Allosteric Regulation Of the Hsp90 Dynamics and Stability By Client Recruiter Cochaperones: Protein Structure Network Modeling

The fundamental role of the Hsp90 chaperone in supporting functional activity of diverse protein clients is anchored by specific cochaperones. A family of immune sensing client proteins is delivered to the Hsp90 system with the aid of cochaperones Sgt1 and Rar1 that act cooperatively with Hsp90 to form allosterically regulated dynamic complexes. In this work, functional dynamics and protein structure network modeling are combined to dissect molecular mechanisms of Hsp90 regulation by the client recruiter cochaperones. Dynamic signatures of the Hsp90-cochaperone complexes are manifested in differential modulation of the conformational mobility in the Hsp90 lid motif. Consistent with the experiments, we have determined that targeted reorganization of the lid dynamics is a unifying characteristic of the client recruiter cochaperones. Protein network analysis of the essential conformational space of the Hsp90-cochaperone motions has identified structurally stable interaction communities, interfacial hubs and key mediating residues of allosteric communication pathways that act concertedly with the shifts in conformational equilibrium. The results have shown that client recruiter cochaperones can orchestrate global changes in the dynamics and stability of the interaction networks that could enhance the ATPase activity and assist in the client recruitment. The network analysis has recapitulated a broad range of structural and mutagenesis experiments, particularly clarifying the elusive role of Rar1 as a regulator of the Hsp90 interactions and a stability enhancer of the Hsp90-cochaperone complexes. Small-world organization of the interaction networks in the Hsp90 regulatory complexes gives rise to a strong correspondence between highly connected local interfacial hubs, global mediator residues of allosteric interactions and key functional hot spots of the Hsp90 activity. We have found that cochaperone-induced conformational changes in Hsp90 may be determined by specific interaction networks that can inhibit or promote progression of the ATPase cycle and thus control the recruitment of client proteins

Differential Modulation Of Functional Dynamics and Allosteric Interactions In the Hsp90-Cochaperone Complexes With P23 and Aha1: A Computational Study

Allosteric interactions of the molecular chaperone Hsp90 with a large cohort of cochaperones and client proteins allow for molecular communication and event coupling in signal transduction networks. The integration of cochaperones into the Hsp90 system is driven by the regulatory mechanisms that modulate the progression of the ATPase cycle and control the recruitment of the Hsp90 clientele. In this work, we report the results of computational modeling of allosteric regulation in the Hsp90 complexes with the cochaperones p23 and Aha1. By integrating protein docking, biophysical simulations, modeling of allosteric communications, protein structure network analysis and the energy landscape theory we have investigated dynamics and stability of the Hsp90-p23 and Hsp90-Aha1 interactions in direct comparison with the extensive body of structural and functional experiments. The results have revealed that functional dynamics and allosteric interactions of Hsp90 can be selectively modulated by these cochaperones via specific targeting of the regulatory hinge regions that could restrict collective motions and stabilize specific chaperone conformations. The protein structure network parameters have quantified the effects of cochaperones on conformational stability of the Hsp90 complexes and identified dynamically stable communities of residues that can contribute to the strengthening of allosteric interactions. According to our results, p23-mediated changes in the Hsp90 interactions may provide molecular brakes\u27\u27 that could slow down an efficient transmission of the inter-domain allosteric signals, consistent with the functional role of p23 in partially inhibiting the ATPase cycle. Unlike p23, Aha1-mediated acceleration of the Hsp90-ATPase cycle may be achieved via modulation of the equilibrium motions that facilitate allosteric changes favoring a closed dimerized form of Hsp90. The results of our study have shown that Aha1 and p23 can modulate the Hsp90-ATPase activity and direct the chaperone cycle by exerting the precise control over structural stability, global movements and allosteric communications in Hsp90

The Degree Distribution of Residue Hubs in the Hsp90 Structures.

<p>The number of hub nodes as a function of the degree of a hub is shown for the solution HtpG structure (in blue); the apo-HtpG structure (in red); the ADP-HtpG structure (in green); the ADP-Grp94 structure (in maroon); the ATP-Grp94 structure (in orange); and the yeast ATP-Hsp90 structure (in brown). The coloring annotation of the distribution is consistent with the annotation of residue hubs in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003679#pcbi-1003679-g007" target="_blank">Figure 7</a>.</p

The Allosterically Communicating Residues in the Hsp90-Aha1 Complex.

<p>The functional dynamics profiles of the Hsp90-Aha1 complexes with the Aha1-N domain (A) and the complete Aha1 molecule (B). A ribbon-based representation of the Aha1 domains is combined with a surface view of the Hsp90 dimer. The color gradient from blue to red indicates the decreasing structural rigidity of protein residues as in previous figures. (C) The distribution of allosterically in the context of the functional dynamics profile. The location and identities of allosterically communicating residues are annotated in spheres colored according to their respective mobility level. The crystal structure of yeast Hsp90 is enveloped in a 50% transparent molecular surface to show the location of the allosterically communicating residues. The three-helix bundle that links the inter-domain regions is shown in ribbon representation.</p

The Network Analysis of the Hsp90 Crystal Structures.

<p>The distribution of hubs (A) and communities (B) in different functional states of Hsp90. The distributions of protein structure network parameters are obtained by averaging computations over MD simulation trajectories. The analysis is based on structurally stable residue interaction networks that remained intact in more than 75% of the simulation snapshots. The distributions are shown for the open solution conformation of HtpG obtained from SAXS studies <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003679#pcbi.1003679-Krukenberg2" target="_blank">[57]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003679#pcbi.1003679-Krukenberg3" target="_blank">[58]</a> (in blue); an apo form of HtpG (PDB ID 2IOQ) <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003679#pcbi.1003679-Shiau1" target="_blank">[56]</a> (in red); an ADP-bound form of HtpG (PDB ID 2IOP) <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003679#pcbi.1003679-Shiau1" target="_blank">[56]</a> (in green); an ADP-bound form of the Grp94 homologue (PDB ID 2O1V) <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003679#pcbi.1003679-Dollins1" target="_blank">[61]</a> (in maroon); an ATP-bound form of the Grp94 homologue (PDB ID 2O1U) <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003679#pcbi.1003679-Dollins1" target="_blank">[61]</a> (in orange); and an ATP-bound conformation of yeast Hsp90 (PDB ID 2CG9) <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003679#pcbi.1003679-Ali1" target="_blank">[55]</a> (in brown).</p

A Model of the Hsp90-ATPase Cycle: The Functional Role of the Cochaperones Sgt1 and Rar1.

<p>In this model, ATP binding to the Hsp90-NTD of apo-Hsp90 in the open form can induce a fast dynamic exchange between a nucleotide-free Hsp90 and an ATP-bound state in which the ATP lids and Hsp90-NTDs are still open. Binding of Rar1-CHORD1 to the Hsp90-NTD intersects the normal progression of the ATPase cycle by preventing the lid enclosure of ATP and inhibiting the formation of the closed Hsp90 dimer. This interaction supports the binding of Rar1-CHORD2 to the other Hsp90-NTD in the other protomer. Upon binding of both Rar1-CHORD domains, SGT1 is recruited to interact with the Hsp90-NTD and Rar1-CHORD2 domain. Cochaperone-mediated arrest of the Hsp90-ATPase conformational cycle in the open form promotes the assembly of the ternary Hsp90-Sgt1-Rar1 complex and recruitment of the NLR clients. In the ternary complex, the fluctuations of the lid segment may allow the catalytic Arg residue from the Hsp90-MD to reach the nucleotide binding site and induce ATP hydrolysis. The Rar1-stimulated hydrolysis of ATP would lead to dissociation of RAR1, SGT1, and NLR client from the Hsp90 dimer. After ATP is hydrolyzed, the Hsp90-NTDs domains dissociate and ADP is released returning Hsp90 to the nucleotide-free open state. The Hsp90 structure is shown in a surface representation with an annotation of structural elements. The Hsp90-NTD is shown in green; the Hsp90-MD is depicted in blue and the Hsp90-CTD is presented in red.</p

Functional Dynamics and Conformational Mobility Profiles of the ATP-bound Hsp90 Dimer and the Hsp90-p23 Complex.

<p>Structural distribution of conformational mobility in the ATP-bound form of yeast Hsp90 (A) and the Hsp90-p23 complex (B) was averaged over three lowest frequency modes obtained from the functional dynamics analysis. A close-up view of the protein mobility profiles for a panel of Ile residues probed in the Ile-targeted NMR experiments of the Hsp90-p23 complex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071936#pone.0071936-Karagz1" target="_blank">[56]</a>. These residues were mapped onto the crystal structure of yeast Hsp90 (PDB ID 2CG9) and depicted in colored spheres according to their mobility. A surface-based protein representation is employed. The color gradient from blue to red indicates the decreasing structural rigidity (or increasing conformational mobility) of protein residues. The numbering of the Ile-probed residues corresponds to the crystal structure of yeast Hsp90. The highlighted Ile residues in yeast Hsp90 (I12, I19, I20, I45, I66, I82, I358, I388, V429, and I471) correspond to the Ile-probed residues in human Hsp90β (I20, I27, I28, I53, I75, I90, I369, I399, I440, and I482 respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071936#pone.0071936-Karagz1" target="_blank">[56]</a>). The three-helix bundle that links the inter-domain N-M and M-C regions and coordinates motions of the regulatory hinges is shown in ribbon representation. The unrelated obstructing features in the foreground were omitted for clarity. The Pymol program was used for visualization of the protein structures (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, and LLC).</p

The Structure of the Full-Length Hsp90 Dimer.

<p>The homodimer architecture of the full-length Hsp9p0 dimer is illustrated by the crystal structure of a closed ATP-bound conformation of yeast Hsp90 dimer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071936#pone.0071936-Ali1" target="_blank">[38]</a>. The structure is shown in a ribbon representation with a detailed annotation of structural elements (left panel) and in a general surface-based protein representation (right panel). The Hsp90-N domain is shown in green; the Hsp90-M domain is depicted in blue and the Hsp90-C domain is presented in red. Left Panel: The ATP lid residues 95–123 are shown in black (95-TIAKSGTKAFMEALSAGADVSMIGQFGVG-123). The catalytic loop residues (371-SEDLPLNLSREMLQQ-385) are shown in cyan with a key catalytic residue R-380 highlighted in cyan sticks. ATP molecule is colored by atoms and is shown in sticks. The three-helix bundle shown in blue ribbons (helix 1: residues 386–408; helix 2: residues 412–431; helix3: residues 435–442) links the inter-domain regions and the regulatory hinges. The N-M inter-domain hinge residues M382 and L383 are shown in cyan spheres; the M-C inter-domain hinge residues E402 and E406 are shown blue spheres. The cyan color of M382/L383 is according to the coloration of the catalytic loop (residues 371–385). The blue color of E402/E406 is according to the coloration of the Hsp90-M domain. In the original crystal structure of yeast Hsp90 the domain annotation for the monomer 1 is: Hsp90-N (residues 2–216), Hsp90-M (residues 262–329, 339–526) and Hsp90-C (residues 527–597, 611–677). In the monomer 2 Hsp90-N (residues 2–216), Hsp90-M (residues 262–526) and Hsp90-C (527–597, 611–677). The structurally unresolved residues are 217–261, 330–338 (only in the first monomer), and 598–610. The Pymol program was used for visualization of Hsp90 structures (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, and LLC).</p

Conformational Mobility Profiling of the Hsp90-Cdc37 Complex.

<p>The NMSF profile of the Hsp90-Cdc37 complex (A) was obtained using the NMR structure (PDB ID 2K5B) of the complex between human Cdc37 (Cdc37-MD, original residue numbering 148–276) and human Hsp90-NTD (original residue numbering 14–223) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086547#pone.0086547-Sreeramulu1" target="_blank">[25]</a>. The residue-based NMSF values were computed by averaging the fluctuations over 20 low frequency modes. The NMSF profile for the Hsp90-NTD residues is shown in blue lines and for the Cdc37-MD residues in red lines. The consecutive residue numbering of the Hsp90-NTD and Cdc37-MD residues is adopted. The original numbering of the Hsp90-NTD (residues 14–223) in the NMR structure corresponds to residues 1–210, and the original numbering of the Cdc37-MD (residues 148–276) corresponds respectively to residues 211–339. (B) The fluctuation distance force constant profile of the Hsp90-Cdc37 complex. The profile is shown in blue lines for the Hsp90-NTD and in red lines for the Cdc37-MD. The consecutive residue numbering is adopted and is consistent with the annotation in (A). The position of the lid motif and the peaks corresponding to functionally important residues are indicated by arrows and annotated. The position of the lid motif (residues 108–138) is highlighted and pointed to by oval circles surrounding the lid. (C) Structural mapping of the conformational mobility in the essential conformational space of the three lowest frequency modes. A surface-based protein representation is employed. The color gradient from blue to red indicates the decreasing structural rigidity of protein residues. The interfacial residues are shown colored spheres according to their mobility. The important functional residues are annotated according to their original crystallographic numbering. (D) The Hsp90-Cdc37 structure is annotated according to the adopted domain coloring. The lid motif in both structures is highlighted and colored in gold; the anchoring residues with the high force constants are indicated.</p

Conformational Dynamics of the Hsp90 Crystal Structures.

<p>The computed B-factors are obtained from MD simulations of apo-HtpG <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003679#pcbi.1003679-Shiau1" target="_blank">[56]</a> (PDB ID 2IOQ) (A); ADP-bound HtpG <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003679#pcbi.1003679-Shiau1" target="_blank">[56]</a> (PDB ID 2IOP) (B); ATP-bound yeast Hsp90 <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003679#pcbi.1003679-Ali1" target="_blank">[55]</a> (PDB ID 2CG9) (C); and ATP-bound Grp94 <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003679#pcbi.1003679-Dollins1" target="_blank">[61]</a> (PDB ID 2O1U) (D). The NTD residues are shown in green, MD residues are in blue, and CTD residues are in red. The residue-based profiles are based on the consecutive residue numbering adopted from the original crystallographic residue annotation. For clarity, the equilibrium profiles are shown only for one monomer of the homodimer. (A) The fluctuation profile of the apo-HtpG crystal structure has the following residue annotation: NTD (residues 1–219); MD (residues 220–474); CTD (residues 475–577). (B) The profile of the ADP-HtpG crystal structure has the following residue annotation: NTD (residues 1–231); MD (residues 232–486); CTD (residues 487–618). (C) The force constant profile of the yeast ATP-Hsp90 has the following residue annotation: NTD (residues 1–215); MD (residues 216–471); CTD (residues 472–609). (D) The force constant profile of the ATP-Grp94 crystal structure has the following residue annotation: NTD (residues 1–179); MD (residues 180–427); CTD (residues 428–573).</p