PROTEINS: Structure, Function, and Genetics 51:167–171 (2003)
Registering ␣-Helices and ␤-Strands Using Backbone
COH. . .O Interactions
S. Kumar Singh, M. Madan Babu, and P. Balaram*
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India
ABSTRACT
The possible occurrence of a novel
helix terminating structural motif in proteins involving a stabilizing short COH. . .O interaction has
been examined using a dataset of 634 non-homologous protein structures (<2.0 Å). The search for this
motif was prompted by the crystallographic characterization of a novel structural feature in crystals of
a synthetic decapeptide in which extension of a
Schellman motif led to the formation of a COH. . .O
hydrogen bond between the T-4 C␣H and the Tⴙ1
CAO groups, where T is the helix terminator adopting a left handed (␣L) conformation. More than 100
such motifs with backbone conformation superposing well with the peptide examples were identified.
In several examples, formation of this motif led to an
approximately antiparallel arrangement of a helical
segment with an extended ␤-strand. Careful examination of these examples suggested the possibility of
registering antiparallel arrangement of helices and
strands by means of backbone COH. . .O interactions with a regular periodicity. Model building
resulted in the generation of idealized ␣␤ and ␤␣
motifs, which can then be generalized to higherorder repetitive structures. Inspection of the antiparallel ␣␤ motif revealed a significant propensity for
Ser, Glu, and Gln residues at the T-4 position resulting in further stabilization using an O. . .HON sidechain– backbone hydrogen bond. Modeling studies
revealed ready accommodation of serine residues
along the helix face that contacts the strand. The
theoretically generated folds correspond to “open”
polypeptide structures. Proteins 2003;51:167–171.
©
2003 Wiley-Liss, Inc.
Key words: polypeptide folds; COH. . .O hydrogen
bonds; helix termination; ␣␤ motifs
INTRODUCTION
Variations in the spatial arrangements of ␣-helices and
␤-sheets give rise to the diversity of polypeptide folds
observed in globular protein structures.1–3 While the
individual structural elements, helices, and sheets are
stabilized by the cooperative formation of multiple hydrogen bonds,4,5 the precise arrangement of secondary structural elements is generally determined by the tertiary
interactions involving amino acid side-chains.6 – 8 The serendipitous observation of an interesting helix termination
motif stabilized by a potential COH. . .O hydrogen bond,
©
2003 WILEY-LISS, INC.
Fig. 1. Superposition of the decapeptide Boc-LUVALUV-DA-DL-UOMe with eight examples of the similar motifs observed in protein
structures (DA ⫽ D-alanine; DL: D-leucine; U ⫽ ␣-aminoisobutyric acid,
Aib). The superposed examples are: 1MROA, Y432 to L441; 1AXN, K59
to E68; 1E2UA, A100 to D109; 1EW4A, Q93 to T102; 1AXN, D215 to
D224; 1HVBA, M169 to S178; 1D5TA, D419 to A428; 1DIXA, N147 to
T156. The observed root-mean-square diviation is 0.65 Å (C␣ atoms only).
in the crystal structure of a synthetic peptide,9 prompted
us to examine the possibility of organizing structures
containing ␣-helices and ␤-strands using backbone
COH. . .O interactions, in order to achieve appropriate
registry. This approach permits the visualization of a
novel class of polypeptide folds in which approximately
antiparallel arrangements of helices and strands may be
achieved purely by favorable backbone interactions.
Figure 1 shows the superposition of the helix termination motif observed in the decapeptide9 Boc-LUVALUV-DA*Correspondence to: P. Balaram, Molecular Biophysics Unit, Indian
Institute of Science, Bangalore 560012, India. E-mail:
pb@mbu.iisc.ernet.in
Received 9 August 2001; Accepted 16 July 2002
168
S.K. SINGH ET AL.
Fig. 2. a: Stick diagram of residues Trp 88 to Phe 105 from the protein CYAY, a member of the frataxin
family of proteins from E. coli (PDB id: 1EW4). Residues Trp 88 to Ala 99 forms the helical segment, Gly 100 is
the T residue and residues Glu 101 to Arg 106 forms the strand segment. Hydrogen atoms on only two labeled
C␣H groups are shown. The bold dotted (horizontal) line represents the COH. . .O hydrogen bonds, whereas
the T-8 to T-4 H␣OH␣ distance in the helix and the T⫹1 and T⫹3 OOO distance in the strand are marked by
the approximately vertical double-edged arrows. The parameters of the two COH. . .O hydrogen bonds are:
T-4 C␣H. . .OAC T⫹1: 2.35 Å, COĤ. . .O: 149°; T-8 C␣H. . .OAC T⫹3: 3.14 Å, COH. . .O: 113°. b: Schematic
view of an idealized antiparallel helix strand motif stabilized by three successive COH. . .O interactions. The
model was generated using a poly-alanine chain with idealized starting values of ␾ ⫽ ⫺65°, ␺ ⫽ ⫺40° for the
␣-helical segment, ␾ ⫽ ⫺120°, ␺ ⫽ 120° for the ␤-strand segment, ␾ ⫽ 60°, ␺ ⫽ 40° for the terminating (T)
residue, and ␾ ⫽ 100°, ␺ ⫽ 0° for the T-1 residue. One hundred cycles of energy minimization in Insight using a
CAO constraint ⱕ3.5 Å yielded the structure illustrated. Twelve residues were used in the helical segment and
six residues in the strand segment.
L-U-OMe (U ⫽ Aib, ␣-aminoisobutyric acid), with eight
similar examples obtained from a dataset of 634 highresolution protein structures. This stereochemical feature
is characterized by the termination of an ␣-helix in a
Schellman motif,10 where the terminating residue “T”
adopts a left-handed (␣L) conformation.11 A short COH. . .O
interaction between the T-4 C␣H and the T⫹1 CAO is a
novel feature of the motif (C. . .O ⱕ3.5 Å) when the T⫹1
residue adopts an extended (␤) conformation. The possible
importance of COH. . .O interactions as a stabilizing
feature in crystals was recognized almost four decades
ago.12 The existence of COH. . .O hydrogen bonds in
collagen was considered in early structural studies,13 with
firm experimental evidence appearing much later.14 Several recent studies have emphasized the role of weak
COH. . .O interactions in organic and biological molecules.15–19
D
RESULTS AND DISCUSSION
A survey of a dataset of 634 non-homologous (ⱕ25%
sequence homology) protein structures20 from the Protein
Data Bank21 (resolution ⱕ2.0 Å) revealed as many as 111
examples of the motif (Babu et al.).22 In almost half these
cases, the ␣-helix was followed by a ␤-strand, with the long
axis of the two elements making an angle less than 40°. A
few examples were identified in which an approximate
antiparallel arrangement of helices and strands was
achieved, wherein two short COH. . .O interactions, T-4
C␣H to T⫹1 CAO and T-8 C␣H to T⫹3 CAO, could be
observed. Figure 2(a) illustrates the structure seen for
residues 88 to 105 in the protein CYAY23 (a member of the
frataxin family from Escherichia coli, PDB id: 1EW4, 1.4
Å). Inspection of the structural feature reveals that the T-8
to T-4 H␣OH␣ distance (5.83 Å) in the helix is approxi-
REGISTERING ␣-HELICES AND ␤-STRANDS
169
Fig. 3. a: Stick diagram of residues Asn 118 to Tyr 136 from the antigen 85C from M. tuberculosis (PDB id:
1DQZ). Residues Asn 118 to Leu 123 forms the strand segment, residues Ser 124 and Met 125 is a slightly
distorted type II⬘ ␤-turn, and Met 125 to Tyr 136 forms the helical segment. The bold dotted (horizontal) line
represents the COH. . .O hydrogen bond between the CAO of Gly 122 and C␣H of Gly 127 (H␣. . .O ⫽ 2.96 Å).
b: A schematic view of an idealized antiparallel strand helix motif stabilized by three successive COH. . .O
interactions. The model was generated using a poly-alanine chain with idealized starting values for the helix
and strand regions as in Figure 2(b). The dihedral angles at the connecting turn segment had ideal type II⬘
values (␾1 ⫽ ⫹60°, ␺2 ⫽ ⫺120°; ␾2 ⫽ ⫺80°, ␺2 ⫽ 0°). Ten residues were used in the helical segment and
seven residues in the strand segment.
mately equal to the OOO distance of 5.82 Å between the
T⫹1 and T⫹3 CAO groups. Encouraged by this, we
considered the possibility of extending the registry observed between a helix and an extended strand to longer
stretches of secondary structures. Using idealized starting
geometries, extremely mild energy minimization (100 cycles
of minimization with the Insight II software, MSI Inc.) was
applied to the starting structure with the proviso that the
appropriate C. . .O distances must be ⱕ3.5 Å. Figure 2(b)
illustrates a stereochemically acceptable alignment of the
helix and strand structures (satisfying these constraints).
No unfavorable short contacts are observed in the model
with all residues lying within the allowed regions of the
Ramachandran map.24 A ␤ hairpin could then be readily
generated, with the N-terminal strand as the template,
using a nucleating type II⬘ ␤-turn.25,26 This results in an
␣␤␤ motif.
We then explored the possibility of registering a strand
and a helix with the opposite polarity. Extraction of the ␤␣
motifs in proteins, in which the connecting loops are
limited to two residues, yielded 202 examples from which
nine examples could be visually identified where the
strand and the helix had a good antiparallel arrangement.
In the case of the antigen 85C from Mycobacterium
tuberculosis27 (PDB id: 1DQZ, 1.5 Å), a potentially useful
short C. . .O distance of 3.75 Å was observed between the
residues Gly 122 and Gly 127 [Fig. 3(a)]. Using this as a
starting model, a stereochemically satisfactory strandhelix motif with registering COH. . .O interactions between residues H-3 CAO and H⫹2 C␣ was made (residue
H is defined as the first residue in the C-terminal helix).
The motif could then be extended as described for the ␣␤
segment [Fig. 3(b)]. Notably, the ␤-strand and the ␣-helix
are linked by a single residue (H-1) adopting a conformation with ␾ ⬃ 67° and ␺ ⬃ ⫺99°, which corresponds to the
i⫹1 residue of the type II⬘ ␤-turn. The first residue of the
helix (␾ ⫽ ⫺79°, ␺ ⫽ ⫺29°) acts as the i⫹2 residue and a
stabilizing 4 3 1 hydrogen bond is formed between the H-2
CAO and the H⫹1 NOH. The H-2 residue, which occupies
the terminal position of the ␤-strand, adopts an almost
totally extended conformation (␾ ⬃ ⫺166°, ␺ ⬃ 161°).
Figure 4(a,b) shows sections of the ␣␤␤ and ␤␤␣ motifs
that are registered exclusively through backbone interactions in a composite ␣␤␤␣ structure. Figure 4(c) is a ribbon
representation of this fold which constitutes an “open”
well-ordered structure. Recent examples of structure determination of classes of RNA-binding proteins have revealed
structures with open motifs, comprising exclusively small
170
S.K. SINGH ET AL.
Fig. 4. Idealized model generated for an ␣␤␤␣ motif using a poly-alanine chain. Idealized values for the
helix and the strand segments are as in Figures 2(b) and 3(b). The ␤ hairpin was constructed using a type II⬘
␤-turn. The strand-helix segment was constructed as described in the text. Views (a) and (b) show the ␣␤␤ and
␤␤␣ segment separately to avoid apparent overlap upon projection of the structure. COH. . .O hydrogen bonds
are indicated as dotted lines. All the hydrogen bonding parameters are within acceptable limits, similar to those
indicated in Figure 1. c: Ribbon diagram of the ␣␤␤␣ motif generated using MOLMOL.28
helical domains.29,30 Examples of open ␤-sheets have also
been reported.31
During the course of our analysis of the helix termination motif illustrated in Figure 1, we observed that in the
examples extracted from the protein database, the residues serine, glutamic acid, and glutamine showed a significant propensity to occur at the T-4 position of the helix.
Inspection of all these examples revealed that an additional side-chain– backbone hydrogen bond between the
O␥ of serine or O⑀ of glutamic acid/glutamine to the
backbone NH of the T⫹3 residue (N. . .O distance ⱕ3.5 Å)
was observed. Whereas the longer side-chain of glutamic
acid or glutamine pushes the ␤-strand away from the
helix, the shorter side-chain of serine can be comfortably
accommodated with a strong O. . .HON hydrogen bond.
Computer modeling studies confirmed that serine could be
comfortably accommodated into positions T-4, T-8, and
T-12 of the N-terminal helix and at positions H⫹2, H⫹6,
and H⫹10 of the C-terminal helix. In the motif generated
in Figure 4(c), the required backbone conformations all lie
REGISTERING ␣-HELICES AND ␤-STRANDS
within the region normally populated by L-amino acids,
with only the i⫹1 position of the type II⬘ ␤-turn requiring a
positive ␾ value, which in proteins is usually achieved by
positioning Gly or Asn residues.32 On the helix faces,
which contact the strands, Gly/Ala/Ser residues can be
accommodated in an almost perfectly antiparallel arrangement, whereas moderate-sized side-chains can be inserted
with some distortions. Although weak backbone– backbone COH. . .O interactions may be used to register
helices with an isolated ␤-strand segment, it is important
to note that the NOH groups on the strand backbone will
not be hydrogen bonded and inaccessible to solvent, a
potentially unfavorable situation. Locating serine residues
on the face of the helix contacting the strand can obviate
this difficulty by forming strong backbone–side-chain
NOH. . .O hydrogen bonds.
CONCLUSION
This approach of connecting ␣-helices and ␤-strands by
means of only one or two connecting residues leads to
arrangements that are distinct from the many ␣␤ motifs
observed thus far in the structures of globular proteins.33
Registry of ␣-helices and ␤-strands by means of backbone–
backbone interactions, sometimes supplemented by stronger side-chain– backbone hydrogen bonds, may provide a
means of creating new polypeptide folds. The invention of
new folds by piecing together persistent structural motifs
observed in peptides and proteins might provide new
dimensions in polypeptide architecture.
ACKNOWLEDGMENTS
This work was funded by the Department of Biotechnology (Government of India) as program support to the area
of “Drug and Molecular Design.” S.K.S. acknowledges
receipt of a senior research fellowship from the Council of
Scientific and Industrial Research (CSIR), Government of
India. M.M.B. acknowledges support from the Indian
Institute of Science Young fellowship and the Indian
Academy of Science summer research fellowship programs.
REFERENCES
1. Thornton JM, Orengo CA, Todd AE, Pearl FM. Protein folds,
functions, and evolution. J Mol Biol 1999;293:333–342.
2. Martin AC, Orengo CA, Hutchinson EG, et al. Protein folds and
functions. Structure 1998;6:875– 884.
3. Brenner SE, Chothia C, Hubbard TJ. Population statistics of
protein structures: lessons from structural classifications. Curr
Opin Struct Biol 1997;7:369 –376.
4. Baker EN, Hubbard RE. Hydrogen bonding in globular proteins.
Prog Biophys Mol Biol 1984;44:97–179.
5. Stickle DF, Presta LG, Dill KA, Rose GD. Hydrogen bonding in
globular proteins. J Mol Biol 1992;226:1143–1159.
6. Ponder JW, Richards FM. Tertiary templates for proteins: use of
packing criteria in the enumeration of allowed sequences for
different structural classes. J Mol Biol 1987;193:775–791.
7. Burley SK, Petsko GA. Weakly polar interactions in proteins. Adv
Protein Chem 1988;39:125–189.
8. Ponder JW, Richards FM. Internal packing and protein structural
classes. Cold Spring Harbor Symp Quant Biol 1987;52:421– 428.
171
9. Aravinda S, Shamala N, Pramanik A, Das C, Balaram P. An
unusual COH. . .O hydrogen bond mediated reversal of polypeptide chain direction in a synthetic peptide helix. Biochem Biohpys
Res Commun 2000;273:933–936.
10. Schellman C. The ␣L conformation at the end of helices in protein
folding. In: Jaenicke R, editor. Protein folding. New York: ElsevierNorth Holland; 1980. p 53– 61.
11. Gunasekaran K, Nagarajaram HA, Ramakrishnan C, Balaram P.
Stereochemical punctuation marks in protein structures: glycine
and proline containing helix stop signals. J Mol Biol 1998;275:917–
932.
12. Sutor DJ. The COH. . .O hydrogen bond in crystals. Nature
1962;195:68 – 69.
13. Ramachandran GN. Structure of collagen at the molecular level.
In: Ramachandran GN, editor. Treatise on collagen. New York:
Academic Press; 1967. p 103–183.
14. Bella J, Berman HM. Crystallographic evidence for C␣OH. . .OAC
hydrogen bonds in a collagen triple helix. J Mol Biol 1996;264:734 –
742.
15. Desiraju GD. The COH. . .O hydrogen bond: structural implications and supramolecular design. Acc Chem Res 1996;129:441–
449.
16. Derewenda ZS, Lee L, Derewenda U. The occurrence of COH. . .O
hydrogen bonds in proteins. J Mol Biol 1995;252:248 –262.
17. Gu Y, Kar T, Scheiner S. Fundamental properties of the COH. . .O
interactions: is it a true hydrogen bond? J Am Chem Soc 1999;121:
9411–9422.
18. Vargas R, Garza J, Dixon DA, Hay BP. How strong is the
COH. . .OAC hydrogen bond? J Am Chem Soc 2000;122:4750 –
4755.
19. Ghosh A, Bansal M. COH. . .O hydrogen bonds in minor groove of
A-tracts in DNA double helices. J Mol Biol 1999;294:1149 –1158.
20. Hobohm U, Scharf M, Schneider R, Sander C. Selection of a
representative set of structures from the Brookhaven Protein
Data Bank. Protein Sci 1992;1:409 – 417.
21. Berman HM, Westbrook J, Feng Z, et al. The Protein Data Bank.
Nucleic Acids Res 2000;28:235–242.
22. Madan Babu M, Kumar Singh S, Balaram P. A COH . . . O
hydrogen bond stabilized polypeptide chain reversal motif at the
c-terminus of helices in proteins. J Mol Biol 2002;322:871– 880.
23. Cho S, Lee MG, Yang JK, Lee JY, Song HK, Suh SW. Crystal
structure of Escherichia coli Cyay protein reveals a previously
unidentified fold for the evolutionary conserved frataxin family.
Proc Natl Acad Sci USA 2000;97:8932– 8937.
24. Ramachandran GN, Ramakrishnan C, Sasisekharan V. Stereochemistry of polypeptide chain configurations. J Mol Biol 1963;7:
95–97.
25. Sibanda BL, Thornton JM. Beta-hairpin families in globular
proteins. Nature 1985;316:170 –174.
26. Gunasekaran K, Ramakrishnan C, Balaram P. ␤ Hairpins in
proteins revisited: lessons for de novo design. Protein Eng 1997;10:
1131–1141.
27. Ronning DR, Klabunde T, Besra GS, Vissa VD, Belisle JT,
Sacchettini JC. Crystal structure of the secreted form of antigen
85C reveals potential targets for mycobacterial drugs and vaccine.
Nat Struct Biol 2000;7:141–146.
28. Koradi R, Billeter M, Wüthrich K. MOLMOL: a program for
display and analysis of macromolecular structures. J Mol Graph
1996;14:51–55.
29. Wang X, Zamore PD, Hall TM. Crystal structure of a Pumilio
homology domain. Mol Cell 2001;7:855– 865.
30. Edwards TA, Pyle SE, Wharton RP, Aggarwal AK. Structure of
Pumilio reveals similarity between RNA and peptide binding
motifs. Cell 2001;105:281–289.
31. Pham TN, Koide A, Koide S. A stable single-layer ␤-sheet without
a hydrophobic core. Nat Struct Biol 1998;5:115–119.
32. Wilmot CM, Thornton JM. Analysis and prediction of the different
types of ␤-turn in proteins. J Mol Biol 1988;203:221–232.
33. Boutonnet SN, Kajava AV, Rooman MJ. Structural classification
of ␣␤␤ and ␤␤␣ supersecondary structure units in proteins.
Proteins 1998;30:193–212.