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Role of K‑Loop Cysteine Residues in the Marburg Virus Protein VP24−Human Keap1 Complex Nisha Bhattarai,† Bernard S. Gerstman,†,‡ and Prem P. Chapagain*,†,‡ †

Department of Physics and ‡Biomolecular Sciences Institute, Florida International University, Miami, Florida 33199, United States

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ABSTRACT: Binding of the Marburg virus protein VP24 to the human Keap1 protein allows the nuclear accumulation of Nrf2, activating cytoprotective antioxidant response pathways during the viral life cycle. We investigate the molecular level details of VP24−Keap1 interactions for both Marburg and Ebola VP24. Our results show that the presence of the cysteine residues in the K-loop region of mVP24 provides strong interfacial interactions with Keap1, including hydrogen bonding and S−H···π interactions, facilitating the formation of a stable complex with Marburg VP24. These cysteine residues are not present in eVP24, which does not form a stable complex with Keap1. These results provide insights into how the Marburg virus, but not Ebola, is able to activate the antioxidant response pathways through direct interactions with Keap1.

1. INTRODUCTION Pathogenic Ebola and Marburg viruses belong to the Filoviridae family.1 There are seven proteins in these viruses: the major matrix protein VP40, the minor matrix protein VP24, transcription factor VP30, nucleocapsid protein VP35, nucleoprotein NP, glycoprotein GP, and the RNA-dependent RNA polymerase L.2 Most of these proteins are known to perform multiple functions during the virus life cycle. For example, VP40 is a transformer protein3,4 which can undergo major structural rearrangements from dimer to hexamer or octamer structures, each with a distinct function.5 VP24 is also a multifunctional protein and has important roles in the formation of the nucleocapsid and viral matrix,6,7 interferon signaling, and host adaptation,8 among others. However, the molecular mechanisms of VP24 functions in the virus life cycle are still poorly understood.9 Some of the protein functions are similar in Ebola and Marburg but interestingly, significant differences exist in other functions. For example, Ebola VP24 (eVP24) is immunosuppressive,6,7,10 whereas Marburg VP24 (mVP24) is not, suggesting that mVP24 may have different roles in the viral life cycle compared to eVP24. A recent study by Edwards et al.11 revealed a novel functional role of mVP24: the activation of the cytoprotective antioxidant response (AR) pathway. Activation of gene expression of AR elements (ARE) occurs through nuclear accumulation of Nrf2. The AR process is suppressed when Nrf2 binds with the human Kelch-like ECH-associated protein 1 (Keap1)12,13 and undergoes degradation in the cytoplasm outside the nucleus via a ubiquitin proteasome pathway.14−17 It was found that Keap1 also interacts strongly with mVP24. With Keap1 engaged with mVP24, Nrf2 is able to accumulate in the nucleus which results in enhanced expression of the AR elements. Therefore, the Marburg virus seems to have evolved © 2018 American Chemical Society

to activate and utilize host cytoprotective gene expression to optimize its replication.11 In contrast, eVP24 does not interact with Keap1 and does not affect the AR pathway. The Keap1− Nrf2 interaction is also found to control Nrf2 ARE activity in both premalignant and malignant cells.18 Other viruses, including hepatitis and influenza are known to activate ARE by triggering oxidative stress or nuclear accumulation of Nrf2 via other signaling pathways.19−25 However, the direct engagement of a viral protein with Keap1 to facilitate the Nrf2 AR seems to be unique to the Marburg virus. Despite the importance of the Keap1−mVP24 complex in the virus life cycle,11 a molecular level detail understanding of the structural basis and mechanism of the Keap1−mVP24 complex formation is still lacking. In general, Keap1 binding motifs on Keap1 partners such as Nrf2 variants contain the sequence DEETGE (also known as the ETGE motif).26 In the mVP24−Keap1 complex, the binding site residues on mVP24 and the binding regions for Keap1 protein are known.11,27 Specifically, residues 205−212 in the linker region of the mVP24 K-loop containing the sequence DIEPCCGE are found to be involved in forming the complex with the binding region of Keap1 from residues 335− 342 and 375−393.27 This Keap1 binding sequence on mVP24 has a pair of Cys residues, DXXTGE, which is lacking in the putative Keap1 high-affinity sequence of Nrf2.11 Therefore, the role of the Cys residues in the Keap1-binding motif is unique to mVP24. Furthermore, it has been observed that mutating the Cys residues affects the binding affinity of the complex in Received: September 14, 2018 Accepted: December 11, 2018 Published: December 28, 2018 18639

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Figure 1. (a) Structure of mVP24 (PDB ID: 4OR8) monomer. The Keap1-binding region of mVP24 (residues 205−212) is highlighted with a circle. (b) Structural alignment of mVP24 (orange) and eVP24 (cyan). eVP24 does not have the protruding K-loop region of mVP24. (c) Structure of human Keap1. Each blade of the Keap1 circular propeller structure is colored differently. The N- and C-terminals of the structure are indicated.

Figure 2. Complex of mVP24-wt (orange) and Keap1 (gray) (a) at 0 ns and (b) at 150 ns. The residues at the mVP24−Keap1 interface are highlighted (cyan: Keap1, purple: mVP24). (c) Residues involved in the interprotein salt-bridges in the mVP24−Keap1 complex. (d) Time evolution of the salt-bridge distance for the salt-bridges formed between mVP24 and Keap1.

mVP2427 but it is also found to affect the protein conformation in other systems.28 In addition, important residues involved in the mVP24− Keap1 complex have been identified,27,29 but the residue pairs involved in the complex formation are not known. Identification of specific amino acid residues that form the interface between a protein and its binding partner is important for understanding the structural features responsible for protein recognition and binding affinity and in exploring the interfaces for drug targets.30,31 In this paper, we performed molecular dynamics (MD) simulations of mVP24 interacting with human Keap1 and investigated the role of various residues in both mVP24 and Keap1. We also investigated Keap1 interactions with eVP24 and found that Keap1 does not bind with eVP24, in agreement with previously reported experimental results.11 Together, we identified the binding site residue pairs between the complexes and investigated why the Cys residues are important in the Keap1 binding motif in mVP24.

outward, but it is missing in eVP24. Figure 1c displays the crystal structure of Keap1, which consists of an N-terminal (broad complex, BTB) domain, a central linker domain, and a C-terminal region. The structure of human Keap1 looks like a circular propeller formed by six blade sections, with each blade composed of four antiparallel beta-sheets.32 2.1. mVP24−Keap1 Complex from Molecular Docking and Simulations. The protein structures used for the molecular docking contained residues 18−241 of mVP24 and 329−609 of Keap1. As the mVP24 K-loop residues 205−212 are known to be involved in the formation of the mVP24− Keap1 complex,11 we used these residues as the known binding site residues for docking. Also, we used the information that the CTD region of Keap1 is the most important region for binding with mVP24.33 The fourth complex generated by ZDOCK consisted of the proper orientation of the K-loop and the contacts in the complex, and we selected this complex for further relaxation with MD. Figure 2a displays the structure of the selected docking complex, which shows significant interprotein interactions. The interactions in the selected complex were further optimized and explored with a 150 ns allatom MD simulation of the complex. Figure 2b shows the structure of the mVP24−Keap1 complex at the end of the 150 ns simulation. The mVP24−Keap1 complex was stable during the entire 150 ns MD simulation (Movie S1). We also ran two

2. RESULTS The mVP24 and Keap1 structures used for protein−protein docking are shown in Figure 1. Figure 1a shows the monomer of mVP24, and Figure 1b highlights the differences between the mVP24 and eVP24 structures. The Keap1-binding K-loop region of mVP24 consisting of residues 205−212 protrudes 18640

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Figure 3. Interprotein hydrogen bonds in the VP24−Keap1 complex for the last 100 ns of simulation; (a) histogram of the number of hydrogen bonds between wt mVP24 and Keap1 (maroon) and the number of hydrogen bonds between the mutant mVP24−C209A/C210A and Keap1 (turquoise). (b) Interaction matrix for the total percentage occupancy of hydrogen bonds between the interprotein pairs of residues in the wt mVP24−Keap1 and (c) for mut-mVP24−Keap1.

experimental studies11 which highlight that the K-loop region in mVP24 is responsible for the mVP24−Keap1 interaction. Apart from these residues, K84, S216, and E217 of mVP24 were also found to be important in stabilizing the complex. Analyzing the residues in human Keap1, we found the major contributing residues to be R415, R380, D389, N387, N382, N414, S602, S555, Y334, and R336. This is consistent with the experimental results11,29 of the importance of Keap1-residues Y334, R380, N382, and R415 in binding with mVP24. For the mVP24-mut, the number of hydrogen bonds was reduced significantly compared to the wild-type. The E207− R415 occupancy was significantly reduced, and the other important residue pairs D205−R380, K84−D389, S216− N382, and Q217−N387 were not present in the mutated complex. This made the mut-mVP24−Keap1 complex less stable compared to the wild-type. This explains why the Cys residues C209/C210 in the mVP24-wt are vital in the formation of the bonds with Keap1 and hence makes the mVP24-wt complex stable. Further roles of the Cys residues will be discussed later. 2.2. Ebola VP24 Interactions with Keap1. Despite the structural similarities between eVP24 and mVP24, they have significantly different binding preferences for Keap1. To compare and quantify the VP24−Keap1 interactions in Ebola and Marburg, we performed 150 ns MD simulation of the eVP24−Keap1 system. The minimized structure of the eVP24−Keap1 complex obtained from docking is shown in Figure 4a. The residues involved in hydrogen bonding and ionic interactions (within 3.5 Å of each other) that keep the complex intact during the MD equilibration process are highlighted. However, during the MD production run, the interprotein interactions start to weaken after 50 ns. By 150 ns, the proteins separate and the complex dissociates (Figure 4a), with only a few interprotein interactions remaining (Movie S2). This shows that the interprotein interactions in the eVP24−Keap1 complex are not able to stabilize the complex. To confirm this, we simulated one additional independent MD run for the eVP24−Keap1, which also resulted in dissociation of the complex. These results agree with the experimental findings,11 which suggested that eVP24 does not bind with Keap1. Comparison of amino acid sequences (Figure 4b) in the Kloop region shows that mVP24 contains two cysteine residues that are not present in four different Ebola virus’ eVP24. Figure 4b shows the sequence alignment using the TCOFFEE

additional independent simulations of 100 ns each, and both runs showed a stable complex. To elucidate the atomic level details of the mVP24−Keap1 interactions in the complex, we investigated the interprotein interactions. As shown in Figure 2c, the mVP24−Keap1 complex features several salt-bridge and hydrogen bond interactions that stabilize the complex. The important saltbridges between mVP24−Keap1 include E207−R415, D205− R380, K84−D389, and E207−R380. Figure 2d shows that these salt-bridges became stronger during the MD simulation. Interestingly, one of the major salt-bridge forming residues, R415 was not in the region specified by the experimental study as the Keap1 binding site,27 which does include some residues that we found important (Figure 3) in the binding region (Y334, R380, N382, N387, and D389). We found that significant hydrogen bonds occurred with the following mVP24−Keap1 residue pairs: S216−N382, I206− N414, C209−S555, and C210−S602. Information about the interprotein hydrogen bonds is displayed in Figure 3. Hydrogen bonds were calculated with a distance cut-off of 3.5 Å and an angle cut-off of 30° using the last 100 ns of the MD simulation. As shown in Figure 3a, a significant number of interprotein hydrogen bonds were formed between the interfacial residues in the mVP24−Keap1 complex. The percentage represents the percent of the last 100 ns of the MD simulation, which had the precise number of interprotein hydrogen bonds. The complex with the wt (wild-type) mVP24 had both a larger most-likely number of hydrogen bonds compared to the mutated mVP24 and a larger maximum number of hydrogen bonds. Figure 3b,c shows the percentage occupancy of the hydrogen bonds and salt-bridges between various residue pairs of mVP24-wt with Keap1 and mVP24-mut with Keap1 during the final 100 ns of the MD simulation. For the calculation of the interaction matrix, the percentage occupancies for all residue pairs were summed. Total occupancies less than 20% are not shown. As shown in Figure 3b, the major contributing residue pairs for hydrogen bonding in the mVP24-wt−Keap1 complex were E207−R415, D205− R380, E207−R380, K84−D389, S216−N382, I206−N414, C210−S602, and C209−S555. The mVP24-wt amino acids involved in the formation of hydrogen bonds were mostly Kloop residues (205−212). Two additional runs for the mVP24−Keap1 complex also resulted in the same major contributing hydrogen bond pairs. This finding agrees with the 18641

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mVP24 significantly reduce the number of hydrogen bonds between the mutant mVP24 and Keap1, making the mutant VP24−Keap1 complex less stable. We also found that the decrease in mVP24−Keap1 H-bonding upon the cysteine to alanine substitution in mVP24 was accompanied by an increase in internal H-bonds in mVP24-mut. Figure 5 displays the loop conformation at the end of 150 ns for both the wt-mVP24− Keap1 complex and the mut-mVP24−Keap1 complex. The mVP24 loop (202−219) undergoes reorientation due to Ala substitution, which causes changes in the hydrogen bonding propensity of several residues. For the wild-type complex, K84 formed H-bonds with Keap1 (D389 and N387), but for the mVP24-mut system, K84 was engaged with internal H-bonds with mVP24 residues V219, E207, and D205 (Figure 5) and not with Keap1 residues. The hydrogen bond occupancy percentage for mVP24-mut K84 with internal mVP24 residues was increased by 66% compared to the wild-type complex. The new internal mVP24-mut H-bond K84−E207 with 68% occupancy might also be responsible for the reduction of the interprotein H-bond between E207−R415, which was a major contributing pair for the wt-mVP24−Keap1 system (Figure 3b). Similar to K84, residues D205, E217, and S218 also increase their internal H-bond occupancy upon alanine substitution (increases of 20, 118, and 10%, respectively). D205 in mVP24-wt makes an interprotein H-bond with Keap1 (Figures 2c and 3b). The repositioning of the K-loop in mVP24-mut results in D205 making more internal H-bonds as shown in Figure 5 and no H-bonds with Keap1 (Figure 3c). This also happens to a lesser extent with E217 and S218. To gain further insight into the mVP24−Keap1 binding at atomic level detail, we examined the interactions of the cysteine residues in the K-loop of wt-mVP24. We observed a πinteraction between C209 of mVP24 and the aromatic residue Y525 of Keap1. This π-interaction is mediated through the sulfur atom of C209 to the aromatic ring of Y525. In Figure 6a, we display the arrangement of the mVP24 C209 and the Keap1 Y525 aromatic ring. This interaction was quite stable throughout the simulation. In general, for the S−H···π interaction to occur, the distance between the center of the aromatic ring and S−H should be under ∼5.5 Å.35,36 Figure 6b shows the plot of the distance between the center of the Y525 aromatic ring and the C209 sulfur atom as a function of time. After 50 ns, the S−H···π interaction stabilizes with an average distance of ∼3.5 Å. Valley et al.35 reported that compared to the X−H···π interactions (where X = C, N, and O), S−H···π is a much stronger interaction.35 Together with the increased internal hydrogen bonding near the K-loop region, the strong S−H···π interaction formed between wt-mVP24 C209 and the

Figure 4. (a) Structure of the eVP24−Keap1 complex at 0 and 150 ns during the simulation. The charged residues at the interface are highlighted in blue (basic) and red (acidic). (b) VP24 sequence alignment of five different species (Marburg and four different Ebola species) in the filovirus family.

webserver34 for VP24 from five different filovirus species: Marburg and four different Ebola species (Zaire, Sudan, Reston, and Tai). The green dotted box highlights the K-loop region that interacts with Keap1. Except for mVP24, the sequence in this region is conserved in all other Filovirus species and the Cys residues are only present in the mVP24 sequence. 2.3. Role of the C209 and C210 Residues in Stabilizing the VP24−Keap1 Complex. A recent study by Johnson et al.27 investigated the role of Cys residues and suggests that mutations C209A/C210A reduce the binding with mVP24, but the exact mechanism and the interaction of C209 and C210 with Keap1 was not known. To elucidate the role of the mVP24-wt residues C209 and C210 in binding to Keap1, we performed additional MD simulations using mutant mVP24−C209A/C210A. We compared the interprotein hydrogen bonds between mutant mVP24 and Keap1 (Figure 3a) and found that the mutations C209A and C210A in

Figure 5. mVP24 K-loop conformation at 150 ns. (a) mVP24-wt (b) mVP24-mut showing the role of K84 in making the intraprotein hydrogen bonds with loop residues. 18642

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work, we investigated the molecular level details of the Marburg VP24 interactions with the human Keap1. Although the eVP24 and mVP24 protein structures are similar, they have quite different binding preferences for Keap1 and this leads to significant physiological differences in the viral life cycle. To understand the molecular mechanisms of Keap1 binding in Marburg and to resolve the differences in Keap1 binding between Marburg and Ebola, we performed MD simulations and investigated the interactions in the complexes. In agreement with the experimental findings,11,27 comparison of the interactions in the mVP24−Keap1 complex and the eVP24−Keap1 complex showed that mVP24 but not eVP24 binds with Keap1. The difference in the Keap1 binding stems from the difference in the Keap1 binding K-loop region, which contains two cysteine residues in mVP24 but not in eVP24 or a mutant mVP24 which was also investigated.27 We specifically identified the amino acid residues in mVP24 and Keap1 that interact to stabilize the bound complex. The important binding pair for the wild-type mVP24 residues was E207−R415, followed by the pairs D205−R380, E207−R380, K84−D389, S216−N382, I206−N414, C210−S602, and C209−S555. The sulfur atom of C209 in mVP24 forms a relatively stable S− H···π interaction with the aromatic ring of Y525, further stabilizing the complex. The interactions with C209 and C210 seem to keep the K-loop in an optimal conformation that promotes the formation of a stable complex. This is shown by both eVP24, which is missing these interactions, and by the mutations C209A and C210A that result in the reorientation of the K-loop because of increased internal hydrogen bonding. Sequence comparison shows that mVP24 is the only filovirus sequence containing the cysteines in the K-loop, suggesting that the ability to activate the host cell’s cytoprotective AR pathway by direct interaction of VP24 with Keap1 appears to be unique to the Marburg virus. These results provide insights into how the Marburg virus is able to utilize the host cells’ AR pathways through Keap1 engagement interactions. Understanding the molecular level details of such interactions is important for developing therapeutics that can target specific proteins to disrupt the virus life cycle.

Figure 6. (a) Arrangement of the wt-mVP24 C209 and the Keap1 Y525 that allows a strong S−H···π interaction. (b) Distance between the center of mass of the Y525 aromatic ring and the C209 sulfur atom. (c) Number of interprotein hydrogen bonds between the wt mVP24 and Keap1 (black curve), mVP24−C209A/C210A−Keap1 (blue curve), and eVP24−Keap1 (red curve). (d) Interprotein interaction energy in three different complexes, mVP24-wt−Keap1 (black), mVP24−C209A/C210A−Keap1 (blue), and eVP24−Keap1 (red).

Keap1 Y525 seems to orient the K-loop in a configuration that enables the formation of a stable complex. Interestingly, the mVP24−C209A/C210A mutant and eVP24, both lacking the “CC” pair in the K-loop region, interact with Keap1 in a similar manner. As shown in Figure 6c, the wt mVP24 and Keap1 form a stable complex with the interface containing the highest number of hydrogen bonds. In contrast, both mVP24−C209A/C210A and eVP24 have a significantly lower number of hydrogen bonds with Keap1. This shows that the VP24 mutations that remove the cysteine residues in the K-loop region diminish the ability of VP24 to bind with Keap1. This is also shown by the interaction energies between the VP24 and Keap1 proteins. Figure 6d shows the total interaction energy (electrostatic plus van der Waals) for the wt-mVP24, the mutant mVP24−C209A/C120A, and the eVP24, respectively, interacting with Keap1. The wt-mVP24− Keap1 complex is the most stable with the lowest total energy throughout the simulation. The mutations C209A and C210A significantly reduced the interfacial interactions and increase the total energy. The eVP24 interactions with Keap1 have the weakest stabilizing interactions (highest energy profile) and result in an unstable complex. Therefore, the presence of cysteine residues in the K-loop region of the wild-type mVP24 seems to be extremely important for the mVP24−Keap1 binding, and thereby inhibiting Keap1−Nrf2 binding and allowing nuclear accumulation of Nrf2 to activate the AR elements.

4. COMPUTATIONAL METHODS Protein structures were obtained from the Protein Data Bank: mVP24 (PDB ID: 4OR837chainA), eVP24 (PDB ID: 3VNF38), and human Keap1 (PDB ID 1U6D32). The PDB crystal structure of mVP24 is a dimer. The mVP24 monomer has 255 residues (residues −13 to 241), and we used chain A from the dimer for our study. The missing residues in the protein structures, including eight residues in the mVP24 Kloop, were added using the Modeller39 software package. The protein structures used for docking contained residues 18−241 for mVP24 and 329−609 for Keap1. Molecular docking was performed with ZDOCK and PatchDock40,41 web-servers. The CHARMM-GUI web-server42 was used to set up input files for all-atom simulations. The system was solvated using the TIP3 water model in a cubic box and was neutralized with counter ions. The final solvated and ionized system for the mVP24− Keap1 complex contained ∼116 000 atoms. Two additional similar systems were set up for the mVP24−Keap1 complex but with C209A and C210A mutations, as well as for the wildtype eVP24−Keap1 complexes. All-atom MD simulations were performed with NAMD 2.1243 using the CHARMM36 force field.44 Each system was minimized for 10 000 steps and equilibrated for 100 ps with backbone and sidechain restraints.

3. CONCLUSIONS As part of the life cycle of the Marburg virus, the host cell’s AR mechanism is enhanced by nuclear accumulation of the human Nrf2 protein. This enhancement occurs because the Marburg virus mVP24 protein interacts with the human Keap1 protein. In the absence of mVP24, Keap1 binds to Nrf2 and prevents Nrf2 from entering the cell’s nucleus. The strong interaction between Keap1 and the Marburg mVP24 does not occur between Keap1 and the Ebola eVP24 and therefore the Ebola virus does not activate the AR pathway of the host cell. In this 18643

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The Ebola virus VP35 protein inhibits activation of interferon regulatory factor 3. J. Virol. 2003, 77, 7945−7956. (9) Bamberg, S.; Kolesnikova, L.; Moller, P.; Klenk, H.-D.; Becker, S. VP24 of Marburg virus influences formation of infectious particles. J. Virol. 2005, 79, 13421−13433. (10) Ding, J.-N.; Zhang, Y.-J.; Zhong, H.; Ao, C.-C.; Li, J.; Han, J.-G. An all-atom molecular dynamics study of the anti-interferon signaling of Ebola virus: interaction mechanisms of EBOV VP24 binding to Karyopherin alpha5. Mol. BioSyst. 2017, 13, 1031−1045. (11) Edwards, M. R.; Johnson, B.; Mire, C. E.; Xu, W.; Shabman, R. S.; Speller, L. N.; Leung, D. W.; Geisbert, T. W.; Amarasinghe, G. K.; Basler, C. F. The Marburg virus VP24 protein interacts with Keap1 to activate the cytoprotective antioxidant response pathway. Cell Rep. 2014, 6, 1017−1025. (12) Magesh, S.; Chen, Y.; Hu, L. Small molecule modulators of Keap1-Nrf2-ARE pathway as potential preventive and therapeutic agents. Med. Res. Rev. 2012, 32, 687−726. (13) McMahon, M.; Itoh, K.; Yamamoto, M.; Hayes, J. D. Keap1dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression. J. Biol. Chem. 2003, 278, 21592− 21600. (14) Cullinan, S. B.; Gordan, J. D.; Jin, J.; Harper, J. W.; Diehl, J. A. The Keap1-BTB protein is an adaptor that bridges Nrf2 to a Cul3based E3 ligase: oxidative stress sensing by a Cul3-Keap1 ligase. Mol. Cell. Biol. 2004, 24, 8477−8486. (15) Furukawa, M.; Xiong, Y. BTB protein Keap1 targets antioxidant transcription factor Nrf2 for ubiquitination by the Cullin 3-Roc1 ligase. Mol. Cell. Biol. 2004, 25, 162−171. (16) Kobayashi, A.; Kang, M.-I.; Okawa, H.; Ohtsuji, M.; Zenke, Y.; Chiba, T.; Igarashi, K.; Yamamoto, M. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol. Cell. Biol. 2004, 24, 7130− 7139. (17) Zhang, D. D.; Lo, S.-C.; Cross, J. V.; Templeton, D. J.; Hannink, M. Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex. Mol. Cell. Biol. 2004, 24, 10941−10953. (18) Kansanen, E.; Kuosmanen, S. M.; Leinonen, H.; Levonen, A.-L. The Keap1-Nrf2 pathway: Mechanisms of activation and dysregulation in cancer. Redox Biol. 2013, 1, 45−49. (19) Cho, H.-Y.; Imani, F.; Miller-DeGraff, L.; Walters, D.; Melendi, G. A.; Yamamoto, M.; Polack, F. P.; Kleeberger, S. R. Antiviral activity of Nrf2 in a murine model of respiratory syncytial virus disease. Am. J. Respir. Crit. Care Med. 2009, 179, 138−150. (20) Burdette, D.; Olivarez, M.; Waris, G. Activation of transcription factor Nrf2 by hepatitis C virus induces the cell-survival pathway. J. Gen. Virol. 2009, 91, 681−690. (21) Schaedler, S.; Krause, J.; Himmelsbach, K.; Carvajal-Yepes, M.; Lieder, F.; Klingel, K.; Nassal, M.; Weiss, T. S.; Werner, S.; Hildt, E. Hepatitis B virus induces expression of antioxidant response elementregulated genes by activation of Nrf2. J. Biol. Chem. 2010, 285, 41074−41086. (22) Ivanov, A. V.; Smirnova, O. A.; Ivanova, O. N.; Masalova, O. V.; Kochetkov, S. N.; Isaguliants, M. G. Hepatitis C virus proteins activate NRF2/ARE pathway by distinct ROS-dependent and independent mechanisms in HUH7 cells. PLoS One 2011, 6, No. e24957. (23) Kesic, M. J.; Simmons, S. O.; Bauer, R.; Jaspers, I. Nrf2 expression modifies influenza A entry and replication in nasal epithelial cells. Free Radical Biol. Med. 2011, 51, 444−453. (24) Lee, J.; Koh, K.; Kim, Y.-E.; Ahn, J.-H.; Kim, S. Upregulation of Nrf2 expression by human cytomegalovirus infection protects host cells from oxidative stress. J. Gen. Virol. 2013, 94, 1658−1668. (25) Ramezani, A.; Nahad, M. P.; Faghihloo, E. The role of Nrf2 transcription factor in viral infection. J. Cell. Biochem. 2018, 119, 6366. (26) Tong, K. I.; Katoh, Y.; Kusunoki, H.; Itoh, K.; Tanaka, T.; Yamamoto, M. Keap1 recruits Neh2 through binding to ETGE and

The particle mesh Ewald method was used for the long-range electrostatic interactions, and the SHAKE algorithm was employed for constraining the covalent bonds. The Nosé− Hoover Langevin method with a piston period of 50 fs and a decay of 25 fs was used to control the pressure. Similarly, Langevin temperature coupling with a friction coefficient of 1 ps−1 was used to control the temperature. A 2 fs time step was used to propagate the simulations. For each system, a 150 ns MD simulation was performed. Image rendering and visualization were done with the Visual MD (VMD)45 software package. The interaction energies between the proteins were calculated with VMD using the namdenergy plugin.43

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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/acsomega.8b02386. Dynamics of the mVP24−Keap1 complex (MPG) Dynamics of the eVP24−Keap1 complex (MPG)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: chapagap@fiu.edu. Phone: 305-348-6266. ORCID

Prem P. Chapagain: 0000-0002-0999-4975 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank nvidia for providing the Titan Xp video cards used for the MD simulations presented in this work. We thank Rudra Pokhrel, Elumalai Pavadai, and Prabin Baral for helpful discussions.

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REFERENCES

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DOI: 10.1021/acsomega.8b02386 ACS Omega 2018, 3, 18639−18645