1 Department of Biology, University of Crete, P.O. Box 2208, GR-71409 Heraklion and 2 Foundation for Research and TechnologyHellas, Institute of Molecular Biology and Biotechnology (IMBB), P.O. Box 1527, GR-71110 Heraklion, Crete, Greece
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Abstract |
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Keywords: 4--helical bundles/
-dihedral angles/rotamer/side chain
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Introduction |
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Side chains in proteins prefer certain conformations as shown by the non-uniform distribution of the -dihedral angles (Janin et al., 1978
). The preferred conformations correspond to energy minima that are generally represented by three regions of
1 (around 60°, 180° and 60°; Janin et al., 1978). Analyses of the distribution of
-dihedral angles by two groups (James and Sielecki, 1983
; Ponder and Richards, 1987
) led to the definition of a rotamer as a dense cluster of points in the
-angle space; furthermore, a rotamer library was developed based upon 19 well-determined protein structures. The relationship between secondary structure and side-chain conformations was subsequently investigated (McGregor et al., 1987
; Summers et al., 1987
). These studies revealed that the rotamer preferences of side chains are strongly affected by the secondary structure. A significant correlation between the backbone
,
values and the side-chain dihedral angles was also found (Dunbrack and Karplus, 1993
). Based on a significantly enlarged data set, Schrauber et al. (Schrauber et al., 1993
) further refined the original rotamer library for globular proteins introduced by Ponder and Richards (Ponder and Richards, 1987
). The influence of backbone conformations was taken into account by grouping the rotamer distributions for each amino acid according to several secondary structure-based classes. Furthermore, the term `rotamericity' of an amino acid was introduced (Schrauber et al., 1993
), defined as the ratio of the total number of occurrences of the specific amino acid in any of the possible rotamers to the total number of occurrences of this amino acid in the sample.
In the present study, the role of the geometric constraints posed by a specific topology to the side-chain dihedral angles was investigated. As a model for protein topology, the 4--helical bundle motif was used. This simple, recurrent tertiary motif consists of four
-helices packed against each other in an antiparallel manner at an angle of about 20° (Figure 1a
) (Weber and Salemme, 1980
; Cohen and Parry, 1986
). The
-helices are usually connected together with loop regions; alternatively, the bundle is formed as an assembly of helices belonging to different polypeptide chains, as is the case with the ColE1 Rop protein (Banner et al., 1987
; Presnell and Cohen, 1989
; Harris et al., 1994
). The amino acid sequences of the helices follow a specific pattern of hydrophilic and hydrophobic residues of the type (a,b,c,d,e,f,g)n (Crick, 1953
). This pattern is repeated every seven residues (heptads). Positions a and d form the core of the bundle and are generally occupied by hydrophobic amino acids (Figure 1b
).
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Materials and methods |
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Results |
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Amino acid composition of the sample
The amino acid composition of the helical parts of the proteins used in this work is presented in Figure 2. Compared with an earlier analysis (Paliakasis and Kokkinidis, 1992
) it shows only minor differences, i.e. (i) a higher occurrence of Leu and (ii) an increase in the Leu to Ala ratio. The composition of internal (a, d) positions (Figure 2
) shows a clear predominance of Leu, Ala, Ile and Val residues, which agrees well with the preferences found by Paliakasis and Kokkinidis.
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The clusters of 1,
2 dihedral angles found for the side chains of our sample are presented in Table III
. The most striking feature of this distribution for the majority of amino acid types is that a large fraction of the side chains belong to a single rotamer. Ile, Val, Thr and Cys are extreme examples, where the dominant cluster contains at least five times more members than the next one. On the other hand, Leu and Arg have two densely populated rotamers whereas Ser and Glu do not show pronounced preferences for a particular rotamer.
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Correlation between frequency of natural mutations and rotamer preferences in the hydrophobic core of the 4--helical bundles
Natural mutations in the hydrophobic core of 4--helical bundles were analyzed from the aspect of rotamer conservation [rotamer conservation in proteins has been reported (Summers et al., 1987
) and forms the basis of molecular modeling by homology]. In this analysis, the sequence alignments described in the Materials and methods section were examined and the pattern of sequence variation at the a and d positions was studied. From a total of 226 pairs of aligned amino acids in the core, 81 substitutions were found. In Table IV
the observed substitutions and the corresponding frequencies for the residues commonly found in a and d positions are presented. An interesting observation is the relatively high frequency of Val
Leu and Met
Leu substitutions. It is worth noting that only four types of substitution account for 40% of the total observed (Met
Leu, Val
Leu, Leu
Ile and Leu
Val). Comparison of Tables III and IV
shows that substitutions occur overwhelmingly between residue types that have their major rotamers in common. For example, the main rotamer of Ile and Met coincides with one of the main rotamers of Leu. As an exception to the above observations, Phe
Ile substitution occurs between residues that do not share any common rotamer while some relatively rare substitutions (e.g. Met
Phe, Tyr
Asp and Tyr
Met) occur between residues which do not share a major rotamer but have at least one less populated
1,
2 cluster in common. For example, the dominant rotamer of Tyr and Phe (
1 = 176°,
2 = 78° and
1 = 180°,
2 = 75°, respectively) coincide with one of the `rare' rotamers of Met (
1 = 175°,
2 = 64°).
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Discussion |
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Previous studies have demonstrated that the secondary structure significantly affects the side-chain conformations by restricting them to a subset of those found in globular proteins (McGregor et al., 1987; Schrauber et al., 1993
). This is confirmed and reinforced by our analysis, which in addition showed that for some amino acids (Tyr, Met, Thr, Cys) the preference of specific side-chain conformations is much more pronounced even when compared to
-helices. This could be an effect of the 4-
-helical bundle topology, which imposes additional constraints to side chains limiting the permitted conformations to a subset of those adopted by
-helices.
Generally, the majority of side chains in the 4--helical bundles do not adopt novel or unusual conformations; side chains are clustered in some of the already known regions (from the globular proteins) of the
1,
2 space. A notable exception to this statement is the Asp residue, several occurrences of which have been classified as a novel rotamer (Figure 5a
). This result was unexpected given that Asp is not subject to the specific constraints of the motif, since it systematically occurs in external or semi-buried positions. Indications of a new rotamer are also present for Asn. An analysis of all Asp and Asn residues in our sample shows that their temperature factors are fairly low and very close to or even lower than the average temperature factor of the structure to which they belong. Thus, the observed behavior is probably not an artifact due to an increased side-chain flexibility. The novel rotamers are not associated with a particular position of Asp/Asn residues (e.g. capping residues) or a particular backbone conformation (all Asp/Asn residues studied have helical
,
angles). Detailed inspection and comparison with the known rotamers of the globular proteins, showed that the novel rotamer places the side chain in an optimal position relative to the protein backbone and the side chains of the neighboring
-helices, so that electrostatic repulsions between charged groups and steric hindrances between the side chains are minimized. However, the size of our sample is too small to go beyond a qualitative discussion of these novel rotamers.
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Side-chain shape, volume, polarity, packing density and cavity volume have been reported as factors that affect the pattern of substitutions in the protein interior (Bordo and Argos, 1990; Vlassi et al., 1999
). Conservation of these structural parameters is important for hydrophobic core mutations. Our present study provides evidence that the pattern of side-chain substitutions in 4-
-helix bundles is also consistent with the conservation of highly populated rotamers. In a previous study, Summers et al. (1987) concluded that for structurally and functionally homologous proteins, there is a high probability that the side-chain conformations are conserved. For amino acid substitutions, the conservation of orientation of both C
and C
atoms, was estimated to be in order of 3575% (Summers et al., 1987
). In the case of 4-
-helical bundles, the frequency of the amino acid substitutions that occur between residues with at least one rotamer in common was estimated to be ~75% and indicates an extensive conservation of rotamers in the core of homologous bundles. This is consistent with recent work by Vlassi et al. (1999), which found a pronounced tendency of 4-
-helical bundles to preserve hydrophobic core packing interactions upon mutations. Exceptions to this behavior (e.g. Phe
Ile substitutions), where rotamer conservation is not possible, occur at the end of the bundles. It is reasonable to assume that the constraints of the motif at those positions are weaker.
The aim of this work, as mentioned, was to examine qualitatively whether steric hindrances posed by the 4--helical bundle topology are reflected in the conformations of side chains. Within this framework, it has been shown that a tertiary motif can affect to some extent the conformations adopted by the side chains of some amino acids. This happens first through additional restrictions imposed to the side-chain conformations, second through the formation of novel
1,
2 clusters and third through some rotamers that appear to be more compatible with internal residues. A natural extension of this work would be a systematic analysis of additional known tertiary motifs. Such a study would provide valuable information for the understanding of protein folding and would find applications in the successful design of mutations and in the homology modeling of new structures.
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Notes |
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References |
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Received September 14, 2000; accepted February 15, 2001.