doi:10.1016/j.jmb.2004.07.002
J. Mol. Biol. (2004) 341, 1327–1341
Analysis of Ordered and Disordered Protein Complexes
Reveals Structural Features Discriminating Between
Stable and Unstable Monomers
Kannan Gunasekaran1*, Chung-Jung Tsai1 and Ruth Nussinov1,2*
1
Laboratory of Experimental
and Computational Biology
Basic Research Program
SAIC-Frederick, Inc.
NCI-Frederick, Frederick
MD 21702, USA
2
Department of Human
Genetics and Molecular
Medicine, Sackler Institute of
Molecular Medicine, Sackler
School of Medicine, Tel Aviv
University, Tel Aviv 69978
Israel
Most proteins exist in the cell as multi-component assemblies. However,
which proteins need to be present simultaneously in order to perform a
given function is frequently unknown. The first step toward this goal
would be to predict proteins that can function only when in a complexed
form. Here, we propose a scheme to distinguish whether the protein
components are ordered (stable) or disordered when separated from their
complexed partners. We analyze structural characteristics of several types
of complexes, such as natively unstructured proteins, ribosomal proteins,
two-state and three-state complexes, and crystal-packing dimers. Our
analysis makes use of the fact that natively unstructured proteins, which
undergo a disorder-to-order transition upon binding their partner, and
stable monomeric proteins, which exist as dimers only in their crystal form,
provide examples of two vastly different scenarios. We find that ordered
monomers can be distinguished from disordered monomers on the basis of
the per-residue surface and interface areas, which are significantly smaller
for ordered proteins. With this scale, two-state dimers (where the
monomers unfold upon dimer separation) and ribosomal proteins are
shown to resemble disordered proteins. On the other hand, crystal-packing
dimers, whose monomers are stable in solution, fall into the ordered
protein category. While there should be a continuum in the distributions,
nevertheless, the per-residue scale measures the confidence in the
determination of whether a protein can exist as a stable monomer. Further
analysis, focusing on the chemical and contact preferences at the interface,
interior and exposed surface areas, reveals that disordered proteins lack a
strong hydrophobic core and are composed of highly polar surface area. We
discuss the implication of our results for de novo design of stable
monomeric proteins and peptides.
q 2004 Elsevier Ltd. All rights reserved.
*Corresponding authors
Keywords: disordered proteins; natively unstructured proteins; protein–
protein interactions; folding mechanisms; two-state and three-state
complexes
Introduction
A large number of proteins perform their
biological functions as oligomers, consisting of
two or more polypeptide chains. The underlying
principle of protein–protein association has been
the subject of many investigations.1,2 Recognizing
Abbreviations used: ASA, solvent-accessible surface
area; SAPs, structurally ambivalent peptides; PDB,
Protein Data Bank.
E-mail addresses of the corresponding authors:
[email protected]; [email protected]
proteins that function only as oligomers is crucial to
the understanding of protein networking, function
and malfunction.3–5 Many attempts have been
made to understand the folding mechanism of
oligomers, specifically dimers, based on energetic
arguments, and surface and interface characteristics.5–9 Efforts have focused on distinguishing
between specific and non-specific binding characteristics of multimers.10–14 Non-specific contacts
between neighboring molecules in crystals are
meaningless from the functional standpoint, and
are considered “artifacts” of crystallization. Nevertheless, the differentiation of functional protein–
protein interfaces from those of crystal-packing
0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
1328
dimers remains a difficult task, especially since
features such as the hydrophobic effect and hydrogen bonding interactions are common to both
biological and crystal contacts.11,15 While proteins
known to exist as multimers only in the crystal form
provide extreme scenarios where interface contacts
are non-specific, natively unstructured proteins
provide examples where complex formation is a
must and interface contacts are obligatory. Natively
unstructured proteins are structured only upon
binding their partner molecule.16–21 Thus, natively
unstructured proteins and crystal-packing complexes provide an excellent opportunity to understand specific and non-specific contacts, and the
structural characteristics of disordered and ordered
monomers.
Here, our goals are twofold. First, to develop a
simple scheme that segregates between disordered
and ordered protein molecules when they are in
their complexed states. Second, to address the
question of whether the difference between the
ordered and disordered proteins arises from
the chemical and contact preferences (hydrophobic
or polar) at the interface or from the composition of
interior (core) and exposed surface residues.
Below, we provide a brief introduction to natively
unstructured proteins and other complexes.
Natively unstructured or disordered proteins
Experiments have shown that a very large
number of proteins and protein domains
exist with little or no ordered well-defined
structure.16–21 Natively unstructured proteins play
roles in cell-cycle control, signal transduction,
transcriptional and translational regulation, and in
macromolecular complexes such as the ribosomes.
These proteins usually have larger hydrodynamic
radii compared to globular proteins.22 The existence
of “disorderness” in these proteins is characterized
by various experimental techniques such as X-ray
crystallography, hetero-nuclear multidimensional
NMR and near-UV and far-UV CD.16 The term
“natively denatured” was introduced in order to
describe the difference between globular proteins
with well-defined tertiary structures, and extremely
flexible proteins. The absence of any or the presence
of only little secondary structure in these proteins
led to the term “natively unfolded” or “disordered”. These proteins appear to lack a degree
of globularity, leading to the absence of tightly
packed cores as in globular proteins.20 Disordered
proteins have no consistency in their size: the
sequence length varies from 50 to nearly 1900
residues for multi-domain proteins.23 Dunker and
co-workers used a neural network method to
predict the occurrence of disordered proteins in
genomes. The network was trained on a dataset
containing sequences longer than 50 residues. They
estimated that a large percentage of cellular
proteins exist in this disordered state: 36–63% in
eukaryotic cells, and up to 33% of bacterial proteins.
Romero et al.24 established that the distributions of
Discriminating Stable and Unstable Protein Monomers
the complexity values for ordered and disordered
sequences overlapped. In general, it is believed that
disordered proteins have numerous uncompensated charged groups and a low content of
hydrophobic amino acid residues.23
Advantages of being disordered
Many researchers have reasoned that the disordered state is advantageous either in binding to
multiple ligands or in reduced sensitivity to
environmental conditions.16,17,25 We proposed that
disordered proteins provide an elegant solution to
the problem of how to have large intermolecular
interfaces, yet have smaller protein, genome and
cell sizes.20 Our proposition was based on the
observation that disordered proteins often have
large intermolecular interfaces, the size of which is
dictated by protein function. For proteins to be
stable as monomers with such extensive interfaces,
the protein size would need to be two to three times
larger. This would either increase cellular crowding
or enlarge the size of the cell by 15–30%, owing to
the increase in sequence length. Recently,
Verkhivker et al.26 have shown that disorder-order
transition is coupled to binding through a detailed
Monte Carlo simulation on an unstructured protein
(p27). The authors propose that functional requirement to form a specific intermolecular interface
dictates the folding mechanism.26
Examples of ordered and disordered complexes
In addition to the natively unstructured proteins,
certain types of oligomers and components of
macromolecular assemblies provide additional
examples of disordered proteins. The oligomers,
mostly dimers and trimers, are observed to fold
through two major paradigms; two-state and threestate mechanisms.7,27 In two-state complex folding,
the unbound monomeric chains do not populate the
native state. Experimentally, they are observed
either in their denatured (D) unbound state or in
their native (N) state when in an oligomeric
complex. In the three-state complex model, the
monomeric chains on their own have a populated
native conformation. These monomers fold independently into a stable structure with a subsequent
dimerization. Thus, monomers of oligomers that
fold via a two-state mechanism are natively
unfolded in their uncomplexed form and can be
compared to the disordered proteins. A large
number of ribosomal proteins are also disordered
when isolated from the ribosome.28,29
Here, we analyze the structural characteristics of
various types of complexes such as the two-state
and three-state multimers, crystal-packing dimers,
ribosomal and the natively unstructured proteins in
order to address the question of whether the
ordered and disordered monomers differ in their
structural properties. To map the features of the
interface, surface and interior, we first analyze the
accessible surface areas of the various complexes.
Discriminating Stable and Unstable Protein Monomers
We find that the per-residue interface and surface
areas of ordered proteins are significantly smaller
than in the disordered monomers. Further analysis
of the polar and hydrophobic chemical characteristics of the interface and surface areas shows that
disordered differ from ordered proteins more
prominently in the composition of their exposed
surface and interior (core) residues and, to a lesser
extent, in the nature of their protein–protein
interfaces. The results presented here provide
means to distinguish two-state and three-state
complexes, and stable and unstable monomers.
Results and Discussion
Dataset
Our aim in the construction of the dataset is to
include as many examples as possible that cover a
range of sequence length, topology, secondary
structure content, and function. Table 1 lists the
selected examples. Our selection of protein complexes is based on experimental studies that have
been reported in the literature. To ensure that the
results are unlikely to be an artifact arising from the
selection of examples, the cases where conflicting
reports about the mechanism (two-state versus
three-state) exist are not included in the dataset.
We included homo- and heterodimers in the dataset
to ensure diversity. Although a large number of
crystal-packing dimers are available in the Protein
Data Bank (PDB),30 we limited their number, since
there are not many documented examples of other
types of complexes (natively unstructured, twostate and three-state complexes) available in the
literature. This assists in ensuring that all complex
types are represented relatively equally. The crystalpacking dimers are defined as pairs of protein
molecules forming interfaces only in the crystal
form. These proteins are documented to be monomeric in solution. Overall, the disordered protein
dataset consists of 44 ribosomal proteins, ten twostate complexes (11 examples, when the monomers
of a hetero complex are treated separately), and five
natively unstructured proteins for which the crystal
structure is known in the complexed state. The
ordered protein dataset consists of ten three-state
complexes (14 examples, when the monomers of
hetero complexes are treated separately) and 16
monomeric proteins that are dimers in crystals.
Two-state complexes are combined with disordered
proteins, since the monomers are unstructured
when separated. Three-state complexes are combined with ordered proteins, since the monomers
can exist in the folded state when separated. A large
number of ribosomal proteins are unstructured in
the uncomplexed form;28,29 therefore, they are
included in the disordered data set. Further
justification for inclusion of proteins in these two
types of dataset becomes clear in the analysis as
described below.
1329
Ordered versus disordered: the per-residue area
Earlier studies on globular proteins have established that a linear relationship exists between
surface area and chain length (number of residues)
in proteins.31,32 We examined the relationship
between surface area and chain length for the
ordered and disordered protein data sets
(Figure 1(a)). Our examination shows that disordered proteins obey a linear relationship between
surface area and chain length, but to a lesser extent
compared to the ordered proteins. A correlation
coefficient of 0.79 is obtained when disordered and
ordered proteins are combined. However, the
correlation improves when the two classes are
treated separately; 0.85 for disordered and 0.96 for
ordered proteins. Further examination of the
relationship between the interface and chain length8
reveals that ordered and disordered proteins cluster
independently (Figure 1(b)). Importantly, both plots
(Figure 1(a) and (b)) indicate that surface and
interface areas of disordered proteins increase
sharply with residue number as compared to
ordered proteins, suggesting that surface and interface topologies of disordered proteins have an
extended shape. However, both plots fail to
distinguish significantly between disordered and
ordered proteins. Subsequently, we examined the
per-residue surface and interface areas (Figure 2).
As can be seen in Figure 2, the per-residue surface
area versus per-residue interface area clearly distinguishes between the two classes of proteins.
Unlike in Figure 1(b), the ordered proteins are more
localized as compared to the disordered proteins,
indicating that they are more compact and globular.
The disordered proteins are distributed sparsely,
suggesting that disordered proteins opt for
extended shapes and larger interface areas, whereas
ordered proteins are more globular and compact.
Figure 3 shows examples obtained from the PDB
depicting this feature.
It is interesting to note here that crystal-packing
dimers, which are monomers in solution, clearly fall
into the category of the ordered proteins that
includes three-state complexes. Thus, this observation shows that these three-state proteins can
exist as stable monomers independently, and their
interface size is similar to that of interfaces observed
for monomers in the crystalline state. Interestingly,
in Figure 2, the maxima of per-residue surface and
interface areas for stable monomers lie around
80 Å2. This finding is particularly useful in de novo
protein design. The three-dimensional structure is
assumed in de novo design prior to the prediction of
sequence that will fit into the conformation.33,34
Therefore, the per-residue surface area can be
calculated for an intended amino acid sequence
from the model building exercises.
We caution that although we see a discontinuity
in Figure 2 between the ordered and disordered
protein clusters with the current dataset, they could
overlap as the data set size becomes larger, since a
continuum is expected. Consequently, there may be
1330
Discriminating Stable and Unstable Protein Monomers
Table 1. The dataset: various types of protein complexes used for analysis
Proteins
A. The disordered proteins (monomer unstable) category
Two-state protein complexes
P53 fragment
GCN4 -p1
b-Nerve growth factor
Trp-aporepressor
Bowman-Birk protease inhibitor
ROP
Arc repressor
Histone H2A-H2B
MetJ
Aggultinin
Natively unstructured protein complexes
Max protein
Cardiac muscle troponin I
Sec9, SNAP-25 like
GAGA factor glutamine-rich
P27
Ribosomal proteins
30 S
50 S
B. The ordered proteins (monomer stable) category
Three-state protein complexes
Triose phosphate isomerase
Glutathione-S-transferase
Aspartate aminotransferase
Tryptophan synthase
Barnase
CD2
SecA
Ascorbate oxidase
Agglutinin (lectin)
Luciferase
Monomers that are dimers in crystal forme
Myohemerythrin
Peptidyl-tRNA hydrolase
Ribonuclease H
Chymosin
Papaya protease
Endonuclease V
Pseudoazurin
Cytochrome P450CAM
Sialidase
Ampc b-lactamase
Bryodin I
Phosphoenolpyruvate carboxykinase
3-Phosphoglycerate kinase
Tonin
Exonuclease III
Periplasmic hydrogenase 1
PDB codea
Complex state
Chain lengthb
Reference
1SAE (A, BCD)
1YSA (C, D)
1BET (A, B)
3WRP (A, B)
1D6R (I, A)
1ROP (A, B)
1ARR (A, B)
1EQZ (AE, BF)
1CMB (A, B)
1BWU (A, DPQ)
Tetramer
Dimer
Dimer
Dimer
Dimer
Dimer
Dimer
Tetramer
Dimer
Tetramer
42
57
107
101
58
56
53
252
104
106
47
48
49
50
51
52
53
54
55
56
1AN2 (A, BCD)
Complex with
DNA
Fragment
147–163
Fragments
Fragment
(protein–DNA
complex)
Fragments
86
57
17
58
138
54
59
60
69
61
1MXL (I, C)
1KIL (CD, ABE)
1YUI (A, B)
1JSU (C, AB)
1FJGc
(17 chains)
1JJ2d (27 chains)
Macro. complex
Varies
62
Macro. complex
Varies
62
1TIM (A, B)
1GLQ (A, B)
1TAR (A, B)
1TTQ (A, B)
1BNI (A, BC)
1HNG (A, B)
1NL3 (A, B)
1AOZ (A, B)
2PEL (A, BCD)
1LUC (A, B)
Dimer
Dimer
Dimer
Dimer
Trimer
Dimer
Dimer
Dimer
Tetramer
Dimer
247
209
401
256
108
175
839
552
232
326
63
64
65
66
67
67
68
69
70
71
2MHR
2PTH
2RN2
3CMS
1PPO
2END
8PAZ
5CP4
3SIL
2BLS
1BRY
1AYL
16PK
1TON
1AKO
1FEH
Monomer
Monomer
Monomer
Monomer
Monomer
Monomer
Monomer
Monomer
Monomer
Monomer
Monomer
Monomer
Monomer
Monomer
Monomer
Monomer
118
193
155
320
216
137
123
406
379
357
247
532
415
227
268
574
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
a
The chain identifiers as deposited in the PDB are indicated in parentheses. The comma indicates how the trimer and tetramer are
treated as dimer for the purpose of the analysis. For example, in the case of Barnase, chains B and C are treated together as one subunit.
The surface area buried at the interface of chain A would be the sum of the interface area between A and B, and A and C.
b
Number of residues.
c
Chains B, C, and F are excluded, since they appear to be stable proteins due to their large size and small interface.
d
Chain F is excluded, for the same reason.
e
These examples are taken from Ponstingl et al.11
some uncertainty associated with cases that fall on
or close to the dividing line. Nevertheless, cases that
fall farther away from the line can be predicted to be
disordered or ordered with greater confidence.
Thus, the plot shown in Figure 2 provides a
quantitative measure of confidence as to whether
a protein can exist as a stable monomer (ordered,
crystal dimers or three-state complexes) or not
(disordered, monomer natively unstructured, twostate complexes, or ribosomal proteins). It is
Discriminating Stable and Unstable Protein Monomers
1331
Figure 1. Plots of (a) surface and (b) interface areas, versus the protein chain length. The interface and surface areas for
all the complexes listed in Table 1 are calculated as described in Methods. The ordered and disordered protein complexes
are represented with the filled circles and triangles, respectively. The disordered proteins have higher rate of growth of
the interface area with respect to the chain length. The correlation coefficient (r) values for the linearly fitted lines are
shown in (a).
interesting to examine the extreme cases of crystalpacking dimers that have a very large interface
similar to that of disordered proteins, as a measure
of control. A recent publication, which provided
substantial improvement in the distinction of
specific (real or functional dimers) and non-specific
(crystal-packing dimers) interfaces, points out three
examples of crystal dimers (hexokinase (1QHA),
creatine kinase (1CKI_1), and MHC class I homologue (1B3J)), which have interfaces near 3400 Å2,
equivalent in size to the average homodimer interface.12 The authors propose a re-investigation of the
1332
Discriminating Stable and Unstable Protein Monomers
Figure 2. Plot of per-residue surface versus per-residue interface areas. The values are calculated by dividing the total
surface or interface area by the number of residues in the monomer (chain length). There is a clear distinction between
ordered and disordered protein structures in the per-residue surface versus interface areas, unlike in Figure 1. The plot
provides a simple scale that measures the confidence with which one can say whether a protein is ordered or disordered:
the farther the point is from the dividing line, the greater the confidence with which a protein can be classified into either
of the classes. The stars indicate three crystal-packing dimers that have very large interface sizes, equivalent to those of
functional homodimers (see the text). As can be seen from this per-residue plot, these extreme cases fall well in the
ordered proteins cluster, indicating that they can be stable monomers (i.e. could exist as a monomer or as a three-state
complex).
oligomeric state for these proteins due to the
unusually large size of the interface. As can be
seen in Figure 2, these extreme cases fall well within
the ordered proteins cluster, indicating that they can
be stable monomers (i.e. these proteins can exist as a
monomer in solution or as a three-state complex).
Thus, the per-residue parameter appears to depict
the globularity and compactness of a stable monomer successfully. Although the per-residue parameter cannot distinguish between crystal-packing
dimers and three-state complexes, it is valuable in
identifying examples such as two-state, disordered,
and ribosomal proteins that are certain to be
functional complexes.
The interfaces of ordered and disordered protein
complexes
Does the nature of the interface in a protein
complex dictate whether the monomer is ordered or
disordered? How different is the interface of
disordered proteins from that of ordered proteins?
In order to address these questions, we examined
the interfaces of the complexes. We find that the
nature of the interface areas are overall similar for
both ordered and disordered protein data sets
(Figure 4).
The polar and hydrophobic chemical composition of surface area buried at the interface of
various complexes is depicted in Figure 4. A notable
disparity seen in the plot is that ribosomal proteins
tend to have equal proportions of polar and
hydrophobic area at the interface, due to their
interaction with nucleic acids.35 On the other hand,
the non-ribosomal proteins (natively unstructured
proteins, two-state and three-state complexes, and
crystal-packing dimers) tend to be biased towards
larger hydrophobic (or non-polar) area at the
interface. Although the bias towards the hydrophobic composition appears slightly larger for the
disordered cases, in most examples the composition
of polar versus hydrophobic area is similar between
the disordered and ordered proteins. For the crystal
dimers, the points tend to cluster due to the smaller
Discriminating Stable and Unstable Protein Monomers
1333
Figure 3. Ribbon representation of examples of ribosomal, two-state, natively unstructured, and three-state proteins
(only monomers shown with PDB code). Disordered proteins (ribosomal, two-state and unstructured) tend to have more
extended shapes leading to a larger interface area compared to the globular and more compact ordered proteins (for
example, three-state). The Figures are drawn using MOLSCRIPT45 and Raster3D.46
size of the interfaces in crystals. Thus, the distinction between the disordered proteins (excluding
ribosomal proteins) and ordered proteins is not
significant in terms of chemical composition at the
interface. This conclusion is further supported by
the analysis provided in the next section. On the
other hand, when we analyze specific versus nonspecific interactions, the inter-molecular residue–
residue contacts at the protein–protein interface
become more revealing. We examined the contacts
at the interface by grouping amino acid residues as
hydrophobic (Ala, Val, Leu, Ile, Phe, Trp, Met, and
Pro) and polar (Ser, Thr, Arg, Lys, His, Glu, Asp, Gln
and Asn). Gly, Tyr, and Cys are included in both
groups, since they have dual nature (both hydrophobic and polar). A residue is considered to be in
contact with a residue from the second subunit if
any of its atoms falls within 5.0 Å from an atom of
the other residue. We also used the van der Waals
radii plus 0.5 Å distance criterion to determine the
contacting residues, and obtained similar results
(data not shown). These definitions are consistent
with earlier studies.36,37 Figure 5 illustrates the
composition of hydrophobic–hydrophobic, polar–
polar and hydrophobic–polar contacts at the
interface. Interestingly, hydrophobic–hydrophobic
contacts are less dominant in ordered proteins as
compared to the disordered proteins. (We note that
earlier analyses have shown that in the case of
functional dimers the interfaces are more hydrophobic than transient/non-obligate associations.8,38
However, the functional dimers as defined by the
earlier studies could include both ordered (for
example, three-state) and disordered (two-state)
proteins.) The composition of the protein–protein
interface of disordered proteins tends to have fewer
polar–polar and hydrophobic–polar contacts. This
observation is surprising, since sequence analyses
1334
Discriminating Stable and Unstable Protein Monomers
Figure 4. Polar and hydrophobic area composition of the interface regions in the complexes. The ribosomal disordered
proteins tend to have larger polar interface area due to their interaction with nucleic acids.35 Although some bias
towards the hydrophobic composition is seen for the non-ribosomal disordered cases, in most cases the chemical
composition is similar for both ordered and disordered proteins.
have suggested that disordered proteins tend to
have a greater content of charged and polar
residues.23 Our work shows that among the polar
residues, the charged residue content is greater. The
greater occurrence of hydrophobic contacts at the
interface, together with the sequence analysis,
suggests that hydrophobic residues, though overall
occurring less frequently in disordered proteins,
might be more important for binding and therefore
might be conserved. Further, it indicates that the
distribution of polar residues in the disordered
proteins are more toward the exposed surface
and/or the interior and less in the interface. The
hydrophobic–hydrophobic contact preferences seen
in the interface of disordered proteins may suggest
a strong role for the hydrophobic groups in the
packing and in determining proteins that can
associate. A recent study has pointed out that
hydrophobic sections of large interfaces, such as in
disordered proteins, are more often juxtaposed than
hydrophobic portions of small interfaces.39
Interface versus surface
Since the features governing the ordered and
disordered protein–protein association are overall
similar, we turned to surface chemical characteristics of the ordered and disordered proteins. Earlier
analyses on globular proteins have found that the
interface in protein–protein complexes is more
hydrophobic than their exposed surface and less
hydrophobic than their buried interior.1,40 We
compared the interface and the exposed surface
area that excludes the area buried at the interface.
The plot shown in Figure 6 indicates the chemical
nature of the interface and exposed surface, in terms
of the fractions of polar and hydrophobic surface
areas. Since the chemical preferences at the interface
of ribosomal proteins are likely to be biased due to
their interaction with nucleic acids (Figure 4), they
are excluded from this analysis. Figure 6 indicates
that the ranges of fraction of hydrophobic area at the
interface overlap (x-axis: interface) for the ordered
and non-ribosomal disordered proteins, although
most disordered examples have greater fractions of
hydrophobic interface area. However, significantly,
the chemical characteristics at the surface show a
larger difference between the ordered and disordered proteins. As can be seen in Figure 6, the
ordered and disordered proteins do not overlap
when the points are projected onto the y-axis
(surface). The disordered proteins have much
smaller fractions of hydrophobic area at the
exposed surface, with the exception of two
examples (1D6R and 1YUI), compared to the
ordered proteins. This feature is reflected in the
polar surface area composition, where most
disordered proteins have much larger fractions
compared to the ordered proteins (Figure 6).
Intriguingly, ordered proteins cluster narrowly
(around 0.5) in the exposed surface area chemical
compositions. This observation suggests that stable
proteins are designed to balance the polar and
hydrophobic composition at the exposed surface
equally. Again, this finding is useful in de novo
protein design where an amino acid sequence
intended to fit a structural model could be
Discriminating Stable and Unstable Protein Monomers
1335
Figure 5. Inter-molecular residue–residue contacts at the interface of ordered and non-ribosomal
disordered (natively unstructured
and two-state) protein complexes.
There are a large number of
ordered protein complexes with
fewer hydrophobic–hydrophobic
contacts as compared to the disordered proteins. Thus, the hydrophobic–hydrophobic contacts are
dominant in the case of disordered
proteins. This is despite the fact
that disordered proteins tend to
have a greater content of polar and
charged residues (Figure 8). Ribosomal proteins are excluded from
the analysis in order to avoid any
statistical bias due to a large
number of ribosomal proteins represented in the disordered proteins
dataset and due to their interactions with nucleic acids.
examined for the exposed surface area composition
through the model-building exercises.
Buried versus exposed
Does the chemical composition of the interior or
exposed area play a role in determining whether a
protein is ordered or disordered? It may be noted
here that a database analysis on the structurally
ambivalent peptides (SAPs) finds that the pattern of
solvent-accessibility differed between two distinct
conformations of the same SAP.41 Figure 7 shows
the comparisons of the nature of the buried versus
the exposed surface areas. In general, ordered
proteins bury hydrophobic area in their interior
four to seven times more than on their exposed
surface, whereas disordered proteins bury much
less hydrophobic area, only about one to five times.
This is despite the fact that disordered proteins
have smaller fractions of hydrophobic surface area
compared to the ordered proteins (Figure 6). These
observations can explain why the disordered
proteins are unstructured in their uncomplexed
form: disordered proteins simply lack a strong
hydrophobic core. Further, ordered proteins bury
polar area equivalent to five to eight times their
exposed polar surface area, while the disordered
proteins bury only one to five times. This is to be
expected, since the surfaces of disordered proteins
are more polar than those of the ordered proteins
(Figure 6). The extended shape of the disordered
proteins could be understood from the tendency to
bury much less polar or hydrophobic area compared to the ordered proteins. Therefore, the
1336
Figure 6. A comparison of fraction of hydrophobic area
composition at the interface and exposed surface (excluding the interface) regions. The fraction of hydrophobic
area buried at the interface is calculated as hydrophobic
area buried at the interface divided by the interface size,
and similarly for the surface fraction. The ordered and
disordered protein cases overlap largely when the points
are projected on the x-axis. However, they can be well
distinguished on the y-axis. A similar Figure can be
generated for the fraction of polar area by 1808 rotation of
the fraction of hydrophobic area plot shown here. The
surface area is, in general, more polar for the disordered
proteins, with exceptions of two examples (1D6R and
1YUI), than for the ordered proteins. Intriguingly, the
ordered proteins cases cluster narrowly when the
chemical characteristics at the surface are considered
(around 0.5; indicated by the stippled rectangle).
Discriminating Stable and Unstable Protein Monomers
order to perceive how relevant this feature is to the
ordered and disordered protein complexes
described here, we analyzed the sequences. We
examined the singlet count of hydrophobic (Ala, Ile,
Leu, Met, Phe, Pro, Trp, Val, Tyr, Cys), neutral polar
(Asn, Gln, Ser, Thr, His, Gly) and charged (Arg, Lys,
Asp, Glu) residues in the sequences of the monomers. Figure 8 plots the ratio of polar to hydrophobic residues versus the ratio of charged to polar
residues. These ratios are calculated in order to
evaluate the preference for the polar, in particular
charged, residues in the disordered and ordered
proteins. With an exception (1D6R), most of the
disordered protein sequences have a large number
of polar residues. In particular, disordered proteins
contain a large number of charged residues. As
found by Uversky et al., the hydrophobic amino
acids are represented less in the disordered protein
sequences compared to the ordered proteins.23 We
calculated the number of possible pairs of hydrophobic–hydrophobic, polar–polar and charged–
charged residues based on the occurrence of the
polar, hydrophobic and charged amino acid residues in the primary structure of the monomers. We
analyzed for a possible distinction between the
ordered and disordered proteins in the short-range
(pairs separated by less than five residues) and in
the long-range (pairs separated by more than five
residues) pairs; however, no difference was seen
(data not shown). It is interesting to recall here the
results of the interface analysis that showed that in
disordered proteins the interface is rich in hydrophobic-hydrophobic interacting pairs, indicating
that hydrophobic residues are largely required for
protein–protein interactions (Figure 5).
Conclusions
distinction between ordered and disordered
proteins lies primarily in the lack of a strong
hydrophobic core and the nature of the exposed
surface area composition. The highly polar nature
of the exposed surface and the weak hydrophobic
core of the disordered proteins may point to a
rugged nature of the energy landscape around the
native conformation.42
Ordered or disordered—how much sequences
can tell us?
Uversky et al. have analyzed the mean hydrophobicities and net charges of 91 disordered and
275 ordered protein sequences.23 On the basis of the
mean net charge versus mean hydrophobicity
analysis, it was suggested that a combination of
low hydrophobicity and large net charge represents
structural features of natively unfolded proteins.
However, an overlapping of disordered and
ordered proteins in the plot of net charge versus
mean hydrophobicity could not be avoided. In
Identifying which proteins need to be present
simultaneously in order to perform a given function
is important for the understanding and prediction
of protein networks and function. The recently
identified natively unstructured proteins, and
monomeric proteins known to exist as dimers
only in the crystal form, represent two extreme
scenarios. In the former, the monomers are unstructured on their own and the contacts at the interface
in the complexed state are obligatory for folding
and binding. In the latter, the monomers are stable
and the contacts at the crystal interface are the
outcome of crystal-packing effects. Hence, this
provides an opportunity to understand why some
protein structures are ordered but others are
disordered in their uncomplexed form. It further
facilitates developing a scheme to distinguish
between the two types, which would be useful for
de novo design and prediction of stable monomeric
structures.
Here, we collect and analyze the interface and
surface size of several types of complexed structures, consisting of natively unstructured proteins,
ribosomal proteins, two-state and three-state
Discriminating Stable and Unstable Protein Monomers
1337
Figure 7. Buried and exposed surface area compositions of disordered and ordered proteins. (a) Disordered proteins
tend to bury one to five times their hydrophobic and polar exposed surface areas (indicated by the red lines), whereas
ordered proteins bury four to seven and five to eight times their exposed hydrophobic and polar surface areas (indicated
by the green lines), respectively. (b) Plot of the ratio of fraction of polar area buried to that of exposed versus ratio of the
fraction of hydrophobic buried to that of exposed. Non-ribosomal disordered proteins tend to have a greater fraction of
polar area buried (consequently, smaller fraction of hydrophobic area) compared to the ordered proteins.
complexes, and crystal dimers. We focus on the
question of whether ordered and disordered monomers differ in their structural properties when they
exist in their complexed form. Our analysis shows
that two-state proteins resemble disordered proteins. On the other hand, the three-state proteins
resemble crystal-packing dimers. The per-residue
interface and surface areas of the ordered proteins is
1338
Discriminating Stable and Unstable Protein Monomers
Figure 8. Analysis of primary structures of disordered (excluding ribosomal proteins) and ordered protein complexes.
The occurrences of the neutral polar, hydrophobic and charged amino acid residues in the ordered and non-ribosomal
disordered proteins are calculated from the sequences. Polar amino acids, in particular charged residues, occur
frequently in disordered proteins. An exception (1D6R) to this general observation is indicated by the arrow.
significantly smaller than that of the disordered
proteins. This provides a simple scale that measures
the confidence with which one can say whether a
given protein in its complexed form can (or cannot)
exist as a stable monomer. Since the calculations are
structure-based, this method can be extended to
proteins with homology-modeled structures.
Further, the maxima of the per-residue surface
area for stable monomers lie around 80 Å2 and this
observation is particularly useful for de novo protein
design. Analysis of the nature of the interfaces
reveals that hydrophobic–hydrophobic intermolecular residue contacts are dominant in the
disordered protein complexes. This is despite the
fact that disordered proteins tend to have a greater
content of polar and charged residues compared to
ordered proteins. Further analysis of the chemical
characteristics shows that disordered proteins differ
from the ordered proteins more prominently in
their composition of the exposed surface area as
compared to the interface. Disordered proteins have
much smaller fractions of hydrophobic area at the
exposed surface, compared to ordered proteins. In
ordered proteins, the hydrophobic and polar nature
of the exposed surface is balanced equally. Furthermore, as expected, the extended shapes of the
disordered proteins bury less hydrophobic and
polar surface area in their cores than stable globular
proteins.
Our analysis further distinguishes between twostate and three-state complexes, with the
disordered proteins falling into the first category.
The prediction method may complement other
means to identify two-state and three-state proteins,
and provide further insight into the distinction
between crystal dimers and functional dimers. We
stress, however, that while here our scales clearly
differentiate between the disordered and ordered
proteins and our analysis illustrates distinct
features, as the data set grows, one should expect
a continuum between the two classes.
Methods
We calculated the solvent-accessible surface area (ASA)
for all proteins using an in-house program that is based
on the Shrake and Rupley algorithm.43 The ASA of a
protein is calculated numerically by discrete spherical
points44 with a probe radius of 1.4 Å. The interface area
buried by a complex is defined as the difference between
the surface area of the dimer and the sum of the surface
areas of the two separate monomers (Interface ASAZ
ASA[monomer 1]CASA[monomer 2]-ASA[dimer]). For
the analysis described here, a trimer is considered as a
dimer with the second monomer consisting of two chains.
This is because we are interested in discriminating
between stable and unstable monomers. In the case of
homodimers, only one monomer is considered in the
calculation of the total surface area. In the case of
heterodimers, the monomers are treated separately. In
order to analyze the nature of the surface, interface and
buried areas, the atom-based polar and hydrophobic
composition of the areas are calculated. Chemical groups
containing nitrogen, oxygen, sulfur and backbone
carbonyl carbon atoms are defined as polar, and those
containing carbon atoms (except the backbone carbonyl
carbon atoms) are treated as hydrophobic. For the
analysis of contacting residues at the interface, we used
a distance cutoff (!5.0 Å) criterion to identify the intermolecular residue pairs. We also used the van der Waals
Discriminating Stable and Unstable Protein Monomers
plus 0.5 Å distance criterion and found that the results
were unchanged.
Acknowledgements
We thank Drs Gavin Tsai, and Jacob V. Maizel for
their helpful discussions. The research of R.
Nussinov in Israel has been supported, in part, by
the Magnet grant, and by the “Center of Excellence
in Geometric Computing and its Applications”
funded by the Israel Science Foundation (administered by the Israel Academy of Sciences), by the
Ministry of Science grant, and by the Tel Aviv
University Basic Research grants. This project has
been funded, in whole or in part, with Federal
funds from the National Cancer Institute, National
Institutes of Health, under contract number NO1CO-12400. The content of this publication does not
necessarily reflect the view or policies of the
Department of Health and Human Services, nor
does mention of trade names, commercial products,
or organization imply endorsement by the US
Government.
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Edited by D. E. Draper
(Received 27 May 2004; received in revised form 1 July 2004; accepted 2 July 2004)