A.D. McLachlan

Tropomyosin coiled-coil interactions: evidence for an unstaggered structure.

Stereochemical arguments based on models of the tropomyosin coiled-coil favour an unstaggered symmetrical form, since this allows the best packing of the hydrophobic groups on the inner face where the two helices interlock. The distribution of polar groups along one side of the helix also shows correlations between positive and negative charges which favour a symmetrical structure stabilized by salt bridges between the helices. The models also show that two symmetrical molecules can join end-to-end by an external overlap of the nonpolar zones at the termini, giving an effective length of 275 amino acids, which fits 14 repeats of Parrys (1974) 19 1/2-residue periodicity. If tropomyosin molecules join in this way without discontinuity of twist, and bind equivalently to all the troponin molecules on the actin helix, the supercoil must take n half-turns in a molecular length, where n must be even for a staggered structure. An unstaggered structure could make seven half-turns relative to the actin helix and present a similar binding surface to all seven actins along its length. Because of the compensating twist of the actin helix the tropomyosin molecule would itself make only six half-turns and have a pitch close to 137 A.

Gene duplications in the structural evolution of chymotrypsin

Chymotrypsin and other members of the serine protease enzyme family have a structure built from two similar domains, each of which is a hydrogen-bonded barrel, containing six antiparallel strands of beta-sheet bonded in the order ABCFED-A …. The folding patterns of the domains have been re-examined by several newly improved shape comparison methods to see whether the barrels could have evolved by gene duplication, as proposed by Matthews and Blow (Birktoft & Blow, 1972). The domains have a similar hydrogen-bond pattern, the same shear number (defined in this paper) for the twist of the barrel, and the cores of their β-sheets can be superimposed so that 46 topologically equivalent α-carbons fit within a root-mean-square distance of 2.43 A and a larger set of 57 α-carbons fit within 3.4 A. These results are highly significant when judged against shape comparisons of many other proteins with themselves, and give strong evidence for gene duplication. The duplication does not include any SS bridges. Both domains have a surprisingly symmetrical structure of two halves ABC, DEF paired round a dyad axis, and the half-domains are each made of two loops twisted in an L-shape, since the second strand (B or E) is bent into two halves B1, B2 or E1, E2. The cores of the four half-domains, each of 23 α-carbons, superimpose in pairs with root-mean-square distances ranging from 1.79 to 2.45 A. In the entire molecule the half-domains are related by a screw dyad which converts domain I strands (ABC) (DEF) into domain II strands (DEF) (ABC) superimposing the six strands with a root-mean-square distance of 2.35 A. These observations suggest that the Chymotrypsin barrel originally evolved from a closely-linked dimer of two intertwined half-domains which became united into one. domain by gene duplication. The enzyme evolved from a second dimer of two full domains and a second duplication. The bacterial protease B from Streptomyces griseus shows the same structural repeats and is consistent with the gene duplication hypothesis. Improved methods for shape comparison of proteins have been developed which are very fast and reliable.

Tests for comparing related amino-acid sequences. Cytochrome c and cytochrome c551

An improved method for testing similarities or repeats in protein sequences is described. It includes three features: a measure of similarity for amino acids, based on observed substitutions in homologous proteins; a search procedure which compares all pairs of segments of two proteins; new statistical tests which estimate the probabilities that observed correlations could have occurred by chance. Calculations show that gene duplication has probably not occurred in plant ferredoxins; phage Qβ and f2 coat proteins may be homologous; and repeats in cytochrome c are not statistically significant. The method predicted an alignment of cytochrome c and c551 sequences which later appeared consistent with Dickersons atomic model of horse cytochrome c.

“Homology” in proteins and nucleic acids: A terminology muddle and a way out of it

“Homology” has the precise meaning in biology of “having a common evolutionary origin,” but it also carries the loose meaning of “possessing similarity or being matched.” Its rampant use in the loose sense is clogging the literature on protein and nucleic acid sequence comparisons with muddy writing and, in some cases, muddy thinking In its precise biological meaning, “homology” is a concept of quality. The word asserts a type of relationship between two or more things. Thus, amino acid or nucleotide sequences are either homologous or they are not. They cannot exhibit a particular “level of homology” or “percent homology.” Instead, two sequences possess a certain level of similarity. Similarity is thus a quantitative property. Homologous proteins or nucleic acid segments can range from highly similar to not recognizably similar (where similarity has disappeared through divergent evolution). If using “homology” loosely did not interfere with our thinking about evolutionary relationships, the way in which we use the term would be a rather unimportant semantic issue. The fact is, however, that loose usage in sequence comparison papers often makes it difficult to know the authors intent and can lead to confusion for the reader (and even for the author). There are three common situations in which hazards arise by using “homology” to mean similarity. The first case is the most obvious offense but perhaps the least troublesome. Here an author identifies sequence similarities (calling them homologies) but claims that the sequences being compared are not evolutionarily related. Some awkward moments occur in such a paper, since the author claims both homology (i.e., similarity) and nonhomology (i.e., lack of a common ancestor). Nonetheless, the author’s ideas are likely to be clear since arguments against common ancestry are presented explicitly. A second case is one in which an author points out similarities (again called homologies) but does not address the issue of evolutionary origins. The reader, seeing the term “homology,” may infer that the author is postulating coancestry when that is not the authors intent. The final case occurs most frequently and is the most subtle and therefore most troublesome. Here, similarities (called homologies) are used to support a hypothesis of evolutionary homology. In this case, the two meanings of homology seem to overlap, and it is almost inevitable that the thinking of author and reader alike will be intrusively distorted as follows. Similarity is relatively straightforward to document. In comparing sequences, a similarity can take the form of a numerical score (O/o amino acid or nucleotide positional identity, in the simplest approach) or of a probability associated with such a score. In comparisons of three-dimensional structures, a typical numerical description is root-mean-square positional deviation between compared atomic positions. A similarity, then, can become a fully documented, simple fact. On the other hand, a common evolutionary origin must usually remain a hypothesis, supported by a set of arguments that might include sequence or three-dimensional similarity. Not all similarity connotes homology but that can be easily overlooked if similarities are called homologies. Thus, in this third case, we can deceive ourselves into thinking we have proved something substantial (evolutionary homology) when, in actuality, we have merely established a simple fact (a similarity, mislabeled as homology). Homology among similar structures is a hypothesis that may be correct or mistaken, but a similarity itself is a fact, however it is interpreted. We believe that the concepts of evolutionary homology and sequence or three-dimensional similarity can be kept distinct only if they are referred to with different words. We therefore offer the following recommendations: *Sequence similarities (or other types of similarity) should simply be called similarities. They should be documented by appropriate statistical analysis. In writing about sequence similarities the following sorts of terms might be used: a level or degree of similarity; an alignment with optimized similarity; the % positional identity in an alignment; the probability associated with an alignment. *Homology should mean “possessing a common evolutionary origin” and in the vast majority of reports should have no other meaning. Evidence for evolutionary homology should be explicitly laid out, making it clear that the proposed relationship is based on the level of observed similarity, the statistical significance of the similarity, and possibly other lines of reasoning. One could argue that the meaning of the term “homology” is itself evolving. But if that evolution is toward vagueness and if it results in making our scientific discourse unclear, surely we should intervene. With a collective decision to mend our ways, proper usage would soon become fashionable and therefore easy. We believe that we and our scientific heirs would benefit significantly.

The 14-fold periodicity in α-tropomyosin and the interaction with actin

A Fourier analysis of the distributions of different types of amino acids in the sequence of tropomyosin shows strong 14th-order peaks in the profiles of both negatively charged and non-polar amino acids, with a period of 1923 residues and an overall repeat length of 275 ± 2 amino acids, which is shorter than the sequence length of 284 amino acids. Both peaks are statistically significant and confirm Parrys work (1974, 1975b). The regularities are analysed in terms of an assumed supercoil structure in which two α-helices lie parallel and in register to form a supercoil with a pitch of 137 A. These molecules are then assumed to overlap end-to-end by eight to nine amino acids so that the periodicity is continuous along an extended filament of linked tropomyosin molecules. The periodic features are stronger in the outer surface of the molecule away from the core of the supercoil. The sequence divides into 14 bands which each have a narrow zone of net positive charge and a broader negatively charged zone. Overlapping every positive zone is a hydrophobic zone which always has at least one non-polar group on the outer surface. Anomalies in the charge distribution are found near the molecular ends and close to Cys190. These are attributed to the end-to-end overlap site and the troponin binding site. In the thin filament the 137 A pitch supercoil would make seven half-twists relative to the twisted actin helix along a 385 A length, so that a pair of adjacent bands would be oriented equivalently with respect to a pair of actins 28 A apart. We therefore suggest that the bands (each containing one zone of each type) should be divided alternately into two series, α and β. Every pair of bands is 3913 residues long and each of the seven pairs corresponds with one segment of the 42-residue gene duplication repeat observed previously in the sequence. The disparity between the periods of 42 and 3913 is overcome by deletions and insertions. The 3913-residue periodicity is not simply a consequence of the supercoil structure or gene duplication but is probably a result of adaptation to the spatial periodicity of the actin helix in muscle. Although the α and β bands are alike in general, they differ systematically in detail and the α bands are more regular than the β. We propose that the seven α and seven β bands are alternative sets of sites which bind equivalently to complementary groups of sites on seven actins in the “relaxed” and “active” states of muscle, respectively. In each band the negative zone probably attaches to actin by magnesium bridges and the hydrophobic zone by direct contacts with the narrow outer edge of the supercoil. Since the supercoil twists 90 ° relative to actin on passing between adjacent α and β bands, a quarter rotation of the whole tropomyosin molecule would detach one set of seven sites and attach the other, allowing a highly co-operative switch mechanism.

Periodic features in the amino acid sequence of nematode myosin rod.

Properties of the amino acid sequence of the nematode myosin rod region, deduced from cloned DNA, are analysed. The rod sequence of 1117 residues contains a regular region of 1094 residues, which has features typical of an alpha-helical coiled coil, followed by a short non-helical tailpiece at the carboxyl end. The hydrophobic amino acids show the expected seven-residue pattern a, b, c, d, e, f, g, which is modulated by a longer repeat of 28-residue zones. In addition, there are four one-residue insertions, or skip residues, at the ends of zones, at positions 351, 548, 745 and 970. Myosin is considerably less hydrophobic than tropomyosin or alpha-keratin and the outer surface of the coiled coil is covered by clusters of positive and negatively charged amino acid side-chains. Molecular models suggest that the coiled coil is continuous throughout the rod, with an approximately uniform left-handed twist, except for a few turns of helix near each skip region, where the twist flattens out to accommodate the extra residue. Fourier transforms of the amino acid profiles show strong periodicities based on repeats of seven residues (7/2 and 7/3) and 28 residues (especially 28/3 and 28/9). The positive and negative charges each have strong 28/3-residue periodicities that are out of phase with one another. The negative charges also show a 196/9-residue modulation frequency, which may reflect the presence of a 196-residue structural unit in muscle, approximately 2 X 143 A long. The distribution of charged amino acids suggests that electrostatic forces are dominant in forming the thick filament structure. Models that allow regular patterns of interacting charges are restricted and the simplest types are discussed.

Repeating sequences and gene duplication in proteins

Abstract The theory that proteins have evolved by repeated internal duplication of short segments of polypeptide chains has been tested by looking for repeats and near repeats in over 50 different proteins, many of them of known structure. The probability that the observed repeats could arise by chance has been calculated. The search does not yield a single new example where the evidence for gene duplication is compelling. No protein shows a unique internally consistent pattern of repeats which both correlates with repeats in the structure and cannot be explained by chance. The evidence is discussed in detail for haemoglobin, chymotrypsin, subtilisin and carboxypeptidase. The evolution of complex large proteins from simple small ones has probably been a process of continuous growth in which chains have been gradually added to the outer surface surrounding an invariable core near the active centre.

Sequence repeats in α-tropomyosin

The amino acid sequence of α-tropomyosin a nearly regular 42-residue pattern which repeats seven times, and probably arises from gene duplication. Although weak it is statistically significant, and persists most strongly on the face of the helix which is paired to its partner in the supercoil. The pattern is related to the non-acidic regions of sequence noted by Stone et al. (1975) . The sequence divides naturally into 41 seven-residue periods, with a regular pattern of polar and non-polar amino acids which is related to the structure of the supercoil. The first three sets of six periods form the 42-residue segments I,II and III in which segments II and III are very similar. In the second half of the sequence periods 24 to 41 form three more segments V, VI and VII, which appear to be a repeat of I,II and III. The alignments suggest gaps between periods 18,19 and 27,28 in segments IV and V, while period 32 appears to be a repeat of 31. These irregularities allow the first six 42-residue repeat segments to keep approximately in step with two lengths of the 19 1/2-residue periodic pattern observed by Parry (1974) .

Three-fold structural pattern in the soybean trypsin inhibitor (Kunitz).

The molecule contains three very similar irregular Y-shaped lobes of antiparallel twisted β-sheet, which are grouped symmetrically round a central axis and linked by hydrogen bonds to form a six-stranded barrel. Each lobe can be superposed on either neighbour by a rotation of approximately 120 °. Of the 160 residues seen in the X-ray electron density map, 101 may be superposed onto other residues within a root-mean-square distance of 2.1 A. The bond which reacts with trypsin lies on a loop between the first two lobes. It is suggested that the protein evolved from a primitive symmetrical trimer of identical subunits by tandem gene triplication.

Fourteen actin-binding sites on tropomyosin?

TROPOMYOSIN plays an important part in the control of muscle contraction. It is a rod-shaped, coiled-coil molecule, about 410 Å long, composed of two parallel α-helical chains which are in register1–4. It lies in the grooves of the actin double helix of all known types of muscle filament and is normally thought to be associated with seven actin units5–7. Calcium regulates the contraction of vertebrate skeletal muscle by its influence on troponin, which in turn leads to a movement of tropomyosin in the actin groove8–10, thereby exposing (in the ‘on’ position) or masking (in the ‘off’ position) the myosin cross-bridge binding areas. The position of the troponin-binding site is known fairly precisely (ref. 11 and review ref. 4), but the actin-binding sites have not yet been identified. Here, we analyse a fourteen-fold periodicity in the amino acid sequence of α-tropomyosin12 from rabbit skeletal muscle and propose that it is associated with seven pairs of quasi-equivalent actin-binding sites. Parry13 and Stone et al.12 first noted several series of amino acid types with a repeat of about 19½ residues, and areas low in acidic residues spaced about 40 residues apart. There is also a slightly irregular 42-residue repeat resulting from gene duplication14 which is in phase with them. We used Fourier analysis to make an objective and systematic search for periodicities in the distributions of acidic, basic and nonpolar groups, and here assess their significance.