Complex structure of human Hsp90N and a novel small inhibitor FS5

Rui Liu· Xiao-Lu Lu · Xian-Hua Huang · Wei He · Jing-Jing Duan ·Jin Zhang · Jian Li,4

Abstract Heat shock proteins (Hsps) are a family of abundantly expressed ATP-dependent chaperone proteins.Hsp90 is an eminent member of Hsp family.Thus far,two primary functions have been described for Hsp90:first,as a regulator of conformational change of some protein kinases and nuclear hormone receptors, and the other as an indispensable factor in cellular stress response. Hsp90 has an essential number of interaction proteins since it participates in almost every biological process and its importance is self-evident. Hsp90 has an inextricable relationship in the pathogenesis of cancer, especially in the proliferation and irradiation of cancer cells, thus being a notable cancer target. Since the discovery of geldanamycin, the first inhibitor of Hsp90,from the bacterial species Streptomyces hygroscopicus, even more attention has been focused toward Hsp90. Many structure-based inhibitors of Hsp90 have been designed to develop an innovative method to defeat cancer. However, already designed inhibitors have various deficiencies, such as hepatotoxicity, poor aqueous solubility, instability, and non-ideal oral bioavailability.Based on the aforementioned reasons and to achieve an optimal performance and fewer side effects,we designed a novel inhibitor of Hsp90, called FS5, and resolved the crystal structure of the Hsp90N-FS5 complex(1.65 A˚,PDB code 5XRB). Furthermore, we compared the complexes Hsp90N,Hsp90N-GDM,and Hsp90N-ATP and suggest that the inhibitor FS5 may compete with ATP for binding to Hsp90, which can be regarded as a potential strategy for the development of novel cancer drugs in the future.

Keywords Heat shock protein 90 · Complex crystal structure · Interactions · Anti-tumor drugs · X-ray diffraction

1 Introduction

Heat shock proteins (Hsps) comprise a conservative ATP-dependent chaperone family, which was first discovered in 1962. Under normal circumstances, Hsps alleviate folding of the normal proteins and prevent proteins from folding incorrectly, as well as to get rid of misfolded or aggregated proteins. Under the toxic conditions, the expression of Hsps increases to adapt the toxicity and improve cell survival rate. Meanwhile, in tumor cells, the level of Hsps also increases for maintaining the balance.Thus, Hsps are not only essential molecular chaperones,but also serve as biomarker of cellular stress response [1].

The Hsp family can be divided into five subfamilies according to the different molecular weight: Hsp110,Hsp90, Hsp70, Hsp60, and several small Hsps [2, 3].Hsp90 is a key factor in Hsps family. In non-stress conditions,it constitutes almost 1–2%proportion of all protein in the eukaryotic cytosol,while under stress conditions,the quantity rises to ～4–6% [4]. In eukaryotes, Hsp90 has two functions: first, as a regulator of conformational change of some protein kinases and nuclear hormone receptors, and the other as an indispensable factor in cellular stress response [5]. Meanwhile, in eukaryotes, Hsp90 can be primarily found in cell nucleus and cytoplasm,playing an indispensable role in gaining valid conformation of more than 200 substrate proteins [6]. Some of them belong to signaling proteins, like kinases, transcription factors, and others [7, 8]. Due to the diversity of its target proteins, Hsp90 participates in many fundamental cellular responses involving signal transduction, cell cycle control,transcriptional regulation, and cellular stress response[9–11], for example human epidermal growth factor receptor (HER2), which has significant role in cancer progression [12–14].

Cancer represents a complex set of diseases that can arise in any cell of the body that is capable of evading normal regulatory mechanisms [15]. Meanwhile, it is known that malignant transformation is extensively dependent on the pathways mediated by Hsp90 [16, 17].Inhibition of Hsp90 leads to the decreased growth and transfer velocity and, ultimately, higher sensibility toward conventional anti-tumor therapy of cancer cells[18,19].At the same time, an inhibitor of Hsp90 is more selective to cancer cells than normal cells and is usually accumulated in cancer tissues while being rapidly eliminated in normal issues [20]. Therefore, Hsp90 is considered to be an important target for combatting cancer.

Hsp90 is a flexible dimer, and its dimerization is very vital for the proper function in vivo, while each monomer consists of three domains [21]. The N-terminal domain(25 kDa),known as the ATP-binding domain,consists of a highly conserved sequence. At the initial step of Hsp90 functioning,ATP binds to its N-terminal domain[22].The middle domain (35 kDa) interacts with the downstream substrate proteins. Upon ATP binding, the middle domain is activated and combines with diverse target proteins to exert a variety of functions. The C-terminal domain(12 kDa) is in charge of the dimerization of Hsp90, whose function is to bind to co-chaperones [23–25]. When the middle domain changes to an open conformation, Hsp90 acquires ATP enzymatic activity. Besides, the middle domain contains a conserved arginine residue, which can interact with γ-phosphate of ATP to provoke ATP hydrolysis and provide energy adapted by Hsp90. When the substrate combines with the middle domain, the conformation of Hsp90 is altered to a ‘closed state.’ Then,Hsp90 can function as a molecular chaperone [26–29].

In human cells, Hsp90 is represented in two isoforms:Hsp90α and Hsp90β.In comparison with Hsp90β,Hsp90α is inessential in mammalian cells and mainly maintains cell homeostasis. Hsp90β, in turn, is expressed at any condition. In cytoplasm, Hsp90 is mainly in the form of homologous dimer, like α-α or β-β. Hsp90α and Hsp90β contain 730 and 724 amino acids, respectively, and share 84%of homology.The critical difference in the amino acid sequence between Hsp90α and Hsp90β is that N-terminal end of Hsp90α contains a penta-amino sequence QTQDQ,while Hsp90β does not. Hsp90α is more sensitive to heat and, therefore, more susceptible to mitosis, which makes this isoform to be more relevant for tumor cell proliferation. Hsp90β, in contrast, is more crucial for cell differentiation and structural construction. In many cancer cells,the expression of Hsp90α is much higher than in normal cells, while Hsp90β shows almost no difference [30, 31].Currently, the selectivity of Hsp90 inhibitors to the particular subtype is under an intensive research aiming to reduce potential side effects.

Generally, in the absence of ATP binding, Hsp90 exists in its natural opened conformation. In this state, Hsp90 forms a so-called ‘V’ shape [32, 33]. Following ATP binding, the N-terminal domain of Hsp90 changes its relative position, and upon dimerization, Hsp90 acquires a closed conformation. Then, two middle domains overlap and react with their target protein. Consequently, the energy of ATP has been employed,and ATP is transformed into ADP and Pi. Finally, two monomers recover to the natural state, the ‘V’ shape, while two middle domains dissociate with their target protein.

The types of Hsp90 inhibitor can be divided into N-terminal inhibitor, C-terminal inhibitor, and middle domain inhibitor. There are a few studies describing the C-terminal and middle domain inhibitors [34]. Geldanamycin(GDM), one of the natural molecules,has been proved to act as a cancer suppressor, which was initially found in Streptomyces hygroscopicus[35].GDM is the first Hsp90 inhibitor to be discovered, and it belongs to the benzoquinone ansamycin class of antibiotics[36].The antitumor mechanism of GDM is based on its ability to occupy ATP-/ADP-binding site in the Hsp90 N-terminal, leading to the conformational change of Hsp90, thereby inhibiting ATP activity. Thus, the binding site is prohibited from the association with the target protein during binding of GDM,which results in loss of function for Hsp90 and subsequent degradation of the substrate[37].At the same time,Hsp90 acts as an ubiquitinase and triggers its own hydrolyzation by proteasome. Ultimately, the dysregulated signaling pathway is blocked, and the tumor development is suppressed [38]. Nonetheless, due to the poor stability and high hepatotoxicity found in animal models, GDM is not used in the clinic as expected[39,40].Since the discovery of GDM, the research on the Hsp90 inhibitor has been the focus of intensive discussions. The analog of GDM,17-(allylamino)-17-demethoxygeldanamycin (17-AAG), is designed to negate the shortcomings of GDM. Its anti-tumor activity is higher than that of GDM,and hepatotoxicity is significantly decreased. Of note, 17-AAG is the first Hsp90 inhibitor to be used in clinical trials[41].However,the modified inhibitor still requires substantial improvement in terms of limited bioavailability, poor water solubility, minor toxicity, and different degrees of other side effects [42].

FS5 is a novel inhibitor of Hsp90, and in the present study, we report on the crystal structure of the complex Hsp90N-FS5 by X-ray diffraction. Our results on comparison of Hsp90N, Hsp90N-GDM, and Hsp90N-ATP complexes revealed that inhibitor FS5 binds to the same site as both GDM and ATP. We believe the analysis of complex structure,and the interactions between FS5 and Hsp90 will provide a rational basis for the design and optimization of novel anticancer drugs targeting Hsp90.

2 Experimental section

2.1 Synthesis of a small molecule

The compound FS5 was prepared according to the methods reported in our previous work [43]. N-(3-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)cyclopropanecarboxamide (9). 1H NMR(400 MHz, CD3OD) δ 7.13 (d, J = 8.4 Hz, 2H), 7.04 (s,1H), 6.90 (d, J = 8.4 Hz, 2H), 6.44 (s, 1H), 3.78 (s, 3H),1.76 (m, 1H), 0.94–0.85 (m, 4H); MS (ESI):400.9[M + H]+(Fig. 1).

2.2 Protein purification and crystallization

The Hsp90 N-terminal domain residues (9–236) were cloned into the vector pET28a to obtain the final plasmid for the overexpression. This plasmid was transformed into competent E. coli cells, Rosetta DE3. The bacteria were grown in 800 mL of LB (Luria–Bertani) broth at 37 °C.Upon reaching OD600 value of 0.6–0.8,200 μM IPTG was added to induce the expression of N-terminal Hsp90 and further incubated for 3–5 h at 30 °C. We then centrifuged the cells at 10,000g for 10 min at 4 °C and resuspended the precipitate in buffer A (100 mM Tris/HCl buffer, pH 7.5,300 mM NaCl, and 5% glycerol) for rupturing the cells with the JNBIO 3000 plus (JNBI). The supernatant containing protein of interest was acquired by centrifugation at 30,000g and 4 °C for 30 min. The supernatant was then transferred into a 5-ml Ni–NTA (Ni2+-nitrilotriacetate)column (GE Healthcare) together with buffer B (100 mM Tris/HCl buffer, pH 7.5, 300 mM NaCl, 100 mM imidazole, and 5% glycerol) for the recovery of Hsp90 protein using imidazole.Superdex 75 PG gel filtration column(GE Healthcare) allows a high yield of the overexpressed protein to be obtained and the removal of imidazole by changing the buffer system to buffer C(100 mM Tris–HCl buffer, pH 7.5, 300 mM NaCl, and 5% glycerol). The positive peak protein was collected and verified by SDSPAGE. Finally, the protein was flash-frozen in liquid nitrogen and stored at -80 °C.

For the experiments, the protein was thawed and adjusted to the concentration of 20 mg/ml in Amicon Ultra-15, 10000Mr cutoff centrifugal concentrator (Millipore). Subsequently, the solution of small molecules was mixed with the protein in a ratio of 5:1 and incubated at 4 °C for 1 h.The hanging drop vapor diffusion method was used to gain crystals at 4 °C using previously described modifications for crystallization(100 mM Tris–HCl buffer,pH 8.5, 20% PEG 4000, 200 mM MgCl2) [44].

2.3 Data collection, structure determination,and refinement.

The crystals were tailored with cryo-loop (Hampton Research,USA)and then flash-frozen in liquid nitrogen for collecting better X-ray data.All datasets were generated at 100 K on macromolecular crystallography beamline 17U1(BL17U1) at Shanghai Synchrotron Radiation Facility(SSRF,Shanghai,China)through an ADSC Quantum 315r CCD detector. The obtained results were processed by the HKL2000 software package. The structures of complex Hsp90N-FS5 were determined by molecular replacement with PHENIX software.The apo structure of Hsp90N(PDB code 3T0H) was considered as a model for the analysis of Hsp90N-FS5 complex[45].The Coot software was used to rebuild the initial model. The final models were refined to resolution limit of 1.65 A˚by using the PHENIX software.The superimposed data were analyzed with PyMOL software package. The complete data collection and statistics of refinement are shown in Table 1.The complex Hsp90NFS5 structure has been deposited in PDB (PDB code 5XRB).

Table 1 Results of the statistical analysis for data processing and model refinement of human Hsp90N-FS5 complexes

3 Results and discussion

3.1 Purification, crystallization, and structure determination of Hsp90N-FS5

The intensively purified protein was collected using metal-chelating chromatography and gel filtration chromatography. The ordinate value of the gel filtration chromatography reached 800 mAu,and a single sharp peak was achieved indicating the high overall purity of the target protein.The collected protein was subjected to SDS-PAGE analysis, which was clearly consistent with the expected band at the molecular weight of 25 kDa, and the purity reached 98%.

After 3–5 days,the complex crystals of Hsp90N-FS5 were obtained by the hanging drop method at 277 K.The form of the crystals is shown in Fig. 2.The mean dimension of the crystals was roughly 250 μm × 150 μm × 50 μm.

The complete information on the crystals was collected for the statistical analysis, which is shown in Table 1. The structure of complex Hsp90N-FS5 has been deposited in PDB (PDB code 5XRB). The diffraction data of crystals were collected at 1.65 A˚resolution and indexed in space groups I222. The unit cell parameters were as follows:a = 67.45 A˚, b = 90.70 A˚, c = 98.28 A˚; α = β = γ =90.00°. The resolved structure of Hsp90N-FS5 included only Val17-Glu223,while no electron density was obtained for residues Asp9-Glu16 and Val224-Glu236 in complex Hsp90N-FS5, which is may be due to the disordered residues in the electron density.

3.2 Analysis of crystal structure and interaction of Hsp90N-FS5

The structure of the Hsp90 N-domain includes a high conservative area for ATP binding, which comprises H2,H4,H7,and L1.This pocket is about 15 A˚in depth and has an α/β sandwich-like structure.The pocket base consists of eight uninterrupted antiparallel β-sheets. According to the crystal structure resolved by X-ray, compound FS5 completely binds to the ATP pocket,which is shown in Fig. 3a,b in two different directions,and the intact electron density of the compound FS5 has been captured and is shown in Fig. 3c.

There is a crucial structure in Hsp90Ntermed ATP lid,which comprises H4–L2–H5–H6 (residues 99–134). This structure can adjust the size of the ligand-binding pocket by flipping its loop in order to regulate the conformation switch between the opened and closed states. Only at the opened conformation can the ligand bind to Hsp90. Subsequently, Hsp90 can activate target proteins and accomplish its function. Thus, ATP lid plays an indispensable role in facilitating the efficient binding of ATP in vivo.

We compared the crystal structures of Hsp90N-FS5(PDB code 5XRB) with other available structures for Hsp90N(PDB code 3T0H), Hsp90N-ATP (PDB code 3T0Z),Hsp90N-GDM(PDB code 1YET)by using PyMOL software [5, 45]. It was clearly observed that the refined crystal structure of Hsp90N-FS5 is different from Hsp90N,Hsp90N-ATP, and Hsp90N-GDM, and this difference is mainly focused on H4 and H5.The crystal packing patterns are also distinct, such as Hsp90N-FS5 (I222), Hsp90N(I222), Hsp90N-ATP (P21), and Hsp90N-GDM (P1211).

The identified differences between Hsp90N-FS5 and Hsp90N, Hsp90N-ATP, and Hsp90N-GDM are described below. As shown in Fig. 4a, we can detect the difference spanning Leu103-Ile128 between Hsp90N-FS5 and Hsp90N, which is a non-overlapping area in superimposition.Meanwhile,as demonstrated in Fig. 4b,we observed a conformational change directly in H4 and H5 following the binding of Hsp90 with compound FS5. At the same time,the L2 loop is rearranged. Because H4, L2, and H5 are parts of the ATP pocket,changes in L2 loop directly lead to the shape alterations of the binding pocket. Specifically,binding of FS5 to Hsp90 induces narrowing of the ligandbinding pocket and its entrance is closed, which makes Hsp90 unreachable for ATP and results in the complete dysfunction of Hsp90. In the same way, as shown in Fig. 4c and Fig. 4e, Met98-Gly137 in Hsp90N-FS5 is different from that in Hsp90N-GDM, and Met98-Met130 in Hsp90N-FS5 is different from that in Hsp90N-ATP. These results illustrate that the conformational changes after Hsp90 binding with the ligand are different between compound FS5 and other classic ligands. As shown in Fig. 4d, f, the most differences between these three complex structures were observed for H4, H5, and L2 loop.

The electrostatic potential surface distribution of complexes Hsp90N,Hsp90N-ATP,Hsp90N-GDM,and Hsp90NFS5 with the ligands is shown in Fig. 5. It was revealed that the electrostatic potential surface surrounding the active pocket in Hsp90N(Fig. 5a) is different from that of Hsp90N-GDM (Fig. 5b), Hsp90N-ATP (Fig. 5c), and Hsp90N-FS5 (Fig. 5d). According to the position of GDM and ATP, we can conclude that compound FS5 binds Hsp90Nin the ligand-binding pocket. There is enough space for optimal FS5 binding.

Furthermore, we compared structures of Hsp90N-FS5 complex with other Hsp90Ncomplexes available for some promising inhibitors together with the comparison of the inhibitor structures.Thus,there is a big difference between Hsp90N-ganetespib (PDB code 3TUH), Hsp90N, and Hsp90N-FS5. The H5 and L2 loops in all three structures demonstrate high discrepancy.As shown in Fig. 6b,the L2 loop in Hsp90Nis turned inward the pocket, indicating the minimal size of the pocket between these three structures.However, the L2 loop in Hsp90N-FS5 is neither reversed nor pronated and is almost flat with H4 and H5. The conformational change of Hsp90Nupon binding of FS5 increases the space in the ligand-binding pocket. The Hsp90N-ganetespib has different structure compared with the two described above. The first half of its L2 loop is turned outward, whereas the second half is turned inward,leading to the significant difference of the shape of the ligand-binding pocket. At the same time, the C-terminal part of H5 of Hsp90N-ganetespib also has some differences with Hsp90Nand Hsp90N-FS5. With regard to its position,the C-terminal of H5 of Hsp90Nis above,C-terminal of H5 of Hsp90N-FS5 is in the middle, and C-terminal of H5 of Hsp90N-ganetespib is at the bottom of the complexes. The major parts of ganetespib and FS5 structures are similar,showing differences only in individual residues (Fig. 6a).

Comparison of Hsp90N-XL888 complex (PDB code 4AWQ) with Hsp90Nand Hsp90N-FS5 also demonstrated essential difference between all three structures[46].At the same time, comparing inhibitor XL888 with ganetespib similarly revealed high structural discrepancy.Notably,the L2 loop of Hsp90N-XL888 is turned more inward than in Hsp90N, which further reduces the space in the ligand-binding pocket.As shown in Fig. 6d,the H5 C-terminal of Hsp90N, Hsp90N-FS5, Hsp90N-ganetespib, and Hsp90NXL888 are different. Moreover, the inhibitor XL888 and FS5 vary significantly. The structure of XL888 inhibitor has a long, almost a bar shape, and the framework is very different from FS5. Indeed, the structure of XL888 differs essentially from all the other inhibitors (Fig. 6c).

Hsp90N-NMS-E973(PDB code 4B7P)is different when compared with Hsp90Nand Hsp90N-FS5 (Fig. 6f) [47].The L2 loop of Hsp90N-NMS-E973 scrolls completely down that is different from Hsp90Nand Hsp90N-FS5 as depicted before. This particular conformation leads to the largest space in the ligand-binding pocket between all the conformations that we discussed in this paper. Likewise,the C-terminal of H5 rotates down as well, which position is different in Hsp90N, Hsp90N-FS5, Hsp90N-ganetespib,and Hsp90N-XL888. Because L2 loop and C-terminal of H5 are both downward, the shape of H4-H5 of Hsp90NXL888 is inverse triangular. As shown in Fig. 6e, the structures of these three inhibitors,NMS-E973,ganetespib,and FS5, are quite different.

Interestingly,the comparison of the crystal structures of Hsp90N, Hsp90N-FS5, Hsp90N-NMS-E973, and Hsp90N-17-DMAG(PDB code 1OSF)indicated that the L2 loop of Hsp90N-NMS-E973 and Hsp90N-17-DMAG is very similar(Fig. 6h) [48]. The orientation, range, and position were almost overlapped. However, there are some subtle differences in C-terminal of H5 between the two complexes so that Hsp90N-17-DMAG drops less than Hsp90N-NMSE973. C-terminal of H5 in the structures of Hsp90N,Hsp90N-FS5, Hsp90N-17-DMAG, and Hsp90N-NMS-E973 is different,and the structure of inhibitor 17-DMAG varies greatly from the both NMS-E973 and FS5 (Fig. 6g).

According to these comparisons,it is noticeable that the binding structures with the inhibitor show remarkable changes in only the L2 and H5 loops,while the other parts are almost similar, suggesting that L2 and H5 are significant for Hsp90 activity.At the same time,being a part of a ligand-binding pocket,the alterations of L2 and H5 directly lead to a change of space in the pocket. Therefore, the design of an appropriate inhibitor should be particularly focused on L2 and H5.

The binding of diverse ligands to Hsp90 induces different conformational changes in the ligand-binding pocket, as shown in Fig. 7. We added another complex structure of Hsp90N-BIIB021 (PDB code 3QDD) to show more changes of the ligand-binding pocket [49]. It is apparent that different ligand structures lead to the corresponding alterations in the shape of the ligand-binding pocket. According to the structure of the pocket and the ligand, and their relative position, we can optimize these inhibitors by modifying functional group to enhance performance and reduce side effects. For better resolution of the inhibitor structures, the 2D diagram of inhibitors is shown in the lower right corner of the corresponding ligand-binding pocket.

As it is shown in Fig. 3d, there are two benzenes in compound FS5, and one of the two benzenes has two hydroxyls. One hydroxyl forms four hydrogen bonds mediated by water with residues:Leu48,Ser52,Asp93,and Thr184(2.98 A˚,2.89 A˚,2.92 A˚,and 3.12 A˚,respectively).The other hydroxyl forms three hydrogen bonds mediated by water with residues Asp93,Thr184,and Gly97(2.76 A˚,2.87 A˚, and 3.01 A˚, respectively), and a direct hydrogen bond with residue Asp93 (2.66 A˚). Meanwhile, the nitrogen in heterocycle forms not only three hydrogen bonds mediated by water with residues Thr184, Asp93, Gly97(2.87 A˚, 2.76 A˚, 3.01 A˚, respectively), but also forms a direct hydrogen bond with residue Thr184 (3.33 A˚).Besides, the other nitrogen in FS5 forms two hydrogen bonds mediated by water with residues Gly97 and Asp102(2.76 A˚and 2.76 A˚, respectively). One oxygen forms one hydrogen bond mediated by water with residue Lys58(2.89 A˚). These hydrogen bonds assure tight binding of FS5 inhibitor to Hsp90. Furthermore, there are pervasive van der Waals forces between compound FS5 surface and Hsp90Nligand-binding pocket. According to the above reasons, inhibitor FS5 can effectively bind to Hsp90.

4 Conclusion

Based on the already available inhibitors, we obtained the complex structure of Hsp90N-FS5 by X-ray diffraction(1.65 A˚), which is deposited in PDB (PDB code 5XRB).The structure demonstrates tight binding of FS5 inhibitor to the ligand-binding pocket of Hsp90, to the binding site of ATP and of other inhibitors. We suggest that FS5 compound can hinder the interaction of Hsp90 with ATP,leading to the loss of function of Hsp90. Our results warrant further studies on FS5 as a potential new drug to conquer cancer. When inhibitor FS5 enters Hsp90Nligand pocket, the L2 undergoes a considerable rearrangement,and H5 of Hsp90 undergoes a significant conformational change.In another perspective,L2 and H5 comprise a vital segment in the ligand-binding pocket, which can directly impact Hsp90 function, as this change is significant for a proper Hsp90 activity.Taking into account the enlargement of the ligand pocket,we can focus on further improvement in the FS5 performance.According to intermolecular force between Hsp90Nand FS5,we can optimize inhibitor-based activity of FS5 by modifying some functional groups to achieve more efficient binding to Hsp90 in order to improve the possibility of clinical application in the future.It is also promising that Hsp90 inhibitors in combination with other valid anticancer therapies provide better results,not only because of their synergistic anti-tumor activities,but also due to the precaution potential in respect of development of drug resistance. The determination of Hsp90N-FS5 complex structure is beneficial for future drug design and has a high relevance for future cancer research.