A comparison of genomic coding sequences for feather and scale keratins: structural and evolutionary implications.

DNA sequences have been obtained for embryonic chick feather and scale keratin genes. Strong homologies exist between the protein coding regions of the two gene types and between the deduced amino acid sequences of the keratin proteins. Scale keratins are larger than feather keratins and the size difference is mainly attributable to four 13-amino acid repeats between residues 77 and 128 which compose a peptide sequence rich in glycine and tyrosine. The strong similarities between the two peptide structures for feather and scale in the homologous regions suggests a similar conformation within the protein filaments. A likely consequence is that the additional repeat region of the scale protein is located externally to the core filament. Tissue-specific features of filament aggregation may be attributable to this one striking sequence difference between the constituent proteins. It is believed that the genes share a common ancestry and that feather-like keratin genes may have evolved from a scale keratin gene by a single deletion event.     

The role of β-sheets in the structure and assembly of keratins

X-ray diffraction, infrared and electron microscope studies of avian and reptilian keratins, and of stretched wool and hair, have played a central role in the development of models for the β-conformation in proteins. Both α- and β-keratins contain sequences that are predicted to adopt a β-conformation and these are believed to play an important part in the assembly of the filaments and in determining their mechanical properties. Interactions between the small β-sheets in keratins provide a simple mechanism through which shape and chemical complementarity can mediate the assembly of molecules into highly specific structures. Interacting β-sheets in crystalline proteins are often related to one another by diad symmetry and the data available on feather keratin suggest that the filament is assembled from dimers in which the β-sheets are related by a perpendicular diad. The most detailed model currently available is for feather and reptilian keratin but the presence of related β-structural forms in mammalian keratins is also noted.     

Solubilization of Chicken Feather Keratin by Ammonium Copper Hydroxide (Schweitzer’s Reagent)

Chicken feather powder was solubilized by Schweitzer’s reagent with shaking in the presence of air and the soluble feather keratin was prepared by dialyzing this extract against running water. Cystine residues in the starting feather keratin was converted to cysteic acid residues in the solubilized derivatives by air oxygen. Copper was bound fairly tightly to the solubilized protein and this copper-protein complex was separated into four fractions by CM- and DEAE-cellulose column chromatography. Each fraction had varied amount of bound copper, having a broad distribution of the molecular weight between 10,000 and 60,000 Sephadex column chromatographically. Although the amino acid composition of all separated feather keratin fractions were quite similar, the different electrophoretic patterns were observed among them by DISC electrophoresis.     

Structure of the three-chain unit of the bovine epidermal keratin filament

The characteristic α-type X-ray diffraction pattern displayed by bovine epidermal keratin filaments can be ascribed to the presence of segments of triple-chain coiled coil α-helix in the repeating three-chain unit of the filaments.Limited proteolysis of filaments polymerized in vitro or a citrate-soluble protein derived from them with crystalline trypsin releases two types of α-helix-enriched particles which provide information on the structure of the three-chain unit. The smaller, particle 2, of molecular weight 42,500, α-helix content of 92% and dimensions of 180 Å × 20 Å, consists of three chains aligned side-by-side that presumably form a coiled coil. The high yields of particle 2 allow the conclusion that all of the α-helix of the epidermal keratin filament is present in the form of these discrete three-chain α-helical segments. The larger, particle 1, recovered during the earlier stages of digestion has a molecular weight of 100,000 to 110,000, α-helix content of 75%, average dimensions of 400 Å × 20 Å and also consists of three chains aligned side-by-side. It contains two α-helical segments corresponding to particle 2 which are located at the amino -terminal and carboxyl-terminal ends and are separated by a region of non-helix. Particle 1 contains all of the α-helix and therefore is the major portion of the three-chain unit of the keratin filament. The products resulting from reaction of intact filament subunits with N-bromosuccinimide suggest that particle 1 is formed during digestion by removal of regions of non-helix from each end of this unit.The structure of the three-chain unit of the bovine epidermal keratin filament may thus be viewed as three polypeptide subunits aligned side-by-side with two discrete coiled coil α-helical segments interspersed with regions of non-helix.     

The cDNA sequence of a human epidermal keratin: divergence of sequence but conservation of structure among intermediate filament proteins

We have determined the DNA sequence of a cloned cDNA that is complementary to the mRNA for the 50 kilodalton (kd) human epidermal keratin. This provides the first amino acid sequence for a cytoskeletal keratin. Comparison of this sequence with those of other keratins reveals an evolutionary relationship between the cytoskeletal and the microfibrillar keratins, but shows no homology to matrix or feather keratins. The 50 kd keratin shares 28%-30% homology with partial sequences of other intermediate filament proteins, which suggests that keratins may be the most distantly related members of this class of fibrous proteins. Our computer analyses predict that the 50 kd keratin contains two long alpha-helical domains separated by a cluster of helix-inhibitory residues in the middle of the protein. These findings indicate that despite major sequence divergence among intermediate filament proteins, they retain sequences compatible with secondary structural features that appear to be common to all of them.     

The structural basis of the two-dimensional net pattern observed in the X-ray diffraction pattern of avian keratin

Feather keratin has a composite structure with a filament-matrix texture, and transmission electron microscopy studies of thin transverse sections of feather rachis by Rogers and Filshie in the early 1960s showed that the filaments have a strong tendency to form sheets. Potentially this could account for the unusual X-ray diffraction pattern noted by Bear and Rugo in the early 1950s, which was interpreted by them as indicating a two-dimensional net structure. Although it is 50 years since these major advances were made the possibility of extracting information on the nature of the filament packing from the diffraction pattern has never been explored. The present contribution shows how, when taken together with current information on the nature of β-sheets in feather keratin, certain features of the X-ray diffraction pattern can now be used to determine the likely arrangement of the filaments in the sheet.     

Three-dimensional modelling of interchain sequence similarities and differences in the coiled-coil segments of keratin intermediate filament heterodimers highlight features important in assembly

Using structural data derived from crystal fragments of vimentin, three-dimensional models have been constructed for the major coiled-coil segments (1A, 1B and 2B) in epidermal and hair keratin intermediate filament molecules. Similarity and difference distributions arising from the heterodimer nature of the keratin molecules have been calculated, colour-coded for ease of observation and represented as movie clips. This approach has enabled the spatial distributions of the charged and apolar residues to be visualized along the seam between the chains and on the surface of the molecule, thus providing new insights into the features of the IF molecule that are important in assembly. An observation of note is that one face of both segment 1A and segment 1B is predominantly apolar and, furthermore, contains the bulk of the differences in the charged residues that occur between the two chains. The face rotated by 180 degrees contains far fewer apolar residues. This suggests the likely internal face of segments 1A and 1B and, hence, those sequence and spatial features that are important in assembly. In addition, the similarity distributions of the acidic and basic residues display a period of about 19 residues over much of each of the two faces of segment 1B. The two 19-residue periods are out of phase with respect to one another, however, thus leading to the previously recorded 9.51 residue period in the axial distributions of the acidic and the basic residues. The apparent doubling of the period arises because 9.51 residues corresponds to a non-integral number of turns of alpha-helical coiled coil.     

Developmentally regulated cytokeratin gene in Xenopus laevis.

We have determined the sequence of cloned cDNAs derived from a 1,665-nucleotide mRNA which transiently accumulates during Xenopus laevis embryogenesis. Computer analysis of the deduced amino acid sequence revealed that this mRNA encodes a 47-kilodalton type I intermediate filament subunit, i.e., a cytokeratin. As is common to all intermediate filament subunits so far examined, the predicted polypeptide, named XK70, contains N- and C-terminal domains flanking a central alpha-helical rod domain. The overall amino acid homology between XK70 and a human 50-kilodalton type I keratin is 47%; homology within the alpha-helical domain is 57%. The N-terminal domain, which is not completely contained in our cDNAs, is basic, contains 42% serine plus alanine, and includes five copies of a six-amino-acid repeating unit. The C-terminal domain has a high alpha-helical content and contains a region with sequence homology to the C-terminal domains of other type I and type III intermediate filament proteins. We suggest that different keratin filament subtypes may have different functional roles during amphibian oogenesis and embryogenesis.     

Solubilization of Feather Keratin by a Copper-amine Complex

Chicken feather keratin was solubilized by cupri-ethylenediamine treatment and the solubilized products were separated into acidic and basic fractions by ion exchangers. In the solubilized products which had a molecular weight between 10,000 and 60,000, all the original cystine residues disappeared and cysteic acid residues were recovered instead of them but partly. The cupri-ethylenediamine reagent which catalyzed air-oxidation of cystine residues in keratin was removable mostly from the products by dialysis against water. The common copper-amine complexes were ineffective to solubilize feather keratin except for Schweitzer’s reagent. One strongly basic, unusual amino acid was detected in the basic solubilized fraction. This amino acid was eluted after arginine by usual column chromatography.     

The complete sequence of the human intermediate filament chain keratin 10. Subdomainal divisions and model for folding of end domain sequences

We present the complete amino acid sequence of the human keratin 10 (type I) intermediate filament chain expressed in terminally differentiated epidermal cells. Comparisons of this sequence with its mouse and bovine counterparts allow us to describe structural features of the functional end domains. First, sections of their respective end domains are highly conserved and permit a redefinition of earlier models for their subdomainal organization. The amino-terminal end domain consists of El, the first 57-58 residues that are basic, glycine-rich, and have been highly conserved among the three species; V1, a region of well-defined quasi repeats of the motif aliphatic-serine/glycinen; and H1, a newly recognized short acidic sequence that has been conserved among the type I keratin family. The carboxyl-terminal end consists of V2 and E2 whose properties but not sequence resemble V1 and E1, respectively. Second, since the E1, H1, and E2 sequences have been highly conserved between the three species, we suggest they are critical elements in defining intermediate filament function. Third, we note that the E and V sequences of the keratin 10 (and other keratin) chains share many properties in common with protein chain turns found in globular proteins. We therefore propose a model in which these sequences form omega loop-like structures (Leszczynski, J. N. & Rose, G. D. (1986) Science 234, 849-855) on the surface of keratin intermediate filaments. This represents the first specific proposal for the end domain structure of any intermediate filament chain.     

Formation of cytoplasmic turns between two closely spaced transmembrane helices during membrane protein integration into the ER membrane

The helical hairpin, two closely spaced transmembrane helices separated by a short turn, is a recurring structural element in integral membrane proteins, and may serve as a compact unit that inserts into the membrane en bloc. Previously, we have determined the propensities of the 20 natural amino acids, when present in the middle of a long hydrophobic stretch, to induce the formation of a helical hairpin with a lumenally exposed turn during membrane protein assembly into the endoplasmic reticulum membrane. Here, we present results from a similar set of measurements, but with the turn placed on the cytoplasmic side of the membrane. We find that a significantly higher number of turn-promoting residues need to be present to induce a cytoplasmic turn compared to a lumenal turn, and that, in contrast to the lumenal turn, the positively charged residues Arg and Lys are the strongest turn-promoters in cytoplasmic turns. These results suggest that the process of turn formation between transmembrane helices is different for lumenal and cytoplasmic turns.     

Do the ends justify the mean? Proline mutations at the ends of the keratin coiled-coil rod segment are more disruptive than internal mutations

Intermediate filament (IF) assembly is remarkable, in that it appears to be self-driven by the primary sequence of IF proteins, a family (40-220 kd) with diverse sequences, but similar secondary structures. Each IF polypeptide has a central 310 amino acid residue alpha-helical rod domain, involved in coiled-coil dinner formation. Two short (approximately 10 amino acid residue) stretches at the ends of this rod are more highly conserved than the rest, although the molecular basis for this is unknown. In addition, the rod is segmented by three short nonhelical linkers of conserved location, but not sequence. To examine the degree to which different conserved helical and nonhelical rod sequences contribute to dimer, tetramer, and higher ordered interactions, we introduced proline mutations in residues throughout the rod of a type I keratin, and we removed existing proline residues from the linker regions. To further probe the role of the rod ends, we introduced more subtle mutations near the COOH-terminus. We examined the consequences of these mutations on (a) IF network formation in vivo, and (b) 10-nm filament assembly in vitro. Surprisingly, all proline mutations located deep in the coiled-coil rod segment showed rather modest effects on filament network formation and 10-nm filament assembly. In addition, removing the existing proline residues was without apparent effect in vivo, and in vitro, these mutants assembled into 10-nm filaments with a tendency to aggregate, but with otherwise normal appearance. The most striking effects on filament network formation and IF assembly were observed with mutations at the very ends of the rod. These data indicate that sequences throughout the rod are not equal with respect to their role in filament network formation and in 10-nm filament assembly. Specifically, while the internal rod segments seem able to tolerate considerable changes in alpha-helical conformation, the conserved ends seem to be essential for creating a very specific structure, in which even small perturbations can lead to loss of IF stability and disruption of normal cellular interactions. These findings have important implications for the disease Epidermolysis Bullosa Simplex, arising from point mutations in keratins K5 or K14.     

Crystallographic analysis of acrosomal bundle from Limulus sperm

The acrosomal process of Limulus sperm contains a bundle of filaments composed of actin and a 102 kDa protein in a 1:1 molar ratio. The structure of the bundle in true discharge was investigated by electron cryomicroscopy, X-ray scattering and crystallographic image analysis. A bundle can be characterized as a quasi-crystal with continuously varying views along the bundle axis. Each segment of the bundle is found to obey the symmetry of space group P1, with a = b = 147 A, c = 762 A, alpha = 90 degrees, beta = 90.6 degrees, gamma = 120 degrees. A unit cell contains a helical repeat of the filament with a selection rule following that of an actin filament. A 24 A projection map based on the h0l view was reconstructed after averaging 5300 unit cells from six electron images. Filaments in this projection are well separated and clearly display a 21 screw symmetry. This screw symmetry results from the helical parameters of the bundle filament and is found to be a non-crystallographic symmetry element present in the unit cell. Our structural analysis has led to the proposal that the assembly of a stable bundle with a defined maximum diameter can be controlled by the crystallographic packing of the twisted filaments.     

Transmembrane insertion of the Colicin Ia hydrophobic hairpin

Colicin Ia is a bactericidal protein that forms voltage-dependent, ion-conducting channels, both in the inner membrane of target bacteria and in planar bilayer membranes. Its amino acid sequence is rich in charged residues, except for a hydrophobic segment of 40 residues near the carboxyl terminus. In the crystal structure of colicin Ia and related colicins, this segment forms an α-helical hairpin. The hydrophobic segment is thought to be involved in the initial association of the colicin with the membrane and in the formation of the channel, but various orientations of the hairpin with respect to the membrane have been proposed. To address this issue, we attached biotin to a residue at the tip of the hydrophobic hairpin, and then probed its location with the biotin-binding protein streptavidin, added to one side or the other of a planar bilayer. Streptavidin added to the same side as the colicin prevented channel opening. Prior addition of streptavidin to the opposite side protected channels from this effect, and also increased the rate of channel opening; it produced these effects even before the first opening of the channels. These results suggest a model of membrane association in which the colicin first binds with the hydrophobic hairpin parallel to the membrane; next the hairpin inserts in a transmembrane orientation; and finally the channel opens. We also used streptavidin binding to obtain a stable population of colicin molecules in the membrane, suitable for the quantitative study of voltage-dependent gating. The effective gating charge thus determined is pH-independent and relatively small, compared with previous results for wild-type colicin Ia. Received: 12 November 1996/Revised: 23 January 1997     

Defining the repeating elements in the cysteine-rich region (CRR) of the CD18 integrin beta 2 subunit

The cysteine-rich region (CRR) of the integrin beta subunits is organised into four repeating elements. By expression of a panel of truncated beta 2 subunits, and CRR segments fused to the C-terminal end of a CD4 soluble fragment, the segment required for the expression of two monoclonal antibody conformational epitopes was determined. This segment, E482-Q574, contains 16 cysteines representing two repeating units. We have thus defined the CRR unit motif of 'xC---C---C---CxCxxCxC---Cx', where 'x' represents a single residue, and '---' represents a stretch of four to 14 residues.     

Studies on the Structure of Feather Keratin: II. A β-Helix Model for the Structure of Feather Keratin

The assumption that the proline residues in feather keratin, which comprise 12 per cent of the total, are periodically located along the polypeptide chain is shown to lead to an essentially unique structure for this fibrous protein. The structure is based on a β-helix; i.e., an extended chain which coils slowly to form a helix of relatively large pitch. Such helices tend to aggregate by hydrogen bonding to form cylindrical units, which in turn can aggregate further into cable-like structures. This model has been tested with respect to its predictions concerning the x-ray diffraction pattern, infrared spectrum, mechanical properties, and chemical behavior of feather keratin. Preliminary results indicate that it is better capable of accounting for the data than previously proposed structures.     

Isolation and molecular weight of pure feather keratin mRNA

Biologically active feather keratin mRNA which directs the synthesis of most or all the keratin polypeptide chains, M.W. 10,000, was prepared either by cellulose chromatography or by dissociation of mRNP particles with Na dodecyl sulphate. Both preparations gave 1 RNA band of MW 250,000 on formamide-acrylamide gels indicating the presence of long untranslated segment(s) whose length has been conserved during the evolution of individual keratin genes.     

Crystal structure of FadA adhesin from Fusobacterium nucleatum reveals a novel oligomerization motif, the leucine chain

Many bacterial appendages have filamentous structures, often composed of repeating monomers assembled in a head-to-tail manner. The mechanisms of such linkages vary. We report here a novel protein oligomerization motif identified in the FadA adhesin from the Gram-negative bacterium Fusobacterium nucleatum. The 2.0 angstroms crystal structure of the secreted form of FadA (mFadA) reveals two antiparallel alpha-helices connected by an intervening 8-residue hairpin loop. Leucine-leucine contacts play a prominent dual intra- and intermolecular role in the structure and function of FadA. First, they comprise the main association between the two helical arms of the monomer; second, they mediate the head-to-tail association of monomers to form the elongated polymers. This leucine-mediated filamentous assembly of FadA molecules constitutes a novel structural motif termed the "leucine chain." The essential role of these residues in FadA is corroborated by mutagenesis of selected leucine residues, which leads to the abrogation of oligomerization, filament formation, and binding to host cells.