Vol. 23 no.
6 2007, pages 717–723
BIOINFORMATICS ORIGINAL PAPER
Structural bioinformatics
On the relationship between sequence and structure
similarities in proteomics
Evgeny Krissinel
European Bioinformatics Institute, Genome Campus, Hinxton, Cambridge CB10 1SD, UK
Received on November 2, 2006; revised on January 8, 2007; accepted on January 11, 2007
Advance Access publication January 22, 2007
Associate Editor: Anna Tramontano
ABSTRACT
Motivation: The underlying assumption of many sequence-based
comparative studies in proteomics is that different aspects of protein
structure and therefore functionality may be linked to particular
sequence motifs. This holds true if sequence similarity is sufficiently
high, but in general the relationship between protein sequence and
structure appears complex and is not well understood.
Results: Statistical analysis of multiple and pairwise structural
alignments of protein structures within SCOP folds is performed.
The results indicate that multiple conservation of residue identity is
not common and that relationship between sequence and structure
may be explained by a model based on the assumption that protein
structure is tolerant to residue substitutions preserving hydropathic
profile of the sequence. This model also explains the origin and
specific value of the sequence similarity threshold, noticed in many
previous studies, below which structural resemblance is not
statistically expected.
Contact: keb@ebi.ac.ukkeb
1 INTRODUCTION
Comparative studies play an important role in bioinformatics.
It is widely assumed that structural resemblance of proteins
implies their functional similarity. This assumption is important
for a range of practical problems addressed by bioinformatics—
from a better understanding of biochemical processes in cell
to drug discovery. It is also widely assumed that structural
features are closely related to sequence composition. Although
a protein with a given sequence may potentially exist in
different conformations, the chances that two close sequences
will fold into distinctly different structures are so small that
they are often neglected in research practice.
There is, however, a limit to which structure and sequence
similarities may be equivalenced. As has been established in
several studies (Kinjo and Nishikawa, 2004; Rost,1999), protein
pairs with a sequence identity higher than 35–40% are very
likely to be structurally similar. Structural similarity in pairs
with a sequence identity of 20–35%, often refered to as ‘twilight
zone’, is considerably less common; less than 10% of protein
pairs with sequence identity below 25% have similar structures.
At the same time, the ‘twilight zone’ is characterized by an
explosion of false negatives (Rost, 1999), which means that
many dissimilar sequences appear to be structural homologues.
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doi:10.1093/bioinformatics/btm006
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Although there are examples of homologous protein pairs with
510% sequence identity (Brenner, et al., 1996; Holm and
Sander, 1996; Hubbard, et al., 1997; Valencia, et al., 1991), it
has been found in many studies (Chotia, 1992; Chotia, and
Lesk, 1986; Hubbard and Blundell, 1987; Krissinel and
Henrick, 2004) that the likelihood of structural homologues
with 520% sequence identity is negligibly small. This is
illustrated by Figure 1, which shows the correlation between
residue conservation C and structure similarity scores (Krissinel
and Henrick,2004):
Ncons
C¼
Nalign
N2align
Q ¼ 
  ð1Þ
1 þ ðRMSD=R0 Þ2 N1 N2
Nalign
Nm ¼
minðN1 , N2 Þ
Here N1 and N2 stand for the total number of residues in two
compared structures, RMSD is r.m.s.d. between Nalign pairs of
residues found in geometrically equivalent positions, Ncons
of which are occupied by pairs of identical residues. The
correlations are represented in the form of reduced density of
probability:

ðx, CÞ

r ðx, CÞ ¼ qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
R xmax R Cmax ffi ð2Þ
0
ð x, C Þdx 0
ð x, C ÞdC
where
ðx, CÞ is the density of probability that a randomly
selected pair of structures in the Protein Data Bank (PDB)
(Berman et al., 2000) will have structure similarity score x and
residue conservation C.
No perfect score for structural similarity has been proposed
so far. Neither r.m.s.d. nor alignment length alone are truly
indicative because one may be always improved at the expense
of another. The Q-score represents a balance between r.m.s.d.
and Nalign, and was found to be a considerably better score
when structural similarity is not self-obvious (Krissinel and
Henrick, 2004). Q-scores range from 0, where no similarity
exists, to 1 where structures are identical. From an empirical
consideration, close structural similarity is suggested by
RMSD
2Å, Nm  0:8 and Q  0:4.
717
E.Krissinel
1.0
Res. conservation C
A B
0.8
0.6
0.4
0.2
0.0
0 0.2 0.4 0.6 0.8 1 0 2 4 6 0 0.2 0.4 0.6 0.8 1
Q-score RMSD [Å]
Fig. 1. Correlations between residue conservation C and structure
similarity scores: (A) Q-score (B) r.m.s.d. (C) normalized alignment
length Nm [cf. Equation (1)], represented as contour maps of reduced
density of probability to find the corresponding pairs of similar
structures in the PDB [cf. Equation (2)], from Krissinel and Henrick,
(2004). The outermost contours correspond to the level of 0.05 from the
maximum. The data suggest that, on average, structures are dissimilar
at C
C0 ¼ 0:2, see discussion in the text.
As may be seen from Figure 1, C0 ¼ 0:2 represents a
threshold, above which all three scores indicate structurally
similar proteins and dissimlar ones at C50:2. The particular
value of C0 has received little, if any, discussion in the above
referenced works, as well as in others. However, one may see a
few intriguing questions arising here. Consider a family of
structurally similar proteins fPgn ¼ fP1 , P2 , . . . , Pn g. If sequence
variations within fPgn were completely random, then having
residue conservation between proteins Pi and Pj,
CðPi , Pj Þ  0:2, and CðPj , Pk Þ  0:2 would not necessarily
mean that CðPi , Pk Þ  0:2. One may think of two types of
sequence relationship within structure families, exemplified in
Figure 2, that may provide CðPi , Pj Þ  0:2 for any Pi, Pj from
fPgn . Multiple residue conservation (type A) is particularly
appealing for bioinformatic applications. It suggests that
protein features, such as fold and functionality, may be due
to the presence of specific sequence motifs in certain structure
positions. This promises a discovery of relationships between
structure, function and sequence evolution, and, in fact, many
bioinformatic studies exploit this sort of largely intuitive
hypotheses. Closed pairwise residue conservation (type B) has
different implications. Here, protein structure can not be so
unambiguously associated with a particular sequence, there-
fore, many different sequences may fold into similar structures
(which, indeed, is the case). As a consequence, protein structure
and function do not appear clearly sequence-related.
Obviously, multiple residue conservation (Fig. 2A) should
not differ significantly from pairwise conservation (Fig. 2B) in
structure families with a high sequence similarity. However, this
case poses little practical value because the requirement of high
a C in comparative studies limits their predictive power. As C
decreases, the sequence–structure relationship is expected to
gradually shift to type B (Fig. 2B). When C falls below a value
of 0.2, as Figure 1 suggests, no sound structure similarity is
statistically expected.
Being relatively clear in outline, the above aspects of
relationship between sequence and structure similarities
remain largely unstudied in details. The ‘critical’ value of
C0 ¼ 0:2 appears confirmed for a sufficiently large number of
different data sets, however, its origin remains unexplained.
718
A B
C
ALA ALA
ALA ALA LEU
LEU VAL
VAL
ALA MET MET
MET LEU
PRO
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Nm MET
MET
LEU
PRO
ILE SER
ILE ILE
TYR
P3 SER P3
P1 P1
ILE ILE
TYR
P2 P2
Fig. 2. Two possible types of sequence relationships in structure
families: (A) multiple residue conservation (B) closed pairwise residue
conservation. Dotted lines denote mapping of identical residues in
structurally equivalent positions. Any protein pair in (A) and any
protein pair in (B) have same number of conserved residues, however,
the character of relationships within the families is completely different.
See discussion in the text.
At the same time, the topic is worth further study because of the
importance of sequence-structure relationship in protein
science. This article attempts to provide an insight into the
sequence and structure relationship by presenting a statistical
analysis of available protein structure families. In particular,
manifestation of multiple and pairwise sequence relationships
in dependence of residue conservation is studied and explained,
and a probable origin of the threshold value C0 is suggested.
2 APPROACH, DATA SET AND METHODS
Multiple and pairwise residue conservation types in particular structure
family fPgn are identified by the corresponding 3D alignment of
proteins Pi. Multiple alignment looks for residues that occupy
geometrically equivalent positions in all n structures, and pairwise
alignment does the same for each of nðn  1Þ=2 protein pairs (Pi , Pj)
separately. A residue is considered conserved if 3D alignment
equivalences it with identical residue(s).
Residue conservation may be expressed by probability distribution

(C) to find Ncons ¼ C Nalign conserved residues from total Nalign
aligned. Distributions
A(C) and
B(C) for multiple and pairwise
conservations, respectively, are calculated from the corresponding 3D
alignments of structure families found in the PDB. The families
should be chosen in a way that allows for sufficient sequence diversity,
on one hand, and to assure a reasonable structure similarity, on the
other. A few protein structure classifications are available. CATH
(Pearl et al., 2003) and FSSP (Holm and Sander, 1996) represent
automatic classifications, while SCOP (Murzin et al., 1995) is a
manually–curated resource. Although no significant differences in
results obtained for different classifications should be expected, SCOP
was chosen in order to avoid any artifacts that might arise from
employing a 3D aligner different from one used for the classification.
SCOP offers a 5-level structure hierarchy (numbers given for release
1.69): 70859 domains grouped into 2845 families, 1539 superfamilies,
945 folds and 7 classes, in order of decreasing structure resemblance.
SCOP folds appear to be the most appropriate set of structure families

for the purposes of the present study, because SCOP families and
superfamilies contain closely related proteins, which implies high
sequence similarity, and structure resemblance between different folds
inside a class is normally very low.
3D pairwise and multiple alignments were performed with algorithms
used in SSM web service at the European Bioinformatics Institute.
Pairwise alignment in SSM is a two-step procedure (Krissinel and
Henrick, 2004). First, seed alignments are generated by matching graphs
built on secondary structure elements (SSE). More than one seed
alignments may be generated if few similar-score alternatives are found.
Then seed alignment(s) are refined by matching pairs of C atoms in
compared structures. A set of different seed alignments and several C
mappings for each of them are explored in order to maximize the Q-score
(cf. Equation 1). Multiple alignment in SSM starts with a set of all-to-all
pairwise SSM alignments in structure family. Using these alignments as a
first approximation, the algorithm probes alternative mappings for those
SSEs that do not have equivalences in all other structures. Each
alternative mapping results in decrease of pairwise scores, but may
increase the multiple alignment score if all SSE equivalences are found.
On the final stage, multiple C mapping is performed using an approach
equivalent to the central star method in multiple sequence alignment
(Gusfield, 1997). A detail description of the multiple SSM algorithm is
given in Krissinel and Henrick (2005). The quality and performance
of the pairwise SSM algorithm have been acknowledged in an indepen-
dent study (Kolodny et al., 2005). Limited comparisons of multiple SSM
and other multiple alignment algorithms [MASS, Dror et al., (2003)
and Combinatiorial Extension, Guda et al., (2004)], performed in
(Krissinel and Henrick, 2005), show good overall agreement.
Because SSM uses secondary structure for obtaining seed alignments,
PDB entries of proteins without secondary structure and those for
which it cannot be calculated (e.g. where only backbone C atoms are
given), were excluded from the dataset. Next, only one structure from
those with sequence identity higher than 50% to each other was left per
SCOP fold in order to focus on the situation where structure similarity
shows a clear dependence on C. The 50% threshold is suggested by the
correlation map between C and Q-score in Figure 1A, which shows that
at C  0:5 Q-score does not depend on C and ranges between 0.8 and 1.
Other structure scores, RMSD and Nm (Fig. 1A,B), do not exhibit this
feature so clearly. Finally, folds with less than three structures remained
were removed from the dataset.
3 RESULTS AND DISCUSSION
The resulting data set included 28 980 protein chains in 506
SCOP folds, which is slightly more than a chain per PDB entry
(a total of 25 973 PDB entries are annotated in SCOP release
1.69). This indicates a reasonable coverage of PDB content,
taking into account that many PDB entries contain non-
crystallographically related copies of the same sequence in the
asymmetric unit.
The obtained distributions
A(C) and
B(C) are shown in
Figure 3. As seen from the figure, the distributions approach
each other at residue conservation C  0:25. A near merge of the
curves at C  0:4 is expected considering that, in families with
high sequence identity, a large fraction of residues is conserved
in both pairwise and multiple alignments. Consider, as an
example, a protein family fPg3 ¼ fP1 , P2 , P3 g. If residue
conservation in pairs (P1, P2) and (P2, P3) is high, then one
can expect that residue conservation in (P1, P3) will be also high.
Then multiple alignment ðP1 , P2 , P3 Þ reduces to the super-
position of all pairwise alignments, thus preserving residue
mapping as in Figure 2A. In this case, both types of residue
Sequence and structure similarities in proteomics
10
Probability distribution r(C)
rA(C)
8
6
rB(C)
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4
2
0 0.1 0.2 0.3 0.4 0.5
Residue conservation C
Fig. 3. Probability distributions
A(C) and
B(C) to find
Ncons ¼ C Nalign conserved residues from total Nalign aligned in multiple
and pairwise alignments of protein families, respectively. See details in
the text.
conservation should occur equally frequently, which indeed is
confirmed by data in Figure 3. As follows from the absolute
values of
A (C) and
B (C) in the high-C end, only a few families
with relatively high sequence identity are found in our data set.
If residue conservation is low,
A(C) and
B (C) may manifest
more differences. For the above example of family fPg3 ,
the fewer identical residues found in pairs (P1, P2) and (P2, P3),
the lesser the chances of finding them conserved across
the whole family ðP1 , P2 , P3 Þ. Indeed, as seen from Figure 3,
in the region of low C,
A(C) and
B (C) behave very differently.
As suggested by the curves, the most probable residue
conservation in multiple alignment is very low (C
0:02). On
the contrary, residue conservation in pairwise alignment
reaches its maximum at C  0:14, while low values of C are
considerably less frequent. This implies a higher statistical
significance of residue conservation in multiple alignment,
which is routinely used in bioinformatic studies. The average
residue conservation, calculated as
Z1
C¼ C
ðCÞ dC ð3Þ
0
equals to CA  0:12 and CB  0:19 in multiple and pairwise
alignments, respectively.
The pronounced decrease in
B (C) at decreasing C
(cf. Fig. 3) indicates that only a fraction ( 16%) of structurally
similar sequence pairs have less than CA  100% conserved
residues, while 50% of multiple alignments fall in this region
of low C. Therefore, most multiple alignments at C
CA
correspond to structure families with a higher pairwise
conservation rate. Therefore, pairwise conservation type
(type B, cf. Fig. 2) prevails at lower sequence similarity.
These results imply that, in general, relationship between
sequence and structure is ambiguous as many different
sequence motifs may correspond to the same structural
feature. Therefore, structural motifs should not be correlated
with any particular residues or their sequences. In this
connection, it is interesting to see whether structural features
may be correlated with particular residue substitutions.
719
E.Krissinel

Statistical significance of residue substitutions is given by the
odds, calculated as described in Henikoff and Henikoff, (1992):
wij ¼ qij =eij
where qij is the observed probability of substitution of residue
type i with residue type j, and eij is the expected probability of
such substitution. Figure 4 shows residue self-odds wii , or
conservation odds, calculated from the results of pairwise
smaller will more readily provoke structural changes than
replacement of similar-size residues. High odds for cysteine
could be attributed to the formation of disulphide bonds:
ð4Þ
breaking such bonds is likely to induce changes in the structure.
The third most conserved residue, histidine, plays an important
role in catalytic triads and might be better conserved for
this reason. Although many different suppositions of this
kind may be given here [and what has been done elsewhere,

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alignments. As seen from the figure, wii is 41 for all residues,
which means that like-residue substitutions are statistically
significant in our data set. However, the odds vary greatly
with the residue type. Tryptophan appears to be the most
conserved residue, which hypothetically might be linked to its
size: replacing a big residue with something considerably
24
20
16
Odds
see, e.g. (Naor et al. 1996)], none of them can be firmly verified
in the framework of statistical studies. In similar studies by
Naor et al. (1996), the most conserved residue is proline,
with cysteine second, while neither tryptophan nor histidine
appear to be particularly conserved. In fact, very little
similarities may be found between data in Figure 4 and those
obtained in the referenced publication. This disagreement could
be attributed to the difference in the data sets: the data set used
in Naor et al., 1996 was considerably smaller (257 structures)
and composed of structurally non-redundant representatives.
The demonstrated dependence on the content of the
data set implies that extensive cross-validation studies are
required in order to conclude on the representation power of

the PDB. One should notice in this connection that, according

12
to the analysis of annotation records in PDB entries, 65% of
protein structures have been solved by molecular replacement
8
(Dr. Alexei Vaguine, University of York, UK, personal
4
communication), which implies a substantial bias towards
already known folds.
0 Figure 5 shows the full log-odds matrix of residue substitu-
tions M, derived from the results of pairwise alignments.
ARG
LYS
GLN
GLU
ASN
ASP
HIS
PRO
SER
THR
GLY
ALA
CYS
TYR
TRP
MET
PHE
LEU
VAL
ILE

Comparison with data in Naor et al. (1996) again shows

a considerable difference in absolute numbers, however,
Fig. 4. Residue conservation odds wii (cf. Equation 6), calculated from the results agree perfectly well on how the amino acid
the results of pairwise alignments. See discussion in the text. residues may be grouped according to their substitution odds.

Fig. 5. The log-odds matrix of residue substitutions M, derived from the results of pairwise alignments. Matrix elements Mij are calculated by truncating
log2 ðwij Þ, where wij represent statistical odds of substitution residue type i with residue type j in structurally similar proteins [cf. Equation (4)]. Diagonal
elements wii represent residue conservation odds shown in Figure 4. Numbers in the downmost line indicate the hydropathy index, introduced by Kyte
and Doolittle, (1982). The order of rows and columns has been chosen such as to select two diagonal sub-matrices with highest substitution odds and two
off-diagonal matrices with lowest values of wij (as divided by vertical and horizontal lines). See discussion in the text.

720

The order of residues in Figure 5 has been obtained with a
nearest-neighbour clustering procedure. This procedure starts
by creating clusters made of 2 residues with maximal values of
wij , and then iteratively merges clusters with maximal substitu-
tion odds wij between their residues. The clustering arrives
at two groups of residues, separated by lines in Figure 5.
Correspondence with empirical hydropathy index, introduced
by Kyte and Doolittle (1982), indicates that, with the exception
of tyrosine and tryptophan, the clustering procedure separates
residues into hydrophilic and hydrophobic types. Since all
alignments were performed between structurally similar
proteins, these results indicate that protein structure is quite
tolerant to residue substitutions that conserve chemical-
physical properties, rather than mere residue identities, in
particular sequence positions.
Similarly to the residue conservation, conservation of hydro-
pathy type is given by the probability distribution ðCH Þ to find
CH Nalign residue substitutions within hydrophobic or hydro-
philic groups of residues from total Nalign aligned sequence
positions. Figure 6 shows distributions A ðCH Þ and B ðCH Þ
calculated from the results of multiple and pairwise alignments,
correspondingly. As seen from the figure, hydropathic proper-
ties appear very well conserved in pairwise alignment,
i.e. between any two structurally similar proteins. The average
H
hydropathic identity is CB  0:69, which means that, on
average, 69% of residue substitutions occur within the
same hydropathy type. This figure is in striking contrast with
the average residue identity conservation found above
(CB  0:19). A relatively well localized distribution B ðCH Þ
(so that only a small fraction of alignments conserves 550%
of hydropathically equivalent residues and no alignments
H
conserve 532% of them) and high value of CB suggest that
hydropathic properties is a major factor that affects protein
folding.
Hydropathy conservation in multiple alignments (or within
structure families) is less pronounced than that in pairwise
comparisons. The corresponding distribution A ðCH Þ appears
)
3
H
Probability distribution h(C
2 residues have an equal occurence rate and substitution
H
hA (C )
1 approaches the following form:
0 B
0 0.2 0.4 0.6
Hydropathy conservation C
Fig. 6. Probability distributions A ðCH Þ and B ðCH Þ to find CH Nalign
hydropathy-conserving residue positions from total Nalign aligned in
multiple and pairwise alignments of protein families, respectively.
See details in the text.
Sequence and structure similarities in proteomics
quite irregular. One should note in this connection that A was
averaged over a considerably fewer number of alignments than
B. Indeed, as only 506 SCOP folds were left in the data set, the
A curve was calculated from 506 multiple alignments. The
curve spans over the region of 0 to 1, divided into 50 bins. This
makes just about 10 counts per bin on average, which is barely
enough for proper averaging. The number of pairwise align-
ments, used for the calculation of B, is much higher: each of
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506 folds contributes nðn  1Þ=2 alignments, where n is the
number of structures in the fold (on average n  57). Besides,
B distribution spans from 0.4 to 1 thus populating almost half
of all bins. These factors help better averaging of the B
distribution. The average multiple hydropathic identity reaches
H
CA  0:51, which is considerably higher than the average
multiple residue conservation CA  0:12. Just as
A(C) and

B(C) (cf. Fig. 3), and for the same reasons, A ðCH Þ and B ðCH Þ
nearly merge at high values of CH 4 0:8, but deviate
substantially in the region of low hydropathy conservation.
The fact that CH may reach very low values in multiple
alignment, but stays high in pairwise comparisons, indicates
that hydropathic properties are conserved in different structure
positions between different members of protein families. It,
therefore, appears that protein structure is relatively insensitive
to how the hydropathy-conserving substitutions are spread
within the sequence, as long as there is a sufficiently high
( 70%) fraction of them. This implies a general prevalence of
pairwise hydropathy conservation, similarly to what was found
above for the conservation of residue identity.
The obtained results suggest that most of structure-preserving
residue mutations should occur within two basic hydropathy
types, which agrees with a general consideration that
hydrophobic interactions play a major role in protein folding.
What can be said about the conservation of residue type C in
this sort of mutations? Obviously, statistically expected value of
C should reach a minimum in the hypothetical extreme case,
when any substitutions within hydropathy types are equally
frequent. Residue conservation above statistically expected may
indicate structural similarity, while lower values are not
indicative. Let us estimate statistical expectancy for residue
hB (C H ) type conservation. Consider an idealized model of sequence
mutations, which assumes that protein structure is conserved at
any number of any residue substitutions within two groups of
10 amino acids each, but is not fully tolerant to substitutions
between the groups. For simplicity, assume also that all
frequency. Then the count matrix of residue substitutions in a
sufficiently large number of structure-conserving mutations
0 1
1  1   
.
B .. . . .
. . .. .. . .
. . .. C
B C
C
B0  1   C
F ¼ f0 B C ð5Þ
0.8 1 B0  0 1  1C
B. . . . . . C
H @ .. . . .. .. . . .. A
0  0 0  1
where the first group includes residues numbered 1 . . . 10 and
the second group - residues 11 . . . 20. f0 and f0 stand for
the number of each residue substitutions within and between
721
E.Krissinel
the groups, respectively. In this matrix, substitutions i ! j and
j ! i are counted in the same element Fi,j, j  i. The total
number of residue substitutions is then given by:
 2  structure similarity in the ‘twilight zone’ the latter is character-
XN X N
N N
jFj ¼ Fij ¼ f0 ð 1 þ Þ þ ð6Þ
i¼1 j¼i
4 2
where N ¼ 20 is the number of aminoacid residues, and the
fraction of conserved residues (or relative number of identical
residue substitutions) equals:
Ncons 1 X N
2
CM ðÞ ¼ ¼ Fii ¼ ð7Þ
Nalign jFj i¼1 1 þ Nð1 þ Þ=2
In the extreme case of  ¼ 0, which corresponds to the
assumption that any residue substitution between the two
groups results in major structural changes, CM ð0Þ  0:18.
This is very close to the empirical threshold C0 derived from
Figure 1. Values of C4CM ð0Þ are achievable with increasing
fraction of like-residue substitutions in evolutionary-related
structures. Such substitutions add to the diagonal elements Fii,
which results in increasing C. Higher values of C correspond to
higher structure conservation, therefore one could expect an
increase in
B(C) at C4CM ð0Þ in Figure 3. However, the higher
residue conservation, the fewer number of different sequences
may be generated. Therefore, the actual decrease in the pairwise
probability distribution (cf. Fig. 3) should be attributed to the
composition of the data set, where close sequences are
underrepresented due to natural reasons as well as a result of
our selection procedure.
In the opposite limit of  ¼ 1, where inter- and intra-
hydropathy group substitutions are equally frequent,
CM ð1Þ  0:095. As seen from Figure 3, CM(1) is close to the
peak in the pairwise probability distribution
B(C). Since CM(1)
corresponds to the situation where identical residue substitu-
tions are as frequent as all others, values of C5CM ð1Þ may be
achieved only by enhancement of unlike-residue substitutions
of both the same and different hydropathy types. Although
such substitutions should result in a larger number of possible
sequence variations, contrary to the situation at C4CM ð0Þ,

B(C) shows a sharp decrease at C5CM ð1Þ (cf. Fig. 3). This
indicates an increased rate of structural changes, so that fewer
number of thus mutated sequences remain in the same structure
family.
Because of many simplifications employed, the described
model cannot pretend to accurately reproduce all results of the
present study. However, it allows for qualitative conclusions
that are in accordance with the results of numerical calcula-
tions. The model demonstrates clearly that conservation of
residue identity C is a very poor indicator of structure similarity
unless the sequences are closely related. The threshold value of
C0  0:2, below which the chance of finding structural
resemblance decreases substantially, is insignificant in terms
of sequence identity, because it is statistically expected in the
situation when residue substitutions are confined only to
hydropathy type. It should be stressed that CM ð0Þ  C0
indicates a principal border line, below which structural
similarity cannot be inferred from sequence comparison.
Therefore, CM(0) should be viewed as the bottom of the
722
‘twilight zone’, and indeed its value nearly coincides with one
found in statistical studies (Kinjo and Nishikawa, 2004; Rost,
1999). Despite principal possibility to correlate sequence and
ized by an explosion of false negatives (Rost, 1999), which
poses considerable difficulties for automatic database searches.
Remarkably, this agrees with the basic assumptions of the
above model of sequence mutations: promiscuous mutations
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within hydropathy groups preserve structure but result in
negative hits in sequence comparison. The importance of
hydropathy type conservation at low sequence identity was also
acknowledged by Kinjo and Nishikawa, 2004, who studied
the sequence–structure relationship by means of eigenvalue
analysis. Therefore, it may be concluded that the described
model generally corresponds to the situation in the ‘twilight
zone’ and that sequence similarity threshold C0 originates from
the existence of two basic hydropathy types of residues. It is
possible that the number of false negatives in the ‘twilight zone’
can be decreased if the residue hydropathy type is taken into
account in sequence comparisons. However, this question is
outside the scope of the present study and requires further
investigation.
4 CONCLUSION
Sequence alignment, based on the matching of residue identity,
is a widely used tool in similarity studies. This is due to many
reasons, the most important of which are the wealth of
methodology developed over many years, a negligibly small
number of solved protein structures (44 000 PDB entries) on
comparison with the number of known protein sequences (some
4 300 000 unique sequences in UniProt database (Leinonen
et al., 2003)), and computational efficiency. While the last
reason becomes less important in view of computer progress
and modern efficient tools for structural alignment, the others
will remain for many years to come.
Results obtained in our study indicate that conclusions made
on the basis of sequence similarity may give far more false
negatives than expected. Although high sequence similarity
almost always correspond to high structure resemblance, the
opposite is far from the truth. Obviously, the same should
apply to substructure motifs, such as active sites and functional
protein interfaces. A bright example is given by nicotinic
receptors (Brejc et al., 2001; Celie et al., 2005). These structures
represent pentameric complexes that function as ion channels
regulated by nicotinic ligands. Few different structures of
the receptors are known, which have been found to be highly
similar topologically yet they show only 33% sequence
similarity. The most conserved part of the structures appears
to be the inner core of monomeric units, while their surface and,
particularly, the pentamer-forming interfaces show no regular
sequence motifs that could help one to identify them by means
of sequence screening.
The ambiguity between sequence and structure similarities
arises from the tolerance of protein structure to residue
substitutions within classes of amino acids having similar
chemical–physical properties. In this study, two classes were
selected on the basis of residue hydropathy type. This allowed
us to propose a model of sequence mutations that explains the

results of structure alignments within protein families selected
on the basis of SCOP folds. In this model, structures remain
similar at the level of sequence similarity that is insignificant
and statistically expected when there is no restrictions on
residue substitutions other than by hydropathy type. This level
is found to be very close to the sequence similarity threshold,
found empirically in many previous studies, below which
structure resemblance becomes more of a casual nature. In the
model’s terms, this corresponds to the inclusion of residue
substitutions between unlike hydropathy types, inducing
significant structural changes.
As a consequence of a relatively high tolerance of protein
structure to amino acid substitutions within the hydropathy
types, residue identity tend to not conserve in sufficiently large
protein families. This may lead to artifacts in analyses based on
multiple sequence alignment, where no structural aspects are
taken into account.
Results of this study confirm earlier findings that structures
from one protein family tend to conserve hydropathic identity
in geometrically equivalent positions (Kinjo and Nishikawa,
2004). It should be stressed that, strictly speaking, this does
not mean that any residue substitutions within the same
hydropathy type would necessarily conserve major structural
features. Obviously, hydropathic properties is not the only
factor that affects protein folding. Stability of protein
structures has been shown to depend on hydrogen bond
pattern (Pace et al., 1996), formation of salt bridges
(Waldburger et al., 1995), disulfide bonds (Betz, 1993),
aromatic interactions (Serrano et al., 1991) and other factors.
Therefore, a mutation that breaks a disulfide bond may
potentially induce a considerable structural change.
Protein functionality is known to be highly structure-specific.
Although each particular structure is determined by its
sequence, the structure–sequence relationship is, as obtained
results show, very ambiguous. Therefore, in general, a firm link
between protein functionality and sequence may not exist.
From this point of view, the significance of sequence in
proteomics is different to that in genomics. In the latter,
nucleotide sequence encodes all aspects of functionality with no
relation to structural features of DNA. In the protein world, it
is not so much sequence of residue identities as that of their
chemical properties and residue chain fold that really matters.
This allows for greater functional diversity and flexibility,
which may have important evolutionary implications.
ACKNOWLEDGEMENTS
This work has been supported by the research grant No. 721/
B19544 from the Biotechnology and Biological Sciences
Research Council (BBSRC) UK. The author would like to
thank Dr Melford John for reading the manuscript and helpful
discussion.
Conflict of Interest: none declared.
Sequence and structure similarities in proteomics
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