Consequences of two different amino-acid substitutions at the same codon in KRT14 indicate definitive roles of structural distortion in epidermolysis bullosa simplex pathogenesis.

Numerous inherited diseases develop due to missense mutations, leading to an amino-acid substitution. Whether an amino-acid change is pathogenic depends on the level of deleterious effects caused by the amino-acid alteration. We show an example of different structural and phenotypic consequences caused by two individual amino-acid changes at the same position. Epidermolysis bullosa simplex (EBS) is a genodermatosis resulting from KRT5 or KRT14 mutations. Mutation analysis of an EBS family revealed that affected individuals were heterozygous for a, to our knowledge, previously unreported mutation of c.1237G>C (p.Ala413Pro) in KRT14. Interestingly, 2 of 100 unrelated normal controls were heterozygous, and 1 of the 100 was homozygous for a different mutation in this position, c.1237G>A (p.Ala413Thr). In silico modeling of the protein demonstrated deleterious structural effects from proline substitution but not from threonine substitution. In vitro transfection studies revealed a significantly larger number of keratin-clumped cells in HaCaT cells transfected with mutant KRT14 complementary DNA (cDNA) harboring p.Ala413Pro than those transfected with wild-type KRT14 cDNA or mutant KRT14 cDNA harboring p.Ala413Thr. These results show that changes in two distinct amino acids at a locus are destined to elicit different phenotypes due to the degree of structural distortion resulting from the amino-acid alterations.

A different conformation for linker L12 in IF molecules in the molecular and filamentous forms: an hypothesis.

The rod domain of IF molecules has been characterized as four alpha-helical coiled-coil segments (1A, 1B, 2A and 2B), three linkers (L1, L12 and L2) and a stutter at the centre of segment 2B. Two of these breaks in coiled-coil continuity (L2 and stutter) have been modelled on the basis of structural data obtained from related proteins. Subsequently, X-ray crystallographic studies on fragments of IF molecules have shown that both models were correct. The third of the breaks - L1 - was predicted to have a flexible structure, consistent with observations that the head domain can fold back over segments 1A and 1B and also unwind into separate strands. Here the structure of the fourth discontinuity (L12) has been modelled. For most IF chain types two conformations are proposed for an eight-residue motif that displays a quasi two-residue repeat based on the presence of apolar residues. In IF it is proposed that the motif will adopt an alpha-helical conformation but that in the molecule the conformation will be beta-like. Thus, assembly will result in or result from a conformational change in L12 thereby attributing L12 a more dynamic and important role in assembly than expected.

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.

Sequence analyses of Type I and Type II chains in human hair and epithelial keratin intermediate filaments: promiscuous obligate heterodimers, Type II template for molecule formation and a rationale for heterodimer formation.

Sequence comparisons have been undertaken for all hair and epithelial keratin IF chains from a single species--human. The results lead to several new proposals. First, it is clear that not only is the chain structure of the molecule an obligate heterodimer but promiscuous association of Type I and Type II chains must occur in vivo. Second, the higher predicted content of alpha-helix in Type II chains in solution relative to that expected for Type I chains suggests that it is the Type II chains that precede their Type I counterparts and that they may serve as templates for molecule formation. Third, heterodimer formation leads naturally to greater structural and functional specificity, and this may be required not only because keratin IF have more interacting partners in its cell type than other types of IF have in theirs but also because hair and skin IF have two distinct structures that relate to the "reducing" or "oxidizing" environment in which they can find themselves. The transition between the two forms may require specific head/tail interactions and this, it is proposed, would be more easily accomplished by a heterodimer structure with its greater in-built specificity.

Human hair keratin-associated proteins: sequence regularities and structural implications.

In this paper, we undertake a sequence analysis of the human keratin-associated proteins (KAP). This analysis has revealed two fundamental pentapeptide quasi-repeats (A and B) of the form C-C-X-P-X and C-C-X-S/T-S/T, respectively. The A repeats are also commonly found in two subforms A1 and A2, -C-C-Q-P-X and C-C-R-P-X, respectively-similar to those found in sheep wool 30-40 years previously. Some high-sulphur and ultra-high sulphur proteins contain predominantly A repeats or B repeats but not regular combinations of them, whereas others are characterised by a contiguous pair of pentapeptide repeats that largely (though imperfectly) alternate to generate decapeptide motifs of the form AB, A1B or A2B. The A and B repeats sometimes occur in complex runs and can generate both 19- and 20-residue repeats of the form BABB' or BA1AA, respectively, where the prime indicates a motif truncated by one residue. Likewise, a 42-residue repeat with BA1BXAAAB (40 residues) separated by a di-serine (two residues) has been observed in an ultra-high sulphur protein from cuticle. To understand the possible conformations adopted by the A and B motifs, a search was initiated of the PDB structural database for a number of overlapping pentapeptide repeats. The total number of matches was 658 and these were found in 451 different proteins. From representative and unique structures the means and standard deviations were calculated for the Phi(i) and Psi(i) angles for the C-C-X-P-X and the C-C-X-S/T-S/T motifs. Molecular modelling has been employed to represent the "average" structure found from crystallographic and nmr data determined for each motif in other proteins. The conformation of consecutive A repeats with proline residues in the cis state is akin to a string of disulphide bond-stabilised pentapeptide knots between which there is relative freedom of rotation about the single bonds that link them. For B pentapeptides, however, the likelihood that a similar disulphide bond is formed appears much lower. This may give additional conformational flexibility to the chain and hence allow the A pentapeptides greater opportunity to interact appropriately with the IF via disulphide bonds, ionic interactions and/or hydrogen bonding.

Modeling effects of mutations in coiled-coil structures: case study using epidermolysis bullosa simplex mutations in segment 1a of K5/K14 intermediate filaments.

The sequence of a protein chain determines both its conformation and its function in vivo. An attempt is made to gain an understanding of the classes of deformations that can arise in an important structural motif, the alpha-helical coiled coil, as a consequence of mutations occurring in its underlying heptad substructure. In order to do so we consider the model structure of segment 1A in intermediate filaments and then investigate the structures arising from each of the 22 mutations observed in cytokeratin K5/K14 molecules that lead to variants of epidermolysis bullosa simplex. These are refined separately using a molecular dynamics protocol. The mutations often result in a significant distortion of the backbone over a turn or so of the alpha helix in either the chain itself or its constituent partner, leading to the likelihood of impaired chain aggregation and hence molecular assembly. One mutant (K14-L143P; 1A-28) gave rise to structural distortion along almost the entire length of segment 1A. The remaining structures showed less deformation, and normal-looking intermediate filaments are likely in vivo. In addition, an identical mutation in the same position in each of the chains in the heterodimer did not necessarily give equivalent structural distortions. Although proline mutations frequently lead to the most severe structural deformations, a non-proline substitution (K14-R125S; 1A-10) gave rise to the largest local structural disruption that was observed. Unexpectedly, mutations in positions a and d were not always of the greatest structural significance, although three in position a were shown by AGADIR to result in a significant increase in alpha-helix stability.

An unusual Ala12Thr polymorphism in the 1A alpha-helical segment of the companion layer-specific keratin K6hf: evidence for a risk factor in the etiology of the common hair disorder pseudofolliculitis barbae.

Pseudofolliculitis barbae (PFB) is a common hair disorder characterized by a pustular foreign body inflammatory reaction that is induced by ingrown hairs of the facial and submental (barbea) regions after regular shaving. It occurs predominantly in black males, while it is rather rare and usually far less severe in Caucasian males. Black individuals have a higher propensity of developing PFB due to their genetic predisposition for curly hair which inherently possesses a much higher risk of growing back into the skin than straight or wavy hair. The PFB process is, however, not gender dependent nor restricted to the face, but can occur in any skin region once regular shaving, plucking, or other traumatic means of hair removal are instituted. Through a family study and a large-scale investigation of randomly sampled PFB-affected and -unaffected individuals, this study demonstrates that an unusual single-nucleotide polymorphism, which gives rise to a disruptive Ala12Thr substitution in the 1A alpha-helical segment of the companion layer-specific keratin K6hf of the hair follicle, is partially responsible for the phenotypic expression and represents an additional genetic risk factor for PFB.

Alpha-helical coiled-coil oligomerization domains are almost ubiquitous in the collagen superfamily.

Alpha-helical coiled-coils are widely occurring protein oligomerization motifs. Here we show that most members of the collagen superfamily contain short, repeating heptad sequences typical of coiled coils. Such sequences are found at the N-terminal ends of the C-propeptide domains in all fibrillar procollagens. When fused C-terminal to a reporter molecule containing a collagen-like sequence that does not spontaneously trimerize, the C-propeptide heptad repeats induced trimerization. C-terminal heptad repeats were also found in the oligomerization domains of the multiplexins (collagens XV and XVIII). N-terminal heptad repeats are known to drive trimerization in transmembrane collagens, whereas fibril-associated collagens with interrupted triple helices, as well as collagens VII, XIII, XXIII, and XXV, were found to contain heptad repeats between collagen domains. Finally, heptad repeats were found in the von Willebrand factor A domains known to be involved in trimerization of collagen VI, as well as in collagen VII. These observations suggest that coiled-coil oligomerization domains are widely used in the assembly of collagens and collagen-like proteins.

Modeling alpha-helical coiled-coil interactions: the axial and azimuthal alignment of 1B segments from vimentin intermediate filaments.

Attempts at predicting the relative axial alignments of fibrous protein molecules in filamentous structures have relied upon representing the (multichain) molecular structure by a one-dimensional sequence of amino acids. Potential intermolecular ionic and apolar interactions were counted and determined as a function of the relative axial stagger between the molecules. No attempts were made to consider the azimuthal aspect of the interacting molecules and neither were apolar or ionic energy terms used. Surprisingly, this simple approach proved remarkably informative and yielded accurate predictions of the axial periods present. However, a more comprehensive analysis involving the energetics of aggregation taking due regard for the relative azimuths of the molecules as well as their separation should decrease the noise level in the calculations and reveal other pertinent information. Toward that end, we have modeled the interaction between two alpha-helical coiled-coil segments in intermediate filament molecules (1B segments from human vimentin). The relative axial alignment and polarity of the molecules is already known from detailed crosslinking studies and this provides a criterion against which the success (or otherwise) of the modeling can be judged. The results confirm that an antiparallel alignment of two 1B segments is preferred over any of the parallel options (as observed experimentally). The calculated axial alignment, however, is not identical to that observed from detailed crosslinking studies indicating that other parts of the molecule (probably the head and tail domains as well as other coiled-coil segments) have a crucial role in determining the precise mode of axial aggregation. The results also show that the apolar interactions seem to be significantly less important in the alignment process than the ionic ones. This is consistent with the observation of a well-defined period in the linear disposition of the charged (but not apolar) residues along the length of the outer surface of the vimentin molecule.

Sequence comparisons of intermediate filament chains: evidence of a unique functional/structural role for coiled-coil segment 1A and linker L1.

A comprehensive analysis of the sequences of all types of intermediate filament chains has been undertaken with a particular emphasis on those of segment 1A and linker L1. This has been done to assess whether structural characteristics can be recognized in the sequences that would be consistent with the role of each region in the recently proposed "swinging head" hypothesis. The analyses show that linker L1 is the most flexible rod domain region, that it is the most elongated structure (on a per residue basis), and that it is the most variable region as regards sequence and length. Segment 1A has one of the two most highly conserved regions of sequence in the rod domain (the other being at the end of segment 2B), with seven particular residues conserved across all chain types. It also contains one of the very few potential interchain ionic interactions that could be conserved across all chain types. However, the aggregation of chains in segment 1A is specified less precisely overall by interchain ionic interactions than are the other coiled-coil segments. The apolar residue contents in positions a and d of the heptad substructure are the highest of any coiled-coil segment in the intermediate filament family. Segment 1A also displays an amino acid composition atypical of not only coiled-coil segments 1B and 2B, but indeed of two-stranded coiled coils in general. Nonetheless, molecular modeling based on the crystal structure of the monomeric 1A fragment from human vimentin shows that coiled-coil formation is plausible. The most extensive regions of apolar/aromatic residues lie at the C-terminal end of segment 2B in the helix termination motif and in segment 1A in and close to the helix initiation motif. The predicted stability of the individual alpha-helices in segment 1A is greater than in those comprising segments 1B and 2B, though potential intrachain ionic interactions are either lacking or are minimal in number. Analysis of the 1A sequence and those regions immediately N- and C-terminal to it has shown that the capping residues are near optimal close to the previously predicted ends, thus adding to the likely stability of the alpha-helical structure. However, a second terminating sequence is predicted in 1A (about 10 residues back from the C-terminus). This allows the possibility of some unwinding of the alpha-helical structure of 1A immediately adjacent to linker L1 when the head domains no longer stabilize the coiled-coil structure. All of these data are consistent with the concept of a flexible hinge at L1 and with the ability of the two alpha-helical coiled-coil strands to separate under appropriate conditions and partly unwind at their C-terminal ends to allow the head domains a greater degree of mobility, thus facilitating function.

A role for the 1A and L1 rod domain segments in head domain organization and function of intermediate filaments: structural analysis of trichocyte keratin.

A dynamic model is proposed to explain how the 1A and linker L1 segments of the rod domain in intermediate filament (IF) proteins affect the head domain organization and vice versa. We have shown in oxidized trichocyte IF that the head domain sequences fold back over and interact with the rod domain. This phenomenon may occur widely in reduced IF as well. Its function may be to stabilize the 1A segments into a parallel two-stranded coiled coil or something closely similar. Under differing reversible conditions, such as altered states of IF assembly, or posttranslational modifications, such as phosphorylation etc., the head domains may no longer associate with the 1A segment. This could destabilize segment 1A and cause the two alpha-helical strands to separate. Linker L1 would thus act as a hinge and allow the heads to function over a wide lateral range. This model has been explored using the amino acid sequences of the head (N-terminal) domains of Type I and Type II trichocyte keratin intermediate filament chains. This has allowed several quasi-repeats to be identified. The secondary structure corresponding to these repeats has been predicted and a model has been produced for key elements of the Type II head domain. Extant disulfide cross-link data have been used as structural constraints. A model for the head domain structure predicts that a twisted beta-sheet region may wrap around the 1A segment and this may reversibly stabilize a coiled-coil conformation for 1A. The evidence in favor of the swinging head model for IF is discussed.