Dynactin, a conserved, ubiquitously expressed component of an activator of vesicle motility mediated by cytoplasmic dynein

Although cytoplasmic dynein is known to attach to microtubules and translocate toward their minus ends, dynein's ability to serve in vitro as a minus end-directed transporter of membranous organelles depends on additional soluble factors. We show here that a approximately 20S polypeptide complex (referred to as Activator I; Schroer, T. A., and M.P. Sheetz. 1991a. J. Cell Biol. 115:1309-1318.) stimulates dynein-mediated vesicle transport. A major component of the activator complex is a doublet of 150-kD polypeptides for which we propose the name dynactin (for dynein activator). The 20S dynactin complex is required for in vitro vesicle motility since depletion of it with a mAb to dynactin eliminates vesicle movement. Cloning of a brain specific isoform of dynactin from chicken reveals a 1,053 amino acid polypeptide composed of two coiled-coil alpha-helical domains interrupted by a spacer. Both this structural motif and the underlying primary sequence are highly conserved in vertebrates with 85% sequence identity within a central 1,000-residue domain of the chicken and rat proteins. As abundant as dynein, dynactin is ubiquitously expressed and appears to be encoded by a single gene that yields at least three alternative isoforms. The probable homologue in Drosophila is the gene Glued, whose protein product shares 50% sequence identity with vertebrate dynactin and whose function is essential for viability of most (and perhaps all) cells in the organism.     

The effect of a side chain-backbone swap on protein stability.

To assess the relative importance of backbone hydrogen bonding (H-bonding) vs. side chain hydrophobicity in protein structural formation, a method called side chain-backbone swap is proposed. Such a method swaps the side chain and backbone portions of certain amino acid residues, such as Asp, Glu, Asn, Gln, Lys, and Arg. Such a swap retains the sequence of a polypeptide and preserves the identity of the backbone linkage. On the other hand, the swap disrupts backbone H-bonding geometry because of the introduction of extra methylene groups into the peptide backbone. In this project, we chose the two-stranded alpha-helical coiled-coil to implement side chain-backbone swap. A pair of 36-residue peptides was designed. The two peptides have identical sequence with four residues in each heptad repeat occupied by glutamyl residues. Each glutamic acid was incorporated either as alpha-glutamyl residue (the peptide is denoted as alpha-Glu-36) or as gamma-glutamyl residue (the peptide is denoted as gamma-Glu-36). The inter-conversion between the two peptides constitutes a side chain-backbone swap. Residues constituting the hydrophobic core of the coiled-coil, however, are left unchanged. The peptide pair was characterized by circular dichroism spectroscopy, reversed-phase liquid chromatography (RPLC), and two-dimensional nuclear magnetic resonance (NMR). The results indicate that alpha-Glu-36 is a two-stranded alpha-helical coiled-coil while gamma-Glu-36 lacks stable structural elements. It is concluded that, at least for coiled-coils where hydrophobic interactions are predominantly long-range, local backbone H-bonding is a required for structural formation, consistent with a hierarchic folding mechanism. The methodological implication of side chain-backbone swap is also discussed.     

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.     

gpwac of the T4-type bacteriophages: structure, function, and evolution of a segmented coiled-coil protein that controls viral infectivity

The wac gene product (gpwac) or fibritin of bacteriophage T4 forms the six fibers that radiate from the phage neck. During phage morphogenesis these whiskers bind the long tail fibers (LTFs) and facilitate their attachment to the phage baseplate. After the cell lysis, the gpwac fibers function as part of an environmental sensing device that retains the LTFs in a retracted configuration and thus prevents phage adsorption in unfavorable conditions. A comparative analysis of the sequences of 5 wac gene orthologs from various T4-type phages reveals that the approximately 50-amino-acid N-terminal domain is the only highly conserved segment of the protein. This sequence conservation is probably a direct consequence of the domain's strong and specific interactions with the neck proteins. The sequence of the central fibrous region of gpwac is highly plastic, with only the heptad periodicity of the coiled-coil structure being conserved. In the various gpwac sequences, the small C-terminal domain essential for initiation of the folding of T4 gpwac is replaced by unrelated sequences of unknown origin. When a distant T4-type phage has a novel C-terminal gpwac sequence, the phage's gp36 sequence that is located at the knee joint of the LTF invariably has a novel domain in its C terminus as well. The covariance of these two sequences is compatible with genetic data suggesting that the C termini of gpwac and gp36 engage in a protein-protein interaction that controls phage infectivity. These results add to the limited evidence for domain swapping in the evolution of phage structural proteins.     

The role of the carboxyl terminal alpha-helical coiled-coil domain in osmosensing by transporter ProP of Escherichia coli

Concentrative uptake of osmoprotectants via transporter ProP contributes to the rehydration of Escherichia coli cells that encounter high osmolality media. A member of the major facilitator superfamily, ProP is activated by osmotic upshifts in whole bacteria, in cytoplasmic membrane vesicles and in proteoliposomes prepared with the purified protein. Soluble protein ProQ is also required for full osmotic activation of ProP in vivo. ProP is differentiated from structural and functional homologues by its osmotic activation and its C-terminal extension, which is predicted to form an alpha-helical coiled-coil. A synthetic polypeptide corresponding to the C-terminus of ProP (ProP-p) formed a dimeric alpha-helical coiled-coil. A derivative of transporter ProP lacking 26 C-terminal amino acids was expressed but inactive. A derivative harbouring amino acid changes K460I, Y467I and H495I (each at the core, coiled-coil 'a' position) required a larger osmotic upshift for activation than did the wild type transporter. The same changes extended, stabilized and altered the oligomeric state of the coiled-coil formed by ProP-p. Amino acid change R488I (also at the 'a' position) further increased the magnitude of the osmotic upshift required to activate ProP, reduced the activity attained and rendered ProP activation transient. Unexpectedly, replacement R488I destabilized the coiled-coil formed by ProP-p. The activity and osmotic activation of ProP were even more strongly attenuated by helix-destabilizing change I474P. These data demonstrate that the carboxyl terminal domain of ProP can form a homodimeric alpha-helical coiled-coil with unusual properties. They implicate the C-terminal domain in the osmotic activation of ProP.     

Cloning and sequencing of the structural gene for the porin protein of Bordetella pertussis

Bordetella pertussis produces a porin protein which is a prominent outer membrane component found in both virulent and avirulent strains. N-terminal amino acid analysis of purified B. pertussis porin was performed and this amino acid sequence was used to design an oligonucleotide that was then utilized to screen a lambda gt11 library containing randomly sheared fragments of DNA from B. pertussis strain 347. One clone, lambda BpPor, was identified and subcloned into pUC18. A portion of the DNA insert in this subclone, pBpPor1, was sequenced and shown to contain the N-terminal region of the structural porin gene. This truncated gene sequence was used to design an additional oligonucleotide that was used to identify a clone, pBpPor2, which overlapped with pBpPor1 and contained a termination codon. The structural gene deduced from this sequence would encode a 365-amino-acid polypeptide with a predicted mass of 39,103 daltons. The predicted product also contains a signal sequence of 20 residues that is similar to that found in other porin genes. The predicted B. pertussis porin protein sequence contains regions that are homologous to regions found in porins expressed by Neisseria species and Escherichia coli, including the presence of phenylalanine as the carboxy-terminal amino acid. DNA hybridization studies indicated that both virulent and avirulent strains of B. pertussis contain only one copy of this gene and that Bordetella bronchiseptica and Bordetella parapertussis contain a similar gene.     

Coiled-coil intermediate filament stutter instability and molecular unfolding

Intermediate filaments (IFs) are the key components of cytoskeleton in eukaryotic cells and are critical for cell mechanics. The building block of IFs is a coiled-coil alpha-helical dimer, consisting of several domains that include linkers and other structural discontinuities. One of the discontinuities in the dimer's coiled-coil region is the so-called 'stutter' region. The stutter is a region where a variation of the amino acid sequence pattern from other parts of the alpha-helical domains of the protein is found. It was suggested in earlier works that due to this sequence variation, the perfect coiled-coil arrangement ceases to exist. Here, we show using explicit water molecular dynamics and well-tempered metadynamics that for the coil2 domain of vimentin IFs the stutter is more stable in a non-alpha-helical, unfolded state. This causes a local structural disturbance in the alpha helix, which has a global effect on the nanomechanics of the structure. Our analysis suggests that the stutter features an enhanced tendency to unfolding even under the absence of external forces, implying a much greater structural instability than previously assumed. As a result it features a smaller local bending stiffness than other segments and presents a seed for the initiation of molecular bending and unfolding at large deformation.     

Importance of secondary structural specificity determinants in protein folding: insertion of a native beta-sheet sequence into an alpha-helical coiled-coil

To examine how a short secondary structural element derived from a native protein folds when in a different protein environment, we inserted an 11-residue beta-sheet segment (cassette) from human immunoglobulin fold, Fab new, into an alpha-helical coiled-coil host protein (cassette holder). This de novo design protein model, the structural cassette mutagenesis (SCM) model, allows us to study protein folding principles involving both short- and long-range interactions that affect secondary structure stability and conformation. In this study, we address whether the insertion of this beta-sheet cassette into the alpha-helical coiled-coil protein would result in conformational change nucleated by the long-range tertiary stabilization of the coiled-coil, therefore overriding the local propensity of the cassette to form beta-sheet, observed in its native immunoglobulin fold. The results showed that not only did the nucleating helices of the coiled-coil on either end of the cassette fail to nucleate the beta-sheet cassette to fold with an alpha-helical conformation, but also the entire chimeric protein became a random coil. We identified two determinants in this cassette that prevented coiled-coil formation: (1) a tandem dipeptide NN motif at the N-terminal of the beta-sheet cassette, and (2) the hydrophilic Ser residue, which would be buried in the hydrophobic core if the coiled-coil structure were to fold. By amino acid substitution of these helix disruptive residues, that is, either the replacement of the NN motif with high helical propensity Ala residues or the substitution of Ser with Leu to enhance hydrophobicity, we were able to convert the random coil chimeric protein into a fully folded alpha-helical coiled-coil. We hypothesized that this NN motif is a "secondary structural specificity determinant" which is very selective for one type of secondary structure and may prevent neighboring residues from adopting an alternate protein fold. These sequences with secondary structural specificity determinants have very strong local propensity to fold into a specific secondary structure and may affect overall protein folding by acting as a folding initiation site.     

Collectin structure: a review

Collectins are animal calcium dependent lectins that target the carbohydrate structures on invading pathogens, resulting in the agglutination and enhanced clearance of the microorganism. These proteins form trimers that may assemble into larger oligomers. Each polypeptide chain consists of four regions: a relatively short N-terminal region, a collagen like region, an alpha-helical coiled-coil, and the lectin domain. Only primary structure data are available for the N-terminal region, while the most important features of the collagen-like region can be derived from its homology with collagen. The structures of the alpha-helical coiled-coil and the lectin domain are known from crystallographic studies of mannan binding protein (MBP) and lung surfactant protein D (SP-D). Carbohydrate binding has been structurally characterized in several complexes between MBP and carbohydrate; all indicate that the major interaction between carbohydrate and collectin is the binding of two adjacent carbohydrate hydroxyl group to a collectin calcium ion. In addition, these hydroxyl groups hydrogen bond to some of the calcium amino acid ligands. While each collectin trimer contains three such carbohydrate binding sites, deviation from the overall threefold symmetry has been demonstrated for SP-D, which may influence its binding properties. The protein surface between the three binding sites is positively charged in both MBP and SP-D.     

Nucleotide sequence of the btuCED genes involved in vitamin B12 transport in Escherichia coli and homology with components of periplasmic-binding-protein-dependent transport systems.

The products of the btuCED region of the Escherichia coli chromosome participate in the transport of vitamin B12 across the cytoplasmic membrane. The nucleotide sequence of the 3,410-base-pair HindIII-HincII DNA fragment carrying a portion of the himA gene and the entire btuCED region was determined. Comparison of the location of the open reading frames with the gene boundaries defined by transposon insertions allowed the assignment of polypeptide products to gene sequences. The btuC product is a highly nonpolar integral membrane protein of molecular weight 31,683. The distribution of hydrophobic regions suggests the presence of numerous membrane-spanning domains. The btuD product is a relatively polar but membrane-associated polypeptide of Mr 27,088 and contains segments bearing extensive homology to the ATP-binding peripheral membrane constituents of periplasmic binding protein-dependent transport systems. Other regions of this protein are similar to portions of the outer membrane vitamin B12 receptor. The btuE product (Mr 20,474) appears to have a periplasmic location. It has the mean hydropathy of a soluble protein but lacks an obvious signal sequence. The cellular locations and structural and sequence homologies of the Btu polypeptides point to the similarity of these three proteins to components of binding protein-dependent transport systems. However, the dependence on a periplasmic vitamin B12-binding protein has not yet been demonstrated.     

Secondary structure of component 8c-1 of alpha-keratin. An analysis of the amino acid sequence.

The amino acid sequence of component 8c-1 from alpha-keratin was analysed by using secondary-structure prediction techniques, homology search methods, fast Fourier-transform techniques to detect regularities in the linear disposition of amino acids, interaction counts to assess possible modes of chain aggregation and assessment of hydrophilicity distribution. The analyses show the following. The molecule has two lengths of coiled-coil structure, each about 20 nm long, one from residues 56-202 with a discontinuity from about residue 91 to residue 101, and the other from residues 219-366 with discontinuities from about residue 238 to residue 245 and at about residue 306. The acidic and basic residues in the coiled-coil segment between residues 102 and 202 show a 9,4-residue structural period in their linear disposition, whereas between residues 246 and 366 a period of 9.9 residues is observed in the positioning of ionic residues. Acidic and basic residues are out of phase by 180 degrees. Similar repeats occur in corresponding regions of other intermediate-filament proteins. The overall mean values for the repeats are 9.55 residues in the N-terminal region and 9.85 residues in the C-terminal region. The regions at each end of the protein chain (residues 1-55 and 367-412) are not alpha-helical and contain many potential beta-bends. The regions specified in have a significant degree of homology mainly due to a semi-regular disposition of proline and half-cystine residues on a three-residue grid; this is especially apparent in the C-terminal segment, in which short (Pro-Cys-Xaa)n regions occur. The coiled-coil segments of component 8c-1 bear a striking similarity to corresponding segments of other intermediate-filament proteins as regards sequence homology, structural periodicity of ionic residues and secondary/tertiary-structure predictions. The assessments of the probabilities that these homologies occurred by chance indicate that there are two populations of keratin filament proteins. The non-coiled-coil regions at each end of the chain are less hydrophilic than the coiled-coil regions. Ionic interactions between the heptad regions of components 8c-1 and 7c from the microfibrils of alpha-keratin are optimized when a coiled-coil structure is formed with the heptad regions of the constituent chains both parallel and in register.     

The structure of the voltage-sensitive sodium channel. Inferences derived from computer-aided analysis of the Electrophorus electricus channel primary structure

A variety of computer-aided analyses was applied to the recently derived amino acid sequence of the Electrophorus electricus sodium channel protein in order to extract structural information such as hydrophobicity, periodicity, and secondary structure predictors. We propose a schematic model for the arrangement and folding of the polypeptide chain within the bilayer. The model consists of 4 homologous regions, each containing 8 membrane-spanning (probably alpha-helical) structures. Several of these structures are amphipathic with a repeat of 3.5 residues, 4 of which (one from each homologous region) are postulated to form a negatively charged channel lining. Gating currents are proposed to arise from voltage-dependent separation of multiple ion pairs buried within the hydrophobic, intramembranous protein interior.     

DNA sequence of the gene scrA encoding the sucrose transport protein EnzymellScr of the phosphotransferase system from enteric bacteria: homology of the EnzymellScr and EnzymellBgl proteins

The nucleotide sequence of the structural gene, scrA, which codes for sucrose-specific EnzymeII(Scr) (EII(Scr)) of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (PTS), was determined. EllScr requires an EnzymeIII, the product of the gene crr, for full activity. The gene scrA is preceded immediately by a classical Shine-Dalgarno sequence (AAGAGGGTA). It contains 1368 nucleotides with an increased GC-content (58%) corresponding to a polypeptide of 455 amino acid residues (Mr 47,500). The protein has the hydropathic profile (average hydropathy +0.82) of an integral membrane protein lacking extended alpha-helical structures and a signal peptide. Comparison with the sequence of the beta-glucoside-specific EnzymeII (EII(Bgl), 625 amino acids, Mr 66,480; Bramley and Kornberg, 1987a; Schnetz et al., 1987) revealed strong homologies between EiI(Scr) and the first 458 residues of EII(Bgl). The 162 carboxyterminal residues of EII(Bgl), however, showed a high homology with the sequence of EnzymeIII (Nelson et al., 1984), a homology also described recently by Bramley and Kornberg (1987b). The evolutionary and functional significance of the similarities with four other EnzymesII is discussed.     

Complete amino acid sequence of streptococcal PepM49 protein, a nephritis-associated serotype. Conserved conformational design among sequentially distinct M protein serotypes

The complete amino acid sequence of PepM49, a peptic fragment of the group A streptococcal type 49 M protein, the antiphagocytic cell surface molecule of the bacteria, is described. This fragment retains the opsonic antibody epitope of the native molecule. The sequence of PepM49, as determined by automated Edman degradations of the uncleaved molecule, and its tryptic and chymotryptic peptides, consists of a total of 143 residues (Mr = 17,187). PepM49, a nephritis-associated M protein serotype, exhibits significant internal homology in its sequence. However, identical sequence repeats of the kind seen in the rheumatic fever-associated serotypes M5, M6, and M24, are absent in PepM49. PepM49 exhibits varying degrees of homology with the M5, M6, and M24 proteins, which is consistent with the existence of variable and conserved regions in the M protein molecule. Predictive analysis as well as CD measurements revealed a high propensity of the PepM49 molecule to assume an alpha-helical conformation. Furthermore, a heptad periodicity of the nonpolar residues, a characteristic of alpha-helical coiled-coil proteins, extends over the entire length of the PepM49 protein. The differences in the nonpolar residue distribution divide the PepM49 sequence into three distinct domains, similar to those seen earlier in the M5 and M6 proteins. Together, these studies establish a conserved conformational design for the sequentially diverse M protein serotypes. However, the pattern of heptad periodicity in the PepM49 protein is quite distinct from that present in the PepM5 and M6 proteins, suggesting distinct differences in structural features among conformationally similar M protein serotypes. This may have relevance to the pathological differences associated with these M protein serotypes.     

Laminins and other strange proteins.

Laminins are large multidomain proteins of the extracellular matrix (ECM) with important functions in the development and maintenance of cellular organization and supramolecular structure, in particular in basement membranes. Each molecule is composed of three polypeptide chains, A (300-400 kDa) and B1 and B2 (180-200 kDa), which together form the characteristic cross-shaped laminin structure with three short arms and one long arm. Many different domains have been identified in laminin by sequence analysis, structural investigations, and functional studies. Each short arm is formed by homologous N-terminal portions of one of the three chains. Structurally, each short arm contains two or three globular domains which are connected by rows of manyfold-repeated Cys-rich "EGF-like" domains. In all three chains this region is followed by a long heptad repeat region similar to those found in many alpha-helical coiled-coil proteins. These parts of the three laminin chains constitute a triple-stranded coiled-coil domain, which forms the extended rodlike structure of the long arm. This is the only domain in the protein which is made up of more than one chain and consequently serves the function of chain assembly. The two B chains are terminated by the coiled-coil domain, but the A chain contains an additional C-terminal segment which accounts for five globular domains located at the tip of the long arm. Several important functions of laminin have been assigned to individual domains in either the short arms or terminal regions of the long arm.(ABSTRACT TRUNCATED AT 250 WORDS)