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Insight into the binding modes of Lassa nucleoprotein complexed with ssRNA by molecular dynamic simulations and free energy calculations a

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Ying Zhang , Hang Chen & Ju-Guang Han a

National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei 230029, People’s Republic of China Accepted author version posted online: 14 May 2014.Published online: 19 Jun 2014.

To cite this article: Ying Zhang, Hang Chen & Ju-Guang Han (2014): Insight into the binding modes of Lassa nucleoprotein complexed with ssRNA by molecular dynamic simulations and free energy calculations, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2014.923785 To link to this article: http://dx.doi.org/10.1080/07391102.2014.923785

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Journal of Biomolecular Structure and Dynamics, 2014 http://dx.doi.org/10.1080/07391102.2014.923785

Insight into the binding modes of Lassa nucleoprotein complexed with ssRNA by molecular dynamic simulations and free energy calculations Ying Zhang, Hang Chen and Ju-Guang Han* National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei 230029, People’s Republic of China Communicated by Ramaswamy H. Sarma

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(Received 6 April 2014; accepted 9 May 2014) Lassa virus (LASV), an arenavirus known to be responsible for a severe hemorrhagic fever, causes thousands of deaths annually and there is no effective vaccine for it so far. The nucleoprotein (NP) of LASV plays an essential role in the replication and transcription of the viral genome. Recent research shows that viral RNA binds in a deep crevice located within the N-terminal domain of NP and suggests a gating mechanism in which NP transforms from a “closed” position to an “open” position to bind RNA. To characterize the molecular mechanisms of how RNA binds to N-terminal domain of NP, two molecular dynamic (MD) simulations of RNA-binding structure and RNA-free structure have been performed. The simulation results show that an important helix α6 interacts with RNA in the “open” conformation, while helix α6 rotates toward the binding crevice and reduces the space of RNA-binding pocket in the “closed” conformation; it appears that helix α6 would clash with RNA while NP is in a “closed” state. In addition, to characterize the role of residues involved in the binding of RNA, the MD simulations of the double-mutant (W164A/F176A) and the single-mutant (G243P) RNA-binding NP complexes have been performed. Our MD simulations and molecular mechanics–generalized born surface area (MM–GBSA) energy calculations exhibit that the three mutant residues increase the binding affinity. Furthermore, we infer that the defect of the replication and transcription of viral genome is possibly due to the change of structural integrity rather than the reduction of RNA-binding affinity. Keywords: Lassa virus; gate mechanism; molecular dynamics simulation; interaction mechanisms; free energy calculation

1. Introduction Lassa virus (LASV), an arenavirus known to be responsible for a severe hemorrhagic fever, causes thousands of deaths annually (Control & Prevention, 2004) and characterizes with muscle aches, sore throat, nausea, and chest and abdominal pain. It is reported that Lassa fever is endemic in West Africa. About 100,000–300,000 case infections and 5000–10,000 deaths occur yearly (Fichet-Calvet & Rogers, 2009; Ogbu, Ajuluchukwu, & Uneke, 2007; Richmond & Baglole, 2003). It has been hypothesized that, in West African population, LASV has been a driver of natural selection. This has been demonstrated by applying tests for selection to genome-wide date (Andersen et al., 2012). Given the high annual incidence rate and the mortality, an effective LASV vaccine is urgently needed. Unfortunately, the pathogenesis of Lassa fever is still not clearly understood (Ogbu et al., 2007). It is found that ribavirin, the antiviral drug, is effective in the treatment of Lassa fever at the beginning of the infection and can reduce the fatality rate (Jahrling et al., 1980; McCormick et al., 1986). But in the later stage, the effectiveness of ribavirin is reduced that comes *Corresponding author. Email: [email protected] © 2014 Taylor & Francis

with potential toxicity and teratogenicity (Fisher-Hoch, Gborie, Parker, & Huggins, 1992; Kochhar, 1990). Hence, ribavirin is not the desirable therapeutic. In addition to the standard treatment with antivirals, some studies have shown higher success rates in vaccine strategies. It is suggested that, if a vaccine can express LASV nucleoprotein (NP) and glycoprotein (Fisher-Hoch, Hutwagner, Brown, & McCormick, 2000; Lukashevich, 2012), it would be an appropriate vaccine candidate to satisfy our goal of triggering the body’s immune system response to defend against the virus. LASV NP is a major component in RNA transcription and a replication process which is synthesized at the early stages of infection. Thus, effective anti-NP immunity during initial stages of the infection will potentially control virus replication. Improving our understanding of NP can promote research and development of vaccines for Lassa fever. LASV has a negative-sense, single-stranded RNA genome containing four genes (Lukashevich, Stelmakh, Golubev, Stchesljenok, & Lemeshko, 1984) that produce four known proteins: a RNA-dependent polymerase (Djavani, Lukashevich, Sanchez, Nichol, & Salvato, 1997;

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Kranzusch et al., 2010), a small matrix Z protein, a NP (Dias et al., 2009; Yuan et al., 2009), and a glycoprotein precursor (Buchmeier, Southern, Parekh, Wooddell, & Oldstone, 1987). The NP associates with the polymerase to encapsulate viral genomic RNAs into ribonucleoprotein (RNP) complexes for RNA replication and transcription (Pinschewer, Perez, & de la Torre, 2003). Qi and colleagues published the X-ray crystallographic structure of Lassa NP at 1.80 Å resolution (Qi et al., 2010) and revealed that a full-length NP (Protein Data Bank (PDB) codes: 3MWP) consist of two distinct domains, the N-terminal domain (residues 7–338) and the C-terminal domain (residues 364–561), which are connected by a flexible linker. The C-terminal domain functions as a 3′-5′exoribonuclease specific for dsRNA (de Silva et al., 2007; Pinschewer et al., 2003; Zuo et al., 2007); the N-terminal domain was proposed as a cap-binding site to provide a deep cavity for snatching m7GTP cap structure of mRNA (Qi et al., 2010). But attempts to co-crystallize NP with an m7GTP analog were unsuccessful (Qi et al., 2010). Also, there is no direct evidence to prove that LASV NP binds cap. Furthermore, mutation of several residues previously proposed to be involved in cap-binding demonstrates that the mutation results in defects in antigenome synthesis rather than defects in mRNA levels (Brunotte et al., 2011). Hence, this deep cavity is likely a binding site for the viral genome, rather than for cap. Hastie and his colleagues obtained crystal structures of the N-terminal domain of LASV NP in complex with ssRNA (PDB codes: 3T5N and 3T5Q) (Hastie et al., 2011). The structure shows that RNA binds in a deep crevice located in the N-terminal domain of NP (NP1–340). According to the X-ray and electron density data, they found that the NP1–340 consists mainly of α-helices and coils. Two important α-helices, α5 (residues 97–109) and the extended 10 residues (residues 112–122) and α6 (residues 130–144), lie across the RNA-binding crevice and prevent the RNA binding. This specific structure suggests a gating mechanism by which NP opens to accept RNA. The replication and transcription of RNA are a viral polymerase-induced conformational transformation process that NP transiently alters its conformation to expose the RNA (Hastie et al., 2011). In the RNA-binding structure, six RNA nucleotides, corresponding to bases 2–7, are resolved (Hastie et al., 2011). Position 3 is always a purine and anchored between R300 and Y308. Bases 2–4 interact with the NP through key residues on the binding pocket. This pocket contains a number of arginine and lysine residues which are responsible for the binding of RNA. RNA bases 4–7 interact with residues of NP through a hydrogen-bond network. In contrast, the RNA-free NP has a more compact structure; the helix α6 is rotated toward the crevice, and the loop connecting α5 and α6 (residues 233–243) is shifted toward the binding pocket

apparently, then a “gate” is formed. When in the closed conformation, the compact structure prevents RNA from binding. In comparison with WT NP, the obtained results propose that mutations of residues that contact RNA bases 2–4 or residues locating on the RNA gate (residues 232– 243) essentially abrogated transcriptional activity (Qi et al., 2010). For example, mutations of W164 and F176 to alanine would prevent transcriptional activity. These residues adjacent to base 2 of RNA forming a hydrophobic pocket (Qi et al., 2010) are a portion of the RNAbinding pocket. Mutation of Gly243 to proline, a residue that lies at the RNA gate region, can also eliminate the expression (Djavani et al., 1997). However, it is unclear that these mutations are important for the structural integrity or RNA binding. In the present study, to facilitate the investigation of the binding mode of RNA to NP and obtain information about the interactions between RNA and the binding pocket, molecular dynamic (MD) simulations have been performed for two WT systems, RNA-binding NP (NP8–339-RNA) and RNA-free NP (NP8–339), as well as two RNA-binding mutation systems: W164A/F176A and G243P complexes. The major objective for the MD simulation is to explore the similarity and distinction for the dynamic behaviors of the two RNA-binding and RNAfree complexes and also to include the mutation systems. For the RNA-binding and RNA-free NP, the analysis indicates that RNA-binding system is more stable. The average distances of some important residues also suggest a closer movement of the binding pocket in the RNA-free NP. Compared to the RNA-binding complex, these movements probably reduce the pocket space of the no bonding NP. The most distinct variation for the RNA-free NP was the movement of the α6 helix. This helix moves toward the pocket and lies in the core, occluding access to RNA. For both mutation systems, the integral structures are changed slightly. This induced the rearrangement of hydrogen-bond networks between RNA and binding pocket when compared to the nonmutation system. To quantitatively describe the influence of the mutant, the MM–GBSA method was used to calculate the absolute binding free energies. MD simulations serve as a link between structure and dynamics. The primary objective for the calculation is to gain an insight into the interaction mechanism between RNA and the binding pocket at atomic level.

2 Materials and methods 2.1. Preparation of ligand–receptor complexes The crystal structures of the NP (PDB id: 3MWP, 3T5N) are obtained from the PDB (Hastie et al., 2011; Qi et al., 2010).

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LASV 3MWP is a trimeric form of RNA-free NP containing the C- and N-terminal domains, whereas 3T5N just contains the N-terminal domain of NP (NP8–339) with six RNA nucleotides (bases 2–7). Previous studies show that the mutations on the C domain (residues 364–561) do not affect the replication and transcription of LASV NP (Hastie et al., 2011; Qi et al., 2010). In addition, our goal is to explore the interactions of ssRNA with the N-terminal domain of NP, so 3T5N is selected as the starting model of our simulation (Li, Li, Chen, & Han, 2013). There is a gap between residues Lys112 and Val163 in 3T5N. 3MWP is used as a template and 3T5N is submitted to I-TASSER tool (Chen, Zhang, Ye, Feng, & Han, 2013; Roy, Kucukural, & Zhang, 2010; Roy, Yang, & Zhang, 2012; Zhang, 2008) to complete the structures. The terminal group of the RNA in 3T5N is a dimethyl phosphate. In order to build a standard nucleic acid system, 5′ terminal phosphate group must be removed (Cieplak, Cornell, Bayly, & Kollman, 1995; Dupradeau et al., 2010). Then the final structure of RNA-binding NP (NP8–339-RNA) was obtained. The RNA-free NP (NP8–339) was obtained by deletion of the RNA from the binding site. Two mutant complexes (W164A/F176A, G243P) were implemented by DeepView (Guex & Peitsch, 1997). All crystal water molecules and two atoms of nickel that were far away from the activity site were removed from the PDB file. 2.2. MDs simulations Four MD simulations, NP8–339-RNA, NP8–339, W164A/ F176A, and G243P complexes were performed using the AMBER11 package (Case et al., 2012). The AMBER ff99SB force field (Duan et al., 2003) was used as the parameter for these systems. All missed H atoms bonded with heavy atoms of proteins and RNA were added using the LEaP module (Case et al., 2012). Each system was solvated in the truncated octahedron periodic box of the TIP3P (Jorgensen, Chandrasekhar, Madura, Impey, & Klein, 1983) water molecules with a cut-off of 10 Å. An appropriate number of Cl− counter-ions was added to neutralize the positive charge of each system. Four steps of minimization were performed for the initial structures. In the first step, the system was minimized by giving restrains of 2.0 kcal/(mol Å2) to all heavy atoms of protein and RNA. In the second step, 2500 cycles of steepest descent method were performed followed by 2500 cycles of conjugate gradient minimization. And then, all restrains were removed. The entire system was optimized by 5000 cycles of steepest-descent method followed by 5000 cycles of conjugate gradient. In the third step, the system gradually heated from 0 to 300 K over 25 ps with a 2 fs time step at a constant temperature and equilibrated at a constant pressure of 1 atm for another 25 ps. In the last step, the whole system was minimized again for 50 ps at the NPT ensemble. In MD simulations, the Particle Mesh Ewald (PME) method

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(Darden, York, & Pedersen, 1993) was applied to calculate long-rang electrostatic interactions. Periodic boundary conditions were applied to all dimensions. The SHAKE method was applied to constrain bonds involving hydrogen atoms. The time step for a MD production run was 2 fs and the coordinates were saved at intervals of 2 ps for analysis. All MD trajectories were performed by PMEMD module and analyzed by the PTRAJ module of AMBER11. All figures were done by the Chimera program (Pettersen et al., 2004).

2.3. MM–GBSA calculations The binding free energies were calculated using the molecular mechanics–generalized born surface area (MM–GBSA) method (Feig, Im, & Brooks, 2004; Kollman et al., 2000). For each system, a total number of 1000 snapshots were taken from the last 20 ns MD trajectory with an interval of 20 ps. The water molecules and counter-ions were stripped by the PTRAJ module of AMBER11 during the calculation. The binding free energies (ΔGbind) of the RNA to the protein were calculated by the following equation: DGbind ¼ Gcomplex  ðGreceptor þ Gligand Þ

(1)

where Gcomplex, Greceptor, and Gligand are the binding free energies of the respective complex, receptor, and the ligand in the solution. ΔG consists of the molecular mechanics free energy (ΔEMM), the solvation free energy (ΔGsol ), and the conformational entropy effect to binding (−TΔS) in the gas phase. It can be expressed as: DDGTOT ¼ DEMM þ DGsol  T DS

(2)

The free energy ΔEMM is further divided into electrostatic interactions ΔEele and van der Waals ΔEvdw in the gas phase, respectively: DEMM ¼ DEele þ DEvdw

(3)

The solvation free energy (ΔGsol ) can be divided into two components, the polar (ΔGGB) and non-polar contributions (ΔGSA), respectively: DGsol ¼ DGGB þ DGSA

(4)

The ΔGsol is calculated with the GB module (IGB = 2) of the AMBER11 suite of programs. In our calculations, the dielectric constant is set to 1.0 inside the solute and 80.0 for the solvent. Atomic radii and charges were in accordance with those used in the MD simulations. The non-polar contribution of the solvation free energy (ΔGSA) is calculated as a function, as follows: GSA ¼ c  SASA þ b

(5)

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where SASA was estimated by the MSMS algorithm, with a solvent probe radius of 1.4 Å. The values of empirical constants γ and β were set to .0072 kcal/(mol Å−2) and .0, respectively. The contributions of entropy (TΔS) to binding free energy were calculated by a NMA with the NMODE module in the AMBER11 program. Because of the entropy calculations for large systems being extremely time-consuming, we extracted only 100 snapshots taken at an interval of 20 ps from the final 20 ns of the MD simulation for the entropy contribution.

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3. Results and discussions 3.1. RNA-binding modes 3.1.1. Root mean square fluctuations (RMSFs) of NP8–339-RNA & NP8–339 In order to understand the relative flexibility of different regions of the NP8–339 models, the RMSFs of Cα atoms with respect to time-averaged positions are presented in Figure 1. The RMSF profiles show that, in both structures, the most rigid regions are located in the α- and β-folded regions, whereas the flexible parts are mainly in the regions that are in contact with the RNA. Compared with RNA-free NP system (NP8–339), the RNA-binding NP system (NP8–339-RNA) shows lower fluctuations, indicating that the presence of RNA can stabilize NP. The reduced mobility of the residues is clearly visible, particularly for the residues in the binding site (residues 211–245). It is suggested that the electrostatic interactions and the hydrogen-bond interactions in the interfaces between RNA and NP significantly produce a stable binding pocket in the complex (for details, see hydrogen-bonds analysis).

Figure 1. The RMSFs of the Cα atoms around their average positions vs. residue number of the NP8–339 and NP8–339-RAN structures.

As for the NP8–339, since there is no RNA in the binding pocket, the whole structure shows higher fluctuations. Especially for the gate region (residues 211–245) (Hastie et al., 2011), the flexible loop displays relatively high RMSF values (2.0–3.2 Å) in comparison with that in RNA-binding NP (1.0 Å). Despite the large fluctuation of NP8–339, two structures in the extended part of helix α5 (resides 112–122) and helix α6 as well as the additional residues on either side of this helix (residues 125–159) show distinct high RMSF values (2.10–3.88 Å for the bound system vs. 2.21–5.26 Å for the free system). Besides, residues 310–330 are more flexible in NP8–339 (~2.0 Å) than in the bound system (~.8 Å). In conclusion, by comparison of the RMSF between the binding and free systems, several residues exhibit different fluctuations, especially for helixes α5 and α6. It is observed that these regions play an important role in controlling the binding of RNA. Hence, the regions mentioned above that have quite different RMSF values may affect the structural fluctuations of the protein. 3.1.2. Stability of trajectories from (root-mean-square deviation) RMSD In order to assess the dynamic stability and evaluate the convergence of the dynamical properties of these systems, the RMSD values from the starting structure of Cα atoms vs. simulation time were calculated. As shown in Figure 2(a), the RMSD of NP8–339 increases steadily during the first 135 ns and reaches to be 3.91 Å. It descends to be 2.66 Å in the following 100 ns. In the next 110 ns, the RMSD rises back to 3.1 Å. After 350 ns simulation, the structure tends to be stable and the system tends to equilibrate. During the whole 410 ns simulation, the RMSD of NP8–339 converges to be 3.10 Å. Different from that of RNA-free system, the RMSD of NP8–339-RNA does not continue to rise or drop down obviously during the simulation course as shown in Figure 2(b), but shakes up and down with values around 1.3–2.7 Å sometimes. As simulation continues, the vibration tends to be reduced. Finally, the RMSD of NP8–339-RNA system converges to be 1.76 Å. The above analysis indicates that the conformation of the RNAbinding system does not change so much. Hence, our results show that RNA-free system undergoes larger fluctuations at the nanosecond time scale than the RNAbound complex; Combined with the RMSF profile, the different equilibrium structures of the two systems are due to the motion of the residues in the binding pocket, especially for the α5 and α6 helixes as mentioned above. 3.1.3. RNA-binding state transits to the free state As far as the local structural differences between RNAbinding and free NP are concerned, it is particularly

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LASV

Figure 2. The RMSDof the NP8–339 (a) and of NP8–339-RNA (b) plot for backbone Cα atoms relative to the starting structure as a function of time.

important to explore the binding pocket movement. According to the NP8–339-RNA structure, we know that RNA occupies a hydrophobic pocket that is composed of residues F176, W164, L172, M54, L120, L239, and I241. It is mentioned above that the RMSF values of α5 helix (residues 112–122); the extended α6 helix (residues 125–159), and the gate region (residues 211–245) in RNA-free NP are obviously higher than in the RNAbinding NP. Therefore, we propose that the α5 and α6 helixes and the gate region in the RNA-free structure may have to change conformation to accommodate the unbound state. Therefore, in order to probe the motion of these regions and the space of the binding pocket, three residues, Met143, Trp164, and Gly243 located within the pocket, were selected. The distances between Met143 and Trp164 and between Met143 and Gly243 were calculated to provide parameters to enquire the changes of these regions and the pocket space. The time series plot and the histogram distributions for respective Met143-Trp164 and Met143-Gly243 distances

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are shown in Figure 3(a)–(d). According to results shown in Figure 3, both Met143-Trp164 and Met143-Gly243 distances in the RNA-free structures have shorter distances than in the RNA-binding structures. As for the Met143Trp164 distance, the mean values (SD) for the RNA-free structure and the RNA-binding structure are 14.48 Å (.90 Å) and 17.93 Å (1.09 Å). As far as the distance of Met143-Gly243 is concerned, the mean values (SD) of RNA-free structure and RNA-binding structure are, respectively, 17.05 Å (2.23 Å) and 22.04 Å (1.94 Å); consequently, the distances between Met143-Trp164 residues and Met143-Gly243 residues in NP8–339-RNA and NP8–339 are apparently different. The smaller distances between the residues in NP8–339 indicate that close movements occur in these regions and probably reduce the space of the binding pocket. For NP8–339, we found that the structure is unstable during the first 135 ns. The RMSD slowly increases to be 3.91 Å and the helix α6 (residues 125–159) shows higher flexibility (with RMSF about 4.59 Å). In these processes, helix α6 distinctly shifts into the binding pocket and the distance between Met143 and Trp164 is decreased. Helix α5 that is linked to α6 also displays a movement toward the crevice. Besides, the gate region (residues 211–245) is mobile and disorderly lies across the binding pocket. It illustrates that the movement of these helixes probably makes the pocket smaller in volume. But, this conformation is unstable because it is not a free energy minimum for the RNA-free system. In the following 100 ns, RMSD descends back to be 2.66 Å with the helix α6 rotating away from the crevice slightly. Then, it reaches to be 3.10 Å in the next 110 ns that comes with α6 helix rotating back to binding pocket slightly. In the last 90 ns, the system goes to equilibrium. The whole conformation presents a fully “closed” state with the α5 and α6 extending across the RNA-binding crevice, occluding access to RNA. The RMSD tends to be converged to be 3.10 Å, indicating that this conformation has a relatively low free energy formed by the closed state keeping the structure stable and in equilibrium. During the entire 410 ns MD simulations, the conformation of NP8–339 transforms from “open” state to “closed” state. This transition involves the rotation of α5 and α6, as well as the movement of gate region (Figure 4). It is suspected that the binding pocket is emptied by the removal of RNA. In the aqueous environment, hydrophobic interactions force the hydrophobic amino acids burying themselves in the hydrophobic center which are away from water. Helix α6 presents a simple pendulum motion toward the hydrophobic binding pocket to prompt the protein folding. Therefore, the hydrophobic interactions act as the main driving force of the transformation. These motions are consistent well with the RMSF analysis

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Figure 3. Figure 3. (a) The Met143-Trp164 Cα distances of the NP8–339 and NP8–339-RNA as a function of time (the top horizontal axis in red represents NP8–339 structure and the bottom horizontal axis in black represents NP8–339-RNA structure); (b) histogram distributions of Met143-Trp164 distance; (c) cartoon representation of the Met143-Trp164 Cα distances; (d) the Met143Gly243 Cα distances (the top horizontal axis in red represents NP8–339 structure and the bottom horizontal axis in black represents NP8–339-RNA structure); (e) histogram distributions of Met143-Gly243 distance; and (f) cartoon representation of the Met143-Gly243 Cα distances. (NP8–339-RNA complex is in gold and NP8–339 complex is in cyan).

(Continued ).

that residues 110–159 and 211–245 undergo high fluctuations in the absence of RNA. The equilibrium structure of NP8–339 displays a more compact conformation. The α6 helix is positioned on the entrance of the binding pocket and helix α5 is shifted toward the pocket. The RNA gate region becomes more flexible and disorderly lies across the deep binding core. All of the changes lock the NP in a closed position to prevent RNA from binding.

LASV

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no direct evidence to prove that the defect in antigenome synthesis is because of the decreasing RNA-binding affinity or the change of structure integrity. In order to facilitate the investigation of RNA-binding mechanism, MD simulations have been performed for double-mutant W164A/ F176A and single-mutant G243P complexes. We take the simulation of non-mutation complex (NP8–339-RNA) as the WT which acts as a control group. The binding free energy and decomposition of free energy calculations, along with the hydrogen-bond interactions, were performed and analyzed to explore the similarities and differences for the dynamic behaviors of the mutation systems by comparing with the WT.

Figure 4. Comparison of the RNA-free (cyan) and RNA-binding (gold) structures of the LASV NP N-terminal domain. (a) Comparison the whole structures of N-terminal domain. (b) Display the distinct motion parts and make other parts transparent.

3.2. The effects of mutations in the residues of binding pocket The previous study suggested that the basic crevice in the N-terminal domain of NP was responsible for the binding of RNA. To characterize the role of residues in binding pocket, a series of mutations to the RNA-binding crevice residues were performed (Hastie et al., 2011; Li et al., 2013). The results show that some of the mutated residues that contact RNA bases could abrogate transcriptional activity compared with WT NP complex and some mutations have no effect on the transcription. For example, W164 and F176 contain long hydrophobic side chains that form a hydrophobic pocket adjacent to the RNA bases; it is proposed that this hydrophobic pocket is a portion of the RNA-binding pocket. Besides, G243, a residue that lies at the gate region, mutating to a rigid proline can also eliminate the transcriptional activity. Indeed, there is as yet

3.2.1. Stability of trajectories for WT and mutant complexes In order to compare the dynamical behavior of doubleand single-mutated systems with WT complex, we performed two MD simulations: 95 ns MD simulation of W164A/F176A double-mutant system and 85 ns MD simulation of G243P single-mutant system. In order to get an insight in mutations of the conformational stability of the RNA-binding NP, the RMSD and RMSF values of NP Cα atoms for the two mutated structures, including the control group, were calculated and are shown in Figure 5. The RMSF profiles show that the two mutant systems share a similar variable trend with the WT structure. From Figure 5(a), we can see that the three structures have the same common highly flexible regions; these regions contain residues 110–122, 125–159 and 211–245. For the simulation of the W164A/F176A structure, the RMSD value of the complex (Figure 5(c)) fluctuates around 1.5 Å in the first 40 ns; the RMSD increases obviously to be about 2.5 Å. According to the simulation trajectory, the fluctuations are mainly due to the slight motion of the gate region and the waving of α6 helix. In the following 35 ns, the RMSD is decent to be 1.58 Å; finally, the RMSD of the trajectory is stable with value of 1.58 Å during the simulations from 75 to 95 ns. In contrast, the calculated results show that the G243P system (Figure 5(d)) is more stable; in the initial 45 ns, the structure fluctuates largely and then, the system achieves equilibrium in the following 40 ns. According to the data obtained from the RMSD values of three complexes, we can conclude that the three simulations share similar characteristics of RMSD with the values of 1.75 Å (WT), 1.58 Å (W164A/F176A), and 1.78 Å (G243P), respectively. The calculated results indicate that the RMSD values of three systems are similar to those of the initial structures during the simulation processes. 3.2.2. Binding free energies estimated by MM–GBSA To obtain more detailed information about the interaction mechanism of RNA and the binding pocket for WT,

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Figure 5. The RMSFs of the Cα atoms around their average positions vs. residue number of the WT, W164A/F176A, and G243P structures. (a) RMSD of the WT, (b) W164A/F176A, and (c) G243P. (d) The plot for backbone Cα atoms during the simulation time.

W164A/F176A, and G243P; absolute binding free energies have been calculated for all the complexes using the MM–GBSA method (Equations (1) and (2)) to complement the structural analysis, and the results were based on the trajectories extracted from the last 20 ns, because the structures of the three systems achieve equilibrium in the last 20 ns. So it is reasonable to calculate the binding free energy and free energy decomposition based on the conformations extracted from these simulations. Absolute binding free energies were calculated as the sum of gas-phase energy (ΔEvdw + ΔEele), solvation free energy (ΔGSA + ΔGGB), and entropy contribution (−TΔS). Table 1 shows the binding free energies and the energy components of the three systems. To investigate the contribution of each energy term to the binding affinity, we compare the four individual energy components and entropy (−TΔS) between the WT complex and the singleor double-mutant complexes. For the three complexes, electrostatic interactions in the gas phase (ΔEele) and van der Waals energy (ΔEvdw) provide the major favorable contributions to the RNA binding. The contributions of electrostatic interactions in the gas phase of the three complexes (ΔEele) are, respectively, −1321.45 (WT), −1384.51 (W164A/F176A), and −1316.35 (G243P) kcal/mol; the contributions of van der Waals (ΔEvdw) of the three complexes are −134.43 (WT), −131.21 (W164A/F176A), and −140.88 (G243P) kcal/mol. Nonpolar solvation energies (ΔGSA) also have favorable contributions to the binding affinity and are similar to each other in all of the three cases (−17.32 (WT), −17.32 (W164A/F176A), and −16.51 (G243P) kcal/mol). Conversely, the entropy components (−TΔS) and electrostatic component of enthalpy of solvation (ΔGGB) contribute unfavorable energy to bind the three systems. The values of conformational entropy (−TΔS) for the three systems are very close (38.94 (WT), 41.51 (W164A/F176A), and 41.51 (G243P) kcal/mol); the values of the electrostatic component of enthalpy of solvation (ΔGGB) are 1332.61 (WT), 1377.34 (W164A/F176A), and 1319.85 (G243P) kcal/mol. Through analysis of the net electrostatic contributions (ΔGele = ΔEele + ΔGGB), we found that the favorable electrostatic interactions in the gas phase (ΔEele) are counteracted by the electrostatic component of enthalpy of solvation (ΔGGB). The calculated net electrostatic contributions (ΔGele = ΔEele + ΔGGB) of these three systems are respective 11.16 (WT), −7.17 (W164A/F176A), and 3.50 (G243P) kcal/mol. It is obvious that the van der Waals interactions are of great importance to the total binding energies. Besides, the difference in the binding free energies between the WT and mutant complexes can be attributed to the net electrostatic (ΔEele + ΔGGB) interactions. Compared to the net electrostatic interactions of WT, the values of W164A/F176A and G243P show a significant increase by 18.33 and 7.66 kcal/mol. Finally,

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Table 1. Binding free energy components for the binding of RNA to the WT, W164A/F176A, and G243P using the MM–GBSA Method. ΔEele, electrostatic energy in the gas phase; ΔEvdw, van der Waals energy; ΔGSA, non-polar solvation energy; ΔGGB, polar solvation energy; TΔS, total entropy contribution; ΔGbind = ΔEele + ΔEvdw + ΔGSA + ΔGGB; ΔΔGTOT = ΔGbind −TΔS. WT Component (kcal/mol)

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ΔEele ΔEvdw ΔGSA ΔGGB ΔGbind −TΔS ΔΔGTOT

W164A/F176A

G243P

Mean

SD

Mean

SD

Mean

SD

−1321.45 −134.43 −17.32 1332.61 −140.58 38.94 −101.64

40.18 6.47 .53 38.66 11.23

−1384.51 −131.21 −17.32 1377.34 −155.7 41.51 −114.19

37.31 6.61 .52 33.56 9.31

−1316.35 −140.88 −16.51 1319.85 −153.89 41.51 −114.01

45.17 5.74 .28 38.79 10.86

11.23

the calculated binding free energies with the contribution of the entropy are −101.64 (WT), −114.19 (W164A/ F176A), and −114.01 (G243P) kcal/mol, which suggests that WT has the weakest binding affinity to bind with RNA. The binding affinity of the double-mutation W164A/F176A and single-mutation G243P increases by 12.55 and 12.37 kcal/mol relative to the WT complex. Based on the above analyses, the binding affinity of the three systems are mainly from the contributions of electrostatic interactions in the gas phase (ΔEele) and van der Waals energy (ΔEvdw). Furthermore, it is exhibited that the mutated complexes display relatively stronger binding affinity compared to WT. The net electrostatic interactions (ΔEele + ΔGGB) are responsible for the differences in the binding free energies between WT and mutant complexes. 3.2.3. Decomposition of binding free energy on per residue In order to better characterize the detailed binding modes about the three complexes, free energy decomposition analysis was performed to decompose the total binding free energies into per residue by MM–GBSA method (Gohlke, Kiel, & Case, 2003; Zoete, Meuwly, & Karplus, 2005). It is helpful for locating important residues that contribute to the receptor–ligand interactions. As indicated in Figure 6(a)–(c) and Table 2, the interaction spectra of three complexes are similar to each other. The residues Phe176, Gly177, Leu239, Leu248, Gly249, Lys253, Lys309, Arg323, and Arg329 have strong interactions with the RNA for all the three complexes. The decomposition of binding free energy of these residues agrees well with the results from the conformation analysis mentioned above. All these residues are located in the binding pocket. In order to obtain the detailed information, ΔG values are decomposed into two parts: the net electrostatic interaction (ΔEele + ΔGGB) and the non-polar interaction (ΔEvdw + ΔGSA). As shown in Figure 6(d)–(e), we found that, for the basic amino acid residues (Lys253,

9.31

10.86

Lys309, Arg323, and Arg329), the main driving forces for the binding of RNA are the net electrostatic interactions (ΔEele + ΔGGB). Take Lys253 as an example, the value of ΔEele in WT system is −114.25 kcal/mol, while it is partly counteracted by the unfavorable interaction ΔGGB (105.43 kcal/mol); eventually, the net electrostatic interactions (ΔGGB + ΔEele) turn into the main forces with value of −8.82 kcal/mol. For the three complexes, as shown in Table 2, the net electrostatic interactions (ΔEele + ΔGGB) are the main driving forces for the binding of basic amino acid residues in the binding pocket. The primary causes of this phenomenon are the electrostatic interactions between backbone phosphate groups of RNA and the positively charged side chains of basic amino acid residues. For the hydrophilic and nonpolar amino acid residues (Phe176, Gly177, Leu239, Leu248, and Gly249), the main driving forces for the binding are the sum of van der Waals and non-polar solvation energies (ΔEvdw + ΔGSA). Such as Phe176, the non-polar interactions (ΔEvdw + ΔGSA) equal to −4.84 kcal/mol, while the net electrostatic interactions (ΔEele + ΔGGB) equal to −3.83 kcal/mol. So, for this kind of residues, the main forces of binding energy are the non-polar interaction, such as van der Waals (ΔEvdw) and the nonpolar contribution of the solvation free energy (ΔGSA), while the polar interactions are secondary. 3.2.4. Hydrogen-bonds analyses As seen from Table 3 and Figure 7(a), four strong hydrogen bonds between RNA and NP existed in all three complexes. First, the NH2 group of Arg329 forms a H-bond with the N7 atom of the adenine in the A3 of RNA strand; the occupancies of the H-bond, N7 (A3)…NH2-HH21 (Arg329), are 97.42% (WT), 99.36% (W164A/F176A), and 99.97% (G243P), respectively. Second, the nitrogen atoms NH1 and NH2 of Arg323 form two H-bonds with the O1P atom in the U4 triphosphate groups of RNA strand; the occupancies of the former H-bonds, O1P (U4)…NH1-HH12 (Arg323), are 85.03% (WT),

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Figure 6.

Figure 6. Decomposition of the total binding free energies per residue for the complexes: (a) WT, (b) W164A/F176A, and (c) G243P and decomposition of total binding free energies on a per-residue basis into contributions from the sum of van der Waals energy and non-polar solvation energy (ΔEvdw + ΔGSA), the sum of electrostatic interactions and polar solvation energy (ΔEele + ΔGGB) for some important residues of (d) WT, (e) W164A/F176A, and (f) G243P.

(Continued ).

53.88% (W164A/F176A), and 80.86% (G243P), and the occupancies of the latter ones are 99.97%(WT), 100% (W164A/F176A), and 99.94%(G243P). Lastly, the hydroxyl oxygen atoms of Ser247 form H-bonds with O1P atom in the U6 triphosphate group of RNA strand; the occupancies of the H-bonds, O1P (C5)…OG-HG (Ser247), are 85.03% (WT), 3.88% (W164A/F176A), and 80.86% (G243P), respectively. The high occupancy percentage of H-bonds indicates that the interactions between the three residues and RNA are relatively stronger. It is suggested that these strong H-bonds between RNA and the residues of the binding pocket are crucial for the stability of all the three structures.

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Table 2. Decomposition the total binding free energies on per-residue basis into contributions from van der Waals energy (vdw), electrostatic energy (ele), polar solvation energy (GB), the non-polar solvation energy (SA) of side-chain atoms (S), backbone atoms (B), and the total (T) of complexes. All values are given in kcal/mol. Residue

Svdw

Bvdw

Tvdw

Sele

Bele

Tele

SGB

BGB

TGB

TSA

TGBTOT

WT TRP164 PHE176 LYS309 LYS253 LEU248 LEU239 GLY249 GLY243 GLY177 ASN174 ARG329 ARG323

−.79 −2.55 −.81 .11 −.68 −2.46 0 0 0 −.58 −1.43 .2

−.11 −1.37 −.2 −.05 −.4 −1.14 −1.38 −.14 −1.72 −.35 −.08 −.06

−.89 −3.92 −1.01 .05 −1.09 −3.59 −1.38 −.14 −1.72 −.93 −1.52 .14

−3.02 −2.9 −109.21 −113.66 −6.72 −6.4 0 0 0 5.45 −72.64 −98.34

1.09 −3.2 1.07 −.59 .22 2.46 −9.06 −2.03 −7.95 −10.17 −.05 1.9

−1.93 −6.1 −108.14 −114.25 −6.5 −3.94 −9.06 −2.03 −7.95 −4.72 −72.69 −96.44

3.15 2.19 102.66 104.77 6.09 6.22 0 0 0 −4.64 68.93 89.38

−1.06 .08 −.83 .66 −2.81 −3.06 5.79 2.08 5.61 4.8 −.01 −1.77

2.09 2.27 101.82 105.43 3.28 3.17 5.79 2.08 5.61 .16 68.92 87.61

−.07 −.29 −.2 −.07 −.03 −.51 −.2 0 −.16 −.14 −.2 −.09

−.8 −8.04 −7.53 −8.84 −4.33 −4.87 −4.85 −.09 −4.21 −5.64 −5.48 −8.78

W164A/F176A TRP164 −.19 SER247 .13 PHE176 −.79 LYS309 −.98 LYS253 .17 LEU248 −1.05 LEU239 −2.9 GLY249 0 GLY177 0 ASN215 −2.46 ARG329 −1.14 ARG323 −.26

−.09 −.86 −1.1 −.15 −.05 −.56 −1.37 −.91 −1.53 −.99 −.1 −.05

−.28 −.73 −1.88 −1.13 .12 −1.61 −4.27 −.91 −1.53 −3.45 −1.23 −.31

−.14 −15.07 .4 −106.9 −111.81 −7.92 −6.08 0 0 .41 −70.99 −95.73

−.07 −1.51 −5.75 1.15 −.74 −1.76 2.32 −11 −9.9 −7.28 −.04 2.01

−.21 −16.58 −5.36 −105.75 −112.54 −9.68 −3.77 −11 −9.9 −6.87 −71.02 −93.72

.12 13.71 −.27 100.72 102.8 6.99 5.98 0 0 .05 66.96 87.75

.11 −1.59 2.59 −.9 .75 −1.69 −2.88 6.62 6.21 4.11 −.03 −1.89

.24 12.12 2.33 99.82 103.55 5.3 3.1 6.62 6.21 4.16 66.93 85.87

−.02 −.15 −.17 −.14 −.07 −.09 −.53 −.22 −.18 −.39 −.23 −.09

−.28 −5.33 −5.08 −7.2 −8.95 −6.08 −5.47 −5.51 −5.39 −6.55 −5.55 −8.25

G243P PHE176 LYS309 LYS253 LEU248 LEU239 GLY249 GLY243 GLY177 ASN174 ARG329 ARG323

−1.28 −.2 −.05 −.8 −1.71 −1.38 −.09 −1.67 −.31 −.13 −.06

−3.87 −1.1 −.41 −1.74 −5.02 −1.38 −.2 −1.67 −.89 −1.36 .01

−2.82 −104.78 −112.9 −6.96 −5.94 0 −14.86 0 6.97 −75.18 −101.13

−3.87 1.29 −.42 −1.05 2.44 −9.21 13.87 −9.85 −10.67 −.17 1.79

−6.69 −103.49 −113.32 −8.01 −3.5 −9.21 −.99 −9.85 −3.7 −75.36 −99.34

2.11 97.64 106.99 6.27 5.78 0 14.59 0 −5.71 71.36 92.29

.92 −1 .42 −1.84 −3.2 5.94 −13.22 6.24 5.54 .07 −1.66

3.02 96.64 107.41 4.43 2.58 5.94 1.37 6.24 −.17 71.43 90.63

−.2 −.17 −.1 −.15 −.48 −.21 0 −.18 −.13 −.18 −.1

−7.73 −8.12 −6.42 −5.46 −6.43 −4.87 .18 −5.45 −4.89 −5.48 −8.8

−2.58 −.9 −.37 −.94 −3.31 0 −.12 0 −.59 −1.24 .07

Compared with WT system, the hydrogen bond networks are changed in both single and double-mutation systems. As for W164A/F176A system, three new strong H-bonds are formed (Figure 7(b)). For example, H-bond is formed between hydroxyl oxygen atoms of Thr216 and O1P atom in the C7 triphosphate group of RNA strand; the occupancy of this H-bond, O1P (C7) …OG1-HG1 (Thr216), is 99.84% and the distance of this H-bond is 2.63 Å. Besides, the NE atom of Arg329 forms a H-bond with the O4 atom of the uracil in the U2 of RNA strand; the occupancy of the H-bond, O4 (U2)…NE-HE (Arg59), is 98.99% and the distance is 2.928 Å. And, the occupancy of the H-bond between

Gly298 and C5 in W164A/F176A system (92.71%) increased significantly compared with WT (12.74%). For the G243P system, it is noted that several new H-bonds are formed between RNA and the protein (Figure 7(c)). For instance, the side chain of Arg300 move toward U4 and a new stable H-bond is formed, O4 (U4)…NH2HH21 (Arg300). The occupancy and distance of this H-bond are 97.99% and 2.821 Å. Compared to WT, the G243P has a more stable H-bond between the O atom of Gly298 and N4 atom of C5; the occupancy of the H-bond is increased to more than 60%. The Tyr213 is located in a loop that is between helix α8 and helix α9; during the simulation course, helix α9 slightly rotates

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Table 3.

Hydrogen bonds between the RNA and NP for the WT, W164A/F176A, and G243P complexes in the last 20 ns trajectory. WT

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Donor U2-O4 Ser121-OG A3-N7 Glu117-OE1 A3-O4’ U4-O1P U4-O1P U4-O4 C5-O1P Gly298-O U6-O1P C7-O1P C7-O2P

Acceptor Arg59-NE-HE U2-N3-H3 Arg329-NH2-HH21 A3-N6-H62 Arg300-NH2-HH22 Arg323-NH2-HH22 Arg323-NH1-HH12 Arg300-NH2-HH21 Ser247-OG-HG C5-N4-H41 Ser237-OG-HG Thr216-OG1-HG1 Tyr213-OH-HH

W164A/F176A

G243P

Occupancy (%)

Distance (Å)

Occupancy (%)

Distance (Å)

Occupancy (%)

Distance (Å)

– 93.02 97.42 75.47 37.24 99.97 85.03 – 79.97 12.74 99.52 – –

– 2.963 3.003 3.056 2.952 2.779 2.957 – 2.627 2.983 2.656 – –

98.99 – 99.36 15.64 49.62 100 53.88 36.28 99.62 92.71 99.9 99.84 –

2.928 – 2.908 3.134 2.975 2.803 3.1 2.995 2.654 2.96 2.703 2.63 –

– 28.27 99.97 – – 99.94 80.86 97.99 99.99 62.77 – – 80.66

– 2.99 2.901 – – 2.775 3.041 2.821 2.6 2.925 – – 2.85

toward the binding pocket (Figure 8), which reduces the distance between side chain of residue Tyr213 and N4 of C5 by 2.85 Å; a new stable H-bond is formed and the occupancy is 80.66%. Except the newly formed H-bonds, there are also several H-bonds that have disappeared. In W164A/F176A system, the distance between oxygen atom of Ser121 and N3 of U2 is shortened; the H-bond, OG (Ser121)…N3-H2 (U2), has disappeared. In G243P mutant system, the distances between hydroxyl oxygen atoms of Ser247 and O1P of U6 increase to 4.24 Å compared to the distance of 2.656 Å in WT; the H-bond, O1P (U6)…OG-HG (Ser237), has disappeared. The important H-bonds always exist stably during the whole simulation in all three complexes. It is supposed that the H-bond networks of these residues are crucial to fix the RNA in the binding crevice. The newly formed H-bonds of the mutant systems are also beneficial for RNA binding with NPs. 3.2.5. Structure binding mode analyses As seen from Table 3 and Figure 7, we observe that the three complexes share some common characteristics. For instance, Arg323 and Arg329 show strong electrostatic interactions with RNA in the three systems. These favorable interactions are supported by hydrogen-bond interactions (Table 3 and Figure 7(a)). For residue Arg329, the energy contributions are −5.48 (WT), −5.55 (W164A/ F176), and −5.48 (G243P) kcal/mol, respectively. As shown in Table 2, the main favorable forces for binding are the net electrostatic contributions (ΔGele = ΔEele + ΔGGB) and then the van der Waals interaction. The net electrostatic contributions (ΔGele) are likely due to the interactions between positively charged side-chain atoms of Arg329 and the negatively charged backbone triphosphate groups of RNA, whereas the van der Waals energy

is likely attributed to the hydrogen bonds between Arg329 and RNA. Like residue Arg329, the binding of Arg323 to RNA is mainly due to the net electrostatic contributions (ΔGele = ΔEele + ΔGGB) and the van der Waals interaction. The energy contributions of the three complexes are −8.78 (WT), −8.25 (W164A/F176), and −8.80 (G243P) kcal/mol, respectively. According to the energy decomposition results, the strong hydrogen-bond interactions are formed between Arg323 and RNA. In addition, a polar amino acid, Ser247, plays a significant role in the binding of RNA. The main favorable forces for the binding are the net electrostatic contributions (ΔGele = ΔEele + ΔGGB); the values are −3.46 (WT), −4.46 (W164A/ F176A), and −3.35 (G243P) kcal/mol. For nonpolar amino acid Leu248, the main force that directs the binding is the van der Waals interactions (ΔEvdw); the values are −3.59 (WT), −4.27 (W164A/F176A), and −5.02 (G243P) kcal/mol, respectively. Observing the location of Leu248, we found that the methyl group on side chain of Leu248 is located in the middle of two pyrimidine rings of RNA (U4 and C5); the C–H…π weakens hydrogen bonds between the alkyl of Leu248 and the pyrimidine rings form the van der Waals energy. These residues play an important role in both the WT complex and the two mutant complexes. Hence, the mutant of these residues does not cause negative influences on the interactions of the binding pocket. By comparing W164A/F176A system with WT, the mutant of residues Phe176 to Ala176 directly reduces the binding affinity by 2.92 kcal/mol. The replacement of phenylalanine with alanine results in a loss of a benzene ring. This change leads to a decrease in van der Waals energy (ΔEvdw) by about 2.04 kcal/mol. The reason of ΔEvdw reduction is supposed to be that the replacement of benzene ring leads to the loss of π − π interaction between the benzene ring of Phe176 and pyrimidine ring in U2 of RNA. For another mutant Trp164Ala, the

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Figure 8. Comparison the structures of WT (gray), W164A/ F176A (pink), and G243P (blue) complexes (helix α8 and α9 are marked).

Figure 7. Major H-bonds between RNA and NP. (a) Common H-bonds of the three complexes. (b) New H-bonds formed by W164A/F176A (red) complexes compared to WT (black). (c) New H-bonds formed by G243P (blue) compared to WT (black). Hydrogen bonds are shown in dashed black line with distances. RNA is indicated in a stick representation and the residues are shown in a line representation.

binding affinity reduces by .52 kcal/mol. This can be mainly attributed to the decrease of side-chain electrostatic interactions (Sele). The Sele decreases from −3.02 to −.14 kcal/mol. The above analysis indicates that the direct effects of the two mutant residues display unfavorable attributions to the binding of RNA. However, besides the two mutant residues, some other residues considerably increase the binding affinity; such as Gly177, which connects to the Phe176, the binding affinity increases by 1.95 kcal/mol. It is because the narrowing of the hydrophobic side chain of Phe176 causes the conformation changes of residues 176 and shortens the

distance between Gly177 and RNA. The residues Lys212, Asn215, and Thr216 also obviously increase the binding affinities of W164A/F176A to RNA. For the residue Lys212, the electrostatic energies of the side chain increases by 27.15 kcal/mol while it counteracts by the unfavorable polar solvation energy (ΔGGB); finally, the net electrostatic contributions (ΔGele = ΔEele + ΔGGB) are increased by 2.41 kcal/mol which mainly results in the increase of binding affinity of Lys212. Based upon the above structural and energetic analyses, we can conclude that, for the two mutant residues alone, the direct effects on the binding affinity are unfavorable. But, after mutation, some other residues except for the two mutant residues obviously increase the binding affinity. As shown in Table 2, the binding free energies are −101.64 kcal/mol (WT) and −114.19 kcal/mol (W164A/F176A), respectively, which indicate that the W164A/F176A complex has stronger binding free energy compared to WT. For the G243P system, the flexible Gly243 lies at the gate region of RNA, and it is replaced by a rigid proline with large hydrophobic side chains. The mutant of Gly243 to Pro243 directly reduces the binding affinity by .27 kcal/mol. As shown in Table 2, the decrease of ΔEele is mainly responsible for the binding loss of residue 243. It was observed that four residues, Ser238, Leu239, Asn240, and Ile241, which are around the mutant residue Gly243, show higher binding affinities compared to the WT complex, and the increased values of binding

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affinities are −.9, −1.56, −1.45, and −1.4 kcal/mol, respectively. The van der Waals energy is the main driving force for the increase in the binding free energy. For the Leu239, with the distances between Leu239 and U4 or C5 decreased, and two methyl groups of Leu239 form C–H…π interactions with the pyrimidine rings of U4 and U5. Thus, the van der Waals energy increases by −1.43 kcal/mol. As seen in Table 2, the binding free energy of the G243P system is −114.01 kcal/mol which increased by 12.37 kcal/mol relative to WT. In general, similar to W164A/F176A system, the single-mutant system also does not defect the binding of RNA, but makes the interactions between RNA and NP stronger. 4. Conclusions MD simulations, MM–GBSA binding free energy predictions, and MM–GBSA binding free energy decomposition analysis are used to explore the binding mode and interaction mechanism of the RNA binding to NP. In order to study the binding modes of RNA, NP8–339RAN and NP8–339 systems are compared. The results reveal a distinct transition of NP conformation from “open” to “closed” state. During the simulation course, helix α6 (residues 125–159) as well as the terminal of helix α5 (resides 112–122) shift toward the core of RNA-binding pocket that acts as a switch. Compared to RNA-binding structure, RNA-free system has a less stable conformation. The distances of Met143-Gly243 and Met143-Trp164 also support the conclusions from above. The distances of Met143 and Gly243 are 14 Å (NP8–339) and 17 Å (NP8–339-RAN), while the distances between Met143 and Gly243 are 18 Å (NP8–339) and 23 Å (NP8–339-RAN), respectively. Compared to NP8–339-RAN, the volume of the binding pocket for RNA-free NP is significantly reduced. In conclusion, in the absence of RNA, the N terminal of NP undergoes a transition from “open” state to “closed” state, preventing the RNA from binding. To figure out the interaction mechanism between RNA and NP-binding pocket, we compare the dynamical behavior of WT and two mutant complexes. The results show that the binding affinity of the two mutant systems increased. The mutant residues themselves result in unfavorable contributions to the binding affinity. But the positions of some residues that interact with RNA in the binding pocket have changed; these residues induce the increase of binding affinity. The van der Waals energy (ΔEvdw) and the net electrostatic contributions (ΔGele = ΔEele + ΔGGB) are supposed to be the key driving forces. In addition, new hydrogen-bond networks make a significant contribution in the two mutant complexes. In conclusion, W164A/F176A and G243P mutant systems do not result in the loss of RNA-binding affinity, but rather make the interaction more stronger. It is supposed that the influence of the residues mutant mainly

reflects on the structural integrity, thus affecting the replication and transcription of RNA. Funding This work is supported by the Chinese National Natural Science Fund [grant number 11179035]; Innovation Program of Shanghai Municipal Education Commission [grant number 14YZ164]; Physical electronics disciplines [grant number 12XKJC01]; and 973 fund of Chinese Ministry of Science and Technology [grant number 2010CB934504].

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