R.D. Bruce Fraser

Fifty years of coiled-coils and α-helical bundles : A close relationship between sequence and structure

alpha-Helical coiled coils are remarkable for the diversity of related conformations that they adopt in both fibrous and globular proteins, and for the range of functions that they exhibit. The coiled coils are based on a heptad (7-residue), hendecad (11-residue) or a related quasi-repeat of apolar residues in the sequences of the alpha-helical regions involved. Most of these, however, display one or more sequence discontinuities known as stutters or stammers. The resulting coiled coils vary in length, in the number of chains participating, in the relative polarity of the contributing alpha-helical regions (parallel or antiparallel), and in the pitch length and handedness of the supercoil (left- or right-handed). Functionally, the concept that a coiled coil can act only as a static rod is no longer valid, and the range of roles that these structures have now been shown to exhibit has expanded rapidly in recent years. An important development has been the recognition that the delightful simplicity that exists between sequence and structure, and between structure and function, allows coiled coils with specialized features to be designed de novo.

Macrofibril assembly in trichocyte (hard α-) keratins

Early electron microscope studies of developing wool and hair established that trichocyte (hard α-) keratin fibers have a composite structure in which filaments, subsequently shown to belong to the class of intermediate filaments (IF), were embedded in a matrix of sulfur-rich proteins. These studies also showed that the IF aggregate in a variety of ways to form what have been termed macrofibrils. Assembly into sheets appears to be an important initial factor in aggregation, and in the present contribution the structural principles governing sheet formation are formulated and specific models for the interaction between neighboring IF in a sheet are proposed, based on existing X-ray diffraction, electron microscope, and crosslinking data. All of the trichocyte keratins so far examined by electron microscopy exhibit similar filament/matrix textures and the mechanism of sheet formation proposed here is likely to have general applicability.

Nucleation and growth of macrofibrils in trichocyte (hard-α) keratins

Abstract The intermediate filaments (IF) in trichocyte (hard-α) keratin form ordered aggregates that are infiltrated by sulfur-rich and tyrosine-rich proteins during fiber development to give a filament-matrix texture, which is stabilized in the later stages by the formation of disulfide linkages. Two polymorphic forms of macrofibril are found in the cortical cells of fine Merino wool. In the first the packing of the IF in the macrofibril is quasi-hexagonal whilst in the second the IF are packed in cylindrical sheets around a central core. In hairs the second type generally predominate. In the present contribution specific models for the mechanisms of nucleation and growth are developed for the two types of macrofibril and their applicability tested by analyzing transmission electron micrographs of wool and hair. Evidence is presented which supports the idea that sheet formation plays an important role in both types of macrofibril assembly and it is suggested that differing intersheet interactions are responsible for the differences between the ortho- and para-types. It is shown that the increase in IF tilt with radius in the ortho-type can be related to the surface lattice of the IF as determined from X-ray diffraction studies. Two possible types of intersheet interaction in the ortho-type are discussed, the first leading to an increase of around 0.5° in IF tilt per layer and the second leading to a much larger tilt of 9.4° per layer. A crude estimate based on the decrease in visibility of the IF with increasing radius in cross-section yielded a value of 0.35°–0.7°.

The role of disulfide bond formation in the structural transition observed in the intermediate filaments of developing hair.

Hair keratin is a composite structure in which intermediate filaments (IF) are embedded in a protein matrix. During the early stages of development in the hair follicle the redox potential is such that the cysteine residues in the IF are maintained in a reduced form. However, at a late stage of development the redox potential changes to produce an oxidizing environment and the IF undergo a structural transition involving both molecular slippage and radial compaction. In our earlier study the changes in the molecular parameters were estimated from knowledge of the sites of artificially induced crosslinks, and it was noted that the changes in these parameters realigned many of the cysteine residues to positions more favorable to disulfide bond formation. As the energy involved in the formation of disulfide bonds is much greater than that of hydrogen bonds or van der Waals interactions the structural transition is likely to be dominated by the requirement that the bonded cysteine residues occur at closely equivalent axial positions. This criterion was used in the present study to obtain more precise values for the molecular parameters in the oxidized fiber than has hitherto been possible. A comparison of the sequences of hair keratins and epidermal keratins suggests that the slippage observed in trichocyte IF during keratinization does not occur in epidermal IF.

Amino acid sequence homologies in the hard keratins of birds and reptiles, and their implications for molecular structure and physical properties.

Avian and reptilian epidermal appendages such as feathers, claws and scales exhibit a filament-matrix texture. Previous studies have established that both components reside within the same single-chain molecule. In the present study the homology in a wide range of aligned sequences is used to gain insights into the structure and function of the molecular segments associated with the filament and with the matrix. The notion that all molecules contain a β-rich 34-residue segment associated with the framework of the filament is reinforced by the present study. In addition, the residues involved in the polymerization of the molecules to form filaments are identified. In the Archosaurs (birds, crocodiles and turtles), and the Squamates (snakes and lizards) segments rich in glycine and tyrosine can be identified in the C-terminal domain. In Rhynocephalians (tuataras) and Squamates a similar segment is inserted at a specific point in the N-terminal domain. In some Archosaurian appendages (both avian and reptilian) segments rich in charged residues and cysteine are found in the N-terminal domain. The likely effect of these segments will be to soften the tissue without compromising its insolubility. The structure and role of the various molecular segments identified in this study and the way in which they might manifest themselves in terms of the physical properties of the particular epidermal appendage in which they appear are also discussed.

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 50years 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.

Reprint of: Keratin intermediate filaments: Differences in the sequences of the Type I and Type II chains explain the origin of the stability of an enzyme-resistant four-chain fragment

Previous studies have shown that a strong interaction exists between oppositely directed 1B molecular segments in the intermediate filaments of trichocyte keratins. A similar interaction has been identified as having a significant role in the formation of unit-length filaments, a precursor to intermediate filament formation. The present study is concerned with the spatial relationship of these interacting segments and its dependence on differences in the amino acid sequences of the two-chain regions that constitute the 1B molecular segment. It is shown that along a particular line of contact both chain segments possess an elevated concentration of residues with a high propensity for dimer formation. The transition from the reduced to the oxidized state involves a simple axial displacement of one molecular segment relative to the other, with no attendant rotation of either segment. This changes the inter-relationship of the two 1B molecular segments from a loosely packed form to a more compact one. After the slippage eight of the cysteine residues in the dimer are precisely aligned to link up and form the disulfide linkages as observed. The two remaining cysteine residues are located on the outside of the dimer and are presumably involved in inter-dimer bonding. The existence of a unique line of contact requires that two chains in the molecule have different amino acid compositions with the clustering of dimer-favoring residues phased by half the pitch length of the coiled coil.

The molecular structure of the silk fibers from Hymenoptera aculeata (bees, wasps, ants)

Silks from the Hymenoptera aculeata (bees, wasps, ants) contain ropes with four α-helical strands, rather than the more usual two strands found, for example, in α-keratin and myosin molecules. Extensive studies of the chemical structure of the silks have shown that each of the four chains in the molecule contains a central coiled-coil rod domain. However, little progress has been made in modeling the three-dimensional structure. X-ray diffraction data on honeybee silk (Apis mellifera), recorded by Rudall and coworkers, has been re-examined in detail and possible structures developed for the various types of filament seen in the silk glands, and for the packing arrangement in the spun fibers. The original X-ray data were re-collected by scanning figures in the original publications, de-screening and averaging perpendicular to the direction of interest, thereby reducing the graininess of the original images. Sufficient numbers of equatorial and meridional reflections were collected to define the axial projection of the base of the unit cell in fibers drawn from the contents of the silk glands, and to suggest that the axial period is different from that suggested by Rudall and coworkers. Models for two types of filament of increasing diameter are developed based on the node-internode packing scheme observed in protein crystals containing four-strand α-helical ropes. The central domains of the four component chains in the molecule are enclosed by N- and C-terminal domains with widely different lengths and compositions. The fibers thus have a composite filament-matrix texture, and possible locations for the matrix are discussed.

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.

Structural Transition of Trichocyte Keratin Intermediate Filaments During Development in the Hair Follicle

The intermediate filaments (IF) in trichocyte (hard α-) keratin are unique amongst the various classes of IF in having not one but two topologically-distinct structures. The first is formed at an early stage of hair development in a reducing environment within the cells in the lower part of the follicle. The second structure occurs at a later stage of hair development in the upper part of the follicle, where there is a transition to an oxidizing environment. Crosslinking studies reveal that molecular slippage occurs within the IF upon oxidation and that this results in many cysteine residues lying in near axial alignment, thereby facilitating disulphide bond formation. The disulphide bonds so formed stabilize the assembly of IF molecules and convert the keratin fibre into a tough, resilient and insoluble structure suitable for its function in vivo as a thermo-regulator and a protector of the animal against its external environment.