Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India 1Present address: Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143-2240, USA
2 To whom correspondence should be addressed. e-mail: ns{at}mbu.iisc.ernet.in
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Abstract |
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Keywords: ß-sheet/ß-strand/extended strand/hydrogen bonding/protein structures
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Introduction |
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The amino acid residue preferences and van der Waals stabilizing interactions are also characteristics of -helices and ß-strands in proteins (Street and Mayo, 1999
). The conformational entropy for the rotation of side chains is suggested to be a key feature in the preference or otherwise of an amino acid type to occur in
-helix or ß-sheet form (Presta and Rose, 1988
; Creamer and Rose, 1992
; 1994; Stapley and Doig, 1997
). For example, interactions between the side chains in positions i and i + 3 (and i + 4) in
-helices (Creamer and Rose, 1995
) and interactions between side chains across ß-strands involved in the formation of a ß-sheet are known to contribute to the stabilization of these structures (Lifson and Sander, 1980
; Otzen and Fersht, 1995
; Smith and Regan, 1995
; Wouters and Curmi, 1995
).
The ß-sheet is generally considered as a secondary structure although it is known to be distinct from the other kinds of regular secondary structures. The distinction stems from the fact that it requires spatially neighbouring regions of the protein, in extended conformation, to become aligned to form the characteristic inter-strand hydrogen bonds. However, it may be inappropriate to refer to the ß-strand as a secondary structure as, unlike other kinds of secondary structure, there are no intra-segment hydrogen bonds. Often, it is tempting to associate the role of formation of a main-chain region in the extended conformation (extended strands or E-strands) with that of ß-sheets.
In this paper, we draw attention to the regions of proteins in extended conformation that are not involved in the formation of a ß-sheet. As the description of an extended strand does not involve the hydrogen bonding of amide and carbonyl groups of the backbone, unless involved in the formation of a ß-sheet, the role of such extended structures in proteins is puzzling. Also, as these E-strands are not participating in the formation of ß-sheet there is no possibility of inter-strand interaction between non-polar residues like the one first observed by Lifson and Sander (1980). We have surveyed a large number of known protein structures and found that such isolated extended strands commonly occur in proteins and share characteristics of loops and ß-sheets in proteins. These E-strands are distinct from the polyproline type II extended conformation whose occurrence in globular protein structures has been extensively studied (Soman and Ramakrishnan, 1983
; Adzhubei et al., 1987a
c; Ananthanarayanan et al., 1987
; Adzhubei and Sternberg, 1993
). The polyproline type II conformation is somewhat similar to that of a single strand of collagen with characteristic (
,
) values of around (65°,140°) and is distinct from that of a ß-strand which has approximate (
,
) values of (115°,130°). Various features of polyproline type II-related structures (also referred to as mobile or M conformations by Esipova and co-workers) as seen in the known crystal structures of proteins have been analysed extensively by Esipova and co-workers (Adzhubei et al., 1987a
c; Vlasov et al., 2001
). In particular, they have made several detailed analyses of length, residue and tetrapeptide sequence distributions and have made comparisons of the extents of occurrence of this structure with that of
-helix and ß-sheet (Adzhubei et al., 1987a
c; Vlasov et al., 2001
). As can be seen during the course of the present analysis, the isolated E-strands described here are distinguished from the polyproline type II-related structures as the (
,
) values of isolated E-strands are closer to those of ß-sheets than polyproline type II structures.
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Materials and Methods |
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A dataset of 250 highly resolved (resolution <2.0 Å) and non-homologous protein structures derived from the Protein Data Bank (PDB) (Bernstein et al., 1977; Berman et al., 2000
) was used for the analysis. In the case of proteins with identical or very similar polypeptide chains, only one of them was considered. The chain used in such cases is shown as a fifth character in the complete list of PDB codes of the proteins used as follows: 1aan, 1aazA, 1abe, 1abk, 1acf, 1acx, 1afgA, 1ahc, 1ak3A, 1alc, 1ald, 1alkA, 1amp, 1ankA, 1aozA, 1apmE, 1arb, 1arp, 1ars, 1ast, 1bbhA, 1bbpA, 1bgc, 1bgh, 1bmdA,1brsD, 1bsaA, 1byb, 1cbn, 1ccr, 1cewI, 1cgt, 1chmA, 1cmbA, 1cot, 1cpcA, 1cpcB, 1cpn, 1cseE, 1cse I, 1csh, 1ctf, 1cus, 1ddt, 1dfnA, 1dmb, 1dri, 1dsbA, 1eca, 1esl, 1ezm, 1fas, 1fdn, 1fgvH, 1fiaA, 1fkf, 1flp, 1flv, 1fna, 1frrA, 1fus, 1fxl, 1fxd, 1gd1O, 1gia, 1gky, 1glqA, 1glt, 1gog, 1gox, 1gp1A, 1gpr, 1hel, 1hip, 1hleA, 1hleB, 1hoe, 1hpi, 1hsbA, 1hsbB, 1hslA, 1huw, 1hvkA, 1hyp, 1iag, 1ifb, 1isaA, 1isuA, 1lcf, 1lec, 1lib, 1lis, 1lldA, 1ltsA, 1ltsC, 1ltsD, 1mba, 1mbd, 1mdc, 1mjc, 1molA, 1mpp, 1nar, 1nbaA, 1nlkR, 1npc, 1nscA, 1olbA, 1onc, 1opaA, 1ovaA, 1pda, 1pgb, 1phc, 1php, 1pii, 1pk4, 1pmy, 1poc, 1poh, 1ppa, 1ppbH, 1ppbL, 1ppfE, 1ppt, 1prn, 1ptf, 1ptsA, 1r69, 1rbp, 1rdg, 1rec, 1ris, 1rnh, 1ropA, 1sacA, 1sbp, 1sgt, 1shaA, 1shfA, 1shg, 1sim, 1sltA, 1smrA, 1srdA, 1stn, 1tca, 1ten, 1tfg, 1tgn, 1tgsI, 1tgxA, 1thbA, 1tml, 1ton, 1trb, 1trkA, 1ubq, 1utg, 1whtA, 1whtB, 1xib, 1ypiA, 256bA, 2acq, 2act, 2alp, 2apr, 2bbkH, 2bbkL, 2bmhA, 2cab, 2ccyA, 2cdv, 2chsA, 2ci2I, 2cmd, 2cpl, 2ctvA, 2cy3, 2cyp, 2end, 2fcr, 2gbp, 2gstA, 2had, 2hbg, 2hmqA, 2lh7, 2lhb, 2ltnA, 2ltnB, 2lzm, 2mcm, 2mltA, 2mnr, 2msbA, 2ohxA, 2ovo, 2pabA, 2pia, 2plt, 2por, 2prk, 2rhe, 2rspA, 2sarA, 2scpA, 2sga, 2sn3, 2spcA, 2trxA, 2tscA, 2wrpR, 2ztaA, 351c, 3app, 3b5c, 3bcl, 3blm, 3c2c, 3chy, 3cla, 3cox, 3dfr, 3dni, 3drcA, 3ebx, 3est, 3grs, 3il8, 3mdsA, 3psg, 3rp2A, 3rubL, 3rubS, 3sdhA, 3tgl, 4azuA, 4bp2, 4cpv, 4enl, 4fxn, 4gcr, 4i1b, 4icb, 4insC, 4insD, 4mt2, 4tnc, 5chaA, 5cpa, 5fd1, 5p21, 5pti, 5rubA, 6ldh, 7acn, 7rsa, 8dfr, 8fabA, 8fabB, 9wgaA.
Identification of secondary structural elements
A stretch of at least four consecutive residues was identified as an E-strand if all the (,
) values in this region lie within the region defined by: 180° <
< 30°, 60° <
< 180° or 180° <
< 150° (Gunasekaran et al., 1998
). A strand in the extended conformation is qualified to be a polyproline II type of structure if the
-values at each of the residues of the segment are greater than 90°. The polyproline II type conformation has a close resemblance to that of a single strand of collagen and is known to occur in globular protein structures (Soman and Ramakrishnan, 1983
; Adzhubei et al., 1987a
c; Ananthanarayanan et al., 1987
; Adzhubei and Sternberg, 1993
; Vlasov et al., 2001
). The E-strands thus picked up were further separated into two classes, namely, isolated (those not in register with another E-strand by means of hydrogen bonding characteristic of ß-sheets) and aligned E-strands (those in register with another E-strand forming a ß-sheet), using an algorithm of secondary structure assignment based on the relative positions of the C
atoms (Ramakrishnan and Soman, 1982
; Soman and Ramakrishnan, 1986
). The E-strands not part of the ß-sheet are referred as isolated solely to reflect the fact that there is no hydrogen bonding interaction between the main-chain polar atoms of the strand with another strand of extended conformation. The aligned E-strands are also referred as ß-strands as they participate in the formation of the ß-sheet. From the ß-strands edge ß-strands were then defined as those segments of extended conformation which are in register with only one other ß-strand, as opposed to inner ß-strands which possess segments in register on either side. Identification of hydrogen bonding is based on the method used by Overington et al. (Overington et al., 1990
) involving distances between putative donors and acceptors and hydrogen bonding interaction energy.
Helices were identified in a manner similar to the E-strands with a criterion that at least four contiguous residues were in the R region (defined by 140° <
< 30°, 90° <
< 45°) (Gunasekaran et al., 1998
). 310 helices were differentiated from
-helices by using the procedure of Ramakrishnan and Soman (Ramakrishnan and Soman, 1982
). Further, a stretch of at least four consecutive residues which does not fall into any of the categories described above was classified as a loop and the remaining non-secondary structural non-loop residues were termed random coil residues. The results of identification of secondary structures using the C
position-based and (
,
)-based methods were very similar to those obtained using other methods such as DSSP (Kabsch and Sander, 1983
).
In the discussions, the symbols ßE, ßB, EI and PPII refer to the edge ß-strand, inner ß-strand, isolated E-strand and polyproline II regions, respectively.
Generation of all the neighbouring molecules in the crystal lattice
We also investigated the interactions, if any, between the isolated E-strands and the neighbouring molecules in the crystal lattice (our dataset contains no NMR structures). For every protein structure with at least one isolated E-strand we generated the fractional coordinates using the cell dimensions given in the coordinate file. Using the space group information, the equivalent points are automatically recognized from the library of equivalent points stored against every space group. The fractional coordinates of all the atoms corresponding to every equivalent point are generated. Further, translations by 1, 0 and +1 are made along each of the fractional x-, y- and z-axes to generate the entire system of neighbouring molecules (including those in the adjacent unit cells) around a given molecule. Finally, all the generated coordinate sets are converted to the original orthogonal ångstroms coordinate system using the cell dimensions. For example, if the space group of a given entry is such that it has four equivalent points [including the original (x, y, z)] and each of the equivalent points can result in a set of 3 x 3 x 3 (= 27) neighbouring molecules to result in the generation of 4 x 27 (= 108) coordinate sets. We cross-referenced our results with those given in PQS server (Henrick and Thornton, 1998) and the results were found to be absolutely consistent. Interaction between the main-chain polar atoms of putative isolated E-strands in the original coordinate set and the neighbouring copies in the crystal lattice was analysed. Further, if a crystal structure has more than one molecule in the asymmetric unit, interaction between the putative isolated E-strand and the other molecule(s) present in the asymmetric unit was also analysed.
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Results and discussion |
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The propensities of the 20 amino acid residues to occur in the various kinds of extended segments and loops were calculated in order to assess the preferences exhibited by individual residues for specific types of structures. The propensities were calculated using the standard ChouFasman approach (Chou and Fasman, 1974). The results are shown in Table II. It can be seen that, in general, the hydrophobic residues are preferred over the polar residues in all the three extended segments, EI, ßE or ßB strands. It is widely known that ß-branched residues such as Val, Ile and Thr show a high propensity to occur in ß-sheets (Chou and Fasman, 1974
; Lifson and Sander, 1979
; Munoz and Serrano, 1994
; Swindells et al., 1995
). Interestingly, the preferences of residues to occur in EI strands also reflect similar characteristics. This strongly reinforces the earlier reports (Swindells et al., 1995
) that strand formation is determined by the intrinsic preferences of amino acid residues (Dinner et al., 1999
). In contrast, as is well known, the loops prefer polar residues.
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Preference for prolines in EI strands
The enhanced preference for proline to occur in EI strands led us to investigate the existence of polyproline type II strands (PPII) (Soman and Ramakrishnan, 1983; Adzhubei et al., 1987a
c; Ananthanarayanan et al., 1987
; Adzhubei and Sternberg, 1993
; Stapley and Creamer, 1999
; Vlasov et al., 2001
) which resemble the EI strands in that the participating residues of the former also possess extended conformation. The PPII regions were recognized as a contiguous stretch of (
,
) values in the polyproline region (see Materials and methods) and did not depend upon the occurrence or otherwise of proline. The search yielded a total of only 56 examples of PPII strands. When the PPII strands were weeded out of the dataset, it was found that these represented only a very small fraction of the extended segments. The recalculated values of the propensities, shown in Table II, after the removal of such strands, still show a striking preference for proline to go into EI strands over the aligned ß-strands.
Close to 42% (N = 216) of the 518 segments classified as EI strands contain at least one proline residue in its sequence. Also, these proline residues are interspersed in the sequence with no specific preference for any particular position within the sequence. These observations lead to two interconnected features that can be conceived as the cause of the high preference for proline in EI strands. First, the lack of an amide hydrogen in the backbone of proline makes it an unsuitable candidate for inclusion into any of the standard secondary structures in which backbone hydrogen bonding plays a crucial role, as in -helices and ß-sheets (Richardson and Richardson, 1988
; Aurora and Rose, 1998
; Gunasekaran et al., 1998
). Second, proline possesses an intrinsic feature of influencing the backbone torsion angles of the residue preceding it to adopt an extended conformation (Gibrat et al., 1991
; MacArthur and Thornton, 1991
; Hurley et al., 1992
). These unique characteristics of proline seem to be the reason for its preferred existence in EI than either the ßE or ßB strands.
Comparison of propensities of occurrence between various kinds of segments
Since EI strands are not part of ß-sheets, secondary structure recognition algorithms usually classify these as loops. Thus, in order to assess their relationship with the PPII, ßE and ßB strands and loops, we calculated Pearsons correlation coefficient (P value) (Minor and Kim, 1994a) between the various pairs of amino acid propensities. The P value was calculated using the equation
P2 = {(xi xav)(yi yav)/[
(xi xav)2
(yi yav)2]
}2
where xi and yi pairs correspond to the amino acid propensities; i represents the index of summations and is the number of amino acid types considered and xav and yav represent mean x and y values, respectively. The P values are listed in Table III. In order to avoid the bias made by the two special residues, the highly flexible Gly and the rigid Pro, they were eliminated from the dataset and the coefficients were recalculated (Swindells et al., 1995). The plots describing these correlations are shown in Figure 3. As we have a reasonably large number of residues in our dataset, the reliability of propensity values is expected to be unaffected by the exclusion of prolyl and glycyl residues from the calculations.
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On the other hand, EI strands show a negative correlation (P = 0.09) with the loop segments. There does not seem to be any drastic change in this value even after the removal of Gly and Pro (P = 0.29). The fact that the residue preferences of EI strands show a strong correlation with the ßE and ßB strands and simultaneously show a negative correlation with loops (in the same way as the ßE and ßB strands; data not shown) induces us to propose that the EI strands resemble the ß-sheet forming ß-strands in terms of the residue preferences (except for Pro) and structure.
Comparison of propensities with other scales
Since the discussion above leads us to believe that EI strands are similar to the other aligned ß-strands, at the level of residue preferences (except for Pro), we compared our propensities with other scales reported in the literature as done, for example, by Finkelstein (Finkelstein, 1995). Since the experimental scales were all derived by hostguest studies by measuring the
G for replacement of one residue with another, the results are reported on a scale relative to one of the amino acid residues, usually alanine or glycine. Also, most of these scales also give an abnormal value of
G for proline. For these reasons, we eliminated all the three residues from our calculations.
The propensities of various amino acids for EI strands correlate best with the ß-sheet propensities derived by Minor and Kim (Minor and Kim, 1994b) (P = 0.75). The comparison of propensities is shown in Figure 4. The same authors also demonstrated the context dependence of amino acid preferences by analysing edge and interior positions (Minor and Kim, 1994a
,
1996) but our propensities show only a very weak correlation with this scale (P = 0.26). We also compared our data with two other scales (Kim and Berg, 1993
; Smith et al., 1994
), but both showed very low correlations of 0.53 and 0.20, respectively.
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Accessibilities of the isolated extended segments
The degree of solvent accessibility of an isolated extended strand was calculated as the ratio of its total accessible surface area (ASA) (Lee and Richards, 1971) as it occurs in the protein to the sum of the ASA of each of the constituent residues as it occurs in an extended conformation (Miller et al., 1987
). It is observed that just over 90% of the 518 segments of ßI strands have accessibilities in the range 050% with about 27% in the range 3040%. Only very few of the segments (13.5%) have low accessibilities (<10%), indicating that most of the EI strands tend to be exposed to the solvent.
In a bid to compare the accessibility profiles of EI strands with both the traditional (aligned) ß-strands and the loops, Figure 5 shows a comparison of the profiles of each of these segments. It can be seen immediately that most of the aligned ß-strand segments have very low accessibility values. Close to 55% of the segments have accessibilities in the range 010% with the population at successive intervals progressively falling. This can also be seen from the inset in Figure 5, which shows the cumulative frequency against the accessibility intervals, where the curve corresponding to the aligned ß-strands reaches a plateau fairly rapidly.
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From the amino acid preferences of EI strands we see that most of the preferred residues are non-polar in nature. However, we also see that these segments of EI strands are exposed to the solvent like the loops. To resolve this dichotomy, we analysed the side-chain accessibilities of the non-polar residues in both the EI strands and loops. The results are shown in Figure 6. It can be seen that the behaviour of non-polar side chains is almost identical in both of these kinds of segments. Close to 45% of the non-polar side chains of EI strands are buried from the solvent indicated by the first peak in Figure 6, indicating that the high accessibility is contributed by polar side chains and main-chain atoms.
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The structural environment of EI strands was analysed by identifying the first occurrence of a secondary structural element before and after these segments. We searched for patterns of the form SaXXXEI strandXXXSb, where S corresponds to a residue in one of the secondary structures and X is a residue which could be part of a regular secondary structure or loop. Table IV shows the frequencies of occurrence of various secondary structural segments at positions Sa and Sb in the vicinity of the isolated E-strands.
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Hence it appears that a stretch of extended conformations is the best type of structure to provide the maximum end-to-end distance for a given number of residues. These EI strands may supplement the long loops that connect secondary structural elements which are spatially well separated.
Hydrogen bonds to the backbone groups of EI strands
Since EI strands are not part of ß-sheets they lack the periodic hydrogen bonding ladder that characterizes a ß-sheet. Hence, the backbone carbonyls and amides of isolated E-strands would have to be satisfied with hydrogen bonds from other protein atoms or the solvent. The 2564 residues participating in EI strands were analysed for hydrogen bonds to or from their backbone polar groups. Of the 2564 residues, 263 were proline residues with only the carbonyl oxygen available as an acceptor for hydrogen bonds. Of these, only 94 examples were hydrogen bonded and the other 169 examples were not. Among the non-prolyl 2301 examples we have four possibilities: a residue could be hydrogen bonded through the amide nitrogen, carbonyl oxygen, both or neither. It was found that 380 examples were hydrogen bonded through the amide nitrogen, 391 through the carbonyl oxygen and 656 through both and 874 examples had no hydrogen bonds to the backbone polar groups.
It can be seen from above that close to 41% of the 2564 residues in EI strands are not hydrogen bonded. Given the solvent-exposed nature of these segments, the hydrogen bonding potential of these polar groups could be satisfied through the surrounding water molecules.
Interaction of EI strands with the adjacent molecules in the crystals
Crystallographic symmetry-related molecules of all the protein structures in our dataset with at least one potential isolated E-strand have been generated as outlined in the Materials and methods section. We investigated the interaction of EI strands with all the adjacent molecules in the crystal lattice. In the cases with more than one molecule in the asymmetric unit interaction between an EI strand and the other chains within the asymmetric unit was also studied.
Of the 518 putative isolated E-strands identified in our analysis only 34 are involved in any prominent interaction with the adjacent molecules in the crystals. At least two hydrogen bonds involving the main-chain carbonyl or amide at the strands and polar groups from the adjacent molecules could be identified in these 34 examples. Eighteen of these examples result from interaction between two molecules in the asymmetric unit of the crystal structure. Some of these examples correspond to the ß-sheet formation with ß-strands coming from different tertiary structures such as seen in the structure of pea lectin. Other examples correspond to interactions between the main-chain carbonyl or amide in the strand with the side-chain polar atoms from a neighbouring molecule.
Based on these observations it is clear that 484 (= 518 34) E-strands in the dataset deemed as isolated by considering a copy of the tertiary structure remain isolated even if the adjacent molecules in the crystals are considered.
Conservation of EI strands in families of homologous proteins
In order to assess the extent to which the EI strands are conserved in homologous proteins, an analysis was carried out on a database of families of aligned homologous protein structures (HOMSTRAD) (Mizuguchi et al., 1998). Considering 97 families of the database that had more than three members, one structure from each family was chosen at random to function as the reference structure. Isolated EI strands present in that structure were identified and their index of conservation was computed amongst the members of that family. The index of conservation (I) of a EI strand from the reference structure was calculated as the percentage ratio of the number of members of the family in which at least 90% of the length of the segment from the reference structure is structurally conserved to the total number of members in that family. The results are shown in Figure 7. It can be seen that about 41% of the 290 examples of EI strands analysed are conserved with a very high value of the index (ranging from 60 to 100%). However, the majority of the examples have low indices of conservation. Thus, the data seem to suggest that these segments are indeed variable in structure, resembling the loop segments of proteins.
|
Isolated E-strands commonly occur in proteins. In spite of the lack of regular hydrogen bonding partners they seem to form stable stretches which are potentially stabilized by the surrounding water molecules and the side chains of polar residues in the protein. It has also been shown that almost all of these isolated E-strands remain isolated even in the context of quaternary structure and interaction of a protein molecule with neighbouring copies in the crystal lattice. In terms of the residue preferences, except for the abundance of proline, they show good similarity to the ß-strands (that are part of sheets), supporting the fact that strand formation is determined by the intrinsic preferences of certain types of residues. On the other hand, they have their other characteristics similar to loops. They seem to be as exposed to the solvent as loops and the hydrophobic groups present in these strands behave in a similar fashion to those in the loops, being buried from the solvent. These extended structures seem to be supplementing the loops in efficiently traversing long distances in the protein with a minimal number of residues. Finally, these observations indicate that isolated E-strands occupy an individual existence with its characteristics shared partly with that of the ß-sheet forming ß-strands and partly with the loops.
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Acknowledgement |
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Received April 8, 2002; revised March 31, 2003; accepted April 8, 2003.