Heat shock protein 90 regulates soluble guanylyl cyclase maturation by a dual mechanism

The enzyme soluble guanylyl cyclase (sGC) is a heterodimer composed of an α subunit and a heme-containing β subunit. It participates in signaling by generating cGMP in response to nitric oxide (NO). Heme insertion into the β1 subunit of sGC (sGCβ) is critical for function, and heat shock protein 90 (HSP90) associates with heme-free sGCβ (apo-sGCβ) to drive its heme insertion. Here, we tested the accuracy and relevance of a modeled apo-sGCβ–HSP90 complex by constructing sGCβ variants predicted to have an impaired interaction with HSP90. Using site-directed mutagenesis, purified recombinant proteins, mammalian cell expression, and fluorescence approaches, we found that (i) three regions in apo-sGCβ predicted by the model mediate direct complex formation with HSP90 both in vitro and in mammalian cells; (ii) such HSP90 complex formation directly correlates with the extent of heme insertion into apo-sGCβ and with cyclase activity; and (iii) apo-sGCβ mutants possessing an HSP90-binding defect instead bind to sGCα in cells and form inactive, heme-free sGC heterodimers. Our findings uncover the molecular features of the cellular apo-sGCβ–HSP90 complex and reveal its dual importance in enabling heme insertion while preventing inactive heterodimer formation during sGC maturation.

The enzyme soluble guanylyl cyclase (sGC, 3 EC 4.6.1.2) participates in numerous biologic signaling cascades by generating cGMP in response to NO (1)(2)(3). Disruption of this pathway contributes to a wide spectrum of pulmonary and cardiovascular diseases (4 -10). Mature, active sGC is a heterodimer com-posed of an ␣-subunit and a ␤-subunit (11)(12)(13). Both subunits are modular and contain an N-terminal H-NOX domain, a middle Per-Arnt-Sim like domain (PAS domain), a coiledcoil domain (CC domain), and a C-terminal catalytic cyclase domain (11)(12)(13). The H-NOX domain of sGC␤, but not that of sGC␣, contains a binding site for iron protoporphyrin IX (heme) (14,15). When loaded onto sGC␤, the heme functions to bind NO and as a nexus for consequent protein structural changes that activate catalysis in the sGC heterodimer (1)(2)(3).
Whereas sGC has been studied extensively for decades, relatively little is known about the processes that form mature, functional sGC␣/␤ heterodimers in mammalian cells. Several studies established that the chaperone heat shock protein 90 (HSP90) plays an important role in sGC maturation and function (6, 16 -18). Antibody pulldown studies first showed that sGC associates with HSP90 in cells, although the reasons for this association were not clear (19 -21). More recent studies showed that HSP90 only associates with the heme-free (apo) form of the sGC␤ subunit (17), thereby helping to drive heme insertion into apo-sGC␤ in an ATP-dependent process (16,17). Once the sGC␤ subunit acquires heme, it dissociates from HSP90 and associates instead with an sGC␣ subunit partner to form a mature sGC␣/␤ heterodimer (16,17). Although clarifying, these cell-based studies did not define the nature of the HSP90-apo-sGC␤ association and thus provided no molecularlevel information.
Our subsequent study with purified apo-sGC␤ and HSP90 proteins revealed that they directly form a complex with low micromolar affinity, in which the middle domain of HSP90 interacts with the PAS domain of apo-sGC␤ (22). This information allowed us to build an energy-minimized structural model of a directly interacting complex (18,22). However, this complex model has not been tested with regard to its accuracy, its formation in cells, or its relevance for heme insertion into apo-sGC␤. To address these gaps, we generated a series of sGC␤ variants that contain model-based point and deletion mutations designed to disrupt the apo-sGC␤-HSP90 interaction. We investigated the ability of each variant to interact with HSP90 both in vitro and in mammalian cells, to undergo heme insertion, to form a heterodimer with sGC␣, and to display cyclase activity. Our findings show that our model for the HSP90 -apo-sGC␤ complex is valid, that the complex forms in live cells, and that HSP90 both drives heme insertion into apo-sGC␤ and prevents premature association of the sGC␣ subunit during the heme insertion process. This improves our molecu-This work was supported by National Institutes of Health Grants P01HL081064 and P01HL103453 (to D. J. S.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Table S1 and Figs. S1-S5. 1  cro ARTICLE lar-level understanding of sGC heterodimer maturation and further defines the key roles played by the HSP90 chaperone.

Generation and general properties of sGC␤ mutant proteins
The deletions and amino acid substitutions that we incorporated into our bovine sGC␤1(1-358) bacterial expression construct are listed in Table S1. All of the sequence changes were located in the C-terminal region that contains the PAS domain, a downstream linker element, and an N-terminal portion of the CC domain (Fig. 1). The deletions and substitutions cluster into three regions. We hypothesized that these regions may interact directly with HSP90, based on our previous hydrogendeuterium exchange MS study of HSP90 -sGC␤ interaction (22) (regions I and III) or on the model that we built of the HSP90 -sGC␤ complex (22) (region II).
All of the His 6 -tagged sGC␤1(1-358) constructs that were generated, including WT, expressed well in Escherichia coli. Following purification, the recombinant proteins were predominantly heme-free, with residual heme content ranging from 0.03 to 0.5 mol of heme/mol of protein (Fig. S1). Adding heme to the purified proteins followed by filtration through a desalting column increased their heme contents, ranging from 0.64 to 0.99 mol of heme/mol of protein. Each hemereconstituted protein exhibited a predominant Soret peak at 431 nm (Fig. S1) and visible spectral features corresponding to predominantly ferrous and five-coordinate heme, as is present in native sGC (23,24). This shows that none of the bacterially expressed and purified apo-sGC␤1(1-358) proteins had a significantly compromised or altered heme binding, consistent with the mutations being located outside of the heme-binding domain. The H-NOX, PAS, and PAS-linker domains of sGC␤ are colored white or teal, with teal indicating regions reported to become protected upon HSP90 binding. The HSP90 homodimer is colored yellow with the N-terminal (N), middle (M), and C-terminal (C) domains of one protomer indicated. B, view of the sGC␤-PAS (white and teal) and HSP90 M (yellow) interacting region. The red highlighted residues are those mutated in the current study. C, portion of the primary sequence and secondary structure of sGC␤ that we targeted for mutagenesis. Dark blue rectangles, sequences that become protected by HSP90 complex formation. The three regions (indicated by Roman numerals) and residues within them targeted for deletion or point mutation are indicated.

Effect of mutations on apo-sGC␤ association with HSP90
We utilized fluorescence polarization to monitor the binding of purified HSP90 with each purified WT or mutant apo-sGC␤1(1-358) protein (22). Fig. 2 shows residual fluorescence polarization traces for each FITC-labeled apo-sGC␤1(1-358) protein upon titration with increasing concentrations of HSP90. The proteins displayed a range of HSP90 interaction capabilities, with R335S/R336S/D342S showing saturation binding and an estimated affinity as good as or slightly better than WT. Two other mutants (L269D/I272S/V275D and H266A/H271A/I272D) showed saturation binding but somewhat poorer estimated affinities, and the other five mutants had HSP90-binding affinities that were either significantly diminished (E291A/E295A/L296D) or undetectable by this method. To exclude labeling artifacts, we repeated the binding experiment using FITC-labeled HSP90 and unlabeled apo-sGC␤1(1-358) proteins. The results were similar and are shown in Fig. S2. Thus, amino acid residues within each of the three proposed binding regions in apo-sGC␤1(1-358) influence its affinity toward HSP90 in the purified protein system. We next introduced the same mutations into a mammalian expression vector that encoded full-length rat sGC␤1(1-619) (17). We transiently expressed each construct in COS-7 cells under heme-deficient conditions, which support the maximum levels of apo-sGC␤-HSP90 interaction (17). We analyzed cell supernatants to assess the expression level and degree of HSP90 association of each mutant. All of the sGC␤ proteins showed similar expression levels but displayed different degrees of HSP90 association (Fig. 3). In general, the rank order of the in-cell HSP90 associations mimicked the HSP90-binding behaviors that we observed for the purified sGC␤ mutant proteins as described in Fig. 2. The good correlation is consistent with apo-sGC␤ directly associating with HSP90 in the cells through an interaction that involves an interface similar to the one depicted in our structural model of the apo-sGC␤-HSP90 complex.

HSP90 interaction is needed for heme insertion into apo-sGC␤ in cells
To assess how HSP90 binding correlates with sGC␤1 heme insertion in live cells, we utilized a construct of rat sGC␤1(1-619) originally developed by Hoffmann et al. (25), which incorporates a tetra-Cys motif (TC) at a specific location near the sGC␤ heme-binding site. Once the TC motif binds the indicator dye FlAsH (FlAsH-TC-sGC␤), it becomes fluorescent and in turn undergoes quenching when heme binds. Such FlAsH-TC-sGC␤ constructs have been used to study heme content and conformational changes in sGC␤ (25,26).
To validate the method, we expressed WT TC-sGC␤1(1-619) and either of two mutant constructs that are defective in heme-binding, TC-sGC␤1(1-619) H105F (17,27) and TC-sGC␤1(1-619) Y135A/R139A (27), in heme-deficient COS-7 cells. We bound FlAsH to their TC motifs and determined how heme addition to the cells impacted their FlAsH fluorescence. Fig. 4 shows that adding heme to cells expressing the WT FlAsH-TC-apo-sGC␤1(1-619) caused a fluorescence decrease that was time-dependent, saturable, and sensitive to HSP90 inhibition, with an initial rate of fluorescence decrease estimated to be Ϫ11.4 Ϯ 0.9 min Ϫ1 . In contrast, adding heme Biological relevance of the sGC␤-HSP90 complex had no effect on the fluorescence signal intensity from cells expressing either of the sGC␤ heme-binding mutants. We thus went on to use the same strategy to evaluate heme insertion into the various apo-TC-sGC␤1(1-619) mutants in the live cells. Fig. 4 shows that the mutant proteins exhibited a range of heme insertion capacities relative to WT, which were also sensitive to HSP90 inhibition. The R335S/R336S/D342S mutant showed heme insertion kinetics similar to or slightly faster than WT, as judged from a fit of its initial fluorescence decrease rate (Ϫ13.1 Ϯ 2.2 min Ϫ1 ), whereas the L269D/I272S/V275D and H266A/H271A/I272D mutants had detectable but 10-fold slower initial rates of heme insertion, as estimated from fits of their fluorescence decreases (Ϫ0.9 Ϯ 0.1 and Ϫ1.0 Ϯ 0.2 min Ϫ1 , respectively). Heme binding to the other five mutant proteins was essentially undetectable by this method. In general, heme insertion into the various sGC␤ proteins expressed in cells correlated well with their different HSP90-binding capabilities, with the exception of the L269D/I272S/V275D mutant, whose heme insertion was somewhat less than what one might expect based on its HSP90 binding capacity. Overall, the results imply that heme insertion into apo-sGC␤ in cells depends on, and is likely limited by, the ability of HSP90 to directly bind to apo-sGC␤ regions I, II, and III.

HSP90 interaction is critical for development of heme-dependent sGC␤ catalytic activity
To independently test the importance of HSP90 complex formation, we compared the ability of each sGC␤ mutant to become catalytically active in cells. sGC␤ can become active in cells in the absence or presence of an sGC␣ partner, although the activity achieved is greater in the latter case (16,17). We first determined the ability of BAY58-2667 (4

-[[(4-carboxybutyl)[2-[2-[[4-(2-phenylethyl)-phenyl]methoxy]phenyl]ethyl]amino]
methyl]benzoic acid hydrochloride; BAY58) to activate catalysis by each mutant when it was expressed in heme-deficient cells with or without sGC␣ co-expression. Under these circum-stances, BAY58 binds within the heme pocket of apo-sGC␤ (28), inducing structural changes that activate catalysis independent of heme or HSP90. Fig. 5 (A and B, left panels) shows the guanylate cyclase activities we obtained when the cell supernatants were given BAY58. All displayed activities approached the level for WT, with the exception of the supernatants containing the two apo-sGC␤ deletion mutants, which were about 50 -60% as active in the absence of sGC␣ co-expression, respectively. All of the sGC␤ proteins expressed in the heme-deficient cells showed similar expression levels (Fig. S3), and as expected, they all had a very poor GTP cyclase activity in response to the NO donor NOC-18 (Fig. S4). The results establish that each sGC␤ mutant protein maintains a robust intrinsic catalytic activity when it is elicited in a heme-and HSP90-independent manner by BAY58.
The GTP cyclase activities of supernatants from similarly transfected cells that were grown in normal medium and in response to the NO donor NOC-18 are shown in the right panels of Fig. 5 (A and B). In this circumstance, the level of cyclase activity depends completely on heme insertion having occurred in sGC␤. The activities varied greatly in response to the NO donor, most were lower than WT, and their rank order was similar with or without the sGC␣ coexpression. In general, the NO-dependent activities of the mutants corresponded well with their capacities for HSP90 complex formation and for achieving heme insertion (as determined above). Thus, heme insertion into sGC␤ and its consequent catalytic activity requires that HSP90 bind to PAS domain regions I, II, and III.

HSP90 prevents sGC␣ binding to apo-sGC␤ during heme insertion
Co-IP studies suggest that sGC␣/␤ heterodimer formation occurs after apo-sGC␤ acquires heme and dissociates its HSP90 chaperone (16,17). Our current study allowed us to test the relative importance of HSP90 association versus heme insertion

Biological relevance of the sGC␤-HSP90 complex
in governing sGC␤ subunit interaction with an sGC␣ partner. We co-transfected heme-depleted COS-7 cells with sGC␣ and each of our sGC␤1(1-619) mutants and then analyzed their associations with sGC␣ versus with HSP90 by performing co-IP on the cell supernatants. Fig. 6A shows that WT apo-sGC␤ associated strongly with HSP90 but minimally with sGC␣, as established previously (16). In comparison, five of the seven apo-sGC␤ mutants with completely or partially impaired HSP90 binding were found to associate much more strongly with sGC␣. The sGC␣-binding capacities of these five mutants correlated inversely with their HSP90 association capacities (Fig. 6B). The remaining two sGC␤1(1-619) mutants with partially impaired HSP90 binding (H266A/H271A/I272D and L269D/I272S/V275D) associated poorly with sGC␣ in the heme-deficient cells, revealing that they are also sGC␣-binding mutants under this circumstance. Co-expression of sGC␣ did not significantly alter the levels of HSP90 association with any of the sGC␤1(1-619) mutants (Fig. S5), suggesting that their heme content is the major factor that determines their HSP90 interaction. Overall, our findings reveal that sGC␤ and sGC␣

Biological relevance of the sGC␤-HSP90 complex
subunits have an intrinsic binding affinity toward one another, even when the sGC␤ subunit is heme-free, but sGC␣ can only bind to apo-sGC␤ when it has defective HSP90 binding. Thus, besides enabling heme insertion, HSP90 may also competitively block sGC␣ from binding to apo-sGC␤ in cells during sGC maturation.

Discussion
Heme insertion during sGC maturation ultimately leads to formation of an NO-responsive heterodimer, but questions remain about the mechanism and regulation of this process. Early studies established that sGC associates with HSP90 in cells and tissues under a variety of conditions. Their association

Biological relevance of the sGC␤-HSP90 complex
usually correlated with an increase in sGC activity, and pulldown and column binding experiments implicated the sGC␤ PAS domain in enabling the HSP90 association (20, 21). More recently, we determined that (i) in cells and tissues, HSP90 associates with only the heme-free form of sGC␤, and not with holo-sGC␤, sGC␣, or the sGC heterodimer (16,17), and (ii) purified apo-sGC␤ and HSP90 interact directly to form a complex (22). Using interaction mapping based on hydrogendeuterium exchange MS and biophysical experiments, existing structural data, and computer-assisted docking, we built a partial model of an sGC␤-HSP90 interaction complex (18,22) (as in Fig. 1). In the present study, we sought to determine whether a complex corresponding to our model actually forms in mammalian cells and, if so, to define its nature and its biological relevance for sGC␤ heme insertion, sGC heterodimer formation, and catalytic activity. Our findings suggest that a direct complex does form between apo-sGC␤ and HSP90 in cells that is similar to our model structure, because model-guided deletions and mutations successfully disrupted the sGC␤-HSP90 complex. Moreover, we found that the complex must form in mammalian cells to drive heme insertion into apo-sGC␤ during maturation. Our study also shows that HSP90, by virtue of binding to apo-sGC␤, prevents premature association of apo-sGC␤ with an sGC␣ partner subunit. In this way, HSP90 prevents formation of a heme-free sGC heterodimer in cells.
To interpret the results of our study, it was important to determine whether the mutations caused unintended disruptions to sGC␤ behavior, such as the ability of its H-NOX domain to bind heme properly or to impact the catalytic activity of sGC. Two lines of evidence suggest that our designed mutations had a negligible impact. (i) All of the purified apo-sGC␤ mutants bound exogenously added heme to generate a common species with spectral features characteristic of native sGC. (ii) When each of the full-length sGC␤ mutants was expressed in heme-deficient mammalian cells, they displayed good catalytic activity upon the addition of BAY58, which binds and activates apo-sGC␤ independent of HSP90 or NO (29,30). Thus, the effects of the mutations appeared to be restricted to PAS domain functions, and their impacts on HSP90 binding, sGC␤ heme acquisition, and NO-dependent catalytic activity in mammalian cells could be clearly interpreted.

Structural findings
The residues that we selected for mutagenesis or deletion were clustered in three regions of apo-sGC␤ that form the HSP90 complex interface in our model structure. Mutant proteins from all three regions either partly or completely impaired HSP90 interaction when tested either as purified constructs or when expressed as full-length sGC␤ proteins in mammalian cells.
Region I residues Phe 265 -His 271 consist of a surface ␣-helix (␣E) that is a common structural element in PAS domain folds (31), followed by a proximal C-terminal loop, which interacts directly with the HSP90 N-M domain interface in our model. Deletion of region I eliminated HSP90 binding and allowed sGC␣ interaction with the heme-free sGC␤. It is unlikely that the deletion grossly perturbed the folding of sGC␤, as the expression level was normal and the purified protein could cor-rectly bind the provided heme. The triple substitutions within region I of surface-exposed His to Ala or of surface-exposed aliphatic side chains to Asp/Ser also induced a partial loss in HSP90 affinity, suggesting that region I of the PAS domain engages in a relatively distributed interaction with HSP90. Interestingly, the heme-free triple mutants were both defective in binding an sGC␣ partner subunit, implying that region I also enables the intrasubunit PAS-PAS domain interactions that are needed for sGC heterodimer formation.
Region II contains an ␣-helix insertion that is not typical of PAS domains (31) along with a relatively extended loop. We designed mutations in this region solely guided by our model of the complex, which suggests that region II contributes to an interaction surface distinct from that of region I and adjacent to that of region III. We found that determinants in region II are also important for HSP90 -sGC␤ interaction. Deletion of the entire region abolished the interaction, whereas the E291A/ E295A/L296D mutant showed substantially weaker affinity toward HSP90. Their location within the loop of region II suggests that this flexible segment is important for the HSP90 interaction while not excluding the possibility that the upstream helical portion also contributes to the binding interface.
Region III corresponds primarily to an unstructured linker region that connects the PAS and CC domains. Mutating aliphatic residues within region III, specifically substituting Leu residues with acidic residues, often but not always abrogated HSP90 binding to apo-sGC␤1(1-358). The HSP90-binding defect was observed for the L333D/L338D/L340D mutant. The L340D/I343D/L345D mutant also showed no detectable HSP90 binding in pure form but did exhibit partial HSP90 binding when expressed in mammalian cells and exhibited some NO-dependent catalytic activity, particularly when co-expressed with sGC␣. In comparison, the region III mutant R335S/R336S/D342S bound well to HSP90 and had normal heme insertion. This is consistent with these particular charged side chains pointing away from the HSP90 interface, as they do in our model complex.
A common feature among all three regions is that surfaceexposed aliphatic residues (e.g. Leu/Ile) make important contributions to the HSP90 interaction. This agrees with the concept that HSP90 prefers to bind flexible, conformation-reporting epitopes that may otherwise be buried in intraprotein or protein-protein interactions (32).
It remains to be elucidated how residues within the three regions may contribute to domain-domain interactions within sGC␤ or between the subunits in the sGC heterodimer. We found that only substitutions within region I antagonized the natural binding affinity that exists between the sGC␤ and sGC␣ subunits. In this regard, it is noteworthy that deleting regions I or II only partially diminished sGC heterodimer activation by BAY58 (Fig. 5B). This suggests that, despite the region I and II deletions, the conformational changes induced by BAY58 binding are for the most part still relayed from the HNOX domain to the catalytic domains of the sGC heterodimer. A conservative interpretation is that interdomain contacts that involve regions I or II help to support, but are not essential for, signal transmission. These concepts can now be further investigated.

Biological relevance of the sGC␤-HSP90 complex A direct HSP90 -apo-sGC␤ interaction drives heme insertion
We were able to monitor heme insertion into FlAsH-TCapo-sGC␤1(1-619) proteins in live cells to determine whether HSP90 binding corresponded to heme acquisition. Overall, we observed that the two processes correlate well. WT apo-sGC␤ incorporated heme over a 15-30-min period, and a mutant with slightly improved HSP90-binding affinity (R335S/R336S/ D342S) showed a slightly faster heme incorporation, suggesting that it is a gain-of-function mutant. In contrast, the four PAS domain mutants with the poorest HSP90 binding affinities (TC-sGC␤1(1-619) ⌬265-271, ⌬284 -301, E291A/E295A/ L296D, and L333D/L338D/L340D) showed no heme insertion over a 90 -180-min period. Two PAS domain mutants with partly compromised HSP90 binding (L269D/I272S/V275D and H266A/H271A/I272D) showed detectable heme incorporation, but at rates slower than the WT. The acquisition of NO-stimulated sGC␤ catalytic activity, which is a heme-dependent function (1), generally correlated well with mutant protein heme insertion and with HSP90-binding capabilities. The correlations between HSP90 binding, heme insertion, and gain of heme-dependent function argue strongly that direct interaction of HSP90 with apo-sGC␤ drives the heme insertion, rather than any other HSP90 interactions that could conceivably be present within a multiprotein heme insertion complex.

Possible mechanism of HSP90 action in heme insertion
We currently hypothesize that HSP90 functions in the heme insertion through its conformational control of the apo-sGC␤ client. If one adds heme to the cells, they will incorporate the heme into the HSP90-bound apo-sGC␤ over a 15-30 min period, and inhibiting HSP90 ATPase activity during this period blocked the heme insertion. Thus, one can imagine HSP90 helping to form and stabilize heme-accepting protein conformations of apo-sGC␤ and facilitating heme insertion in an ATP-dependent manner or, similarly, HSP90 facilitating shifts between protein conformations (partial folding/ unfolding) that ultimately facilitate heme insertion in an ATP-dependent manner. In this way, we propose that HSP90 drives heme insertion into apo-sGC␤ through an ATP-dependent process.
Besides sGC␤, it is intriguing to speculate that heme insertion into other known HSP90 clients, such as globins (33) and NO synthases (34), may also involve a direct complex formation with HSP90. These clients do not contain PAS domains, so their interactions with HSP90 would need to involve distinct protein regions. Our current study provides a template to investigate how HSP90 interaction facilitates heme insertion at the molecular level and how this process may be regulated by co-chaperones or post-translational modifications.

HSP90 controls two steps of sGC maturation
Heterodimer formation is the final step in sGC maturation and is critical because both the ␣ and ␤ subunits provide residues that form the active site and the substrate-binding site (11,12). Heterodimer formation also permits cross-subunit communication that regulates catalytic activity in response to NO binding, S-nitrosation, and other signaling mechanisms within cells (12,21,(35)(36)(37). Our previous work confirmed that two sGC␤ subpopulations (heme-free and heme-bound) exist in cells and revealed that each has a distinct protein binding partner: apo-sGC␤ is bound to HSP90, whereas holo-sGC␤ either self-associates or forms a heterodimer with a sGC␣ subunit partner (17). Our current study clarifies how sGC␤ interactions with HSP90 versus sGC␣ are governed.
Surprisingly, most of the apo-sGC␤ mutant proteins with defective HSP90 binding now associated with sGC␣ in cells to form heme-free heterodimers. This behavior has not been observed for WT apo-sGC␤ (17) and implies that (i) unlike many other HSP90 client proteins (38 -41), apo-sGC␤ does not strictly require HSP90 binding to stabilize it or protect it from degradation in mammalian cells, and (ii) apo-sGC␤ is inherently capable of binding with sGC␣ but does not do so in mammalian cells because it is instead sequestered by HSP90. Thus, in the context of sGC maturation, a heterodimer forms after heme insertion not because heme binding itself enables sGC␤ to bind sGC␣, but because HSP90 blocks premature heterodimer formation until heme is inserted into the apo-sGC␤. It is likely that heme insertion promotes conformational changes in sGC␤ that favor HSP90 dissociation. The ability of HSP90 to block sGC␣-sGC␤ heterodimerization is consistent with it interacting directly with the sGC␤ PAS domain, because the PAS domains of sGC␤ and sGC␣ interact to form the heterodimer (42,43). Thus, HSP90 and sGC␣ may compete for binding to the PAS domain of sGC␤. The fact that HSP90 also drives heme insertion into apo-sGC␤ means that it governs sGC maturation in two ways: HSP90 blocks formation of a heme-free heterodimer and drives the heme insertion reaction of apo-sGC␤. This presents new regulatory points to modulate the total cellular pool of active sGC. A model for sGC maturation that incorporates these concepts is shown in Fig. 7.
Our work raises questions regarding the possible functions of apo-sGC in vivo. Prior studies showed that heme could be removed from purified sGC heterodimer with a weak detergent and that the basal activity of the heme-free sGC was greater than its heme-bound counterpart (23,44,45). Our work questions whether such heme-free sGC heterodimers could accumulate in mammalian cells, as we always find that apo-sGC␤ is complexed with HSP90 and unable to associate with sGC␣. When the H105F sGC␤ heme-binding mutant was expressed in mammalian cells, it was found in complex with HSP90 and remained so even after the cells were provided heme (17). Tissues from knock-in mice bred to express the H105F sGC␤ mutant in place of the WT exhibited a basal sGC activity that was higher than that of WT tissues, consistent with expression of heme-free sGC␤ (46). Unfortunately, it was not determined whether the H105F sGC␤ in the tissues was present as a homodimer, as a heterodimer, or in complex with HSP90. Of note, our previous study (17) showed that sGC activators like BAY58, which fill the heme-binding site, drive sGC homodimer or heterodimer formation in cells independent of heme or of HSP90. This also appeared to occur in the H105F sGC␤ knock-in mice, as judged by an observed increase in tissue sGC activity upon treatment with BAY58 (46). These considerations provide a strong incentive for further studies of the biological Biological relevance of the sGC␤-HSP90 complex nature and behaviors of apo-sGC␤ in cells, tissues, animal models, and human physiology.

Conclusions
We tested the reliability of an HSP90 -sGC␤ model complex that could become useful to study structure-function aspects of heme insertion into apo-sGC␤. Further, we determined that when the complex forms in cells, it drives heme insertion while actively preventing sGC␣ from binding to apo-sGC␤ during sGC maturation until heme has been inserted. In this way, HSP90 may directly prevent the formation of NO-unresponsive, heme-free sGC heterodimers and further regulate the NO and cGMP signaling pathways.

General methods and materials
The TC-FlAsH In-Cell Tetracysteine Tag Detection Kit was obtained from Invitrogen. All other reagents and materials were obtained from sources reported elsewhere (17).

Molecular biology
Bovine sGC␤1(1-358) DNA (which codes for the H-NOX domain, the PAS domain, and the N-terminal portion of the coiled-coil domain) (22) was subcloned into pMAL-c2X vector on the N-terminal side of maltose-binding protein (MBP) for bacterial expression. pET20b vector containing rat sGC␤1(1-385) with a C-terminal His 6 tag was a generous gift from Dr. Michael Marletta (University of California, Berkeley). pCMV5 mammalian expression plasmid containing full-length rat sGC␣1(1-690) or rat sGC␤1(1-619) (H-NOX domain, PAS domain, coiled-coil domain, and catalytic domain) was described previously (17). The bovine and rat sGC␤1 amino acid sequences are 98% identical, and none of the residues we modified for our study differ between the bovine and rat sequences. pCMV5 mammalian expression plasmid containing rat sGC␤1(1-690) with a CCPGCC sequence in residues 239 -244 (TC-sGC␤1) was made by Genscript (Piscataway, NJ). Designed mutations listed in Table S1 were prepared by site-directed mutagenesis using primers (Invitrogen) with Q5 DNA polymerase (New England Biolabs, Ipswich, MA) on pMAL-sGC␤1(1-358), pET20b-sGC␤1(1-385), pCMV5-sGC␤1(1-619), and pCMV5-TC-sGC␤1(1-619). The sequences of mutations were confirmed by DNA sequencing at the Cleveland Clinic Genomics Core Facility. The pET15MHL plasmid containing full-length human HSP90␤(1-724) was described previously (22). When HSP90 binds to apo-sGC␤, it drives heme insertion into apo-sGC␤ while also preventing its premature interaction with sGC␣ to form a heme-free, nonfunctional sGC heterodimer. Once heme insertion is complete, HSP90 dissociates from sGC␤, and this allows it to partner with sGC␣ to form a mature and functional sGC heterodimer. WT and mutant sGC␤1(1-358),  sGC␤1(1-385), and HSP90␤ in E. coli pMAL-sGC␤1(1-358) WT and mutants were each transformed into E. coli BL21 (DE3) for expression and purified by a method described previously with modifications (22). Briefly, sGC␤-MBP recombinant proteins were first purified on an amylose column using a method described elsewhere (47), and their purity was checked by SDS-PAGE. In some cases, further anion-exchange chromatography on a Q-Sepharose column with a liner gradient of 50 mM to 1 M NaCl in 40 mM EPPS, 10% glycerol, pH 8.5, buffer was used to further purify sGC␤-MBP and mutant proteins (22). pET20b-sGC␤1(1-385) was grown and purified using a method reported previously (24) with modifications. Briefly, WT and mutant sGC␤1(1-385) constructs were each transformed into BL21(DE3)-Rosetta cells for expression. Overnight cultures were grown at 37°C in Luria broth and used to inoculate 500-ml expression cultures grown in Terrific broth. Expression cultures were grown until A 600 ϳ0.5 and subsequently cold-shocked for 10 min in an ice bath. Protein expression was induced with 250 M isopropyl-D-1thiogalactopyranoside for 20 h at 25°C at 250 rpm. Cells were harvested by centrifugation and then resuspended in 40 mM EPPS, 150 mM NaCl, 10% glycerol, pH 7.5, buffer supplemented with protease inhibitors, benzonase, and lysozyme. Resuspended cells were lysed by sonication and clarified by centrifugation. Lysate was then purified with a gravity nickel-nitrilotriacetic acid column with a method described previously (22). Human HSP90␤(1-724) with an N-terminal histidine tag was transformed into BL21 colonϩ cells for expression and purified with a gravity column with nickel-nitrilotriacetic acid-agarose as described previously (22).

Western blotting and immunoprecipitation
Western blotting and immunoprecipitation were performed by methods described previously (16). For immunoprecipitation, 500 g of total cell supernatant protein was precleared with Protein G beads (20 l) (16).

In vivo labeling of TC-sGC␤ with FlAsH
TC-sGC␤1(1-619) expressed in COS-7 cells was labeled with FlAsH using a method described elsewhere (25) with modifications. Briefly, transfected cells grown in fluorescence 96-well plates were washed three times with phenol red-free DMEM containing 1 g/liter glucose. Cells were then incubated with a mixture of FlAsH/1,2-ethanedithiol made in Opti-MEM (final concentration 0.5 M/12.5 M) for 1 h at room temperature. Afterward, cells were washed three times with 250 M 1,2-ethanedithiol in phenol red-free DMEM ϩ 10% FBS and then three times with phenol red-free DMEM ϩ 10% FBS. Phenol red-free DMEM ϩ 10% serum and 5 M heme was then added to the cells for monitoring heme insertion.

Measurement of fluorescence of FlAsH-TC-sGC in cells
We transiently transfected COS-7 cells that grew in fluorescence 96-well plates in heme-depleted medium in the presence of succinyl acetone to enable the production of mutant and WT TC-apo-sGC␤1(1-619). Two heme-binding mutants, TC-sGC␤1(1-619) H105F (16) and TC-sGC␤1(1-619) Y135A/R139A (27), were also used as negative controls. After 24 h of transfection, we labeled the TC-sGC1(1-619) proteins with FlAsH, added heme, and monitored the heme insertion over 3 h by following the fluorescence intensity of each FlAsH-TC-sGC protein with a Flexstation 3 microplate reader (Molecular Devices, San Jose, CA). FlAsH-TC-sGC proteins were excited at 488 nm, and emission was detected at 528 nm. 100 readings were taken for signal integration every 1 or 2 min. cGMP enzyme-linked immunosorbent assay cGMP formation and its concentration in cell supernatant assays was determined by a method described previously (17) with modifications. Briefly, immediately after lysing COS-7 cells, different amounts of cell supernatant were added to a reaction mixture containing 500 M GTP alone or with 30 M NO donor NOC-18, and these mixtures were incubated for 30 min at 37°C. In some groups, 10 M BAY58 was added in place of NO donor. The cGMP in the cell supernatant reactions was then quantified with a cGMP ELISA kit (Cell Signaling Technology, Danvers, MA).