1 Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, 2 Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK
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Abstract |
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Keywords: asparagine/aspartic acid/carbonyl stacking/propensity/Ramachandran plot
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Introduction |
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Amino acid Ramachandran preferences |
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The empirical rules of amino acid conformational propensities calculated from known structures have been examined many times and with a large number of different structural data sets (Chou and Fasman, 1978; McGregor et al., 1987
; Swindells et al., 1995
). However, while many features are well documented, most have not been explained in terms of the interactions that stabilize them. In this context asparagine is interesting (Richardson, 1981
) because it has a high preference for the partially allowed regions of the Ramachandran plot (Chou and Fasman, 1974
) and in particular for the left-handed
-helical region (
-L) (Srinivasan et al. 1994
). Aspartic acid also shows a high preference for the partially allowed regions of the Ramachandran plot. Its propensity for the
-L region is lower than that of asparagine but is still well above average for the amino acids (Chou and Fasman, 1978
).
Why any amino acid residue is found in the partially allowed Ramachandran regions is a question of interest. One possibility is that an amino acid may tolerate small deviations from its ideal conformation in order to optimize stabilizing tertiary interactions in the protein, such as hydrogen-bonding patterns or keeping hydrophobic residues buried or interactions with the substrate or ligand at the active site (Moult and Herzberg, 1991). Thus, the unfavourable energy of these small local distortions can be compensated by the energetic favourability of the overall structure. Amino acids with a high propensity for partially allowed Ramachandran regions should therefore display some form of stabilization with respect to all other amino acids in that conformation to explain this relative preponderance.
The -L is a small region with positive
in the Ramachandran plot labelled area E in Figure 2
. For non-glycyl residues it has a higher energy than the larger right-handed
helical region with negative
. L-Amino acids in the
-L conformation exhibit a close approach of the backbone carbonyl oxygen to the ß-carbon of the side chain, a situation which does not occur in the right-handed
-helical region.
-L has particularly unfavourable steric clashes for ß-branched amino acids such as Thr, Val and Ile. Asparagine adopts conformations in
-L and other partially allowed regions more readily than any other non-glycyl residue. It has often been assumed that this preference is due to hydrogen bonding by the asparagine side chain back to a preceding main-chain carbonyl, but this hydrogen-bonding pattern does not occur very frequently (C.M.Deane, unpublished results).
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Carbonylcarbonyl interactions |
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MacCallum et al. (1995a,b) examined the importance of Coulombic interactions between backbone carbonyls in proteins as a stabilizing factor in -helices, ß-sheets and the right-handed twist often observed in ß-strands. Their calculations indicate an attractive interaction energy of ~-8 kJ/mol in specific cases and they remark that, within their computational model, these interactions are ~80% as strong as the backbone hydrogen bonds in proteins.
In this paper, we examine the possibility of asparagine and aspartic acid side-chain carbonyls interacting with neighbouring backbone carbonyls in this fashion. We show that such interactions can stabilize these two amino acid types in partially allowed conformations in a way that is not available to other amino acids.
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Results and discussion |
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Stabilizing interactions
An examination of the 45 examples of asparagine and the 14 examples of aspartic acid in area E, from test set structures having resolutions better than 1.5 Å, was carried out to search for possible stabilizing interactions that might account for the high propensity of asparagine in this region. This revealed that the asparagine/aspartic acid side chains are frequently solvent exposed or hydrogen bonded to distant parts of the protein chain (not involved in hydrogen bonding back to the neighbouring backbone atoms). If this distant hydrogen bonding were the only stabilizing interaction, glutamic acid and glutamine should have similarly high propensities since they have the same hydrogen bonding possibilities. However, an interaction was noted that might add extra stability to those asparagine and aspartic acid in area E; this is a carbonylcarbonyl interaction. This stabilization is local and theoretically available to four of the amino acids, asparagine, aspartic acid, glutamine and glutamic acid, as they all possess carbonyls in their side chains. The percentage of residues involved in an interaction between the side-chain carbonyl of X to the backbone carbonyl of X or X 1 was calculated for each of the areas and overall for the residue types asparagine, aspartic acid, glutamic acid and glutamine, as shown in Figure 1B. Glutamine and glutamic acid show very little carbonyl stacking locally, presumably owing to steric constraints. Of the asparagine side-chain residues in area E, 79% are involved in carbonylcarbonyl interactions at a carbonoxygen separation of
4 Å. The 4 Å cut-off was selected as at this range there is still an appreciable attractive interaction energy out to van der Waals + 0.5 Å (Allen et al., 1998
). The same ratio between the areas and the different amino acids is observed with variation in the distance cut-off between 3.5 and 4.2 Å.
Carbonylcarbonyl stacking of the side chain over main chain
These data show that asparagine and aspartic acid have far greater percentages of carbonylcarbonyl interactions per residue than glutamine and glutamic acid and that asparagine shows the greatest percentage of all. Carbonylcarbonyl interactions can be seen in all the regions for asparagine and aspartic acid but are least prevalent in area A (Figure 1B).
Two types of carbonylcarbonyl interaction were observed: type 0, stacking of the asparagine/aspartic acid side chain over its own backbone carbonyl; and type 1, stacking of the asparagine/aspartic acid side-chain carbonyl over the backbone carbonyl of the previous residue. An example of each is shown in Figure 3. These can generally be correlated with the two most common
1 of asparagine/aspartic acid. Type 0 is usually associated with a
1 in the trans (180°) conformation and type 1 is associated with
1 in the g+ (60°) conformation. The two types have slightly different percentages of occurrence in the five different areas. They also have very different average distances of interaction: using the shortest carbonoxygen separation, type 0 is about 0.4 Å shorter than type 1 on average.
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The remaining question in this analysis is why asparagine has a much greater propensity than aspartic acid for area E and for the partially allowed regions in general, when both have the possibility of forming carbonylcarbonyl interactions. The carbonylcarbonyl interaction is seen less for aspartic acid and particularly in area E. It would seem that the interaction is weaker for aspartic acid, probably because of greater repulsion between the charged oxygen atoms of the side chain when they are close to the backbone oxygen atoms.
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Conclusion |
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This predominance is explained by a stabilization that can only be achieved by asparagine and aspartic acid. This is a carbonylcarbonyl interaction between the side-chain carbonyl and local backbone carbonyls, specifically the backbone carbonyl of the asparagine/aspartic acid residue in question or the one previously. The interaction energy of this dipoledipole interaction has previously been calculated and shown to be of comparable strength to that of a hydrogen bond. This means that there is stabilization energy available to asparagine/aspartic acid for the partially allowed regions of the Ramachandran plot in all structural contexts that is not available to any other amino acid. Hence it is energetically more favourable for an aspartic acid or asparagine to be found in these partially allowed regions than any other non-glycyl amino acid.
The theme of this analysis emphasizes the fact that close examination of unusual propensities in the Ramachandran plot can reveal possible stabilization for these features which can lead to further understanding more generally of the dependence of protein tertiary structure on primary structure.
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Materials and methods |
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A value P > 1 indicates a preference and a value P < 1 indicates disfavour for a region. The calculations were done with and without secondary structure as defined by Kabsch and Sander (1983), and also with and without Pro and Gly as they have unique conformational features (Kabsch and Sander, 1983). Propensities were also calculated for regions outside regular secondary structure replacing the number in an area of the plot by the number not involved in secondary structure.
The Protein Data Bank (PDB) (Abola et al., 1987, 1997
) files to be used for calculation of these propensities were selected using PDB_SELECT (Hobohm et al., 1992
; Hobohm and Sander, 1994
) to collect 253 PDB chains with an R factor below 30%, a resolution better than 1.8 Å and sequence identity less than 35%. The PDB chains were then `cleaned' (Morris et al., 1992
). These chains contain a total of 51151 residues with 3164 aspartic acid and 2525 asparagine residues; 43% of all residues are involved in secondary structure as defined by Kabsch and Sander (1983).
The overall environments of the individual asparagine and aspartic acid residues in these regions were then inspected for interesting features. Carbonylcarbonyl interactions between the asparagine/aspartic acid/glutamine/glutamic acid side chain and the backbone carbonyl of the asparagine/aspartic acid/glutamine/glutamic acid residue or the preceding one were then calculated. A carbonylcarbonyl interaction was presumed to occur if the shortest distance between the carbon/oxygen of the side chain to an oxygen/carbon of a backbone carbonyl was below a threshold. The distance cut-off for the analysis of these interactions was varied between 3.5 and 4.2 Å.
In the X-ray crystal structures of proteins defined at resolutions >1.5 Å, the electron density map cannot distinguish between carbon, nitrogen and oxygen atoms and the hydrogen atoms can rarely be seen. For the majority of side chains the atoms can be uniquely identified from the shape of the electron density, but asparagine, glutamine and histidine side chains appear symmetrical in the electron density and some specific atoms can only be identified on the basis of their interactions, principally hydrogen bonds. In the cases of asparagine and glutamine, the problem is to distinguish between the side-chain nitrogen and oxygen atoms. This relates directly to the analysis of the carbonylcarbonyl interaction described here. In this study the asparagine and glutamine sidechains were allowed to occur in either conformation and that which confirmed a carbonylcarbonyl interaction was used in the calculation.
The type of interaction motif was defined by calculation of the four angles A (C1O1C2), B (O1C2O2), C (C2O2C1) and D (O2C1O1) (subscript 1 indicating the side-chain carbonyl and 2 the backbone carbonyl).
The three ideal motifs as described by Allen et al. (1998) are as follows: (a) an anti-parallel motif with two short carbonoxygen interactions and ideal angle pattern 90, 90, 90, 90°; (b) a perpendicular motif with only one short carbonoxygen interaction and angle pattern 75, 15, 180, 90°; and (c) a parallel motif with only one short carbonoxygen interaction and angle pattern 90, 90, 55, 55°.
If all angles were within 15° of these ideal patterns (carbonyl bond length 1.2 Å, carbonoxygen contact 3.5 Å and no shearing) and at least the expected number of short carbonoxygen interactions were present in any interacting pair, they were considered to form that motif. The angle pairs A,C and B,D are interchangeable for calculation of similarity to the angle pattern.
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Notes |
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References |
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Received February 3, 1999; revised March 19, 1999; accepted March 24, 1999.