Thomasin A. Smith

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 α‐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 α 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 α‐helix stability. Proteins 2004.

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.

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.

Modeling α‐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 α‐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. Proteins 2003;50:207–212.

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.