Department of Biophysics, Bose Institute, P-1/12 CIT Scheme VIIM, Calcutta 700 054, India
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Abstract |
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Keywords: 310-helix/-helix/secondary structure/structural motif
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Introduction |
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Since the majority of 310-helices in proteins are very short, comprising of three residues (or one-turn) only, the results of PDB analyses of 310-helices have almost always been determined by these single-turn helices. However, with increasing number of structures in PDB, the number of longer (two-turn or more) 310-helices have grown to a small but a finite size. Therefore, as the number of known protein structures grow, there is a need to re-examine the role of 310-helices in proteins, especially the longer ones, in the context of their immediate structural environment. The present analysis, where we focus exclusively on two-turn and longer 310-helices in proteins, establishes that unlike the single-turn 310-helices, two-turn and longer 310-helices in proteins mostly occur independent of any contiguous -helix or other secondary structural element (SSE), and often as part of a super-secondary structural motif (SSSM), defined as a set of sequence-contiguous SSEs that pack into well-defined three-dimensional super-secondary structural or folding units (Efimov, 1984
).
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Materials and methods |
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Results and discussion |
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The average backbone dihedral angles of the 40 two-turn and longer 310-helices were found to be = 69.3 (±20.9) and
= 18.1 (±19.7). This compares well with a recent analysis with a larger dataset (Smith et al., 1996
) where the corresponding values were
= 62.8 (±38.0) and
= 16.5 (±34.7). The average backbone dihedral angles for the N-cap and the C-cap residues were [
= 90.1 (±60.3),
= 82.3 (±73.5)] and [
= 68.5 (±63.6),
= 67.5 (±87.2)] respectively. The average pitch of the helices were found to be 5.88(±0.29) Å. These average values as well as an inspection of individual backbone dihedral angles for the helices show that these are indeed well formed 310-helices and not just a distorted
-helix.
Amino acid composition of 310-helices
The larger culledpdb data set, consisting of 1085 protein chains, was used to determine the amino acid composition of 310-helices. The number of three, four, five, six, seven, eight, nine and 10 residue 310-helices present in this set are 2365, 396, 108, 94, 21, 8, 4 and 5 respectively. Here we summarize the results for amino acid over-representation where the helices are classified as short (35 residues) and long (6 and more residues). N-cap (N0): Asp, Asn, His, Pro (short) and Asp, Ser (long). N1: Pro, Ala, Gly, Trp (short), Pro, Met (long). N2: Glu, Asp, Ser, Asn, His, Trp (short), Asp, Glu, Gly, Pro (long). N3: Asp, Glu, Phe, His, Leu, Lys, Asn, Gln, Tyr, Ser, Trp, Ala (short), Trp, Tyr, Asp, Glu, Met (long). C-cap (C0): Leu, Ile, Phe, Val, Cys, Gly (short), Phe, Leu, Ile (long). The results on the short 310-helices are almost in accordance with a previous study (Karpen et al., 1992) performed exclusively on 77 three-residue 310-helices. While an amino acid composition study is important, because of its statistical nature, its application to a limited number of 310-helices is prone to errors. However, our results confirm the reported amino acid composition trend of the more abundant short 310-helices (Karpen et al., 1992
) and points to the interesting variation of amino acid composition as a function of chain length, which should be looked at more carefully only when a larger dataset becomes available before any definite conclusion can be made.
Two-turn and longer 310-helices present in SSSM
When the 310-helices from the small dataset were examined carefully, in more than 50% of cases (25 structures) they showed an unprecedented tendency to occur as part of a SSSM. A summary of the observed SSSMs are shown schematically in Figure 1 and a few representative 310-helices are shown in Figure 2
in the context of their immediate structural environments. The remaining 15 helices were found to occur as termini of
-helices (6), termini of the protein chain (1) or part of a long loop (8). Table I
summarizes all the 310-helices and proteins studied in this work.
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The 310-helix was found contiguous with an -helix in five proteins. In peroxidase, the contiguous
-helical and 310-helical stretches in the entire helix are of equal length. The 310-helix contributes the distal histidine coordinating the heme, suggesting an important indirect structural role related to protein function as shown in Figure 2a
. In acetohydroxy acid isomeroreductase only one turn of the
-helix was contiguous to the two turn 310-helix. This is unusual as more often the
-helix is longer than the 310-helix. Three other cases of
-helical termini were more typical of a long
-helix with a short 310-helical termini. Unlike the above case, in phosphoribosyl anthranilate isomerase, the two turn 310-helix is contiguous with one more turn of 310-helix. In only one protein was a C-terminal 310-helix found, that in 2Fe-2S ferredoxin.
As an independent SSE, the occurrence of 310-helices in long loops have been mentioned earlier (Martin et al., 1995). We found eight cases where 310-helices were present in long loops. In half of these, the 310-helix acts as a connector between two SSSMs. For example, in glutathione synthetase, a 310-helix connects two SSSMs. When not a connector, the loops are often part of a SSSM and exhibit a functional role, like the metal binding site in leucine aminopeptidase.
SSSMs containing a 310-helix
The simplest SSSM containing a 310-helix is the helixhelix motif comprising of one - and one 310-helix. These occur either as a 310/
corner (motif II.1) or a 310/
hairpin (motif II.2). The II.1 motif is mostly present in heme proteins with a globin-like fold exhibiting strong co-planar interaction with the heme. A typical case, that of myoglobin, is shown in Figure 2b
. The only non-heme protein containing the II.1 motif is glycogen phosphorylase where, unlike in the globin counterparts, the 310-helix is attached to the N-terminal end of the
-helix. Of the four cases where the motif II.2 was found, no SSEs were found close to the motif (sequence or spatial) suggesting inherent stability of the motif, as an independent folding unit. Only in one case, that of endoglucanase V, did a disulphide bond covalently connected the two helices.
A single 310-helix also forms a SSSM with two other SSEs. The 310-helix occurs as an anti-parallel ß-strand connector (motif III.1) in three proteins. In streptavidin, the anti-parallel ß-strands are part of a ß-barrel with Trp120 from the 310-helix being part of the binding site. In T-cell surface glycoprotein, the anti-parallel ß-strands are part of a larger ß-sandwich super structure, and in pseudoazurin, His81, part of the 310-helix, and Cys78 and Met86, flanking the helix, bind the copper ion. Two proteins contain the 310-helix as the `turn' in an -ß-hairpin motif (motif III.2)rubisco and triosephosphate isomerase, both sharing the ß/
(TIM) barrel fold. The other motif, 310-helix as a parallel ß-strand connector (motif III.3), occurs in endo-ß-N-acetylglucosaminidase where, strictly speaking, the 310-helix is part of the long loop described earlier.
A 310-helix was also found to occur in SSSMs containing more than three SSEs as described here. In old yellow enzyme and aconitase, a pair of 310- and -helices pack against a pair of parallel ß-strands (motif IV.1). In both cases, a loop connecting the SSEs in the motif exhibits strong binding activity or is part of the active site. Figure 2c
shows the interaction of motif IV.1 of aconitase with the Fe4-S4 cluster. In cellulase CelC, an
-helix310-helix hairpin motif forms a single layer SSSM with a pair of short antiparallel ß-strands (motif IV.2). Cellulase CelC exhibits yet another two layer motif (motif V.1) containing three parallel ß-strands and a pair of 310- and
-helices which recurs in Klebsiella aerogenes urease as well. In both cases several residues that are part of the motif's loops and turns participate in the active site.
Deoxyribonuclease I exhibits a two layer motif (motif VI.1) containing two pairs of anti-parallel ß-strands and a pair of 310- and -helices where several loop residues and a ß-strand residue are involved in oligopeptide binding. Four parallel ß-strands along with three helices, two
-helices and one 310-helix, form a two layer motif (motif VII.1) in dienelactone hydrolase, where, like the previous motifs, two residues in the loop of the motif form part of the active site. The same set of SSEs, with different connectivity, form a three layer motif (motif VII.2) in flavodoxin where a part of the motif, away from the 310-helix, is involved in FMN binding. Three pairs of anti-parallel ß-strands form a pseudo barrel SSSM (motif VII.3) along with a 310-helix in ascorbate oxidase, where three His residues, contributed by a ß-strand and a turn, are part of the active trinuclear copper site. In xanthine-guanine phosphoribosyltransferase, three parallel ß-strands, each 90° bent via a turn, form a motif (motif VIII.1) along with a pair of 310- and
-helices, where Asp89 in the turn is the magnesium ion interaction site, as shown in Figure 2d
.
Implications for SSSMs containing a 310-helix
Although we report only a small number of SSSMs containing two-turn and longer 310-helices in this work, upon analyzing the larger culledpdb dataset with less stringent structural quality, the number of SSSMs containing 310-helices grew. The larger dataset contained 132 two-turn and longer 310-helices where 63% 310-helices occur as part of a SSSM. The remaining 310-helices were found to occur as termini of -helices (18%), termini of the protein chain (4%) or part of a long loop (15%). This clearly establishes the high propensity of two-turn and longer 310-helices to be present in SSSMs. It should be noted that these SSSMs, especially IIIVIII, are not meant to represent typical and frequently observed motifs containing a 310-helix. Rather, they represent structural motifs within which the independent 310-helices occur, and therefore, even for single occurrences, they demonstrate the diversity of structural environment in which two-turn and longer 310-helices may occur. On the other hand, SSSM II can be considered as a `conserved' SSSM that occurs again and again in unrelated proteins. A simple sequence and structural analysis of these helices did not yield any clear statistically significant trend dictating their occurrences or stabilities, probably due to the small dataset used. Specific interactions, from within the protein and from solvent and ligand molecules, possibly account for the stability of these 310-helices. Only a protein-specific and case-by-case detailed atomic level examination would reveal if the presence of the 310-helices imposes any crucial structural constraints that ultimately translates into a functional role.
These motifs are novel in that they contain a 310-helix in place of the more expected -helix. It is remarkable that in the majority of the representative SSSMs illustrated here, either the 310-helix itself (direct) or some part of the motif (indirect) was found to have some functional role. A natural question that arises at this point is: `Why did a particular motif substitute a 310-helix for an
-helix?' As demonstrated from computer simulations (Tirado-Rives et al., 1993
; Basu et al., 1994
; Huston and Marshall, 1994
), energetically the two helical forms are very close, both in terms of inter-helical free energy difference
G° and free energy of activation
G
. Experimental data also suggest that they are inter-convertible under suitable conditions (Li et al., 1997
; Rigby et al., 1997
) or that the 310-helix may be an intermediate in
-helix formation (Sundaralingam and Sekharudu, 1989
). At the same time, structural motifs are thought to be independent folding units (Efimov, 1984
, 1997
). This is because identical structural motifs are often found to occur in unrelated proteins. Recent NMR studies of protein folding intermediates also lend support to such hypothesis (Barber et al., 1996
). In the context of folding and stability, these motifs seem to have stabilized some conformational intermediate containing a 310-helix during their folding pathway. Further, since the motifs often exhibit direct or indirect functional roles, they could also be special in that they can undergo local conformational relaxation (310-helix
-helix) under structural constraints arising from their environment like binding a ligand. It will be interesting to see, perhaps from computer simulations, the conformational pathways of folding of these motifs and if these motifs indeed show a 310-helix
-helix transition under suitable conditions.
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Acknowledgments |
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Notes |
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References |
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Received April 23, 1999; revised July 2, 1999; accepted July 2, 1999.