Mechanistic Model for the Hsp90-Driven Opening of
Human Argonaute
The assembly of RNA-induced silencing complex (RISC) is a key process in small RNA-mediated gene silencing. Loading of small RNAs into Argonaute (Ago), the key player protein in the process, has been shown to depend on the Hsp90 chaperone machinery. Experimental single-molecule data indicate that ATP binding to the chaperone facilitates the conformational changes leading to the open state of Ago essential to form a complex with small-RNA duplexes. Yet, no atomic-level description of the dynamic mechanisms and protein–protein interactions underpinning Hsp90-mediated Ago conformational activation is available. Here we investigate the functionally oriented structural and dynamic features of Hsp90-human Ago (hAgo2) complexes in different ligand states by integrating protein–protein docking techniques, all-atom MD simulations, and novel methods of analysis of protein internal dynamics and energetics. On this basis, we develop a structural-dynamic model of the mechanisms underlying the chaperone-assisted human RISC assembly. Our approach unveils the large conformational variability displayed by hAgo2 in the unbound vs the Hsp90-bound states. In this context, several hAgo2 states are found to coexist in isolation, while Hsp90 selects and stabilizes the active form. Hsp90 binding modulates the conformational plasticity of hAgo2 (favoring its opening) by modifying the patterns of hAgo2 intramolecular interactions. Finally, we identify a series of experimentally verifiable key sites that can be mutated to modulate Hsp90-mediated hAgo2 conformational response and ability to bind RNA.
Introduction<!>Structures Examined in
This Study<!><!>Structures Examined in
This Study<!>Building Models of the
hAgo2-Hsp90 Complex<!><!>Characterization of the Structural Dynamics
of the Different
Models and Comparison with the Apo state: Functional Implications<!><!>Characterization of the Structural Dynamics
of the Different
Models and Comparison with the Apo state: Functional Implications<!><!>Characterization of the Structural Dynamics
of the Different
Models and Comparison with the Apo state: Functional Implications<!><!>The Determinants of Differential Dynamics: Insights from Energetic
Analysis of hAgo2 in Different Complexes<!><!>The Determinants of Differential Dynamics: Insights from Energetic
Analysis of hAgo2 in Different Complexes<!>Connecting Internal Energetics
and Dynamics: Implications for
Recognition Mechanisms<!><!>Connecting Internal Energetics
and Dynamics: Implications for
Recognition Mechanisms<!>Conclusions<!>MD Set
Up<!>Protein–Protein Docking<!>Collective
Motion Analysis<!>Energy Decomposition Method<!>Distance Fluctuation Analysis<!>Center of Mass (COM) Distances Analysis<!>
<p>Small RNAs—small
interfering RNAs (siRNAs) and microRNAs
(miRNAs)—can silence the expression of their complementary
target messenger RNAs (mRNA) through the formation of the effector
RNA-induced silencing complex (RISC). The effector complex, in turn,
fine-tunes gene-regulation in diverse organisms contributing to cellular
homeostasis in a number of diverse physiological processes. The Argonaute
(Ago) family of proteins is the core component of RISC, acting as
the catalytic engine for the endonucleolytic cleavage of target mRNAs
(known as RNA interference [RNAi]) and/or as a molecular platform
for their translational repression, deadenylation, and degradation.1−3</p><p>RISC assembly can be divided at least into two main successive
steps: (1) duplex loading, in which small RNA duplexes are inserted
into Ago proteins to form pre-RISC, and (2) passenger ejection or
unwinding, in which the two strands are separated within the Ago protein
and one of them is ejected from Ago. The resulting functional complex
is called mature RISC or simply RISC.</p><p>The binding between mRNAs
and Ago takes place through several contact
sites. Alanine scanning experiments and spectroscopic analyses allowed
to identify the N-domain of human Ago2 (hAgo2) as the initiator of
duplex unwinding during RISC assembly. This event was shown to be
coupled to N-domain conformational changes:4 indeed, the human N-domain can assume different poses in the context
of the full length protein depending on the exact step of RISC assembly,
similarly to what already observed in bacterial structures for the
relative positioning of the Ago N-domain in the different states of
the complex (apo, binary bound to the RNA guide or ternary in a protein-guide-target
complex).5−7 Deletion of amino acids 53–135 compromises
the stability of the protein, which is still able to load siRNA albeit
more slowly than WT, while no detectable mature RISC (unable to unwind)
is observed.8</p><p>Loading of small RNAs
into Argonaute has been shown to require
the intervention of the chaperone machinery, which entails heat shock
proteins Hsp70 and Hsp90: the relevance of chaperones during duplex
loading has been confirmed in different species and isoforms (fly
Ago1 and Ago2, mammalian Ago2, and plant Ago1, Ago4 and Ago7).9−15</p><p>The current model of functioning is based on the hypothesis
that
Hsp90 stabilizes a high-energy, RNA-free Argonaute, which upon loading
of a duplex, unwinding, and ejection of the passenger strand, stabilizes
in a low-energy, guide-RNA-sequestered conformation, whereby the latter
steps are likely favored by the intrinsic energy difference between
the high-energy RNA-free Argonaute and a mature, guide-bound RISC.3</p><p>Recent studies have demonstrated that the
overall domain organization
of Ago proteins is highly flexible in their RNA-free form. In particular,
it is hypothesized that in the absence of the chaperone machinery,
the binding cavity of Ago2 is too narrow to accommodate small RNA
duplexes, as observed in studies of the fly protein.16 ATP hydrolysis by the heat shock protein triggers the set
of conformational changes needed by Ago proteins to load otherwise
too bulky and sterically hindered RNA duplexes. Yet, chaperones are
dispensable in the subsequent steps of RISC cycle, i.e., binding,
cleavage, and release of the complementary target RNAs by RISC.16−18 In this framework, besides inducing a set of structural changes
in the client protein Ago, the chaperone machinery itself undergoes
a set of structural changes tightly connected to specific steps of
its ATP hydrolysis cycle.19 The strong
crosstalk between the mode of action of the chaperone system, its
own structural behavior, and the structure/thermostability of client
proteins is currently a hot subject of research and debate.20,21</p><p>In mammalian RISC assembly, duplex loading is an ATP-dependent
reaction and Hsp90β binds to the client in its ATP-bound form.22,23 Recent evidence showed that the ATPase mutant E42A, unable to hydrolyze
ATP, significantly reduced target cleavage activity highlighting an
additional point of chaperone control over RISC dynamical assembly.24 Importantly, the in vitro reconstitution
of the chaperone-mediated Ago2-RISC assembly has been extensively
described for Drosophila melanogaster by Tomari and
co-workers.12,18,25 Single-molecule analysis provided information on the conformational
ensembles of the intermediate states in RISC biogenesis and target
cleavage mechanisms. Moreover, the authors gained insights into the
mammalian assembly of human RISC further confirming the high conservation
of the duplex loading mechanism and the requirement for the intervention
of chaperones to incorporate bulky RNAs into Ago.24</p><p>Despite these fundamental advances, understanding
the structural
basis of chaperone-mediated RISC assembly, in other words how Hsp90
selects, binds and stabilizes the conformation competent for duplex
loading (the active state), represents a challenging task. The structural
organization of this state remains elusive, and no atomistic-resolution
data are available concerning domains or regions of Argonaute that
are connected by chaperones. Moreover, questions still remain on which
of these interactions are essential for determining the active form
of Argonaute and for RNA-loading.3,18</p><p>The
aim of this work is to develop a structural model that can
provide atomistic insights into the mechanisms underlying the chaperone-assisted
human RISC assembly, rationalizing experimental evidence, and laying
the foundations for the design of approaches to control/tune RNA silencing
processes. To achieve these goals, here we combine protein–protein
docking calculations with extensive MD simulations and novel methods
of analysis of structural dynamics and energetic stabilization of
complexes to generate a model of the Hsp90 and human Ago2 complex,
recapitulating the determinants of the functionally oriented aspects
of the interaction. Moreover, we present a detailed investigation
of the internal dynamics of hAgo2 free and hAgo2 in complex with the
chaperone. In this framework, we pinpoint hAgo2 single-point mutants
acting as key hotspots driving the interaction with Hsp90 that may
ultimately modulate RNA loading mechanism. Our computational approach
offers new insights into the role of Hsp90 in RISC assembly and contributes
experimentally verifiable key sites for the investigation of Argonaute-mediated
gene silencing mechanisms.</p><!><p>Recently, the crystal
structure of human Ago2 bound to a mixed population of siRNA was solved,
showing that the overall domain organization and mode of siRNAs binding
are similar to those of prokaryotic Argonautes, which have been more
extensively studied (Figures 1 and S1).1,6,7,17,26−30 Herein, small RNA binding plays an important role, locking the flexible
apo form of the eukaryotic Argonaute protein into a single dominant
conformation. Indeed, the high conformational variability of apo hAgo2
is likely the reason why no X-ray structure of the full length apo
protein has been determined to date.17,30,31</p><!><p>Human Argonaute. 3D structure of hAgo2 is shown in cartoons
and
ghost surface. N-terminal, PAZ, MID and PIWI, connected by two linker
domains (L1 and L2), are indicated.</p><!><p>From a structural point of view, hAgo2 consists of four conserved
domains: N-terminal, Piwi/Ago/Zwille (PAZ), MID, and P-induced winpy
testis (PIWI), connected by two linker domains (L1 and L2) (Figure 1). Overall, the protein
appears arranged in two main lobes, where the nucleic acid-binding
channel runs across the four domains. The loaded strand serves as
the spine of the Ago protein: limited proteolysis with thermolysin
suggested that, in the absence of the RNA strand, the MID domain is
hinged toward the PIWI domain and adopts an open conformation whereby
the interface between the two is exposed to solvent while the C-terminal
region remains unfolded.15,17,28</p><p>On the other side, Hsp90 displays a multipartite structure
where
different domains control distinct functions, connecting ATP processing
in the N-terminal domain and client binding/remodelling in the middle
domain. Recent results unveiled an intricate array of correlated functional
motions among distant regions of Hsp90 depending on specific binding
states, which helped gain insights into the complex mechanisms used
by the chaperone to assist the formation of multiprotein assemblies.21,32−35 In this scenario, investigating the internal dynamics pattern of
the chaperone-hAgo2 assembly can shed light on the traits of internal
dynamics of the complex that are connected to its functional role
in the RISC conformational cycle.</p><!><p>We first
set out to characterize the conformational ensemble of the apo protein
(apo hAgo2) to detect distinct dynamic and energetic properties of
the unbound state that may be perturbed upon Hsp90 binding. Extensive
all-atoms molecular dynamics (MD) of the unbound hAgo2 protein were
run at different temperatures, 300, 350, and 400 K. Nonphysiological
temperatures were used to speed up structural sampling toward local
unfolding events and expand conformational ensembles. Importantly,
it has been shown that the highly dynamic character of the apo form
of hAgo2 is dramatically reduced when small RNAs bind the protein
and that this process is assisted by the chaperone machinery.16 In order to investigate the effects of the chaperone
on Ago internal dynamics and their effect on Ago structural modulation,
we modeled the structural complex of the interaction between Hsp90
and hAgo2 and studied the dynamic and energetic patterns of the complex.
As no direct experimental knowledge on the interacting faces of the
two proteins is available to guide the docking simulation of hAgo2-Hsp90
complex, we run different (blind) predictions to select the representative
complex.</p><p>It is worth noting here that the Hsp90 structure selected
for these calculations is the closed one, which corresponds to the
chaperone conformation in complex with the Cdc37 cochaperone and the
Cdk4 client kinase in the cryoEM structure solved by the Agard group.
As an important caveat, it must be mentioned that alternative, more
open, Hsp90 conformations could have been used for our calculations.
In this picture, the chaperone would offer larger contact surfaces
to the recognition of hAgo2. Our working hypothesis here is that this
Hsp90 conformation corresponds to the most likely activated state,
responsible for productive binding and reshaping of clients, as observed
in the Agard structure. In this framework, we reasoned that running
our docking experiments on open Hsp90 structures modeled on SAXS or
FRET data may have suffered from limitations due to low resolution
and introduced a level of variability that could expectedly have affected
the results.</p><p>Specifically, predictions of hAgo2-Hsp90 complex
were carried out
using a combination of docking methods, ClusPro, Zdock, and Patchdock,
based on rigid-body protein–protein protocols (see Methods): the results of the three different approaches
were combined to identify consensus complex poses by in-depth structural
analyses, visual inspection, and evaluation of consistency with available
data. At the end of the process, four different models of the hAgo2-Hsp90
complex were selected (see Methods), where
Hsp90 preferably binds the N-domain of hAgo2, consistent with recent
experimental evidence on hAgo2 N-domain mutants with compromised RISC
cycle, but no direct association with chaperone-mediated conformational
opening.8 In three out of four models (complexes 1, 2, 4), at least one of the Hsp90
N-domains is engaged in a stable interaction with hAgo2 (top interactions, Figures 2 and S2). In complex 1 Hsp90 N-domains
interact with residues of PIWI domain, whereas in complexes 2 and 4 the binding concerns hAgo2 N-domain (Figures 2 and S2). In the remaining model (complex 3), one of the two Hsp90 M-domains binds the hAgo2 N-domain, resulting
in a highly asymmetric unit (side interaction, Figures 2 and S2).</p><!><p>hAgo2-Hsp90
complexes. Selected docking poses for hAgo2-Hsp90 interaction
(see Methods). hAgo2 is shown in cartoons
(N → green, PAZ → orange, PIWI → yellow, MID
→ lime, L1 → purple, L2 → pink) and ghost surface.
Blue and gray solid surface representation is used for Hsp90 subunits.</p><!><p>Extensive MD simulations were run for each predicted complex to
gain insights into the structural stability of the complexes and/or
evaluate the occurrence of local rearrangements. The dynamic evolution
of the unbound hAgo2 at different temperatures and in complex with
Hsp90 immediately shows the extremely flexible nature of Ago, which
is modulated by chaperone binding. To clarify the dynamic behavior
of hAgo2, we built a unique hAgo2 meta-trajectory obtained by the
concatenation of all the single MD runs from both the apo and the
Hsp90-bound states. Cluster analysis (performed applying the Gromos
clustering algorithm36) was applied to
the meta-trajectory, considering the backbone atoms of hAgo2 (RMSD
cutoff set to 0.5 nm, to account for the expectedly large structural
variations of the protein). Figure 3 shows that in the absence of Hsp90, hAgo2 largely
adopts one preferential conformation (cluster #1), which corresponds
to a closed structure not competent for RNA loading. In contrast,
in hAgo2-Hsp90 complexes, hAgo2 substantially populates distinct conformational
ensembles, with the exception of complex 1, for which
the conformations collected in cluster #1 (corresponding mostly to
the apo state) are accessible. Indeed, complexes where hAgo2-Hsp90
interaction takes place at the hAgo2 N-domain (complexes 2, 3, and 4) share common structural dynamics
traits, with Ago populating mainly cluster #2, representative of open
conformations of the client. Moreover, a secondary effect related
to the orientation (top/side interaction) of hAgo in the binding pose
can be observed. While complexes 2 and 4 stably adopt cluster #2 conformations, complex 3 shows
larger dynamic variability. These preliminary investigations of the
hAgo2-Hsp90 interaction models suggest that Hsp90 can stabilize a
set of open hAgo2 conformations that may be only marginally visited
by the apo protein.</p><!><p>Cluster analysis on hAgo2 meta-trajectory. Cluster id
and counts
per cluster are indicated on x- and y-axes, respectively. Cluster composition is displayed and colored
per system. See Methods.</p><!><p>Specifically, it is worth noting that among the four hAgo2-Hsp90
complexes, complex 1 appears to be the least compatible
with experimental/biochemical observations: in this model, Hsp90 does
not significantly affect hAgo2 dynamics compared to the apo state,
stabilizing a conformation that is largely superimposable to the unbound
state. Complex 3 represents an intermediate case; even
though chaperone binding modulates the dynamic behavior of hAgo2,
compared to the apo form, the observed high conformational plasticity
of the complex (given the occupancy of clusters 2, 4, 5, and 6 in Figure 3) indicates a relatively unstable
interaction. In this framework, complexes 2 and 4 appear to be the models that best capture, at a qualitative
level, the general features of Hsp90 and hAgo2 binding that may be
related to the functional properties of the complex. In particular,
the two models suggest a role for Hsp90 in the preferential selection
of stable hAgo2 open structural states, preorganized for efficient
polynucleotide binding. This result is consistent with the experimental
observations that point to the presence of the chaperone as a necessary
requirement for RNA loading by the client. It is tempting to suggest
that Hsp90s role is to stabilize a functional conformation of the
client that would not be populated in the uncomplexed state. The chaperone
does not act as a foldase but rather as a regulator/promoter of the
functions of other proteins.8,16</p><p>To characterize
the main protein motions explored in the different
cases, we next performed a principal component analysis on hAgo2 protein
(CÎ± atoms) along the meta-trajectory (Figure 4). First, PCA confirms that, once promoted
by the contact with Hsp90, the open states of hAgo2 are stable: indeed,
in complexes 2 and 4, hAgo2 spans a limited
ensemble of conformational space. In contrast, apo hAgo2 is characterized
by a much broader distribution of conformations (colored spots in Figure 4). We observe that
the presence of Hsp90 pushes hAgo2 to sample different regions of
the essential space, compared to the unbound case (different values
of the first vector describing the principal motions on x-axis). Along these lines, the temperature increase in apo hAgo2
simulations favors the transition of the protein to compact structures
where the RNA cleft is narrowed and PAZ and MID domains can freely
move closer to each other. In contrast, even at higher temperatures,
hAgo2 explores more open conformations, competent for duplex loading,
in association with the chaperone.</p><!><p>PCA analysis. Principal component analysis
on hAgo2 in the complexes
(A) and at different T (B). hAgo2 covariance matrix
has been built on CÎ± atoms and used for individual system trajectory
projection. The first two principal components (eigenvectors 1 and
2 on x- and y-axes) are used for
the projection. Extreme projections along the meta-trajectory on the
average structure corresponding to hAgo2 in the complexes (C) and
apo hAgo2 (D) are rendered in surface views.</p><!><p>At the domain level, the differential dynamic modulation induced
by Hsp90 is reflected by the diverse structural domains rearrangements
of hAgo2. Specifically, we measured the distance between the center
of mass (COM) of PAZ and PIWI domains along the simulation time, as
an index of the reciprocal positioning of the two lobes. This analysis
(further confirmed by the evolution of the angle spanned by the two
lobes of Ago protein, see Figure S3 in the
Supporting Information) demonstrates that the presence of the chaperone
favors the opening and "freezes" Ago in a structural
organization where the two lobes are well separated (Figure 5). This wider conformation
presents a larger cavity for RNA recognition and loading. In this
context, a broader distribution of COM distances is further observed
for the apo hAgo2 simulations (300 K), as shown in Figure 5, consistent with previous
analyses. Importantly, the larger COM distances we report are qualitatively
consistent with experimental distances (56 Å) measured by FRET
analysis between PAZ and MID-labeled domains in the Ago2-RISC, where
small RNA loading prompts Ago2 to adopt a more open conformation.17,18</p><!><p>PAZ–PIWI
distances. Statistical distribution of the distances
between the center of mass (COM) calculated on PIWI and PAZ domains
(see Methods) along the simulation time of
hAgo2 in the apo form at 300 and 400 K and bound to Hsp90 in complex 2 and 4. On the y-axis, histograms
counts are normalized to 1.</p><!><p>As the presence
of Hsp90 is shown to tune the global conformational dynamics of hAgo2,
we investigated whether distinct internal patterns of residue-pair
interactions can be identified for hAgo2 in the complexes and in the
apo form. The underlying hypothesis is that differential combinations
of pair interactions may stabilize hAgo conformations in isolation
or in the complex. The functional relevance of distinct specific patterns
of predicted intraprotein interactions determined by Hsp90 being bound
or unbound is experimentally verifiable by, e.g., site-directed mutagenesis
and analysis of mutation impact on the stability of the complexes.</p><p>To this end, we have used the energy decomposition method (EDM).37,38 Our approach is based on the concept of domains as compact and independent
folding units (i.e., stability cores) and on the analysis of the residue–residue
energy interactions obtainable through classical all-atom calculations.
In particular, starting from the analysis of the residue-based nonbonded
pair-interaction energy matrix associated with a protein, our method
filters out and selects only those specific subsets of interactions
that define possible independent stability cores within a complex
protein structure. This allows grouping different protein fragments
into energy clusters that are found to correspond to the stabilization
cores of domains in specific conformations. EDM is in fact designed
to identify specific regions that contribute the most to the stabilization
of a certain conformational ensemble, through eigenvector decomposition
and simplification of the residue pair-interactions matrix.</p><p>Comparative analysis of EDM matrices for the apo and bound forms
demonstrate that Hsp90 binding reorganizes the energetics of internal
interactions in hAgo2 (Figure S4). Upon
complex formation, we observe a redistribution of the stabilization
nuclei that eventually span various regions of hAgo. New hotspots
(brighter points) of increasing stabilization appear on top of the
PIWI domain. To identify which residues contribute the most to the
new energy pattern in the complex, we analyzed the components of the
eigenvector that recapitulates most of the nonbonded energy (this
eigenvector is the one associated with the first eigenvalue of the
EDM matrix; see Methods). In the Hsp90-bound
conformations, the stabilization energy distribution for hAgo redistributes
from PIWI (that represents the principal stability core in the apo
state at room temperature) to other regions. In particular, in complex 2 (and to a smaller extent in 4) Hsp90 appears
to favor the formation of stability cores in the N, PAZ, and MID domains,
at the expense of the PIWI region (Figure 6). In this view, a more diffuse organization
of stabilizing interactions, induced by the chaperone, may facilitate
access to open hAgo2 states alternative to the closed ones observed
in the apo state. More open conformations ought to be expectedly more
prone to RNA binding.</p><!><p>EDM profile. hAgo2 nonbonded interactions energy along
the sequence
described by the first component. Higher peaks correspond to the regions
that recover most of the stabilization energy. On the x-axis, hAgo2 domains are indicated as colored bars along the following
sequence: N-domain (green), PAZ (orange), MID (gray), and PIWI (yellow).</p><!><p>Biochemical studies demonstrate that small-RNA
loaded hAgo2 is
more stable than empty Ago.17 From our
analyses, the perturbation triggered by RNA binding that involves
mainly PIWI and PAZ domains could significantly impact on the whole
protein stability in a state where the stabilizing nucleus is mainly
located in the PIWI domain. In contrast, chaperone binding may activate
an energy redistribution that results in a more plastic system capable
of functional adaptation in which the interaction with RNA would not
compromise the global stability.</p><!><p>On the basis of these findings, we
next asked whether this energetic modulation could be associated with
specific internal dynamic properties. In order to gain insight into
the intrinsic dynamics and flexibility of the protein, we analyzed
the residue-pair distance fluctuation along the simulation time (see Methods), which allows the identification of patterns
of coordination between physically distant regions.</p><p>The presence
of Hsp90 also reverberates on the internal dynamics of hAgo2. The
yellow areas in Figure S5, associated with
flexible regions, decrease when the chaperone binds the client. In
particular, this event significantly reduces the flexibility of PAZ
domain. This differential modulation becomes clearly evident when
a principal component analysis is performed on hAgo2 CÎ± atoms
in each system. The characterization returns a remarkable difference
of PAZ dynamics for the unbound and complexed Ago systems (see Figure S6). The presence of the chaperone, anchoring
PIWI and N domains, limits their dynamics with respect to the unbound
form, especially for the N domain. This may favor a coordinated motion
of N and PAZ domains, as indicated in the matrix obtained as the difference
between the DF patterns in the unbound and complex states (see Figure S5), where regions identifying protein
residues that increase the dynamical coordination upon Hsp90 binding
are evidenced. It is interesting that also the reciprocal motion of
PAZ and MID domains turns to be affected by the presence of the chaperone.
The internal dynamics analyses suggest indeed that the stabilization
of a more open accessible Ago state is driven by the reduced flexibility
of PAZ domain, that is linked to a decreased flexibility of the N
and more importantly MID domain, where the first event of RNAs anchoring
is known to occur. This result directly links chaperone mechanisms
to hAgo2 RNA binding and loading.</p><p>Taken together, the differential
energetic and dynamics fingerprints
of the protein in the various states were used to guide the definition
of those areas of the interacting surfaces that may be crucial in
the chaperone recognition process. From the analysis of interacting
regions of hAgo2 and Hsp90 in complex 2 and 4, we defined a consensus stretch of amino acids present in both interfaces:
in particular, hAgo2 residues bonded to Hsp90 were selected and analyzed.
Hence, by combining these hotspots on the common interface between
complexes 2 and 4 with oncogenic mutational
mapping from the COSMIC database (https://cancer.sanger.ac.uk/cosmic), we were able to identify specific residues that have a high probability
of modulating hAgo2-Hsp90 interaction, whose mutation results in a
phenotypic functional impact. Specifically, we focused on those amino
acids at the interface that are involved in key contacts (salt bridges
or hydrogen bonds interactions) between the two binding partners and
that played a special role in complex stabilization (see also Figure 6). We identified
as important interactors residues R126 and E186 from the hAgo2 N-domain,
and residues K276, R384, and P392 in the PAZ domain (Figure 7), which are found to correspond
to oncogenic point mutations in the COSMIC database.</p><!><p>hAgo2-Hsp90 complex.
Localization of the computed hotspots (red
spheres) mapped onto hAgo2 in the unbound state (A) and in the complex
with Hsp90 (B). hAgo2 and Hsp90 are displayed in gray and blue cartoons,
respectively. For clarity, only complex 2 is displayed.</p><!><p>To further validate our predictions, we investigated
additional
N-domain hAgo2 mutants that have been proven deficient in RNA-duplex
loading (chaperone-dependent activation) from in vitro RISC assembly experiments.7,8 Thus, we modeled G32A,
L40A, R126A, E186A, K276E, R384E, and P392K mutants and analyzed the
impact of these mutations on protein complex stability (ΔΔG). Two structure-based predictors were used, I mutant (http://folding.biofold.org/i-mutant/i-mutant2.0.html) and SDM (http://marid.bioc.cam.ac.uk/sdm2/), both taking advantage of the large structural information available
on the effect of single point mutations on native protein folds (ProTherm
and Homstrad+Toccata databases, respectively). I mutant is a neural-network-based
approach able to estimate changes in protein stability from either
sequence or structure descriptors, relying on an energy-based FOLD-X
algorithm.39 The second method, SDM, is
based on a statistical potential energy function derived by environment-specific
substitution frequencies tables, providing a stability score prediction
and estimation of disease association.40</p><p>This cross-validation revealed that the two major and opposite
effects concern L40A, as the most destabilizing substitution for both
predictors (ΔΔG = −2.3 kcal mol–1 (I mutant) and −3.9 kcal mol–1 (SDM) on average for the two complexes) and K276E as the least destabilizing
mutant. It is worth underlining that K276E is predicted to slightly
stabilize the complex (SDM) or very unlike to occur (as indicated
by a specific reliability score provided by I mutant).39,40 The remaining mutations exhibit an intermediate behavior between
these two cases, yet displaying some destabilization effect. Protein
stability changes upon mutations are given in Table
S1: normalized ΔΔG values calculated
by the two servers are given to improve comparability and point out
relations between the two (Figure S7). SDM
and I mutant showed a good correlation in the predictions of energy
changes, with R2 = 0.82 and R2 = 0.9 for complexes 2 and 4, respectively: indeed, mutations on complexes 2 and 4 yielded to very comparable trends, whereby E186A and R384E,
localized at the hAgo2 L1 and L2 linkers respectively, turned out
as the more difficult point mutations to predict, likely because of
their interfacial position.</p><p>In light of these findings, all
the designed mutations seem to
alter the complex stability except K276E. This result can be explained
by the high solvent accessibility in this position: the smaller impact
on the complex could rely on the enhanced conformational freedom of
the side-chain.</p><p>Summarizing, the detailed analyses of the complex
interfaces have
highlighted critical points of interaction between the two binding
partners. Consistently, one of the most destabilizing mutations predicted,
G32A mutant, showed a large defect in loading the siRNA duplex, lower
for microRNA duplex, supporting recent observation that the more flexible
RNA duplex (mismatch-containing microRNA) is slightly less dependent
on the Hsp90 ATPase activities compared to the perfectly complementary
and more rigid siRNA duplex, which would require a complete/functional
hAgo2 opening. This picture provides a structural basis to the effect
of the G32A mutant, which can be reconnected to a defect in the chaperone-dependent
activation.8,24</p><p>At this stage and based
on the limited knowledge concerning the
hAgo2-Hsp90 complex, though we cannot rule out the possibility of
a different structural rearrangement between the two proteins, these
data seem to corroborate the reliability of the designed model interface:
additional support to our comparative approach may be seen in the
chaperone mutational profile reported by the COSMIC database, where
Hsp90 amino acids involved in hAgo2 key interactions show a high degree
of mutability (i.e., Y56C, N101Y, T110A, H149Y, D151N, H171Y, D228N,
and E232K).</p><!><p>By combining protein–protein
docking techniques, extensive
MD simulations, and novel methods of analysis of protein conformational
stability, we have developed a structural model of the chaperone-assisted
RISC assembly that proves able to recapitulate the salient traits
of dynamics and interactions underlying complex formation. It is important
to stress that the simulations and analyses of internal energetics
discussed here have been based on the use of one specific version
of the Amber force field.41 Recent studies
have underlined the dependence of dynamic features in NMR ensembles
of protein conformations. While it would be desirable to test the
models of the large complexes described here with different force
fields, it is important to underline that our calculations show a
clearly differentiated dynamics of hAgo2 in the absence or presence
of Hsp90. In this context, it is important to underline that the models
are able to unveil hotspots whose functional relevance had already
been proven experimentally. Being corroborated by independent experimental
findings, our results provide an atomic-resolution mechanistic view
on the roles of Hsp90-hAgo2 binding in RNA loading. This model demonstrates
that the presence of the chaperone is able to modulate the conformational
plasticity of hAgo2 favoring its opening. Such motion is compatible
with a state of the protein primed to load its substrate, thus capable
to bind RNAs. Moreover, our model demonstrates that Hsp90 binding
modifies the energetic patterns of hAgo2: we could identify the regions
of the interacting surfaces essential for a stable interaction. In
this framework, we defined single-point mutations that could potentially
compromise/weaken the recognition mechanism. These results pinpoint
a key role for Hsp90 in RNA loading.</p><p>Recent advances in Ago2
conformational activation in Drosophila showed a
coordinated mechanism triggered the Hsp70/Hsp90 chaperone
machinery. By smFRET experiments, Tsuboyama et al.,18 underscored the pivotal role of Hsp70 in priming the opening
of fly Ago2 with the subsequent recruitment of Hsp90, which is required
to intercept and stabilize the open and effector form of Ago2 to assemble
functional RISC. An equivalent function has been recently clarified
for Hsp90 in the cryoEM structure of the Hsp90-Cdc37-Cdk4 complex,
where the chaperone binds and stabilizes the kinase domain in a more
open shape.42,43 Furthermore, in a previous work,
we carried out an in-depth analysis of internal energy fingerprint
of several kinase domain that concurred to reconcile the Hps90 ability
to bind specific kinase proteins with client thermostability.21,44,45</p><p>From a structural perspective,
our selected hAgo2-Hsp90 models
(complexes 2 and 4) display interacting
surfaces that significantly overlap with the binding mode that Hsp90
shows with another client.</p><p>The comparison of our predicted models
with the Hsp90-Sgt1 complex
(PDB 2JKI),
reveals indeed that both the clients engage Hsp90 N-terminal domain
through very similar interaction sites (Figure
S8B). In contrast, a different positioning is observed for
the client kinase in Hsp90-Cdc37-Cdk4 complex, even if the key anchoring
role of the N-domain of the chaperone in the recruitment of the cochaperone-client
is maintained (Figure S8C,D). The differences
in the interaction sites of Hsp90 are in line with its chaperone functionality
and the broad set of different client proteins that Hsp90 binds and
stabilizes. Nevertheless, our data suggest that key interactions on
the N–domain could be conserved among different binding modes
in different complexes.</p><p>Structures of prokaryotic Argonaute
complexes show a highly flexible
N-domain able to move freely with respect to the rest of the protein
depending on the size of the RNA duplex bound within the central cleft.7,8 Furthermore, it has been proposed that this structural element might
be conserved, given that a fully complementary duplex modeled onto
human Ago2 structures would show a similar clash.3</p><p>Altogether, our data demonstrate that the stabilization
of a more
open hAgo2 conformation is fundamentally promoted by chaperone binding:
Hsp90 could induce structural rearrangements to open hAgo2 cleft to
accommodate RNA duplex or select and bind states that are only minimally
present in the unbound hAgo2 conformational ensemble. Consistent with
this, Jiang and collaborators identified a metastable open state of
hAgo2, able to accommodate microRNA, in rapid equilibrium with other
states.46</p><p>Our results reinforce the
hypothesis that hAgo2 recruitment operated
by the chaperone may occur at the very first stage of RISC cycle.
In Drosophila, Hsp70 is dedicated to initiate the
conformational opening of Ago whereas Hsp90 stabilizes the opened
form; Hsp90 alone was unable to produce the active, open form of Ago.
Our simulations of apo-hAgo2 carried out at different temperatures,
and subsequently used to build the interacting complex with Hsp90,
were designed to expand the structural ensemble of Argonaute in its
unbound state and, therefore, to mimic the effect of external cofactors,
such as the cochaperone Hsp70.18 Our interaction
model reconciles the coexistence of several hAgo2 states from which
the chaperone system selects and stabilizes the active state. In this
light, molecular dynamics simulations highlight the large conformational
variability displayed by hAgo2 in the unbound form compared to the
complex, thus providing a structural hypothesis for the conformational
organization of the two proteins in the complexed state. Furthermore,
our studies allowed us to characterize the energetic effect driven
by the interaction with Hsp90 and to suggest mutants that can be tested
experimentally.</p><p>Well-tempered regulation of small RNA levels
is critical for diverse
biological processes in various organisms. A better understanding
of the sequential dynamic conformational changes of interacting partners
during RISC assembly can ultimately illuminate the molecular bases
of disease mechanism and could pave the way for new strategies to
tune AGO proteins toward gene silencing tools and RNAi therapeutics.</p><!><p>The starting X-ray model of hAgo2 was retrieved
from the Protein Data Bank with access number 4Z4C, removing target
RNA. Full-length hAgo2 is made by 838 residues, namely N-domain (aa.
36–166), L1 (aa. 176–226), PAZ (aa. 231–365),
L2 (aa. 374–420), MID (aa. 429–511) and PIWI (aa. 496–797).
Protein refinements were carried out using Maestro (Schrödinger
Release 2016-4, LLC, New York, NY).</p><p>The MD simulation package
Amber v1247 was used to perform computer
simulation by applying the Amber-ff99SB force field.48 The systems were solvated, in a simulation box of explicit
water molecules (TIP3P model),49 counterions
were added to neutralize the system, and periodic boundary conditions
were imposed in the three dimensions. After minimizations, systems
were subjected to an equilibration phase where water molecules and
protein heavy atoms were position restrained, and then, unrestrained
systems were simulated for a total of 8 Î¼s, in a NPT ensemble; a Langevin equilibration scheme and a Berendsen thermostat
were used to keep constant temperature and pressure (1 atm), respectively.
Electrostatic forces were evaluated by the particle mesh Ewald method50 and Lennard-Jones forces by a cutoff of 8 Å.
All bonds involving hydrogen atoms were constrained using the SHAKE
algorithm.51</p><p>An ionic strength of
0.150 M NaCl was reproduced based on experimental
settings.</p><p>To enhance sampling independent replicas of 500 ns
(3 × 0.5
Î¼s at 300 K, 0.5 Î¼s at 350 K, and 0.5 Î¼s at 400
K Î¼s) were run for each system with different initial velocities
at 300, 350, and 400 K temperatures.</p><p>The same MD settings were
applied to the simulations of hAgo2-Hsp90
complexes and run on Acellera (ACEMD). Two independent replicas of
500 ns were run for each complex (4 complexes × 0.5 × 2
= 4 Î¼s).</p><p>MD analyses were carried out on a meta-trajectory
(heavy atoms),
obtained by concatenating all the trajectories of the apo form of
hAgo2 at different temperatures and the hAgo2 from hAgo2-Hsp90 complexes.</p><p>Figures are created using VMD, Pymol, and Chimera.52−54</p><!><p>Docking
experiments
were carried out using the refined Hsp90 structure from the cryoEM
complex (5FWK pdb access code43) and different RNA-free
hAgo2 structures (X-ray structure, PDB code 4Z4C,30 and two representative structures of the open and closed
form from MD simulations) in order to consider the large structural
variability displayed by the hAgo2 apo form. ClusPro (https://cluspro.org),55 Zdock(https://zlab.umassmed.edu/zdock/),56 and PatchDock (https://bioinfo3d.cs.tau.ac.il/PatchDocK/),57 were used as protein–protein docking algorithms
to model the interaction in hAgo2-Hsp90. The three docking softwares
predict protein–protein interactions by means of rigid-body
docking using the structural information derived from the two interacting
partners individually. No prior information about the binding site
and/or additional energy options were added in our search. Different
scoring functions are implemented in each of the docking algorithm
to rank low-energy docked complexes. Docking predictions were performed
either constraining or not the interaction at the N/M domains of the
chaperone, based on literature data.9,15,16 A total of 180 complexes were generated, filtered
according to the software scoring function, and therefore subjected
to careful structural analyses and considerations. In particular,
a cluster analysis on backbone atoms (cutoff: rmsd 3 nm) was applied
to the full set of predicted complexes and a visual inspection guided
the screening of the most representative complexes. Significantly,
from a statistical perspective, 29% of the 180 docked complexes presented
a preferred interaction surface localized at the N-domain level of
the Argonaute protein, in line with experimental evidence.8 In the end, four hAgo2-Hsp90 complexes were selected
for molecular dynamics simulations as reported above.</p><!><p>To extract functionally
relevant movements, a principal component analysis (PCA) has been
applied to MD simulations in order to filter global, collective motions
from local, fast motions.58 The concerted
motions associated with the largest collective atomic fluctuations
(i.e., that account for the largest contribution to the atomic root
mean deviations) are recovered by the principal eigenvectors (essential
modes) of the covariance matrix of the given dynamic ensemble. PCA
analysis was carried out on CÎ± atoms of hAgo2 protein along
the meta-trajectory. A total of 5000 snapshots per trajectory per
system are projected on the essential subspace described by the first
two eigenvectors responsible for the maximum variation in conformation
observed along molecular simulation. hAgo2 simulation in its apo state
is used as reference and the conformational space sampled by hAgo2
bound to Hsp90 in the four complexes is projected onto apo-hAgo2 essential
subspace. Therefore, the comparison among the conformational space
spanned by hAgo2 in the apo state and in different interaction complexes
can be evaluated. The two first eigenvectors account for the 55% and
12% of the total variance of the simulation.</p><!><p>The energy decomposition
method (EDM)37,38 yields an interaction matrix Mij, obtained by averaging the
interaction energies between residue pairs, comprising all the nonbonded
inter-residue atomic energy components (namely, van der Waals and
electrostatic), calculated over the structures visited during a MD
trajectory or the representative conformation of the most populated
cluster. The method builds on a simplified picture of the most relevant
residue–residue interactions in a certain fold. For a protein
of N residues, this calculation produces an N × N matrix. The matrix Mij can be diagonalized and re-expressed
in terms of eigenvalues and eigenvectors, in the formwhere N is the number of
amino acids in the protein, Î»k is an eigenvalue, and wik is the ith component of the associated normalized
eigenvector. The matrix of the pair energy-couplings corresponding
to the first eigenvector is chosen to filter the contact map and identify
all inter-residues low-energy contributions.</p><!><p>Distance fluctuation
DFji is defined as the time-dependent
mean square fluctuation of the distance rij between CÎ± atoms of residues i and j:where brackets indicate the time-average
over
the trajectory. Low DF values indicate highly coordinated residues.32</p><!><p>Distance length
evolution between the two hAgo2 subdomains centroids were analyzed
by the VMD tools package.52 Centroids were
defined as the center of mass of PIWI (amino acids 496–797)
and PAZ domains (amino acids 231–365). The distance between
the two obtained geometric centers was calculated along simulation
time for complexes 2 and 4.</p><!><p>Figure S1, 3D structure
of h human argonaute bound to
target RNA; Figure S2, selected docking poses for hAgo2-Hsp90 complexes
in different orientations; Figure S3, statistical distribution of
the rotational angle described by PIWI and PAZ domains along the simulation
time; Figure S4, nonbonded pair-interactions energies of hAgo2 in
the apo form (at 300 K) and in complex 4; Figure S5,
distance fluctuations analysis of the hAgo2 in the apo form (at 300
K) and in complex 4; Figure S6, extreme projections
on principal modes derived from ED analysis in the unbound protein
at 300 K and in complex 4; Figure S7, effect of mutations
on protein stability; Figure S8, structural comparison among Hsp90-client
complexes; and Table S1, changes in protein stability upon mutations
(PDF)</p><p>Coordinates
of hAgo2-Hsp90_1 (PDB)</p><p>Coordinates of hAgo2-Hsp90_2 (PDB)</p><p>Coordinates of hAgo2-Hsp90_3 (PDB)</p><p>Coordinates
of hAgo2-Hsp90_4 (PDB)</p><p>ci0c00053_si_001.pdf</p><p>ci0c00053_si_002.pdb</p><p>ci0c00053_si_003.pdb</p><p>ci0c00053_si_004.pdb</p><p>ci0c00053_si_005.pdb</p><p>The authors
declare no
competing financial interest.</p>