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Allosteric sites: remote control in regulation of protein
activity
Enrico Guarnera1 and Igor N Berezovsky1,2
The presence of multiple allosteric sites in proteins motivates
development of allosteric drugs — modulators of protein
activity with potentially higher specificity and less toxicity than
traditional orthosteric compounds. A quest for allosteric control
of any protein starts from the identification and characterization
of allosteric sites. Protein dynamics is the basis for allosteric
communication. Binding of effector molecules to allosteric sites
modulates structural dynamics, thus affecting activity of
remote functional sites. We review here theoretical concepts
and experimental approaches for exploring allosteric sites,
their role in allosteric regulation, and ways to assess their
druggability. Key steps of the design procedure aimed at
obtaining allosteric drugs with required agonistic/antagonistic
effect are proposed, and their computational and experimental
elements are discussed.
Addresses
1
Bioinformatics Institute (BII), Agency for Science, Technology and
Research (A*STAR), 30 Biopolis Street, #07-01, Matrix ,
138671 Singapore, Singapore
2
Department of Biological Sciences (DBS), National University of
Singapore (NUS), 8 Medical Drive, 117579 Singapore, Singapore
Corresponding author: Berezovsky, Igor N (igorb@bii.a-star.edu.sg)
Current Opinion in Structural Biology 2016, 37:1–8
This review comes from a themed issue on Theory and simulation
Edited by Modesto Orozco and Narayanaswamy Srinivasan
http://dx.doi.org/10.1016/j.sbi.2015.10.004
0959-440/# 2015 Elsevier Ltd. All rights reserved.
Introduction
Allosoteric sites are the ‘‘other objects’’, from Greek allos
( lloz, other) and stereos (stere z, object), which regulate
activity of proteins by remotely affecting their active sites.
Molecular mechanisms of allosteric communication are
rooted in the dynamic nature of proteins [1–3] — a com-
mon trait of all types of molecules from small single-
domain structures to complex oligomeric enzymes [4],
receptors (e.g. GPCR [5,6]), huge molecular machines such
as chaperones [4,7] and AAA+ proteases [8]. The physics
of protein structure and dynamics is, therefore, a corner-
stone in the study of causality and energetics of allosteric
signaling [9,10,11]. The biological implications of alloste-
ric effects were envisioned by Monod, Changeux, and
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Jacob in their pioneering work ‘Allosteric proteins and
cellular control systems’ [12]. On the basis of the assump-
tion that ‘the allosteric effector does not need to bear any
chemical or metabolic relation of any sort with the sub-
strate’, the authors hypothesized that ‘the absence of any
inherent obligatory chemical analogy or reactivity between
substrate and allosteric effector appears to be a fact of
extreme biological importance’ [12]. It took about half a
century before the significance of allosteric regulation was
fully recognized, and it became apparent that potential
allosteric drugs are free from major drawbacks of traditional
orthosteric compounds. In particular, non-competitive ac-
tion [13–15] via modulation of protein structural dynamics
[4,16,17] is among the major advantages of prospected
allosteric drugs. GPCRs and protein kinases are two groups
that make up to about half of the current drug targets.
Allosteric inhibitors of kinases [18,19] do not affect non-
specifically conserved ATP binding sites, avoiding off-
target inhibition and preventing drug toxicity. In the case
of GPCRs [5,6], allosteric modulators do not cause receptor
desensitization typical of treatment with orthosteric acti-
vators. Additionally, allosteric effectors provide functional
selectivity allowing the regulation of different subsets of
downstream signaling pathways via binding of specific
effector molecules to distinct locations of the same recep-
tor. Detection of communication between the allosteric
and functional sites, as well as design of effector molecules
with required agonist/antagonist activities are the key steps
in obtaining allosteric drugs [15,20]. Here, we review
major theoretical concepts, computational models, and
state-of-the-art experimental approaches currently used
in the study of allosteric regulation. We also briefly discuss
future tasks and sketch a generic approach for design of
allosteric effectors with required agonist/antagonist char-
acteristics.
From phenomenological models to atomic
level description of allostery
The view of allostery has undergone significant transfor-
mation since it was first introduced in phenomenological
models with conformational selection (Monod–Wyman–
Changeux model [21], MWC) and induced fit (Kosh-
land–Nemethy–Filmer [22], KNF) scenarios. The energy
landscape concept [1,2,23] supported by vast experimental
data [24–26] unraveled the omnipresence of allosteric con-
trol [3] in all types of proteins [24]. On the basis of accumu-
lated NMR data, the original model of protein as a
fluctuation around the averaged structure [27] was further
elaborated into a more general conformational equilibrium
view with configurational states coexisting in a structural
Current Opinion in Structural Biology 2016, 37:1–8
2 Theory and simulation
ensemble [10]. Using statistical physics arguments Cooper
and Dryden showed that the allosteric free energy due to
ligand binding can be chiefly entropic, and the minor
changes in fluctuations (one per cent RMS per atom)
may result in a large free energy change (of the order of
kT per molecule). NMR studies substantiated this view
[26,28], unraveling, for example, major role of entropy in
regulation of CAP transcriptional activity [29,30], in re-
laxation dynamics of calmodulin [31,32], and in regulation
of proteins with intrinsically disordered regions [11,33].
A recent comprehensive phenomenological model of
allostery accounts for the switching between agonist
and antagonist effects via a population shift in the pro-
tein’s conformational ensemble [34], opening an oppor-
tunity for design of protein switches [35]. At atomic level
resolution, simple harmonic models implemented in the
context of normal mode analysis (NMA) [36–38] provide a
proper description of the protein dynamics modulated by
allosteric effectors. A coarse grained elastic network mod-
el (ENM) approach was used for predicting the allosteric
effects due to backbone fluctuations in CRP/FNR family
transcription factors, CAP and GlxR, followed by the
experimental verification via X-ray and calorimetric anal-
ysis [39]. Ultimately, modeling and experimental anal-
ysis of allosteric regulation should properly take into
account the balance between the background contribu-
tion from the structural enthalpy and the conformational
entropy modulated as a consequence of the ligand bind-
ing. In order to obtain the required agonist/antagonist
mode of the activity modulation [40], atomic level
description of the ligand–protein interactions is a prereq-
uisite.
Experimental identification and
characterization of allosteric sites
The starting point in a search for allosteric control of any
protein is an identification of the protein’s binding exosites
[6,13–15,20,41,42,43]. Existence of multiple latent allo-
steric sites has been recognized on many occasions in
different proteins, albeit a systematic approach to their
detection and characterization is yet to be developed. A
wealth of the proteomic data combined with libraries of
potential ligands has made high-throughput [44] and frag-
ment-based screening [45] integral parts of pharmaceutical
research, detecting target binding sites and providing
primary compounds in drug discovery projects. Site-direct-
ed approaches, such as alanine scanning [46], disulfide
trapping [40,47], hydrogen/deuterium exchange mass
spectrometry [48,49] (HDXMS), fluorescent [50] and
photoaffinity [51,52] labeling are complementary to the
high-throughput techniques. Given a protein of interest,
the latter methods allow one to find allosteric sites and to
investigate their function-related conformational dynam-
ics. HDXMS and disulfide trapping are particularly suit-
able for exploring the allosteric sites and the modes of
their regulation. Specifically, it has been shown that upon
Current Opinion in Structural Biology 2016, 37:1–8
binding to the same regulatory site different disulfide-
containing fragments can occupy individual subsites
and, thereby, generate more potent activating or inhibiting
effects [40,47]. Therefore, disulfide trapping can be an
efficient and systematic protocol in hypothesis-driven li-
gand discovery, addressing the question of agonism and
antagonism in the function regulation [40]. Analysis of
protein–ligand interactions and allosteric signaling in the
cAMP/protein kinase A [48] shows that time-dependent
HDXMS monitoring can be instrumental in exploring
conformational transitions related to function and its allo-
steric regulation [49]. It is worth noting that standard
biochemical experiments and work with biased ligand
libraries are typically expensive and time consuming.
Given the strong demand for investigation and screening
of a rapidly growing number of allosteric drug targets
[14,15], computational in silico methods have become
indispensable part of current research in allostery
[13,41,43,53].
Computational approaches for prediction of
allosteric sites, and web-based databases
and servers for allostery
Sequence-based/structure-based approaches developed
for detecting allosteric sites are reviewed elsewhere
[41]. In brief, sequence-based approaches utilize conser-
vation of the protein sequences, which reflects similarity
of the corresponding 3D structures. For example, statis-
tical coupling analysis [54] of multiple sequence align-
ments was recently shown to be helpful in detecting the
communication between allosteric and functional sites.
Structure-based models rely upon analysis of known
binding pockets and ligands [41]. A major drawback of
these approaches is the static nature of the objects under
consideration complemented with a bias towards traits
typical for binding sites in the training set. Allosteric
modulation of protein activity is non-competitive, which
is manifested in a low evolutionary pressure on allosteric
sites and in their distinct pharmacophoric characteristics
compared to those of orthosteric pockets. For example,
while allosteric sites can be very shallow, structure-based
approaches are focused around deep surface pockets
typical of ‘traditional’ drug targets. The problem of shal-
low binding pockets is addressed in DARC (Docking
Approach using Ray-Casting) by matching topographies
of the surface pockets observed within the protein and
obtained by viewing potential ligands from the same
vantage point [55].
The major future challenge is a detection and drugg-
ability assessment of the latent allosteric sites. It can be
resolved only by exploring the dynamics of the structure,
because latent sites are active in certain structures of the
conformational ensemble, determined by the structural
dynamics, binding of other ligands, and mutations
[39,56,57]. A key characteristic of any allosteric site
is its ability to couple to the intrinsic dynamics of the
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 Allosteric regulation of protein activity Guarnera and Berezovsky 3
protein [16,17], which, in turn, underlies communica-
tion with relevant functional sites through coherent col-
lective motions [4] in the whole protein. Using a
combination of Monte Carlo (MC) simulations for map-
ping binding hot spots with elastic network-based NMA
for surveying dynamics of the structure, the concept of
binding leverage — a measure of the strength of coupling
between the ligand binding and protein dynamics that
can predict potential allosteric sites [16] — was intro-
duced. Binding leverage does not depend on sequence/
structure conservation of the site and/or deepness/shal-
lowness of the binding pocket, reflecting only the effect
of binding on the protein dynamics. Therefore, it can be
calculated for any single structure without a preliminary
knowledge of its function and mode of regulation [16].
A measure of the strength of allosteric communication,
leverage coupling, was also introduced, and it is calculat-
ed as a dot product of binding leverages of communicat-
ing sites [4]. Communication is strong if the dynamics of
corresponding sites is governed by the coherent degrees
of freedom, which is schematically illustrated in
Figure 1. Leverage coupling was shown to be an effective
indicator of allosteric communication in a wide range of
proteins (different sizes and functions, regulated by li-
gand binding or post-translational modification) and huge
molecular machines such as chaperones. Comparative
analysis of allosteric mechanisms and negative/positive
Figure 1
A Several state-of-the-art computational approaches and
F Database (ASD), which is a manually curated resource
F with comprehensive information on hundreds of allosteric
A
Current Opinion in Structural Biology
Illustration of communication governed by the coherent degrees of
freedom in multidomain/oligomeric structure of protein. Two degrees
of freedom are illustrated here with grey and greenblue arrows. ‘Grey
movements’ of quarters of the structure (shown by grey arrows)
provide communication between neighboring allosteric (A) and
functional (F) sites. ‘Greenblue movements’ of left and right halves of
the structure (greenblue arrows) determine communication between
allosteric sites.
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cooperativity was performed for whole structures of bac-
terial GroEL-GroES, human CCT, and archaeal thermo-
some chaperones [4]. It was shown that leverage
coupling is instrumental in detecting positive intra-ring
cooperativity in GroEL-GroES and thermosome, nega-
tive inter-ring cooperativity in GroEL-GroES, as well as
non-cooperative mechanism in CCT (Figure 2).
Investigation of structural dynamics has recently become
common in prediction and characterization of allosteric
sites. Molecular dynamics (MD) simulations are frequently
used for the search of allosteric binding sites, estimation of
the protein–ligand binding affinities, and detection of the
allosteric communication. Bahar et al. detected allosteric
sites and assessed their druggability via binding dynamics
of the probe molecules, followed by the characterization of
the sites’ binding affinities [58]. A combination of MD
simulations and principal component analysis was used to
study effect of mutations to glycine in the heterodimeric
nuclear receptor complex in order to identify dynamically
important residue–residue contacts and to illustrate the
role of dynamical frustration at the effector binding site
[59]. Two state apo/holo Go-like models were recently
proposed for MD simulations as a general framework for
identifying the allosteric binding sites [17], characterizing
the allosteric communication [60], and effect of mutations
[56]. The effect of ATP binding to allosteric sites on
structural dynamics [61] as well as evolution of these sites
are of special interest, since ATP molecules can be either
endogenous substrates or allosteric effectors, or even play a
dual role of the allosteric effector and substrate [62].
collections of experimental data are implemented in
publically available web-servers and databases reviewed
in [14,41,43,56]. The data and computational tools of
these resources [63,64–66] provide a solid foundation for
prediction and analysis of allosteric sites and design of
allosteric effectors [13]. Zhang et al. created the allosteric
proteins, thousands of modulators, and the data on allo-
steric diseases [66]. On the basis of a nonredundant set
[42] from ASD, the same authors implemented an SVM
classifier for predicting allosteric sites in the Allosite web-
server [64]. They also compiled two benchmark sets in
the ASbench database [65]. The concepts of local close-
ness [67], binding leverage [16], and leverage coupling
[4] were implemented in the web-resource SPACER
[63] — a server for predicting allosteric communication
and effects of regulation. SPACER can be used as a
starting point in experimental efforts, providing predic-
tion of potential allosteric sites via local closeness or
binding leverage. It also allows one to explore the
strength of communication between the allosteric and
functional sites, where, in addition to known sites, a user
can specify a site of interest and analyze its communication
Current Opinion in Structural Biology 2016, 37:1–8
4 Theory and simulation

Figure 2

GroEL - GroES CCT Thermosome

1 1 1
TE GroES

Intermediate Equatorial
Equat.
TI

Interm.
TA
CE

Apical
Apical
CI
ATP CA

ATP
ATP

0 0 0
ATP CA CI CE TA TI TE GroES ATP Apical Interm. Equat. ATP Apical Intermediate Equatorial

Current Opinion in Structural Biology

Allosteric communication in chaperones. Top row shows surface representations of bacterial GroEL-GroES, human CCT, and archaeal
thermosome chaperones. The surface is colored in a gradient from cyan to magenta, displaying the coupling/communication between one ATP
site and the rest of the structure [4] from the weakest (cyan) to strongest (magenta). Bound ADP molecules are displayed as orange spheres
throughout. Bottom row contains matrices of normalized leverage coupling Cpq (for explanations see [4]): apical, intermediate, and equatorial
domains are separated in cis and trans units in GroEL-GroES (CA/CI/CE and TE/TI/TA, respectively), and shown together in CCT and thermosome.
This figure is rearrange and complemented from Figures 7 and 8 in [4].

with others. The utility of SPACER has been shown in
predicting latent allosteric sites in a family of structurally
conserved matrix metalloproteases (MMPs) [68] and in
rationalization of the systematic mapping of the protein
mutational space [69]. Table 1 provides a brief description
of the most advanced and reliable web-servers and data-
bases developed so far.
Current status and future directions
A strong driving force of the current allostery research is a
keen interest in prediction of allosteric sites and analysis
of their druggability. Prospected allosteric drugs are ad-
vantageous compared to orthosteric ones, as they can
modulate protein function, not shutting it off completely.
The non-competitive nature of allosteric action results in
a higher specificity of regulation along with lower toxicity
[13–15,53]. Keeping in mind many challenges, such as
limitation of experimental techniques and physical
potentials currently used in modeling [13,41,43,47],
disregarding the role of water [13,70], difficulties in
experimental verification, one can propose a sketch of
the generalized procedure for selecting the target alloste-
ric sites and for characterizing their druggability in a
protein of interest.
A quest for allosteric control of a protein or protein
complex/receptor/molecular machine [4,5–8] should in-
clude the following key steps: first, identification and
characterization of the allosteric sites; second, detection
of the communication between allosteric and functional
sites; and third, quantification of the interactions between
the allosteric sites and the effector molecules in order to
achieve required agonistic/antagonistic effect and degree
of modulation of the native protein activity. First, increas-
ing evidence suggests that latent allosteric sites can be
found in practically any protein. These sites are active in
certain states of the protein’s conformational ensemble, or
they emerge as a result of protein modification caused by

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 Allosteric regulation of protein activity Guarnera and Berezovsky 5

Figure 3

Allosteric communication

Detection of allosteric sites

Allosteric
site

Functional
site

Current Opinion in Structural Biology

From exploring the allosteric communication to identifying the allosteric sites. Top row shows process of allosteric signaling upon binding of the
effector molecule. Given a functional site of interest, computational detection of the relevant allosteric site is proposed via surveying the reverse
communication upon binding the substrate molecule to the functional site (bottom row).

the binding of other ligands or by mutations. Therefore,
in order to explore all allosteric sites, the screening of a
protein surface should be aimed at the quantification of
the effect of binding on the protein structural dynamics in
different states of the protein. Second, given a functional
site of interest, the relevant allosteric site can be identi-
fied based on the communication detected between
them. Figure 3 illustrates an idea for affecting the func-
tional site, for example, mimicking the binding of a
substrate molecule, in order to locate corresponding allo-
steric sites via feedback communication. Third, the re-
quired agonist/antagonist activity can be achieved in the
detected allosteric site via adjustment of the set of
interactions between the effector molecule and the atoms
of the site. Screening of ligand libraries complemented by
mutations in the allosteric site in addition to computa-
tional predictions is a way to obtain desired regulatory
effect.
While both experimental and computational approaches
are proven assets in drug design, the balance between
them in the search for allosteric drugs is yet to be
established. In particular, the non-competitive nature
of allosteric effectors requires specific protocols for
computational prediction of candidate allosteric sites.
HDXMS has the potential to discriminate between func-
tional and allosteric sites, thus providing complementary
experimental verification. Modeling of the substrate
binding that unravels a sought allosteric site via reverse
communication is an essential computational element,
which does not have a matching experimental counter-
part. Finally, optimization of the effector–protein inter-
actions can provide a starting template, which would be
instrumental in reducing the size of the experimentally
screened compound library. Disulfide trapping can delin-
eate the agonist/antagonist nature of individual contacts,
providing the data for theoretical estimates and verifying

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6 Theory and simulation

Table 1

Currently available web-servers and databases recommended for use

Name, reference, URL Methods, models, and major features

Allosite [64] Structural pocket-based analysis and support vector machine classifier
mdl.shsmu.edu.cn/AST Predicts location of allosteric sites in protein

AllosMode [56] Molecular dynamics simulations sample ligand-induced conformational transitions

salilab.org/allosmod Machine-learning algorithm predicts effect of mutations on allosteric conformational equilibrium

SPACER [63] Monte Carlo simulations of ligand binding

allostery.bii.a-star.edu.sg Normal mode analysis of protein dynamics
Detects native and latent allosteric sites
Quantifies communications between allosteric and functional sites

ASD [66] Collection of experimentally determined sites

mdl.shsmu.edu.cn/ASD Description of allosteric mechanisms
Biochemical experiment is a source of the data

ASBench [65] Contains benchmark sets of allosteric sites:

mdl.shsmu.edu.cn/asbench ‘Core set’ with 235 unique sites
‘Core-diversity set’ with 147 structurally diverse sites

effects of predicted ligands. Together with mutations in
the binding site, these are the crucial elements of a
combined theoretical/experimental strategy for the de-
sign of allosteric drugs with required agonistic or antago-
nistic effects.
Conflict of interest statement
No conflict of interests.
Acknowledgements
The authors thank Birgit Eisenhaber and Westley Sherman for critical
reading of the manuscript, valuable comments, and editing.
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