Crystallogenesis of Steroid-Converting Enzymes and Their

on a Thieno[2,3-d]pyrimidin-4(3H)-one Core Applying Molecular Dynamics Simulations and Ligand–Protein Docking. Sampo Karkola , Annamaria Lilienk...
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CRYSTAL GROWTH & DESIGN 2007 VOL. 7, NO. 11 2206–2212

ReViews Crystallogenesis of Steroid-Converting Enzymes and Their Complexes: Enzyme–Ligand Interaction Studies and Inhibitor Design Facilitated by Complex Structures† Mausumi Mazumdar,‡ Ming Zhou,‡ Dao-Wei Zhu, Arezki Azzi, and Sheng-Xiang Lin* Laboratory of Oncology and Molecular Endocrinology, CHUL Research Center (CHUQ) and LaVal UniVersity, 2705 BouleVard Laurier, Ste-Foy, Quebec G1V 4G2, Canada ReceiVed July 27, 2007; Accepted August 20, 2007; ReVised Manuscript ReceiVed August 20, 2007

ABSTRACT: Crystal growth of steroid enzymes in complex with hydrophobic ligands is hindered by the low solubility of both partners. However, their interaction can promote highly soluble complexes, leading to well diffracting crystals and high-quality structures. The moderate amphiphilic property of polyethylene glycol increases steroid solubility and facilitates complex formation, but altered orders of soaking can lead to a variety of complex formations and different structures. Understanding the latter mechanism is important for inhibitor design. Thus, different enzyme–ligand complex structures have provided detailed pictures of steroid alternative binding and multispecificity of the enzymes, as, for example, in the case of human 17β-hydroxysteroid dehydrogenases (17β-HSD) type 1 due to C19 steroid pseudosymmetry or in human 17β-HSD type 5 due to the spatial binding site. These complex structures also provide insights into the dynamics of the enzyme binding and catalytic process. Hybrid inhibitors constituted by the steroid and adenine cores of the native substrate and cofactor can properly occupy their original binding sites in 17β-HSD1, leading to an inhibition constant at the nanomolar level. Altered complex crystallization and even a modified order of soaking may result in different complex structures, shedding light on detailed protein–ligand interactions as well as on the enzyme reaction mechanism. These results highlight the important contribution and high significance of the crystal growth mechanism to protein structure determination and function study, as well as to drug design for medicinal applications.

1. Introduction: Toward a Rationale for Steroid Enzyme Crystallization In recent decades protein crystallography has emerged as an essential tool to study the binding geometry and interaction patterns of protein–ligand and protein–protein complexes. To understand the structure–function relationship of various proteins in the postgenomic era, crystallography remains one of the most fundamental tools. The determination of accurate complex structures is an essential input for structure-based drug design, and crystallization of complexes remains a critical step. Understanding the factors involved in the formation of such complex crystals implicates an understanding of fundamental physicochemical mechanisms. It is believed that hydrophobic interactions constitute the main thermodynamic driving force for the binding of small molecule ligands to their cognate protein targets.1 It was reported that when a ligand binds to a solvated protein, water in the binding cavity is expelled into the bulk. In many cases of protein–ligand † Part of the special issue (vol 7, issue 11) on the 11th International Conference on the Crystallization of Biological Macromolecules, Que´bec, Canada, August 16–21, 2006 (preconference August 13–16, 2006). * Corresponding author. E-mail: [email protected]. ‡ These authors have made similar contributions to this work.

binding, the energy of interaction between water in the binding cavity and the protein is roughly comparable to the energy of the interaction between the complexed ligand and protein. Hydrophobic enclosures found in ligand–protein systems aid recognition by perturbing the solvation of the binding cavity, which in turn results in a relative stabilization of the bound complex. Such hydrophobic regions are compelling targets for drug design because it is possible, with suitable ligands, to obtain exceptionally large enhancements of potency with a minimal change in molecular mass.2 When the interaction between a protein molecule and that of a ligand is hydrophobic, for example, in the case of steroid enzymes, it poses a challenge in complex crystal formation. The steroid converting enzymes have highly hydrophobic active sites, in accordance with the hydrophobic nature of their substrates. The concentration of the protein used for crystallization is generally elevated, and conversely the hydrophobic ligand has a very low solubility. The solvents of choice that are used to increase ligand solubility (e.g., DMSO, ethanol, etc.) are in general not favored by proteins. In this review, we attempt to analyze the crystallizations of representative steroid enzymes in complex with their multiple substrates to evaluate the implication and contribution of different crystal growth procedures and whether or how the crystallization

10.1021/cg700709k CCC: $37.00  2007 American Chemical Society Published on Web 11/07/2007

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Crystal Growth & Design, Vol. 7, No. 11, 2007 2207 Table 1. Detailed Crystallization Strategies by Vapor Diffusion of Various 17β-HSD1 Complexes Solved To Datea

PDB-ID

enzyme complexed with-

reference

crystallization method cocrystallization cocrystallization (low protein concentration in PEG at high concentration) cocrystallization (low protein concentration in PEG at high concentration) cocrystallization combined cocrystallization and soaking combined cocrystallization and seeding combined cocrystallization and soaking soaking with gradual ligand additions over a time rang soaking with gradual ligand additions over a time range soaking with gradual ligand additions over a time range soaking with gradual ligand additions over a time range

1IOL 1FDS

17β-estradiol 17β-estradiol

5 6

1FDT

estradiol and NADP+

6

1EQU 1DHT 3DHE 1I5R 1JTV 1QYV 1QYX 1QYW

equilin complexed with NADP+ dihydrotestesterone dehydro-epiandrosterone EM-1745 testosterone NADP androstenedione and NADP androstanedione and NADP

a

22 23 23 24 25 26 26 26

initial protein concentration (mg/mL) 15 4.5 4.5 not available 10 10 16 17 12–15 12–15 12–15

Crystallization assays were carried out at 22 or 27 °C (for refs 23 and 26).

methods result in altered protein–ligand structures and different interactions between the partners. This turns out to be critically important to our study of enzyme function as well as to that of inhibitor design. Human 17β-hydroxysteroid dehydrogenases (17β-HSDs) are responsible for the last step in biosynthesis or the first step of inactivation of various active estrogenic and androgenic steroids. The first member of this family to be crystallized was 17βHSD13 more than a decade ago. Following the apo-enzyme structure,4 the first complex structure,5 and the ternary complex were reported.6 To date more than a dozen enzyme–steroid complex structures for type 1 and eight for type 5 (17β-HSD5/ AKR1C3) have been solved. These structures combined with functional studies clearly illustrate the interactions between the enzymes and their ligands. High-affinity inhibitors were designed based on the structural information obtained. In some cases, varying the crystallization process yielded different complexes and structures, demonstrating modified interactions between the macromolecule and the ligands. The 3R-hydroxysteroid dehydrogenases (3R-HSDs) are members of another steroid converting enzyme family. 3R-HSDs inactivate the most potent androgen 5R-dihydrotestosterone (5RDHT) to 5R-androstane-3R,17β-diol (3R-diol).7 To date, a total of seven 3R-HSD structures have been reported. The human 3R-HSD structure (3R-HSD type 3) was solved at 1.25 Å in complex with testosterone (T) and NADP+.8 The structures of its complex with NADP+/citrate acetate and with NADP+/ ursodeoxycholate (3.0 Å),9 as well as that with NADP+ (1.9 Å),10 were also published. The additional three structures are from rat (3R-HSD/NADP+/T11) and Comamonas testosteroni (apoenzyme and 3R-HSD/NADP+12). Currently, there are three structures of human type 1 11βhydroxysteroid dehydrogenase (11β-HSD1) available in the Protein Data Bank13,14 (in preparation) and one from guinea pig.15 The enzyme modulates ligand access for glucocorticoids and mineralocorticoids. The glucocorticoid hormones (cortisol in humans) play essential roles in adaptation to stress, regulation of metabolism, and inflammatory responses; thus, specific inhibition of tissue-specific glucocorticoid activation by 11βHSD1 constitutes a novel and promising concept in the treatment of metabolic syndromes and cardiovascular diseases.

2. Cocrystallization Cocrystallization is one of the most commonly applied methods to grow crystals of protein complexes, in which the ligand and protein partners are present together during crystallization.16,17 This process facilitates protein crystal growth. In practice, one starts from low concentrations of ligand and

protein to form the complex in the solution followed by concentration of the complex. As binding neutralizes the hydrophobicity on both protein and ligand, the total ligand (free and bound) concentration in the solution can become much higher than the free ligand solubility. Complex formation will permit new introduction of steroids into the protein solution, thus gradually saturating the binding sites of all protein molecules. In such a way, complex formation stabilizes floppy looped structures, often observed at the protein binding site, and consequently favors crystal growth. Indeed, introduction of a ligand to an unstable protein before crystallization, during purification, or before protein expression stabilizes the conformation of the produced protein, thus helping expression, purification, and crystallization of the protein. This has been demonstrated in the crystallization and structure determination of complexes between 17β-HSDs,3,18–20 11β-HSDs,21 and their steroid ligands and analogues (examples in Table 1). The first complex crystal of 17β-HSD1, i.e., that with E2, was obtained by cocrystallization. The enzyme/steroid complex was formed with a gradual concentration of diluted enzyme solution in the presence of a starting concentration of 25 µM E2. When E2 was bound to the enzyme, new steroid molecules could be dissolved leading to the further occupancy of the binding sites of other enzyme molecules. In this way, a final stoichiometry of 1:2 of enzyme (300 µM) versus E2 (600 µM) was reached, as verified by 14C-labeling on the steroid. The E2 concentration in the complex was more than 20 times higher than the solubility of the free steroid, indicating the close interaction of both hydrophobic partners, the binding site of the enzyme, and the steroid. This mixture was then crystallized using the vapor diffusion method, and structure solution of the first complex of 17β-HSD15,27 was obtained. The complex crystals diffracted to 2.3Å, with data collection on an in-house facility (with a Rigaku R200 generator and R-axis II image plate detector, ref5). In the 17β-HSD1/E2 complex structure, the enzyme binding site is a narrow hydrophobic tunnel highly complementary to the steroid. The total buried surface for E2 is 229 Å2, accounting for 92% of the surface area of the free steroid. Accordingly, such buried surface for the enzyme binding site is 340 Å2, 71.5% of which is hydrophobic and contributes the main thermodynamic force for steroid binding. The clear electronic density of E2 in the complex, demonstrated by the Fo – Fc omit map contoured at 2.8 σ, confirms the full occupancy of the binding site through cocrystallization (Figure 1), in accordance with the obtainable high complex concentration verified by radioisotope assay. For the crystal growth and structure determination of the type 5 17β-HSD enzyme, the specific cofactor NADP+ was introduced to the Escherichia coli cells expressing the enzyme just before cell disruption to enhance the purification yield. No

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Figure 1. (A) Cartoon representation of a monomer of 17β-HSD1 with 17β-estradiol positioned at its binding site (prepared using SETOR). The estradiol molecule and side chains of residues Ser 142, Tyr 155, Lys 159, His 221, and Glu 282 in the active site are shown in white. (B) View of the calculated final Fo – Fc electron density omit map contoured at 2.8σ and the final refined model for the 17β-estradiol molecule (generated using the program O). The figure is adapted from ref 5.

cofactor was added to the succeeding chromatographic columns or during the concentration or crystallization steps.28 Two crystal forms were obtained, whose structures were determined demonstrating ternary enzyme complexes with androstenedione (4dione) and testosterone. It was only after structure determination that it became clear why NADP+ remained bound in the binding site all the way from cell disruption to crystal preparation. Indeed, it has been demonstrated that two salt bridges are formed between Glu222 from the flexible loop-C and Lys270 from β-strand 266–271, between Glu222 and Tyr24 from the β-strand 19–24. Such a safety-belt seems to shift the loop-C much closer to the cofactor, significantly enhancing the cofactor affinity.29 This was confirmed by enzyme steady-state kinetics showing a Km of about 7 nM for NADP+.29 It is a good example illustrating how understanding the crystal growth process can contribute to that of the enzyme mechanism. Another interesting case concerns preparation of crystals of the 17β-HSD5/inhibitor (EM1404) complex. Here, the first attempts consisted of soaking the enzyme/NADP+ crystals with EM1404, making use of the increased solubility of the steroids or analogues in PEG (polyethylene glycol) solution.29 Surprisingly, this approach resulted in a 17β-HSD5/PEG fragment/NADP+ complex after crystal structure determination. The method was subsequently modified so that to gradually increase the concentration of the enzyme–steroid solution and to cocrystallize the inhibitor complex. Because of the significantly lower solubility of EM1404 compared to other steroids, an initial solution containing 2.5 µM inhibitor and a similar enzyme concentration was gradually concentrated in the presence of NADP+, which was then diluted again and followed by introduction of new EM1404 molecules. This repetition permitted the saturation of the enzyme resulting in the complex crystallization and structure determination.28,29 The EM1404 complex formation is again shown by its clear electron density. In fact, the solved structure demonstrates important hydrophobic interactions and some hydrogen bonding of the inhibitor at the enzyme binding site, in spite of the ligand occupation of only part of the site. In the complex structure, a wall around the middle of the binding pocket is lined with several hydrophobic residues that form a cavity complementary to the shape of EM1404. Such hydrophobic

interactions explain the formation of the enzyme steroid complex at 300 µM after binding that is about 100 times the inhibitor solubility. In the 17β-HSD5/PEG fragment/NADP+ complex crystal a symmetrically related enzyme molecule is located near loopC. This may force the loop to move closer to the binding site entrance, thus blocking the exchange of PEG and EM1404 when soaking the enzyme/NADP binary complex. This effect may additionally be enhanced by the real low solubility of EM1404 as compared to other steroids.30 The above example suggests that cocrystallization, in which the molecule complexes are formed in the presence of both ligand and cofactor at low concentration followed by gradual concentration, can lead to more success in obtaining the protein–ligand complex crystals corresponding to the true protein mechanism in solution. The ternary complex crystals of human type 3 3R-HSD/ NADP+/T were also obtained by the cocrystallization method with gradual concentration mentioned above.31 Its complex structure was resolved at 1.25 Å with clear electron densities of the cofactor NADP+ and the substrate T8 revealing detailed interactions between the enzyme and its ligands. The two ternary complex structures reported for human 11βHSD113 and one binary complex structure of 11β-HSD1/NADP from guinea pig were again obtained from cocrystallized crystals.15 As indicated by Hosfield and his colleagues, the steroid corticosterone was added even earlier during the protein expression step.13 Once the protein/ligand complex formed, it stabilized the protein conformation and neutralized hydrophobicities on both the protein binding site and steroid. The structures were solved at 1.55 and 1.8 Å for the enzymes of human origin and that from guinea pig was resolved at 2.5 Å. An additional example is provided by DHEA-sulfotransferase (DHEA-ST). The production of ternary complex crystals proceeded more easily by mixing DHEA-ST and the nonsulfonated cofactor analogue 3′-phosphoadenosine-5′-phosphate (PAP) and the well solution, followed by the addition of small volume solution of DHEA, which has a higher solubility than other steroids in solution.32

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Crystal Growth & Design, Vol. 7, No. 11, 2007 2209

This series of examples clearly shows the close relationship between the mechanism of crystal growth and the protein–ligand interactions.

3. Crystal Soaking Soaking is another frequently used method to obtain protein/ ligand(s) complex crystals. In this method, the apo-protein, or mother crystals (possibly in a binary complex) can be made first. Then ligands will be introduced into the crystallization drops under suitable conditions, followed by diffusion and binding to the protein molecule resulting in the formation of various complex crystals. The size and configuration of the channels within the lattice of apo-protein crystals will determine the maximum size of the ligands that may diffuse in.33 In comparison with cocrystallization, one advantage of soaking is that it allows us to make just one batch of apo-enzyme crystals followed by the introduction of different ligands into separate drops, yielding different complex crystals. This is necessary as the steroid enzymes often possess multispecificity and can bind different steroids. To obtain crystals of DHEA and DHT in complex with 17β-HSD1, apo-enzyme crystals were obtained which were then soaked with either 1 mM of DHEA or DHT in PEG 3500. The complex crystals diffracted to 2.3 and 2.24 Å resolution, respectively.23 In the case of the 17βHSD1/T complex, the addition of the ligand was carried out gradually during successive rounds extending over a few days to reach the final testosterone concentration of 1 mM in the crystallization drop. Complex crystals were thus obtained, and the structure was solved at a resolution of 1.54 Å. Addition of as high as 1 mM T during one attempt led to crystal cracking followed by its final dissolution.25 The low solubility of steroids or their analogues is often a limiting factor in the acquisition of enzyme/steroid complexes by soaking. For example, in another attempt to obtain the 17β-HSD1/ E2 complex,6 the enzyme concentration was reduced to 4.5 mg/ mL from 10 to 15 mg/mL, and the concentration of PEG 4K was increased from 24 to 32% compared with other reported crystallization conditions. At the higher concentration of PEG, the solubility of E2 increased and the presence of three molecules of steroid per enzyme-binding site in the crystallization drop was attained. Two crystal structures were solved by these authors, with one data set collected at room temperature and the other at low temperature (-150 °C). The crystals grown at room temperature diffracted at 1.7 Å and showed one E2 molecule along with 127 water molecules and residues 1–285. The low temperature crystals diffracted to 2.2 Å, and the electron density of the loop residues, NADP+ along with E2 was obtained. 17β-HSD1’s preference of NADP over NAD was used to significantly improve the purification and led to the first enzyme crystals.3,34 This was demonstrated structurally in the ternary complex,6 in accordance with kinetics studies.35,36 The role of Lys159 in aiding the catalytic reaction via the cofactor that had been proposed in the binary complex structure by Azzi and colleagues5 was also confirmed with the crystal structure of the ternary complex. Soaking failed however when an attempt was made to crystallize the 17β-HSD1 binary complexes with 3β-androstanediol and 5R-androstenediol (Mazumdar et al., unpublished results). Many X-ray data sets were obtained but with no electron density of the ligand. An attempt to introduce the cofactor NADP+ prior to soaking with the substrates resulted in the successful solution of the ternary complex structures involving these steroids. Thus, during the complex crystal growth, the sequence of different ligand additions may be

important. In most steroid dehydrogenases (i.e., short chain alcohol dehydrogenases), the cofactors bind first37 to the enzyme and the reaction obeys the bi bi mechanism. The introduction of substrate and cofactor into the protein may result in the reactive state of the enzyme. When an attempt was made to obtain DHT and T ternary complexes with 17βHSD1 and the cofactor NADP+, the crystallization resulted in the ternary structure of their oxidizing products, namely, androstanedione (A-dione) and 4-dione, respectively,26 demonstrating the favored reaction under the crystallization condition. This is a very important point to note while planning complex crystal formation.

4. Combined Cocrystallization and Soaking or Seeding As mentioned above, soaking provides a convenient means, but it does not always yield the desired complex crystals. In the case of the crystallization of 17β-HSD1 in complex with inhibitors EM519 and EM-553 (ref 38, Figure 2), soaking the fully grown crystals with the inhibitors led to the cracking of the crystals. Finally, the combined method was employed, i.e., soaking the very small starting crystals with the inhibitors led to successful complex crystal production. In fact, this is between the soaking and the cocrystallization method mentioned above, as the nucleus formation and crystal growth are under different conditions. The combination approaches often yield good results. The 17β-HSD1 binary complexes with dihydrotestosterone (DHT) and 20R-hydro-progesterone (20R-OH-P) were also obtained by seeding the apo-enzyme crystals into the cocrystallization drop; for example, first very small apo-enzyme crystals were obtained (0.2 × 0.16 × 0.06 mm3) which were then removed to drops containing both protein (10 mg/mL) and steroid (1 mM). The protein and precipitant concentrations were lower than in the starting crystallization conditions (corresponding to the metastable zone). The small apo-enzyme crystals continued to grow under the cocrystallization condition with a substantial increase in volume and high-quality crystals were eventually obtained. The crystals diffracted to 2.2 Å resolution.39 As shown in the above examples, the order of ligand introduction is especially important when soaking is applied. Kinetic study often shows that some enzymes sequentially bind cofactor and substrate. After binding with the first ligand, the enzyme would process a conformational change, which facilitates the binding of the second ligand. As reported in ref 38, when small crystals of apo-17β-HSD1 appeared in the drop, 4 to 5 additions of NADP+ were carried out to reach a final concentration of 5 mM, followed by the addition of the inhibitors EM519 and EM553. Both inhibitors had a steroidal backbone with extremely low solubility, and their final concentrations reached about 1 mM in the presence of PEG. The additions were carefully made at the edge of the drop so that the ligands reached the crystals by diffusion during crystal growth with the least possible perturbation of the crystal lattice.

5. Crystal Structures and Enzyme–Ligand Interaction The first binary complex structure of 17β-HSD1 showed that O3 of E2 formed an H-bond with the Nε2 atom of His221 (3.1 Å) and Oε2 of Glu282 (2.7 Å). The hydroxyl at O17 of E2 formed two H-bonds, one with the hydroxyl oxygen of Tyr155 (3.5 Å) and the other with Ser142 (3.1 Å). This demonstrated the strong interaction of E2 with the enzyme, in addition to the hydrophobic interactions that account for the major binding energy. The large hydrophobic surface contributes to the main thermodynamic forces and is provided by hydrophobic and

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Figure 2. Chemical structures of the ligands E1, EM-519, EM-553, EM1404, and EM1745 (drawn using Chemdraw). Table 2. Crystal Structures of 17β-HSD1 Complexes with Various Ligands and the H-Bond Interactions They Form with Specific Protein Residuesa PDB-ID

substrate

Tyr155

Ser142

1FDS 1IOL 1FDT 3DHE 1DHT 1JTV 1QYX 1QYW

E2 E2 E2/NADP DHEA DHT testosterone A-dione/NADP 4-dione/NADP

O17 (3.37) O17 (3.5) O3 (1.17)

O17 (3.2) O17 (3.1) O3 (1.15) O1 (3.33) O17 (3.38)

a

Glu282 O3 (2.7) O3 (3.32) O3 (3.27) O17 (2.66)

His221

crystallization method

O3 (3.14) O3 (3.10) O3 (2.96) O3 (2.50) O17 (2.67) O17 (2.67)

soaking cocrystallization soaking seeding and cocrystallization soaking soaking soaking in the reactive state condition soaking in the reactive state condition

O17 (2.65)

The H-bond distances in Å units are given in parentheses.

aromatic residues Val143, Leu149, Pro187, His221, Val225, Phe254, Phe259, Leu262, and Leu263 (Table 2, ref 5). Furthermore, the complex structures with C19 steroids explained the critical role of Lys149 in the recognition of C-18 and C-19 steroids, which was also confirmed by variation studies.23 The complex structures with DHT and DHEA showed the absence of an H-bond with Tyr155 but that the interaction with Glu282 was still present, suggesting that Glu282 possibly plays a role in substrate recognition during binding. From the binary structure of the 17β-HSD1/T complex (Table 2), a preference for the reversible mode of binding was observed that shed light on the mechanism for recognition of various ligands by 17β-HSD1. It has often been observed that crystal growth and structure determination work together with other physicochemical studies of the proteins to confirm the experimental results. In fact, the distance 24.7 Å between the bound NADPH and the sole tryptophan (Trp46) in a 17β-HSD1 subunit was revealed in the enzyme’s structure, and the results turned out to be in excellent agreement with that obtained from fluorescence energy transfer experiments (26.9 Å), suggesting a similar enzyme conformation in the crystal and in solution.40 In the case of 17β-HSD5 four large loops, namely, Loop-A (24–33), Loop-B (117–143), Loop-C (217–238), and Loop-D (301–323) form the substrate and cofactor binding sites of the enzyme (Figure 3). Potential hydrogen bonding partners from the enzyme are located either at the base or at the entrance of

the binding pocket and include Tyr24, Ser118, Ser129, Asn167, and Arg226. The type 5 enzyme differs significantly from the type 1 enzyme by possessing a spacious and flexible steroid binding site (Figure 3). This is estimated to be about 960 or 470 Å3 in the 17β-HSD5 ternary complex with T or 4-dione, respectively, indicating a significant conformation change in the two substrate complexes. The binding site is considerably more flexible and spacious than that of the type 1 enzyme, which measures about 340 Å3, and remains almost identical in various complexes.41 From a comparison of both the enzymes, recognition and distinction of C18 and C19 steroids are very important in the mechanism of the type 1 enzyme, but for type 5 the binding is “non-positional and non-stereoselective”.42 In the 17βHSD5 structure, a safety belt was found, indicating a very strong binding of the cofactor. Similarly, after solving human 3RHSD3/NADP+/T crystals, the authors subsequently attempted to make crystals in the absence of cofactor, but without success.8 Similar to 17β-HSD5, NADP+ has a very high affinity to the enzyme, and the cofactor may come from the cells itself and remain bound with the enzyme through different steps. In the 17β-HSD1 complex, the cofactor NADP+ is in the extended sys-conformation but in type 5 it is oriented in the extended anticonformation. The transfer of hydrogen from the NADP+ to the steroid is at 4-pro(S) from the C-4 position of the nicotinamide in the case of type 14, but for type 5 it is at 4-pro(R) from the C-4.29

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combined with minimal energy method to model the intermediate state, can demonstrate the dynamics of the catalysis.29,30,43 In a recent study by Steuber and colleagues,44 preliminary work explored the effect of soaking time and methods of crystallization on crystal complex formation with aldose reductase and zopolrestat. The study showed that varying the soaking time could result in different interaction patterns in the formed complexes, while time variation during cocrystallization did not affect the interactions. More studies will be required to conclude the effect and to understand the mechanism. Again, we need to pay special attention to the choice of soaking conditions.

7. Complex Crystal Structures Facilitating Inhibitor Design X-ray crystallography is essential as it provides the structure of the target protein allowing detailed insights into its interaction with a bound ligand. Prerequisite for such a structure-based approach is correct model building and error-free interpretation of electron density maps. However, even in the case of successful crystal structure determination, it is not necessary to provide a definite answer with respect to binding mode corresponding to the in vivo situation. The flexibility of a protein as seen from the B-factors renders a special challenge in the design of inhibitors. A complex formation before introduction of other hydrophobic reagents into the crystallization system is likely to result in a complex closer to the solution condition under which the steroid enzyme does the catalysis. The information obtained from the binary and ternary complexes of 17β-HSD1 was very supportive in the design of hybrid- inhibitors, with some modeling studies that finally approved an initial idea to make inhibitors interacting with the enzyme in double sites and led to the synthesis of EM-1745 (Figure 2), with nanomolar level affinity. The 17β-HSD1/EM1745 complex crystallization39 was obtained by soaking. The electron density map showed clear density of the inhibitor with the steroidal core and the adenine core in their respective binding sites.24 Many enzyme–inhibitors have been designed based on crystal complex structures45–47 but have not resulted in enough effective compounds as often occurs for most enzymes. One of the reasons could be that a single complex structure with a particular ligand in the case of multispecific enzymes does not tell the whole dynamic interaction story. Thus, crystal structure information, the most valuable input for inhibitor design, needs to be scanned using more intensive crystallogenesis studies. Figure 3. Stereoviews of the different orientations of the substrate binding site in type 5 17β-HSD. (A) 17β-HSD5/4-dione/NADP structure, 4-dione is in magenta. (B) 17β-HSD5/EM1404/NADP, EM1404 is in cyan. (C) 17β-HSD5/testosterone/NADP, testosterone is in brown. (D) Superimposition of 17β-HSD5/EM1404/NADP and 17β-HSD5/PEG/NADP complexes near the substrate entrance. The blue dash line represents the CR residues (131–133) in Loop-C of 17βHSD5/EM1404/NADP complex with weak electron density and is thus not included in the final refined PDB coordinates. The green dash line represents those unmodeled residues (127–137) in 17β-HSD5/PEG/ NADP complex. The figure is adapted from ref 30.

6. Dynamics of the Enzyme Binding and Catalytic Processes As a functional molecule in the crystal form, enzyme binding and catalysis are dynamic processes. A complex structure can only demonstrate one snapshot at a particular condition. In accordance with the kinetics, a series of enzyme–ligand (substrate, product, and cofactor) binary and ternary complexes,

8. Conclusions From this review, we can see that protein crystal growth and design contribute significantly to macromolecular structure determination. The choice of the crystallization methods can be very important for the structural study, and cocrystallization is often favorable to lead to the desired structural information describing the true protein mechanism in solution. During cocrystallization, the interactions between the enzyme and the ligands are closer to the natural environment, but in soaking the crystals are already formed and we should pay attention to the possible side effects by new ligand introduction. From the above discussion, we can clearly see the intimate relationship between protein crystal growth, structure–function study, and inhibitor design. In fact, they are different aspects of a central theme, that is, the physico-biochemistry of the macromolecule. Thus, the understanding of one aspect can often facilitate that of the other, or verify the results from the other. A combination of these approaches is necessary to advance our

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study synergistically, leading to a profound understanding of the protein structure and function, facilitating the new development of structural biology and protein science.

Appendix A technical summary for the crystal growth of steroid enzyme complexes: (1) Introducing the steroid during protein expression and purification can stabilize protein conformation, thus helping crystallization. (2) Concentrating the protein together with steroid starting from low concentration can overcome steroid solubility problems to achieve cocrystallization. (3) As the steroid converting enzymes often have multispecificities, soaking with different steroid substrates with apo-enzyme crystals simplifies the work, especially good for crystal growth of binary complexes. (4) PEG can increase the solubility of steroids but may occupy the hydrophobic binding pocket. So steroid should be present from the initiation of crystal growth to facilitate complex formation. (5) The addition of the steroid has to be done slowly (many times in multiple rounds) in the crystal drop for soaking. (6) Ligands can be introduced to drops with small apo-enzyme crystals for a combined cocrystallization and soaking. (7) Using small seeds from apo-enzyme crystals in a cocrystallization drop is another way to obtain crystals of enzyme complexes, as a combined cocrystallization and seeding. (8) If the affinity of the steroid is low for the particular enzyme, introduction of the cofactor prior to adding the steroids can help in the complex crystal formation.

Abbreviations DHEA DHT 4-dione DMSO E1 E2 HSD NAD(P)+ PEG T

dehydroepiandrosterone dihydrotestosterone androstenedione dimethyl sulfoxide estrone estrodiol hydroxysteroid dehydrogenases nicotinamide adenine dinucleotide (phosphate) polyethylene glycol testosterone

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CG700709K