, or is a general characteristic of nephritis-associated serotypes, the amino acid sequence of M57, another nephritis-associated serotype, has now been examined. The gene encoding M57 was amplified by PCR, cloned into pUC19 vector, and sequenced. The C-terminal half of M57 is highly homologous to other M proteins (conserved region). In contrast, its N-terminal half (variable region) revealed no significant homology with any of the M proteins. Heptad periodicity analysis of the M57 sequence revealed that the basic design principles, consisting of distinct domains observed in the M6 protein, are also conserved in the M57 molecule. However, the heptad motif within the coiled-coil subdomain I of M57 was distinct from M5 and M6 but similar to M49. Similar analyses of the heptad characteristics within the reported sequences of M1, M12, and M24 proteins further confirmed the conservation of the overall architectural design of sequentially distinct M proteins. Furthermore, the heptad motif within subdomain I of the AGN-associated serotypes M1 and M12 was similar to M49 and M57, whereas that of the RF associated M24 was similar to the M5 and M6 proteins. These results clearly demonstrate a correlation between the heptad motifs within the distal coiled-coil subdomain of the M proteins from different streptococcal serotypes and their epidemiological association with the sequelae AGN and RF.     

Tropomyosin shares immunologic epitopes with group A streptococcal M proteins

Tropomyosin is an alpha-helical coiled-coil protein with structural similarities to the streptococcal M protein. In order to show serologic cross-reactivity between streptococcal M proteins and tropomyosin, we selected from a panel of murine mAb those which reacted with M proteins and tropomyosins in the ELISA. Western blots were used to study the reactions of each mAb with human and rabbit cardiac and rabbit skeletal tropomyosins. The antibodies were further characterized for their reactions with the additional autoantigens myosin, actin, keratin, and DNA. Five mAb were found which reacted with either PepM5 or ColiM6 protein and tropomyosin in Western blots or ELISA. Two of the tropomyosin positive mAb were also antinuclear antibodies and were inhibited with DNA. In Western blots of cardiac tropomyosins, the mAb reacted with either the 70-kDa dimer of tropomyosin, the 35-kDa monomer, or both. Some differences were observed in the reactions of the mAb with the different tropomyosins in Western blots. The heart cross-reactive epitopes shared between M proteins and tropomyosin were in most instances shared with cardiac myosin. Differences were observed among the reactions of the mAb with the different tropomyosins. This report constitutes the first evidence of serologic cross-reactivity between streptococcal M proteins and tropomyosins.     

Solution NMR structure of the junction between tropomyosin molecules: implications for actin binding and regulation

Tropomyosin is a coiled-coil protein that binds head-to-tail along the length of actin filaments in eukaryotic cells, stabilizing them and providing protection from severing proteins. Tropomyosin cooperatively regulates actin's interaction with myosin and mediates the Ca2+ -dependent regulation of contraction by troponin in striated muscles. The N-terminal and C-terminal ends are critical functional determinants that form an "overlap complex". Here we report the solution NMR structure of an overlap complex formed of model peptides. In the complex, the chains of the C-terminal coiled coil spread apart to allow insertion of 11 residues of the N-terminal coiled coil into the resulting cleft. The plane of the N-terminal coiled coil is rotated 90 degrees relative to the plane of the C terminus. A consequence of the geometry is that the orientation of postulated periodic actin binding sites on the coiled-coil surface is retained from one molecule to the next along the actin filament when the overlap complex is modeled into the X-ray structure of tropomyosin determined at 7 Angstroms. Nuclear relaxation NMR data reveal flexibility of the junction, which may function to optimize binding along the helical actin filament and to allow mobility of tropomyosin on the filament surface as it switches between regulatory states.     

The structure of the carboxyl terminus of striated alpha-tropomyosin in solution reveals an unusual parallel arrangement of interacting alpha-helices

Coiled coils are well-known as oligomerization domains, but they are also important sites of protein-protein interactions. We determined the NMR solution structure and backbone (15)N relaxation rates of a disulfide cross-linked, two-chain, 37-residue polypeptide containing the 34 C-terminal residues of striated muscle alpha-tropomyosin, TM9a(251-284). The peptide binds to the N-terminal region of TM and to the tropomyosin-binding domain of the regulatory protein, troponin T. Comparison of the NMR solution structure of TM9a(251-284) with the X-ray structure of a related peptide [Li, Y., Mui, S., Brown, J. H., Strand, J., Reshetnikova, L., Tobacman, L. S., and Cohen, C. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 7378-7383] reveals significant differences. In solution, residues 253-269 (like most of the tropomyosin molecule) form a canonical coiled coil. Residues 270-279, however, are parallel, linear helices, novel for tropomyosin. The packing between the parallel helices results from unusual interface residues that are atypical for coiled coils. Y267 has poor packing at the coiled-coil interface and a lower R(2) relaxation rate than neighboring residues, suggesting there is conformational flexibility around this residue. The last five residues are nonhelical and flexible. The exposed surface presented by the parallel helices, and the flexibility around Y267 and the ends, may facilitate binding to troponin T and formation of complexes with the N-terminus of tropomyosin and actin. We propose that unusual packing and flexibility are general features of coiled-coil domains in proteins that are involved in intermolecular interactions.     

Conformational characteristics of the complete sequence of group A streptococcal M6 protein

M protein is considered a virulence determinant on the streptococcal cell wall by virtue of its ability to allow the organism to resist attack by human neutrophils. The complete DNA sequence of the M6 gene from streptococcal strain D471 has allowed, for the first time, the study of the structural characteristics of the amino acid sequence of an entire M protein molecule. Predictive secondary structural analysis revealed that the majority of this fibrillar molecule exhibits strong alpha-helical potential and that, except for the ends, nonpolar residues in the central region of the molecule exhibit the 7-residue periodicity typical for coiled-coil proteins. Differences in this heptad pattern of nonpolar residues allow this central rod region to be divided into three subdomains which correlate essentially with the repeat regions A, B, and C/D in the M6 protein sequence. Alignment of the N-terminal half of the M6 sequence with PepM5, the N-terminal half of the M5 protein, revealed that 42% of the amino acids were identical. The majority of the identities were "core" nonpolar residues of the heptad periodicity which are necessary for the maintenance of the coiled coil. Thus, conservation of structure in a sequence-variable region of these molecules may be biologically significant. Results suggest that serologically different M proteins may be built according to a basic scheme: an extended central coiled-coil rod domain (which may vary in size among strains) flanked by functional end domains.     

Altered affinity of CBF beta-SMMHC for Runx1 explains its role in leukemogenesis

Chromosomal translocations involving the human CBFB gene, which codes for the non-DNA binding subunit of CBF (CBF beta), are associated with a large percentage of human leukemias. The translocation inv(16) that disrupts the CBFB gene produces a chimeric protein composed of the heterodimerization domain of CBF beta fused to the C-terminal coiled-coil domain from smooth muscle myosin heavy chain (CBF beta-SMMHC). Isothermal titration calorimetry results show that this fusion protein binds the Runt domain from Runx1 (CBF alpha) with higher affinity than the native CBF beta protein. NMR studies identify interactions in the CBF beta portion of the molecule, as well as the SMMHC coiled-coil domain. This higher affinity provides an explanation for the dominant negative phenotype associated with a knock-in of the CBFB-MYH11 gene and also helps to provide a rationale for the leukemia-associated dysregulation of hematopoietic development that this protein causes.     

Proteolytic activity of YibP protein in Escherichia coli

Escherichia coli YibP protein (47.4 kDa) has a membrane-spanning signal at the N-terminal region, two long coiled-coil regions in the middle part, and a C-terminal globular domain, which involves amino acid sequences homologous to the peptidase M23/M37 family. A yibP disrupted mutant grows in rich medium at 37 degrees C but not at 42 degrees C. In the yibP null mutant, cell division and FtsZ ring formation are inhibited at 42 degrees C without SOS induction, resulting in filamentous cells with multiple nucleoids and finally in cell lysis. Five percent betaine suppresses the temperature sensitivity of the yibP disrupted mutation. The mutant has the same sensitivity to drugs, such as nalidixic acid, ethidium bromide, ethylmethane sulfonate, and sodium dodecyl sulfate, as the parental strain. YibP protein is recovered in the inner membrane and cytoplasmic fractions, but not in the outer membrane fraction. Results suggest that the coiled-coil regions and the C-terminal globular domain of YibP are localized in the cytoplasmic space, not in the periplasmic space. Purified YibP has a protease activity that split the substrate beta-casein.     

Crystal structure of a coiled-coil domain from human ROCK I

The small GTPase Rho and one of its targets, Rho-associated kinase (ROCK), participate in a variety of actin-based cellular processes including smooth muscle contraction, cell migration, and stress fiber formation. The ROCK protein consists of an N-terminal kinase domain, a central coiled-coil domain containing a Rho binding site, and a C-terminal pleckstrin homology domain. Here we present the crystal structure of a large section of the central coiled-coil domain of human ROCK I (amino acids 535-700). The structure forms a parallel α-helical coiled-coil dimer that is structurally similar to tropomyosin, an actin filament binding protein. There is an unusual discontinuity in the coiled-coil; three charged residues (E613, R617 and D620) are positioned at what is normally the hydrophobic core of coiled-coil packing. We speculate that this conserved irregularity could function as a hinge that allows ROCK to adopt its autoinhibited conformation.     

The troponin tail domain promotes a conformational state of the thin filament that suppresses myosin activity

In cardiac and skeletal muscles tropomyosin binds to the actin outer domain in the absence of Ca(2+), and in this position tropomyosin inhibits muscle contraction by interfering sterically with myosin-actin binding. The globular domain of troponin is believed to produce this B-state of the thin filament (Lehman, W., Hatch, V., Korman, V. L., Rosol, M., Thomas, L. T., Maytum, R., Geeves, M. A., Van Eyk, J. E., Tobacman, L. S., and Craig, R. (2000) J. Mol. Biol. 302, 593-606) via troponin I-actin interactions that constrain the tropomyosin. The present study shows that the B-state can be promoted independently by the elongated tail region of troponin (the NH(2) terminus (TnT-(1-153)) of cardiac troponin T). In the absence of the troponin globular domain, TnT-(1-153) markedly inhibited both myosin S1-actin-tropomyosin MgATPase activity and (at low S1 concentrations) myosin S1-ADP binding to the thin filament. Similarly, TnT-(1-153) increased the concentration of heavy meromyosin required to support in vitro sliding of thin filaments. Electron microscopy and three-dimensional reconstruction of thin filaments containing TnT-(1-153) and either cardiac or skeletal muscle tropomyosin showed that tropomyosin was in the B-state in the complete absence of troponin I. All of these results indicate that portions of the troponin tail domain, and not only troponin I, contribute to the positioning of tropomyosin on the actin outer domain, thereby inhibiting muscle contraction in the absence of Ca(2+).     

Partial deduced sequence of the 110-kD-calmodulin complex of the avian intestinal microvillus shows that this mechanoenzyme is a member of the myosin I family

The actin bundle within each microvillus of the intestinal brush border is laterally tethered to the membrane by bridges composed of the protein complex, 110-kD-calmodulin. Previous studies have shown that avian 110-kD-calmodulin shares many properties with myosins including mechanochemical activity. In the present study, a cDNA molecule encoding 1,000 amino acids of the 110-kD protein has been sequenced, providing direct evidence that this protein is a vertebrate homologue of the tail-less, single-headed myosin I first described in amoeboid cells. The primary structure of the 110-kD protein (or brush border myosin I heavy chain) consists of two domains, an amino-terminal "head" domain and a 35-kD carboxy-terminal "tail" domain. The head domain is homologous to the S1 domain of other known myosins, with highest homology observed between that of Acanthamoeba myosin IB and the S1 domain of the protein encoded by bovine myosin I heavy chain gene (MIHC; Hoshimaru, M., and S. Nakanishi. 1987. J. Biol. Chem. 262:14625- 14632). The carboxy-terminal domain shows no significant homology with any other known myosins except that of the bovine MIHC. This demonstrates that the bovine MIHC gene most probably encodes the heavy chain of bovine brush border myosin I (BBMI). A bacterially expressed fusion protein encoded by the brush border 110-kD cDNA binds calmodulin. Proteolytic removal of the carboxy-terminal domain of the fusion protein results in loss of calmodulin binding activity, a result consistent with previous studies on the domain structure of the 110-kD protein. No hydrophobic sequence is present in the molecule indicating that chicken BBMI heavy chain is probably not an integral membrane protein. Northern blot analysis of various chicken tissue indicates that BBMI heavy chain is preferentially expressed in the intestine.     

Tropomyosin lysine reactivities and relationship to coiled-coil structure

We have carried out a detailed analysis of tropomyosin structure using lysines as specific probes for the protein surface in regions of the molecule that have not been investigated by other methods. We have measured the relative reactivities of lysines in rabbit skeletal muscle alpha, alpha-tropomyosin with acetic anhydride using a competitive labeling procedure. We have identified 37 of 39 lysines and find that they range 20-fold in reactivity. The observed reactivities are related to the coiled-coil model of the tropomyosin molecule [Crick, F.H.C. (1953) Acta Crystallogr. 6, 689-697; McLachlan, A.D., Stewart, M., & Smillie, L.B. (1975) J. Mol. Biol. 98, 281-291] and other available chemical and physical information about the structure. In most cases, the observed lysine reactivities can be explained by allowable interactions with neighboring amino acid side chains on the same or facing alpha-helix. However, we found no correlation between reactivity and helical position of a given lysine. For example, lysines in the outer helical positions included lysines of low as well as high reactivity, indicating that they vary widely in their accessibility to solvent and that the coiled coil is heterogeneous along its length. Furthermore, the middle of the molecule (residues 126-182) that is susceptible to proteolysis and known to be the least stable region of the protein also contains some of the least and most reactive lysines. We have discussed the implications of our results on our understanding the structures of tropomyosin and other coiled-coil proteins as well as globular proteins containing helical regions.