Construction of ATP-Switched Allosteric Antioxidant Selenoenzyme

Letter pubs.acs.org/acscatalysis

Construction of ATP-Switched Allosteric Antioxidant Selenoenzyme Tiezheng Pan, Yao Liu, Chengye Si, Yushi Bai, Shanpeng Qiao, Linlu Zhao, Jiayun Xu, Zeyuan Dong, Quan Luo,* and Junqiu Liu* State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, 2699 Qianjin Road, Changchun 130012, China S Supporting Information *

ABSTRACT: Rational redesign of allosteric protein offers an efficient strategy to develop switchable biocatalysts. By combining the computational design and protein engineering, a glutathione peroxidase (GPx)-like active center that contains the catalytic selenocysteine (Sec) residue and substrate-binding Arg residue was precisely incorporated into the allosteric domain of adenylate kinase (AKe). The engineered selenoenzyme shows not only high GPx activity but also adenosine triphosphate (ATP)-responsive catalytic property, which is regulated by its opened to closed conformational change upon ATP binding. Theoretical and mutational analysis reveals that the synergistic effect of electrostatic interactions and van der Waals (vdW) interactions for substrate recognition is a major contribution to the high activity. The mitochondrial oxidative damage experiment further demonstrated its antioxidant ability at the subcellular level, offering a potential application toward controllable catalysis in vivo. KEYWORDS: artificial selenoenzyme, enzyme design, adenylate kinase, allosteric switches, antioxidant

T

compounds at the expense of glutathione (GSH).8 Notably, the residues surrounding the catalytic site (e.g., Arg and Gln) prominently contribute to the specific binding of GSH through electrostatic interactions and salt bridges, which provide the substrate recognition capacity to ensure the high activity of GPx. The catalytic cycle is illustrated in Scheme 1.

he significant protective effect of glutathione peroxidase (GPx) is generally regarded as a vital defense in organisms against reactive oxygen species (ROS)-mediated oxidative damage.1 Given the high cost and poor stability of native GPx, the development of efficient GPx mimics has attracted increasing attention during the past few decades.2 Especially, the simple GPx mimic ebselen (PZ51) has already been evaluated clinically for the treatment of stroke.3 Recently, a new strategy, the rational redesign and engineering of protein molecules such as monoclonal antibodies4 and homogeneous enzyme families,5 has also been confirmed to be extremely effective in the construction of artificial GPx enzymes for rivalling the high efficiency and specificity of natural ones. In contrast to synthetic molecules, proteins as a natural source undoubtedly present the most versatile scaffold to design GPx mimics because of their flexible, robust, adaptable, and enzymelike structures. Computational design further pushes this strategy forward by extending the frameworks to nonhomogenous proteins through precisely orienting the catalytic groups and establishing a proper microenvironment (e.g., hydrogen-bonds, hydrophilicity/hydrophobicity, and electrostatic complementarity) in a completely new designed active site for synergistic substrate recognition and catalysis.6 This allows molecular design of bioinspired antioxidant with broader properties to meet diverse demand.7 As a well-known antioxidant selenoenzyme, the structure and catalytic mechanism of GPx have been resolved. GPx contains the rare amino acid selenocysteine (Sec) for the reduction of excessive hydroperoxides (ROOH) into harmless hydroxyl © 2017 American Chemical Society

Scheme 1. Catalytic Cycle of Native GPx

Nevertheless, ROS not only cause macromolecular damage but also act as critical signaling molecules.9 This dual property of ROS requires switchable antioxidant enzymes that can precisely regulate the redox conditions according to the ROS level. In light of the fact that there are close relationships Received: November 17, 2016 Revised: January 7, 2017 Published: February 6, 2017 1875

DOI: 10.1021/acscatal.6b03274 ACS Catal. 2017, 7, 1875−1879

Letter

ACS Catalysis between ROS level and many physiological indexes, we previously developed a Ca2+-activated GPx model based on an allosteric recoverin protein as the Ca2+-rich mitochondrial membrane is constantly attacked by ROS that are formed during aerobic respiration.10 Besides, the depletion of ATP has been detected along with extreme oxidative damage in the process of ischemia−reperfusion injury,11 showing a pressing need for novel ATP-responsive antioxidant enzyme. Because natural allosteric proteins are probably the most reliable dynamic scaffolds given their advantages of robust conformational switches and supersensitive responsiveness, one feasible way to implement this model is to engineer both the catalytic group and substrate binding site into an allosterically regulated protein, and then put it under the control of a conditional trigger by coupling the function of the active site with conformational switches.12 In this work, we report that ATP, an important biogenic stimulant used in cells for biological function activation, can serve as a new trigger for the design and construction of switchable selenoenzymes based on AKe from E. coli (Figure 1). AKe is an allosteric protein which can switch between open

Figure 2. (A) “Open” and “Closed” states of seleno-AKe-F137R (PDB code: 4AKE for the Open form and 1AKE for the Closed form). (B) The GSH-binding cleft of seleno-AKe-F137R. (C) Intermolecular interactions between GSH and seleno-AKe-F137R. (D,E) Electrostatic potential distribution of binding pocket of seleno-AKe and selenoAKe-F137R, respectively.

Figure 1. Design of an ATP-switched artificial GPx based on the protein adenylate kinase.

and closed states through an ATP-driven conformational change.13 Therefore, the utilization of AKe as a scaffold functionalized with a GPx-like active site could provide a unique way to control the activity of an artificial selenoenzyme in response to ATP stimuli. Using molecular docking calculations, all possible GSH-binding sites in the allosteric domain of AKe were screened out. As shown in Figure S1, two specific hotspot areas in the open AKe model have been determined, and the lowest binding energy conformation was chosen from the largest cluster. Structural analysis revealed that the predicted substrate-binding region is surface-enriched with positively charged residues such as Arg119, Arg123, Arg131, Arg156, and Arg167, which may facilitate the recognition of two carboxylate groups of GSH. Other residues like Ser10, Val132, His134, and Asn138 also contribute to van der Waals (vdW) interactions with GSH (Table S1). On the basis of the catalytic mechanism of native GPx, Ile120 was first selected as the target to generate the catalytic Sec site because this site is exposed on the surface of AKe in the ATP-free state, while buried inside the protein in the ATP-bound state (Figure 2A). Also, its particular spatial position is beneficial to the nucleophilic attack of GSH (Figure 2B,C). Then, AKe-GSH recognition can be enhanced by F137R replacement to change the electrostatic potential distribution of active site for improved GPx activity (Figure 2D,E). Site-directed mutagenesis at the designed two positions, as well as their surrounding positions, were performed to evaluate this switchable GPx model and to determine the critical factors responsible for its high activity.

With the active site created by two site-specific modifications, the AKe-based artificial GPx exhibited high activity as expected (Figure 3A). The substitution at position 120 (Ile-to-Sec) endowed AKe with the new enzymatic function to catalyze the reduction of H2O2 by GSH. The catalytic activity was measured to be 120.2 μmol·min−1·μmol−1, which approaches the magnitude level of human plasma GPx activity (Table 1).14 In contrast to other GPx mimics, the catalytic efficiency of ATP-free seleno-AKe is approximately 120-fold higher than that of seleno-organic compound ebselen,15 whereas no activity was detected with wild-type AKe, AKe-I120C/F137R, ATPbound seleno-AKe, or just ATP as control experiment. This indicated that Sec120 was successfully incorporated into a favorable microenvironment and can accelerate the enzymatic reaction. A subsequent F137R mutation (seleno-AKe-F137R) caused a moderately increased activity of 143.1 μmol· min−1·μmol−1, which is consistent with the initial hypothesis that the improved electrostatic environment for GSH recognition has a positive effect on enzyme catalysis. Theoretical analysis also confirmed that F137R mutation avoids the steric interference of Phe137 and promotes the electrostatic interactions between GSH and seleno-AKe-F137R (Table S1). The functional roles of other amino acid residues in the designed active site were identified by a series of mutations on seleno-AKe that might influence substrate binding based on computational analysis of these residues with relatively high 1876

DOI: 10.1021/acscatal.6b03274 ACS Catal. 2017, 7, 1875−1879

Letter

ACS Catalysis

suggested that the synergistic effect of electrostatic interactions and vdW interactions played a crucial role in the high-affinity binding mechanism of seleno-AKe with GSH. Therefore, the designed artificial selenoenzyme showed significant GPx activity because a high affinity for substrate indicated a low Michaelis constant (Km) to enhance the rate of enzymatic reaction. The allosterically regulated activity of seleno-AKe-F137R was first evaluated at different concentration levels of ATP to determine its threshold concentration needed for ATPactivated conformational switches. As shown in Figure 3B, ATP was able to inhibit the activity significantly with an IC50 value of 8 μM. With the increase of ATP concentration up to 1 mM, the same level in a cell,16 the activity of seleno-AKeF137R was almost fully turned off compared to its original level. Because of the irrelevance of ATP to GPx activity demonstrated in the control experiment, it is concluded that the dramatically decreased activity can be attributed to the conformational change of seleno-AKe-F137R upon ATP binding, which mediates the interconversion of the active site from open state to closed state and thereby enables its activity to be delicately controlled by ATP concentration. The repeatable switching of the GPx activity of seleno-AKe-F137R was then performed by using a hexokinase-coupled reaction, during which hexokinase consumed ATP to facilitate the ATP release to reproduce the open-state AKe (Figure 3C).17 The result indicated that the activity is able to recover after the conformational change triggered by the ATP release from seleno-AKe-F137R (Figure 3D). This process can be repeated for multiple cycles with a moderate decrease of activity recovery, showing a robust reversibility of the designed ATPswitched antioxidant selenoenzyme. In order to understand the enzymatic properties of selenoAKe-F137R, a kinetic assay was carried out by varying the concentration of one substrate while keeping the other substrate at a fixed concentration. Double-reciprocal plots of the initial velocity versus substrate concentration yielded a series of paralleled linear plots, which revealed a ping-pong mechanism (Figure 4). As shown in Table S2, the kinetic

Figure 3. (A) Catalytic curves of ATP-bound and ATP-free selenoAKe-F137R. (a) Negative control containing no seleno-AKe-F137R; (b) ATP-bound seleno-AKe-F137R; (c) ATP-free seleno-AKe-F137R. (B) Catalytic activity of seleno-AKe-F137R with different ATP concentrations. (C) Schematic representation of the recovery mechanism of seleno-AKe-F137R activity. (D) Reversible switching of the catalytic activity of seleno-AKe-F137R controlled by ATP.

Table 1. Catalytic Activity of Artificial Selenoenzymes catalysta

activity (μmol·min−1·μmol−1)

wild-type AKe AKe-I120C/F137R ATP-bound seleno-AKe ATP-free seleno-AKe ATP-free seleno-AKe-F137R ATP-free seleno-AKe-R119G ATP-free seleno-AKe-R123G ATP-free seleno-AKe-V132G ATP-free seleno-AKe-H134G ATP-free seleno-AKe-S10G Ebselen15 Sec22 pGPx (human plasma)13

NDb ND ND 120.2 ± 1.5 143.1 ± 2.7 11.6 ± 2.2 25.4 ± 0.6 87.7 ± 3.1 71.1 ± 0.8 74.9 ± 5.3 1 0.05 302

a

The GPx activities of all the catalyst were determined under the same experiment condition. (For more details, please see Supporting Information.) bND: no detectable GPx activity.

intermolecular interaction energies (Table S1). Site-directed mutants at positions 119 and 123 showed a dramatically changed enzyme activity. As shown in Table 1, 10.4-fold and 4.7-fold decreases were observed for seleno-AKe-R119G and seleno-AKe-R123G, respectively. The calculated structure and energy (Figure 2C and Table S1) revealed that the guanidine groups of both arginines were very close to the negatively charged carboxyl groups of GSH and provide strong electrostatic interactions (−4.37 and −4.02 kcal/mol) for substrate recognition. On the other hand, the vdW interactions of Val132 and His134 (−1.03 and −2.02 kcal/mol) also partially contributed to the binding process. The catalytic activities of V132G and H134G mutants were found to decrease by 27.0% and 40.8% when removing the side chains of the two residues, which resulted in the local instability of binding between AKe and one side of GSH. Furthermore, a similar result of 37.7% decreased activity were demonstrated for S10G mutant because Ser10 provided a considerable vdW interaction energy (−1.24 kcal/mol) to stabilize the other side of GSH. All these findings

Figure 4. Double reciprocal plots of the reduction of H2O2 by GSH under the catalysis of seleno-AKe-F137R ([E0] = total enzyme concentration). (A) [E0]/V0 versus 1/[GSH] (mM−1) at [H2O2] = 0.25 (▲), 0.50 (●) and 1.00 mM (■). (B) [E0]/V0 versus 1/[H2O2] (mM−1) at [GSH] = 0.25 (▲), 0.50 (●) and 1.00 mM (■).

constants kcat/Km H2O2 and kcat/Km GSH are 105 M−1·min−1 and 106 M−1·min−1, respectively. These constants are lower than those of native GPxs but comparable to those of some GPx mimics, such as Se-4 and protein-imprinted GPx mimics.18 In addition, the kcat and Km parameters of each mutants have been determined (Table S3). The results indicated that the activity variation of these mutants was mainly due to their binding affinity with GSH, which is consistent with the contribution of each mutated residue for substrate stabilization 1877

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oxidative damage such as ischemia−reperfusion injury in pathophysiology.

by theoretical analysis. Moreover, the optimal pH and temperature were determined to be 8.0 and 28 °C, respectively (Figure S9), while native GPx has an optimal pH of 8.8 and optimal temperature of 50 °C.19 Finally, the antioxidative effect of seleno-AKe-F137R was evaluated by its ability to protect mitochondria against oxidative damage. The integrity of mitochondria under heavy oxidative stress was studied by the swelling assay that was monitored by the decrease in absorbance at 520 nm.20 The oxidative damage of mitochondria was induced by incubating with sulfate/ ascorbate and then measured by light scattering. When ATPbound seleno-AKe-F137R was added, the absorbance of the swelling mixture is close to that of the damaged mitochondria (Figure 5A). In contrast, ATP-free seleno-AKe-F137R sig-

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscatal.6b03274. Experimental details including theoretical calculation, structure comparison, enzyme engineering, SDS-PAGE, ESI-MS, circular dichroism, ITC, activity assays, and the antioxidant effect of the selenoenzyme characterized by mitochondria damage models (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Zeyuan Dong: 0000-0001-6509-9724 Junqiu Liu: 0000-0002-8922-454X Notes

The authors declare no competing financial interest.

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Figure 5. Determination of mitochondria swelling (A) and lipid peroxidation (B) by oxidant. (A) Control with no oxidant (a); 1 μM and 0.5 μM of ATP-free seleno-AKe-F137R (b,c); 1 μM of ATPbound seleno-AKe-F137R (d); mitochondrial damage in the presence of ferrous sulfate/ascorbate and no GPx mimics (e). (B) Control with no oxidant (a); 1 μM and 0.5 μM of ATP-free seleno-AKe-F137R (b,c); 1 μM of ATP-bound seleno-AKe-F137R (d); mitochondrial damage in the presence of ferrous sulfate/ascorbate and no GPx mimics (e).

ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (no. 21420102007, 21574056, 91527302, 21234004, 21221063, and 21474038), the Chang Jiang Scholars Program of China, the Science and Technology Development Program of Jilin Province (20140101047JC and 20160520005JH), and project 2016014 Supported by Graduate Innovation Fund of Jilin University.

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nificantly prevented mitochondria from swelling and displayed a concentration-dependent effect. This finding demonstrated that seleno-AKe-F137R has good biocompatibility and ability to protect organelles from oxidative damage at subcellular level. In the process of mitochondrial oxidative damage, malondialdehyde (MDA) was generated as the final product of free-radicalmediated lipid peroxidation, whose accumulation is an important biomedical indicator and can be determined by the thiobarbituric acid (TBA) assay.21 As shown in Figure 5B, adding ATP-bound seleno-AKe-F137R was unable to prevent MDA from accumulation, and the final amount of MDA was at almost the same level as that of the damage group. However, once ATP-free seleno-AKe-F137R was added in the reaction system, it exhibited obvious inhibition to MDA accumulation in a concentration-dependent manner. The results revealed that the biological effect of seleno-AKe-F137R is also regulated by ATP through the allosteric mechanism of AKe. In conclusion, a novel ATP-responsive switchable selenoenzyme was constructed on the basis of the allosteric AKe scaffold through computational design and protein engineering with a GPx-like machinery. The resulting seleno-AKe-F137R exhibited not only high enzymatic activity and antioxidative effect to compete the native GPx but also reversibly regulated behavior with the excellent sensitivity of μM level to ATP when its conformation shifts upon ATP binding. Furthermore, some key amino acid residues such as Arg119, Arg123, Val123, His134, and Ser10 have been determined to elaborate the high-affinity binding mechanism of this artificial selenoenzyme contributed to its high activity. This study may have potential application prospect in the case of low ATP concentration with extreme

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DOI: 10.1021/acscatal.6b03274 ACS Catal. 2017, 7, 1875−1879