Segmented alpha-helical coiled-coil structure of the protein giardin from the Giardia cytoskeleton

The parasitic flagellate Giardia is the source of a filamentous protein, giardin, which binds to microtubules. The primary sequence of one giardin chain has been decoded from the base sequences of cDNAs isolated by antibody screens of a library constructed in the expression vector lambda gt11. The amino acid sequence favours a continuous alpha-helical fold for the protein without any inserts of a non-helical character. Analysis of apolar residue positions revealed 35 repeating heptads consistent with coiled-coil structure. This conformation relates giardin to the alpha-type fibrous proteins (k-m-e-f class) like tropomyosin and myosin (also found in Giardia). The giardin sequence has a regular series of skip residues like those at certain positions in the rod section of nematode myosin where the internal apolar seam of the coiled coil is shifted on the helix surface. The skips divide the giardin coil into quasi-equivalent structural segments about 4 nm in length, which might be domains for combining with tubulin subunits in the microtubule surface lattice.     

Role of the Kinesin Neck Region in Processive Microtubule-based Motility

Kinesin is a dimeric motor protein that can move along a microtubule for several microns without releasing (termed processive movement). The two motor domains of the dimer are thought to move in a coordinated, hand-over-hand manner. A region adjacent to kinesin's motor catalytic domain (the neck) contains a coiled coil that is sufficient for motor dimerization and has been proposed to play an essential role in processive movement. Recent models have suggested that the neck enables head-to-head communication by creating a stiff connection between the two motor domains, but also may unwind during the mechanochemical cycle to allow movement to new tubulin binding sites. To test these ideas, we mutated the neck coiled coil in a 560-amino acid (aa) dimeric kinesin construct fused to green fluorescent protein (GFP), and then assayed processivity using a fluorescence microscope that can visualize single kinesin–GFP molecules moving along a microtubule. Our results show that replacing the kinesin neck coiled coil with a 28-aa residue peptide sequence that forms a highly stable coiled coil does not greatly reduce the processivity of the motor. This result argues against models in which extensive unwinding of the coiled coil is essential for movement. Furthermore, we show that deleting the neck coiled coil decreases processivity 10-fold, but surprisingly does not abolish it. We also demonstrate that processivity is increased by threefold when the neck helix is elongated by seven residues. These results indicate that structural features of the neck coiled coil, although not essential for processivity, can tune the efficiency of single molecule motility.     

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.     

Structural determinants for EB1-mediated recruitment of APC and spectraplakins to the microtubule plus end

EB1 is a member of a conserved protein family that localizes to growing microtubule plus ends. EB1 proteins also recruit cell polarity and signaling molecules to microtubule tips. However, the mechanism by which EB1 recognizes cargo is unknown. Here, we have defined a repeat sequence in adenomatous polyposis coli (APC) that binds to EB1's COOH-terminal domain and identified a similar sequence in members of the microtubule actin cross-linking factor (MACF) family of spectraplakins. We show that MACFs directly bind EB1 and exhibit EB1-dependent plus end tracking in vivo. To understand how EB1 recognizes APC and MACFs, we solved the crystal structure of the EB1 COOH-terminal domain. The structure reveals a novel homodimeric fold comprised of a coiled coil and four-helix bundle motif. Mutational analysis reveals that the cargo binding site for MACFs maps to a cluster of conserved residues at the junction between the coiled coil and four-helix bundle. These results provide a structural understanding of how EB1 binds two regulators of microtubule-based cell polarity.     

C-terminal domains implicated in the functional surface expression of potassium channels

A short C-terminal domain is required for correct tetrameric assembly in some potassium channels. Here, we show that this domain forms a coiled coil that determines not only the stability but also the selectivity of the multimerization. Synthetic peptides comprising the sequence of this domain in Eag1 and other channels are able to form highly stable tetrameric coiled coils and display selective heteromultimeric interactions. We show that loss of function caused by disruption of this domain in Herg1 can be rescued by introducing the equivalent domain from Eag1, and that this chimeric protein can form heteromultimers with Eag1 while wild-type Erg1 cannot. Additionally, a short endoplasmic reticulum retention sequence closely preceding the coiled coil plays a crucial role for surface expression. Both domains appear to co-operate to form fully functional channels on the cell surface and are a frequent finding in ion channels. Many pathological phenotypes may be attributed to mutations affecting one or both domains.     

The predicted coiled-coil domain of myosin 10 forms a novel elongated domain that lengthens the head

Myosin 10 contains a region of predicted coiled coil 120 residues long. However, the highly charged nature and pattern of charges in the proximal 36 residues appear incompatible with coiled-coil formation. Circular dichroism, NMR, and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single alpha-helix (SAH) domain in solution and does not dimerize to form a coiled coil even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin 10 containing the motor, the three calmodulin binding domains, and the full-length predicted coiled coil showed that it was mostly monomeric at physiological protein concentration. In dimers the molecules were joined only at their extreme distal ends, and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin 5 heavy meromyosin obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus the predicted coiled coil of myosin 10 forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins 6 and 7a and MyoM and could function to increase the size of the working stroke.     

Model structure of the prototypical non-fimbrial adhesin YadA of Yersinia enterocolitica

Non-fimbrial adhesins, such as Yersinia YadA, Moraxella UspA1 and A2, Haemophilus Hia and Hsf, or Bartonella BadA represent an important class of molecules by which pathogenic proteobacteria adhere to their hosts. They form trimeric surface structures with a head-stalk-anchor architecture. Whereas head and stalk domains are diverse and appear (frequently repetitively) in different combinations, the anchor domains are homologous and display the properties of autotransporters. We have built a molecular model for the prototypical non-fimbrial adhesin, YadA, by combining the crystal structure of the head (PDB:1P9H) with theoretical models for the stalk and the anchor. The head domain is a single-stranded, left-handed beta-helix, connected to the stalk by a conserved trimerization element (the neck). The stalk consists of a right-handed coiled coil, containing ten 15-residue repeats with a C-terminal stutter (insertion of four residues). The stalk continues into the conserved anchor domain, which is formed by four heptads of a left-handed coiled coil, followed by four transmembrane beta-strands. Our model of the YadA coiled coil, generated with the program BeammotifCC, combines these periodicities into a structure that starts with a pronounced right-handed supercoil and ends with a canonical, left-handed conformation. The last two heptads of the coiled coil are located within a 12-stranded beta-barrel, formed by trimerization of the four transmembrane beta-strands in each monomer. We propose that this pore assembles in the outer membrane to form the opening through which the monomer chains exit the cell. After export is completed, the fiber folds and the pore is occluded by the coiled coil. Our model explains how these proteins can act as autotransporters in the absence of any homology to classical, single-chain autotransporters.     

Cloning and sequencing of rat plectin indicates a 466-kD polypeptide chain with a three-domain structure based on a central alpha-helical coiled coil

We have determined the complete cDNA sequence of rat plectin from a number of well-characterized overlapping lambda gt11 clones. The 4,140-residue predicted amino acid sequence (466,481 D) is consistent with a three-domain structural model in which a long central rod domain, having mainly an alpha-helical coiled coil conformation, is flanked by globular NH2- and COOH-terminal domains. The plectin sequence has a number of repeating motifs. The rod domain has five subregions approximately 200-residues long in which there is a strong repeat in the charged amino acids at 10.4 residues that may be involved in association between plectin molecules. The globular COOH-terminal domain has a prominent six-fold tandem repeat, with each repeat having a strongly conserved central region based on nine tandem repeats of a 19-residue motif. The plectin sequence has several marked similarities to that of desmoplakin (Green, K. J., D. A. D. Parry, P. M. Steinert, M. L. A. Virata, R. M. Wagner, B. D. Angst, and L.A. Nilles. 1990. J. Biol. Chem. 265:2,603-2,612), which has a shorter coiled-coil rod domain with a similar 10.4 residue charge periodicity and a COOH-terminal globular domain with three tandem repeats homologous to the six found in plectin. The plectin sequence also has homologies to that of the bullous pemphigoid antigen. Northern blot analysis indicated that there is a significant degree of conservation of plectin genes between rat, human, and chicken and that, as shown previously at the protein level, plectin has a wide tissue distribution. There appeared to be a single rat plectin gene that gave rise to a 15-kb message. Expression of polypeptides encoded by defined fragments of plectin cDNA in E. coli has also been used to localize the epitopes of a range of monoclonal and serum antibodies. This enabled us to tentatively map a sequence involved in plectin-vimentin and plectin-lamin B interactions to a restricted region of the rod domain.     

Simultaneous formation of right- and left-handed anti-parallel coiled-coil interfaces by a coil2 fragment of human lamin A

The elementary building block of all intermediate filaments (IFs) is a dimer featuring a central α-helical rod domain flanked by the N- and C-terminal end domains. In nuclear IF proteins (lamins), the rod domain consists of two coiled-coil segments, coil1 and coil2, that are connected by a short non-helical linker. Coil1 and the C-terminal part of coil2 contain the two highly conserved IF consensus motifs involved in the longitudinal assembly of dimers. The previously solved crystal structure of a lamin A fragment (residues 305-387) corresponding to the second half of coil2 has yielded a parallel left-handed coiled coil. Here, we present the crystal structure and solution properties of another human lamin A fragment (residues 328-398), which is largely overlapping with fragment 305-387 but harbors a short segment of the tail domain. Unexpectedly, no parallel coiled coil forms within the crystal. Instead, the α-helices are arranged such that two anti-parallel coiled-coil interfaces are formed. The most significant interface has a right-handed geometry, which is accounted for by a characteristic 15-residue repeat pattern that overlays with the canonical heptad repeat pattern. The second interface is a left-handed anti-parallel coiled coil based on the predicted heptad repeat pattern. In solution, the fragment reveals only a weak dimerization propensity. We speculate that the C-terminus of coil2 might unzip, thereby allowing for a right-handed coiled-coil interface to form between two laterally aligned dimers. Such an interface might co-exist with a heterotetrameric left-handed coiled-coil assembly, which is expected to be responsible for the longitudinal A_CN contact.     

Coiled-coil trigger motifs in the 1B and 2B rod domain segments are required for the stability of keratin intermediate filaments

Many alpha-helical proteins that form two-chain coiled coils possess a 13-residue trigger motif that seems to be required for the stability of the coiled coil. However, as currently defined, the motif is absent from intermediate filament (IF) protein chains, which nevertheless form segmented two-chain coiled coils. In the present work, we have searched for and identified two regions in IF chains that are essential for the stability necessary for the formation of coiled-coil molecules and thus may function as trigger motifs. We made a series of point substitutions with the keratin 5/keratin 14 IF system. Combinations of the wild-type and mutant chains were assembled in vitro and in vivo, and the stabilities of two-chain (one-molecule) and two-molecule assemblies were examined with use of a urea disassembly assay. Our new data document that there is a region located between residues 100 and 113 of the 2B rod domain segment that is absolutely required for molecular stability and IF assembly. This potential trigger motif differs slightly from the consensus in having an Asp residue at position 4 (instead of a Glu) and a Thr residue at position 9 (instead of a charged residue), but there is an absolute requirement for a Glu residue at position 6. Because these 13 residues are highly conserved, it seems possible that this motif functions in all IF chains. Likewise, by testing keratin IF with substitutions in both chains, we identified a second potential trigger motif between residues 79 and 91 of the 1B rod domain segment, which may also be conserved in all IF chains. However, we were unable to find a trigger motif in the 1A rod domain segment. In addition, many other point substitutions had little detectable effect on IF assembly, except for the conserved Lys-23 residue of the 2B rod domain segment. Cross-linking and modeling studies revealed that Lys-23 may lie very close to Glu-106 when two molecules are aligned in the A(22) mode. Thus, the Glu-106 residue may have a dual role in IF structure: it may participate in trigger formation to afford special stability to the two-chain coiled-coil molecule, and it may participate in stabilization of the two-molecule hierarchical stage of IF structure.     

The affinity of the dynein microtubule-binding domain is modulated by the conformation of its coiled-coil stalk

The microtubule-binding domain (MTBD) of dynein is separated from the AAA (ATPase with any other activity) core of the motor by an approximately 15-nm stalk that is predicted to consist of an antiparallel coiled coil. However, the structure of this coiled coil and the mechanism it uses to mediate communication between the MTBD and ATP-binding core are unknown. Here, we sought to identify the optimal alignment between the hydrophobic heptad repeats in the two strands of the stalk coiled coil. To do this, we fused the MTBD of mouse cytoplasmic dynein, together with 12-36 residues of its stalk, onto a stable coiled-coil base provided by Thermus thermophilus seryl-tRNA synthetase and tested these chimeric constructs for microtubule binding in vitro. The results identified one alignment that yielded a protein displaying high affinity for microtubules (2.2 microM). The effects of mutations applied to the MTBD of this construct paralleled those previously reported (Koonce, M. P., and Tikhonenko, I. (2000) Mol. Biol. Cell 11, 523-529) for an intact dynein motor unit in the absence of ATP, suggesting that it resembles the tight binding state of native intact dynein. All other alignments showed at least 10-fold lower affinity for microtubules with the exception of one, which had an intermediate affinity. Based on these results and on amino acid sequence analysis, we hypothesize that dynein utilizes small amounts of sliding displacement between the two strands of its coiled-coil stalk as a means of communication between the AAA core of the motor and the MTBD during the mechanochemical cycle.     

Association of the leukocyte plasma membrane with the actin cytoskeleton through coiled coil-mediated trimeric coronin 1 molecules

Coronin 1 is a member of the coronin protein family specifically expressed in leukocytes and accumulates at sites of rearrangements of the F-actin cytoskeleton. Here, we describe that coronin 1 molecules are coiled coil-mediated homotrimeric complexes, which associate with the plasma membrane and with the cytoskeleton via two distinct domains. Association with the cytoskeleton was mediated by trimerization of a stretch of positively charged residues within a linker region between the N-terminal, WD repeat-containing domain and the C-terminal coiled coil. In contrast, neither the coiled coil nor the positively charged residues within the linker domain were required for plasma membrane binding, suggesting that the N-terminal, WD repeat-containing domain mediates membrane interaction. The capacity of coronin 1 to link the leukocyte cytoskeleton to the plasma membrane may serve to integrate outside-inside signaling with modulation of the cytoskeleton.     

Angiopoietin-1 and -2 coiled coil domains mediate distinct homo-oligomerization patterns, but fibrinogen-like domains mediate ligand activity.

Activity of endothelial Tie2 receptor tyrosine kinase is modulated by two naturally occurring, secreted ligands, angiopoietin-1 and -2, which have opposing effects on its phosphorylation. Receptor tyrosine kinase activation requires receptor dimerization/multimerization, which, for many receptors, is mediated by homo-oligomeric ligands binding to and bridging receptor molecules. We show here that angiopoietin-1 and -2 form distinct arrays of disulfide-linked homo-oligomeric complexes. Their mobilities on nonreducing gels suggest that angiopoietin-2 exists predominantly as a homodimer but also forms higher order multimers. In contrast, angiopoietin-1 forms some homotrimers, but predominantly exists in higher order multimers. These two structurally related, 60% homologous ligands are predominantly composed of an amino-terminal coiled coil domain and a carboxyl-terminal fibrinogen-like domain. We show that their distinct oligomerization patterns are determined by their coiled coil domains and, furthermore, that their coiled coil domains, but not their fibrinogen-like domains, are sufficient to mediate formation of disulfide-linked homo-oligomers. In contrast, the differential effects of these ligands on endothelial Tie2 phosphorylation is mediated by their fibrinogen-like domains. We conclude from these studies that the coiled coil and fibrinogen-like domains of the angiopoietins have distinct functions with the coiled coil domain mediating ligand homo-oligomerization and the fibrinogen-like domain mediating ligand activity.     

Striated microtubule-associated fibers: identification of assemblin, a novel 34-kD protein that forms paracrystals of 2-nm filaments in vitro

Microtubule-associated fibers from the basal apparatus of the green flagellate alga Spermatozopsis similis exhibit a complex cross-striation pattern with 28-nm periodicity and consist of 2-nm filaments arranged in several layers. Fibers enriched by mechanical disintegration and high salt extraction (2 M NaCl) of isolated basal apparatuses are soluble in 2 M urea. Dialysis of solubilized fibers against 150 mM KCl yields paracrystals which closely resemble the native fibers in filament arrangement and striation pattern. Paracrystals purified through several cycles of disassembly and reassembly are greatly enriched (greater than 90%) in a single protein of 34 kD (assemblin) as shown by SDS-PAGE. A rabbit polyclonal antibody raised against assemblin labels the striated fibers as shown by indirect immunofluorescence of isolated cytoskeletons or methanol permeabilized cells and immunogold EM. Two-dimensional electrophoresis (isoelectric focusing and SDS-PAGE) resolves assemblin into at least four isoforms (a-d) with pI's of 5.45, 5.55, 5.75, and 5.85. The two more acidic isoforms are phosphoproteins as shown by in vivo 32PO4-labeling and autoradiography. Amino acid analysis of assemblin shows a high content of helix-forming residues (leucine) and a relatively low content of glycine. We conclude that assemblin may be representative of a class of proteins that form fine filaments alongside microtubules.     

Unusual ultrastructures of the Branchiostoma IF protein C2 containing heptads in the tail

Branchiostoma intermediate filament (IF) protein C2 contains a long tail domain consisting of several degenerate repeats which display a heptad repeat pattern. This unique tail sequence is predicted to constitute a long coiled coil domain in C2, which is separated from the rod by a glycine-rich linker L3. The recombinant IF protein C2 shows, in electron microscopy (EM), parallel rodlike dimers of 66.7 nm decorated by a larger globule on one side and a smaller globule on the other side. In contrast, the length of the tailless C2 dimers, decorated by only one small globule, is about 26 nm shorter. These results indicate that both the rod domain and the newly predicted coiled coil segment 3 participate in the formation of a double-stranded coiled coil dimer. Moreover, the two to four C2 dimers are able to associate via their globular tail domain into multiarm oligomers, an ability not seen by the tailless C2 mutant or the other currently known protostomic and vertebrate IFs.     

The single nuclear lamin of Caenorhabditis elegans forms in vitro stable intermediate filaments and paracrystals with a reduced axial periodicity

The lamins of the tunicate Ciona intestinalis and the nematode Caenorhabditis elegans show unusual sequence features when compared to the more than 35 metazoan lamin sequences currently known. We therefore analyzed the in vitro assembly of these two lamins by electron microscopy using chicken lamin B2 as a control. While lamin dimers usually appear as a rod carrying two globules at one end, these globules are absent from Ciona lamin, which lacks the central 105-residue region of the tail domain. The deletion of 14 residues or two heptads from the coiled coil rod domain of the single C.elegans lamin results in a 1.5-nm shortening of the dimer rod. Similarly, the paracrystals assembled from the C.elegans lamin exhibit a 3.1-nm reduction of the true axial repeat compared to that of chicken lamin B2 paracrystals. We speculate that the banding pattern in the C.elegans lamin paracrystals arises from a relative stagger between dimers and/or a positioning of the globular tail domain relative to the central rod that is distinct from that observed in chicken lamin B2 paracrystals. Here we show that a nuclear lamin can assemble in vitro into 10-nm intermediate filaments (IFs). C.elegans lamin in low ionic strength Tris-buffers at a pH of 7.2-7.4 provides a stable population of lamin IFs. Some implications of this filament formation are discussed.     

Hyperthermostable Surface Layer Protein Tetrabrachion from the ArchaebacteriumStaphylothermus marinus: Evidence for the Presence of a Right-handed Coiled Coil Derived from the Primary Structure

The scaffold of the surface layer covering the hyperthermophilic archaebacteriumStaphylothermus marinusis formed by an extended filiform glycoprotein complex, tetrabrachion, which is anchored in the cell membrane at one end of a 70 nm stalk and branches at the other end into four arms of 24 nm length. The arms from a canopy-like meshwork by end-to-end contacts, enclosing a “quasi-periplasmic space”. The primary structure of the complex, obtained by an approach based entirely on the polymerase chain reaction, shows that the light and the heavy chains are encoded in this order in a single gene and are generated by internal proteolytic cleavage. One light chain associates with the N-terminal part of a heavy chain to form one of the four arms of the complex, comprising about 1000 residues. Following a glycine-rich linker of about ten residues, the C-terminal 500 residues of the four heavy chains converge to form a four-stranded parallel coiled coil, which ends in a transmembrane segment. The sequence of the coiled coil is exceptional in that the heptad repeat of hydrophobic residues typical for left-handed coiled coils shifts to an undecad repeat after an internal proline residue, indicating that the C-terminal part of the sequence forms a right-handed coiled coil. Such a periodicity has not been detected in coiled coils to date. The almost flawless pattern of aliphatic residues, mainly leucine and isoleucine, throughout the hydrophobic core of the stalk provide one explanation for its exceptional stability.     

Plectin: a cytolinker by design

Plectin is a cytoskeletal protein of >500 kDa that forms dumbbell-shaped homodimers comprising a central parallel alpha-helical coiled coil rod domain flanked by globular domains, thus providing a molecular backbone ideally suited to mediate the protein's interactions with an array of other cytoskeletal elements. Plectin self-associates and interacts with actin and intermediate filament cytoskeleton networks at opposite ends, and it binds at both ends to the hemidesmosomal transmembrane protein integrin beta-4, and likely to other junctional proteins. The central coiled coil rod domain can form bridges over long stretches and serves as a flexible linker between the structurally diverse N-terminal domain and the highly conserved C-terminal domain. Plectin is also a target of p34cdc2 kinase that regulates its dissociation from intermediate filaments during mitosis.