1 Molecular Biophysics Unit and 3 Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
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Abstract |
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Keywords: peptide flip/Ramachandran angles/ß-turn/Walker motif
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Introduction |
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In view of growing interest in the proteins containing a segment with Walker sequence, the Brookhaven Protein Data Bank (Berman et al., 2000) was searched and 649 polypeptide chains were found to have such a sequence. Many of these proteins do not bind or use nucleotides in their reactions. Therefore, it appeared that the sequence of the variant quartet and the specific loop structure might have a role in nucleotide binding. To fill the lacunae of information, conformations of the backbone of the peptide fragments of GXXXXGKT (S) were examined using Ramachandran angles. The data analysis in this paper indicates that different foldings are possible for the Walker sequences and only in the nucleotide-binding proteins they have a distinctive loop structure.
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Materials and methods |
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Results of data analysis |
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Search for the sequence GXXXXGKT (S) in the Protein Data Bank (April 2001 release) revealed 649 entries having this sequence, occurring in 395 protein structures with a resolution of 4 Å or better. Out of the 204 combinations of sequence possible for the variable region XXXX, only 92 were found to occur, of which 18 had only one entry. The present analysis is limited to these data
The Ramachandran angles of Walker sequence
Groups having more than one entry were examined from the structural viewpoint. The mean and r.m.s. values of the Ramachandran angles and
were computed at the eight residues of the segment. Should the same sequence give the same structure, as is widely believed, the r.m.s. values for a group would be small. Using a liberal upper limit of 40°, dissimilar structures were found to be present in 10 of these groups, as revealed by the high r.m.s. values for some of the Ramachandran angles. Using similarity of the Ramachandran angles as the criterion, these were divided into further sub-groups. The various sequences and location of the segment in the protein of the group thus obtained are given in Table I
, along with the PDB code, chain identifier, resolution of the structure and r.m.s. for those groupings with more than one entry (the protein names are not included in Table I
owing to the large number of examples; however, they are included in Table II
, which gives the selected set). The sub-groups with same sequence are indicated by suffixes A, B and C, to the group number. It can be seen that the r.m.s. values are now reasonably small. Some sequences assume more than one conformation: two for six sequences (005 GAGALGKT, 012 GLRSDGKT, 016 GLPAIGKT, 030 GATGTGKT, 058 GTAFEGKS and 077 GLYRTGKS); three for three sequences (006 GHVDHGKT, 033 GPTGVGKT and 059 GKGGIGKS); and four for one sequence (003 GGAGVGKT). These data implied that highly localized conformational variants are possible in these segments retaining overall structural similarity.
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The next step was the grouping of the conformations irrespective of the sequence of the variable region of the Walker segment. This was done as follows: (1) for those groups in Table I which had only one entry, the choice was unambiguous; (2) for those groups having more than one entry, one with the best resolution shown in bold face in Table I
had been picked up as the representative of the group/sub-group. These collectively gave 107 examples which were regrouped solely on the basis of similarity of the Ramachandran angles (
,
). Of the new sets thus obtained, 53 (out of a total of 107) entries constituted the major set. Another set had five entries, while seven others had two entries each. The last set comprised 35 entries, without any structural similarity among them. In any particular set, proteins with high overall sequence homology could be found, although the sequences of the variable region were different. These are as follows: (1AYL, 1OEN); (1A4R, 1MH1); (1DPF, 1TX4); (1CIP, 1AS0), (1AGP, 1CTQ, 1RVD, 421P, 821P); pairs [(2CYP, 1CCG); (1MHY (D), 1MTY (D)], as well as [1DT0 (A), 1ISA (B)]. Since the structures in such cases are expected to be similar, the entries that had the best resolution were retained. These were 1AYL, 1MH1, 1TX4, 1CIP, 1CTQ, 2CYP, 1MTY (D) and 1ISA (B). The final grouping thus obtained is given in Table II
, which has 45 proteins in set I, five in set II, two each in sets III, IV, V and VI and the remaining 38 in set VII. The mean and r.m.s. (
,
) values of sets IVI are given in Table III
and these are small enough to warrant structural similarity among the members. The r.m.s. has no relevance for the last set (set VII).
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Structural differences between segments having the same Walker sequence are also perceivable from the foregoing data. There are nine such examples in the present data set. The PDB codes along with the Ramachandran angles at the eight positions of Walker sequence of these nine pairs are given in Table IV. Large differences in Ramachandran angles are observed at four different locations within the segment (shown in bold face in the table). These are as follows: (i) 1VOM2MYS (A), (ii) 1F5N (A)1DG3 (A), (iii) 1FP6 (A)1G20 (E) and (iv) 1EFT1EFU (A) all at locations 3/4; (v) 1MMO (D)1MMO (E) at locations 4/5; (vi) 1ISA (A)1ISA (B), (vii) 1D9X (A)1D9Z (A) and (viii) 1BMF (D)1BMF (E) at locations 5/6; and (ix) 1G3I (A)1G3I (S) at locations 6/7. These large changes arise owing to a flip of the peptide unit spanning the two residues.
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The last entry in Table IV corresponds to the sequence GPTGVGKT occurring in the two chains A and S of the protein 1G3I and the conformations are different. In this case the peptide unit between locations 6 and 7 show a rotation of
90° about the virtual C
C
bond, instead of a flip, as is found in the other examples.
The ball and stick diagrams of these nine examples with a flipped peptide unit shown within a box are given in Figure 3. The overlap of the polypeptide backbones appears good. The examples of pairs ivi correspond to the flip occurring at the middle peptide unit of the well-known 4
1 hydrogen-bonded ß-turns of types I and II (Venkatachalam, 1968
; Gunasekaran et al., 1998
). However, the flip of the peptide unit observable in pairs viiix does not correspond to the ß-turn flip as the values of (
,
) are far different from those characteristic of ß-turn ranges. Further, the 4
1 hydrogen bond is also absent. Notwithstanding the flip, the same overall backbone structure is retained.
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In the case of proteins with oxygen-related reactions, the difference appears to be present in the polypeptides as isolated. The two proteins of methane monooxygenase, showing a flip at position 4/5, are derived from two organisms. The Walker sequence is present only in the Fe-form of superoxide dismutase and the two identical subunits of this enzyme protein exhibit this flip of a peptide unit. It is possible that the O=O group may act as the P=O in nucleotide phosphate in proteinsubstrate interactions. No relationship has so far been found between the peptide flips in Walker sequences and the activities of these proteins.
The secondary structures flanking the Walker sequence
The foregoing analysis indicated that the variable region is unlikely to determine the conformation of the Walker sequence A found in many nucleotide-binding proteins. The characteristic loop structure of the Walker sequence in these proteins is known to be preceded by a ß-strand and followed by an -helix (see, for an example, Abrahams et al., 1994
). It was therefore of interest to examine the occurrence of the flanking secondary structure of Walker sequences in proteins listed in Tables I and II
. For this purpose, segments of eight residues on either side of Walker sequences were examined for the presence of secondary structures (
=
-helix; ß = ß-strand;X = neither
nor ß; W = Walker sequence A). All nine possible combinations do occur and their distribution is given in Table V
. The majority of the examples fall into the category of ßW
. This structural motif is present in all cases in the sets 1, 2 and 6 and some in set 7 of Table II
. Interestingly, each of these proteins can bind to nucleotides leading to hydrolysis of the terminal phosphate to provide energy for accompanying reactions (e.g. ATPases) in a large number of cases and in some cases transfer the phosphate to acceptors (kinases). This is true of the examples of proteins in the miscellaneous set 7. Hence it appears that the structural motif ßW
, but not W alone, is the determining factor for nucleotide binding. The examples in sets 3, 4 and 5 of Table II
, although small in number (only two each), show distinctive motifs of XWß,
Wß and
W
, respectively.
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Discussion |
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The second observation in this study is the sharing of a common loop structure in proteins of the major group which use and bind nucleotide phosphates. These include kinases, phosphatases, ATPases, heat shock proteins, transfer/transport ATPases, permeases, myosin motor domain and elongation factor. The variable quartet (XXXX) has little influence on the bend as seen from the minor variation of overlap in this region (Figure 1). Indeed, the variable quartet is so highly random in sequence that it gives no clue on the looping. Of these, G (13.3%), A (11.9%), S (9.8%), V (8.4%) and T (5.9%) occur more commonly than other amino acids, but no sequence can be identified with a set or a sub-set of proteins. Thus the formation of the ß-turn loop seems to depend less on this sequence and more on the polypeptide chains on either side of the P loop, characteristically a ß-sheet at the N-terminus and an
-helix at the C-terminus. The absence of the classical 4
1 hydrogen bond in these loop structures appears to provide more room to surround and manipulate the phosphate chain of nucleotides for exchanging terminal phosphate.
Finally, the minor, local differences in the structures with the same Walker sequence, in our opinion, are of importance as they offer possibilities of participation in the functions of these proteins. These relate to the flip of peptide units in four positions (34, 45, 56, 67 in Table IV) in these sequences. The large differences in Ramachandran angles indeed brings to light these structural variants. Three examples are noted in the pairs that show these flips: the same enzyme protein from two different organisms (methyl monooxygenase), the two subunits of a homodimer protein (Fe-superoxide dismutase) and the binding of nucleotide to one of the two subunits (F1-ATPase, ß-subunit). The last example is a case with possible interaction of the substrate and the backbone structure of the enzyme active site and offers interesting mechanistic possibilities. Details of this have been reported elsewhere (Ramasarma and Ramakrishnan, 2002
).
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Notes |
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Acknowledgments |
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References |
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