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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 Jacob in their pioneering work ‘Allosteric proteins and
development of allosteric drugs — modulators of protein cellular control systems’ [12]. On the basis of the assump-
activity with potentially higher specificity and less toxicity than tion that ‘the allosteric effector does not need to bear any
traditional orthosteric compounds. A quest for allosteric control chemical or metabolic relation of any sort with the sub-
of any protein starts from the identification and characterization strate’, the authors hypothesized that ‘the absence of any
of allosteric sites. Protein dynamics is the basis for allosteric inherent obligatory chemical analogy or reactivity between
communication. Binding of effector molecules to allosteric sites substrate and allosteric effector appears to be a fact of
modulates structural dynamics, thus affecting activity of extreme biological importance’ [12]. It took about half a
remote functional sites. We review here theoretical concepts century before the significance of allosteric regulation was
and experimental approaches for exploring allosteric sites, fully recognized, and it became apparent that potential
their role in allosteric regulation, and ways to assess their allosteric drugs are free from major drawbacks of traditional
druggability. Key steps of the design procedure aimed at orthosteric compounds. In particular, non-competitive ac-
obtaining allosteric drugs with required agonistic/antagonistic tion [13–15] via modulation of protein structural dynamics
effect are proposed, and their computational and experimental [4,16,17] is among the major advantages of prospected
elements are discussed. allosteric drugs. GPCRs and protein kinases are two groups
Addresses that make up to about half of the current drug targets.
1
Bioinformatics Institute (BII), Agency for Science, Technology and Allosteric inhibitors of kinases [18,19] do not affect non-
Research (A*STAR), 30 Biopolis Street, #07-01, Matrix , specifically conserved ATP binding sites, avoiding off-
138671 Singapore, Singapore
2
Department of Biological Sciences (DBS), National University of
target inhibition and preventing drug toxicity. In the case
Singapore (NUS), 8 Medical Drive, 117579 Singapore, Singapore of GPCRs [5,6], allosteric modulators do not cause receptor
desensitization typical of treatment with orthosteric acti-
Corresponding author: Berezovsky, Igor N (igorb@bii.a-star.edu.sg) vators. Additionally, allosteric effectors provide functional
selectivity allowing the regulation of different subsets of
Current Opinion in Structural Biology 2016, 37:1–8
downstream signaling pathways via binding of specific
effector molecules to distinct locations of the same recep-
This review comes from a themed issue on Theory and simulation
tor. Detection of communication between the allosteric
Edited by Modesto Orozco and Narayanaswamy Srinivasan 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
http://dx.doi.org/10.1016/j.sbi.2015.10.004
major theoretical concepts, computational models, and
state-of-the-art experimental approaches currently used
0959-440/# 2015 Elsevier Ltd. All rights reserved.
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.

Introduction From phenomenological models to atomic


Allosoteric sites are the ‘‘other objects’’, from Greek allos level description of allostery
( lloz, other) and stereos (stere z, object), which regulate The view of allostery has undergone significant transfor-
activity of proteins by remotely affecting their active sites. mation since it was first introduced in phenomenological
Molecular mechanisms of allosteric communication are models with conformational selection (Monod–Wyman–
rooted in the dynamic nature of proteins [1–3] — a com- Changeux model [21], MWC) and induced fit (Kosh-
mon trait of all types of molecules from small single- land–Nemethy–Filmer [22], KNF) scenarios. The energy
domain structures to complex oligomeric enzymes [4], landscape concept [1,2,23] supported by vast experimental
receptors (e.g. GPCR [5,6]), huge molecular machines such data [24–26] unraveled the omnipresence of allosteric con-
as chaperones [4,7] and AAA+ proteases [8]. The physics trol [3] in all types of proteins [24]. On the basis of accumu-
of protein structure and dynamics is, therefore, a corner- lated NMR data, the original model of protein as a
stone in the study of causality and energetics of allosteric fluctuation around the averaged structure [27] was further
signaling [9,10,11]. The biological implications of alloste- elaborated into a more general conformational equilibrium
ric effects were envisioned by Monod, Changeux, and view with configurational states coexisting in a structural

www.sciencedirect.com Current Opinion in Structural Biology 2016, 37:1–8


2 Theory and simulation

ensemble [10]. Using statistical physics arguments Cooper binding to the same regulatory site different disulfide-
and Dryden showed that the allosteric free energy due to containing fragments can occupy individual subsites
ligand binding can be chiefly entropic, and the minor and, thereby, generate more potent activating or inhibiting
changes in fluctuations (one per cent RMS per atom) effects [40,47]. Therefore, disulfide trapping can be an
may result in a large free energy change (of the order of efficient and systematic protocol in hypothesis-driven li-
kT per molecule). NMR studies substantiated this view gand discovery, addressing the question of agonism and
[26,28], unraveling, for example, major role of entropy in antagonism in the function regulation [40]. Analysis of
regulation of CAP transcriptional activity [29,30], in re- protein–ligand interactions and allosteric signaling in the
laxation dynamics of calmodulin [31,32], and in regulation cAMP/protein kinase A [48] shows that time-dependent
of proteins with intrinsically disordered regions [11,33]. HDXMS monitoring can be instrumental in exploring
conformational transitions related to function and its allo-
A recent comprehensive phenomenological model of steric regulation [49]. It is worth noting that standard
allostery accounts for the switching between agonist biochemical experiments and work with biased ligand
and antagonist effects via a population shift in the pro- libraries are typically expensive and time consuming.
tein’s conformational ensemble [34], opening an oppor- Given the strong demand for investigation and screening
tunity for design of protein switches [35]. At atomic level of a rapidly growing number of allosteric drug targets
resolution, simple harmonic models implemented in the [14,15], computational in silico methods have become
context of normal mode analysis (NMA) [36–38] provide a indispensable part of current research in allostery
proper description of the protein dynamics modulated by [13,41,43,53].
allosteric effectors. A coarse grained elastic network mod-
el (ENM) approach was used for predicting the allosteric Computational approaches for prediction of
effects due to backbone fluctuations in CRP/FNR family allosteric sites, and web-based databases
transcription factors, CAP and GlxR, followed by the and servers for allostery
experimental verification via X-ray and calorimetric anal- Sequence-based/structure-based approaches developed
ysis [39]. Ultimately, modeling and experimental anal- for detecting allosteric sites are reviewed elsewhere
ysis of allosteric regulation should properly take into [41]. In brief, sequence-based approaches utilize conser-
account the balance between the background contribu- vation of the protein sequences, which reflects similarity
tion from the structural enthalpy and the conformational of the corresponding 3D structures. For example, statis-
entropy modulated as a consequence of the ligand bind- tical coupling analysis [54] of multiple sequence align-
ing. In order to obtain the required agonist/antagonist ments was recently shown to be helpful in detecting the
mode of the activity modulation [40], atomic level communication between allosteric and functional sites.
description of the ligand–protein interactions is a prereq- Structure-based models rely upon analysis of known
uisite. binding pockets and ligands [41]. A major drawback of
these approaches is the static nature of the objects under
Experimental identification and consideration complemented with a bias towards traits
characterization of allosteric sites typical for binding sites in the training set. Allosteric
The starting point in a search for allosteric control of any modulation of protein activity is non-competitive, which
protein is an identification of the protein’s binding exosites is manifested in a low evolutionary pressure on allosteric
[6,13–15,20,41,42,43]. Existence of multiple latent allo- sites and in their distinct pharmacophoric characteristics
steric sites has been recognized on many occasions in compared to those of orthosteric pockets. For example,
different proteins, albeit a systematic approach to their while allosteric sites can be very shallow, structure-based
detection and characterization is yet to be developed. A approaches are focused around deep surface pockets
wealth of the proteomic data combined with libraries of typical of ‘traditional’ drug targets. The problem of shal-
potential ligands has made high-throughput [44] and frag- low binding pockets is addressed in DARC (Docking
ment-based screening [45] integral parts of pharmaceutical Approach using Ray-Casting) by matching topographies
research, detecting target binding sites and providing of the surface pockets observed within the protein and
primary compounds in drug discovery projects. Site-direct- obtained by viewing potential ligands from the same
ed approaches, such as alanine scanning [46], disulfide vantage point [55].
trapping [40,47], hydrogen/deuterium exchange mass
spectrometry [48,49] (HDXMS), fluorescent [50] and The major future challenge is a detection and drugg-
photoaffinity [51,52] labeling are complementary to the ability assessment of the latent allosteric sites. It can be
high-throughput techniques. Given a protein of interest, resolved only by exploring the dynamics of the structure,
the latter methods allow one to find allosteric sites and to because latent sites are active in certain structures of the
investigate their function-related conformational dynam- conformational ensemble, determined by the structural
ics. HDXMS and disulfide trapping are particularly suit- dynamics, binding of other ligands, and mutations
able for exploring the allosteric sites and the modes of [39,56,57]. A key characteristic of any allosteric site
their regulation. Specifically, it has been shown that upon 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- cooperativity was performed for whole structures of bac-
tion with relevant functional sites through coherent col- terial GroEL-GroES, human CCT, and archaeal thermo-
lective motions [4] in the whole protein. Using a some chaperones [4]. It was shown that leverage
combination of Monte Carlo (MC) simulations for map- coupling is instrumental in detecting positive intra-ring
ping binding hot spots with elastic network-based NMA cooperativity in GroEL-GroES and thermosome, nega-
for surveying dynamics of the structure, the concept of tive inter-ring cooperativity in GroEL-GroES, as well as
binding leverage — a measure of the strength of coupling non-cooperative mechanism in CCT (Figure 2).
between the ligand binding and protein dynamics that
can predict potential allosteric sites [16] — was intro- Investigation of structural dynamics has recently become
duced. Binding leverage does not depend on sequence/ common in prediction and characterization of allosteric
structure conservation of the site and/or deepness/shal- sites. Molecular dynamics (MD) simulations are frequently
lowness of the binding pocket, reflecting only the effect used for the search of allosteric binding sites, estimation of
of binding on the protein dynamics. Therefore, it can be the protein–ligand binding affinities, and detection of the
calculated for any single structure without a preliminary allosteric communication. Bahar et al. detected allosteric
knowledge of its function and mode of regulation [16]. sites and assessed their druggability via binding dynamics
A measure of the strength of allosteric communication, of the probe molecules, followed by the characterization of
leverage coupling, was also introduced, and it is calculat- the sites’ binding affinities [58]. A combination of MD
ed as a dot product of binding leverages of communicat- simulations and principal component analysis was used to
ing sites [4]. Communication is strong if the dynamics of study effect of mutations to glycine in the heterodimeric
corresponding sites is governed by the coherent degrees nuclear receptor complex in order to identify dynamically
of freedom, which is schematically illustrated in important residue–residue contacts and to illustrate the
Figure 1. Leverage coupling was shown to be an effective role of dynamical frustration at the effector binding site
indicator of allosteric communication in a wide range of [59]. Two state apo/holo Go-like models were recently
proteins (different sizes and functions, regulated by li- proposed for MD simulations as a general framework for
gand binding or post-translational modification) and huge identifying the allosteric binding sites [17], characterizing
molecular machines such as chaperones. Comparative the allosteric communication [60], and effect of mutations
analysis of allosteric mechanisms and negative/positive [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
Figure 1
endogenous substrates or allosteric effectors, or even play a
dual role of the allosteric effector and substrate [62].

A Several state-of-the-art computational approaches and


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
F Database (ASD), which is a manually curated resource
F with comprehensive information on hundreds of 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-
A
ness [67], binding leverage [16], and leverage coupling
Current Opinion in Structural Biology [4] were implemented in the web-resource SPACER
[63] — a server for predicting allosteric communication
Illustration of communication governed by the coherent degrees of and effects of regulation. SPACER can be used as a
freedom in multidomain/oligomeric structure of protein. Two degrees starting point in experimental efforts, providing predic-
of freedom are illustrated here with grey and greenblue arrows. ‘Grey tion of potential allosteric sites via local closeness or
movements’ of quarters of the structure (shown by grey arrows) binding leverage. It also allows one to explore the
provide communication between neighboring allosteric (A) and
functional (F) sites. ‘Greenblue movements’ of left and right halves of
strength of communication between the allosteric and
the structure (greenblue arrows) determine communication between functional sites, where, in addition to known sites, a user
allosteric sites. can specify a site of interest and analyze its communication

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

Current Opinion in Structural Biology 2016, 37:1–8 www.sciencedirect.com


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

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