Dynamozones are the most obvious sign of the evolution of conformational dynamics in HIV-1 protease.

Proteins are not static but are flexible molecules that can adopt many different conformations. The HIV-1 protease is an important target for the development of therapies to treat AIDS, due to its critical role in the viral life cycle. We investigated several dynamics studies on the HIV-1 protease families to illustrate the significance of examining the dynamic behaviors and molecular motions for an entire understanding of their dynamics-structure-function relationships. Using computer simulations and principal component analysis approaches, the dynamics data obtained revealed that: (i) The flap regions are the most obvious sign of the evolution of conformational dynamics in HIV-1 protease; (ii) There are dynamic structural regions in some proteins that contribute to the biological function and allostery of proteins via appropriate flexibility. These regions are a clear sign of the evolution of conformational dynamics of proteins, which we call dynamozones. The flap regions are one of the most important dynamozones members that are critical for HIV-1 protease function. Due to the existence of other members of dynamozones in different proteins, we propose to consider dynamozones as a footprint of the evolution of the conformational dynamics of proteins.

Molecular insights into the behavior of the allosteric and ATP-competitive inhibitors in interaction with AKT1 protein: A molecular dynamics study.

AKT1 is a family of serine/threonine kinases that play a key role in regulating cell growth, proliferation, metabolism, and survival. Two significant classes of AKT1 inhibitors (allosteric and ATP-competitive) are used in clinical development, and both of them could be effective in specific conditions. In this study, we investigated the effect of several different inhibitors on two conformations of the AKT1 by computational approach. We studied the effects of four inhibitors, including MK-2206, Miransertib, Herbacetin, and Shogaol, on the inactive conformation of AKT1 protein and the effects of four inhibitors, Capivasertib, AT7867, Quercetin, and Oridonin molecules on the active conformation of AKT1 protein. The results of simulations showed that each inhibitor creates a stable complex with AKT1 protein, although AKT1/Shogaol and AKT1/AT7867 complexes showed less stability than other complexes. Based on RMSF calculations, the fluctuation of residues in the mentioned complexes is higher than in other complexes. As compared to other complexes in either of its two conformations, MK-2206 has a stronger binding free energy affinity in the inactive conformation, -203.446 kJ/mol. MM-PBSA calculations showed that the van der Waals interactions contribute more than the electrostatic interactions to the binding energy of inhibitors to AKT1 protein.

Investigation of the inhibitory behavior of XFE and mitoxantrone molecules in interaction with AKT1 protein: a molecular dynamics simulation study.

The PI3K/Akt/mTOR pathway is one of the important pathways in many cancers. Akt is a serine-threonine kinase protein identified as a drug target for cancer treatment. Therefore, anticancer drugs are essential therapeutic targets for this pathway. In the current study, the inhibitory effect of two anticancer molecules, XFE and mitoxantrone, on AKT1 protein that can impact the activity of the AKT1 protein was investigated by using molecular docking and molecular dynamics (MD) simulations. The molecular docking results presented a relatively higher binding affinity of the mitoxantrone molecule in interaction with AKT1 than the XFE molecule. These results were validated by the MM/PBSA technique that was performed on obtained trajectories of 25 ns MD simulations. The mitoxantrone molecule has an intense binding energy of - 880.536 kcal/mol with AKT1 protein, while the XFE molecule shows a binding energy value of - 83.569 kcal/mol. Our findings from molecular dynamics simulations indicated that both molecules have favorite interactions with AKT1 protein. Other analyses, such as RMSF and hydrogen binding on trajectories obtained from MD simulations, indicated that the mitoxantrone molecule could be a relatively potent inhibitor for AKT1. Based on the results of this study and the structure of mitoxantrone, it is expected to be a good candidate for cancer treatment as a (PI3K)/Akt/mTOR inhibitor.

Design and simulation of the liposomal model by using a coarse-grained molecular dynamics approach towards drug delivery goals.

The simulated liposome models provide events in molecular biological science and cellular biology. These models may help to understand the cell membrane mechanisms, biological cell interactions, and drug delivery systems. In addition, the liposomes model may resolve specific issues such as membrane transports, ion channels, drug penetration in the membrane, vesicle formation, membrane fusion, and membrane protein function mechanism. One of the approaches to investigate the lipid membranes and the mechanism of their formation is by molecular dynamics (MD) simulations. In this study, we used the coarse-grained MD simulation approach and designed a liposome model system. To simulate the liposome model, we used phospholipids that are present in the structure of natural cell membranes (1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)). Simulation conditions such as temperature, ions, water, lipid concentration were performed based on experimental conditions. Our results showed a liposome model (ellipse vesicle structure) during the 2100 ns was formed. Moreover, the analysis confirmed that the stretched and ellipse structure is the best structure that could be formed. The eukaryotic and even the bacterial cells have elliptical and flexible structures. Usually, an elliptical structure is more stable than other assembled structures. The results indicated the assembly of the lipids is directed through short-range interactions (electrostatic interactions and, van der Waals interactions). Total energy (Van der Waals and electrostatic interaction energy) confirmed the designed elliptical liposome structure has suitable stability at the end of the simulation process. Our findings confirmed that phospholipids DOPC and DOPE have a good tendency to form bilayer membranes (liposomal structure) based on their geometric shapes and chemical-physical properties. Finally, we expected the simulated liposomal structure as a simple model to be useful in understanding the function and structure of biological cell membranes. Furthermore, it is useful to design optimal, suitable, and biocompatible liposomes as potential drug carriers.

Thermostability of Ctenophore and Coelenterate Ca2+-Regulated Apo-photoproteins: A Comparative Study.

The stabilities of Ca2+-regulated ctenophore and coelenterate apo-photoproteins, apo-mnemiopsin (apo-Mne) and apo-aequorin (apo-Aeq), respectively, were compared biochemically, biophysically, and structurally. Despite high degrees of structural and functional conservation, drastic variations in stability and structural dynamics were found between the two proteins. Irreversible thermoinactivation experiments were performed upon incubation of apo-photoproteins at representative temperatures. The inactivation rate constants (kinact) at 50 °C were determined to be 0.001 and 0.004 min-1 for apo-Mne and apo-Aeq, respectively. Detailed analysis of the inactivation process suggests that the higher thermostability of apo-Mne is due to the higher activation energy (Ea) and subsequently higher values of ΔH* and ΔG* at a given temperature. According to molecular dynamics simulation studies, the higher hydrogen bond, electrostatic, and van der Waals energies in apo-Mne can validate the relationship between the thermal adaptation of apo-Mne and the energy barrier for the inactivation process. Our results show that favorable residues for protein thermostability such as hydrophobic, charged, and adopted α-helical structure residues are more frequent in the apo-Mne structure. Although the effect of acrylamide on fluorescence quenching suggests that the local flexibility in regions around Trp and Tyr residues of apo-Aeq is higher than that of apo-Mne, which results in it having a better ability to penetrate acrylamide molecules, the root-mean-square fluctuation of helix A in apo-Mne is higher than that in apo-Aeq. It seems that the greater flexibility of apo-Mne in these regions may be considered as a determining factor, affecting the thermal stability of apo-Mne through a balance between structural rigidity and flexibility.

An experimental investigation on the influence of various buffer concentrations, osmolytes and gold nanoparticles on lysozyme: Spectroscopic and calorimetric study.

Considering importance and several industrial applications of lysozyme, including natural antibiotic and preservative, identifier for the diagnosis of diseases, and extraction purposes, its reversibility and stability studies can be very important. In this paper, the role that buffer and osmolytes concentrations play on the thermodynamic stability of lysozyme denaturation process, that is a new simple and inexpensive method, was evaluated by Nano-DSC III, far- and near-UV CD and fluorescence techniques. In thermal denaturation study, RI and ΔG of protein increased from 25.62% to 58.82% and 48.87 to 63.63 kJ mol-1 with the increment of buffer and osmolytes concentrations, respectively. These changes showed a significant increase of 129.59% in RI and 28.16% in ΔG. The effect of buffer and osmolytes concentrations on the secondary and tertiary structures of protein was also investigated. The results indicated that increment of buffer and osmolytes concentrations increase rigidity and thermodynamic stability of protein. Also, structure of protein may be changed by its interaction with GNPs. Hence, interaction of lysozyme with GNPs was studied at the buffer and osmolytes concentrations that gives the maximum RI and ΔG, respectively. The results showed that molten globule-like state was formed by lysozyme in the presence of GNPs.

A molecular dynamics investigation on transporting mechanism of glucose through a cyclic peptide nanotube.

Cyclic peptide nanotubes (CPNTs) are open-ended, hollow, and tubular structures that are made of several cyclic peptide rings. These structures can act as a transmembrane channel and transport ions or small molecules. In this work, we studied the stability of a cyclic peptide nanotube 8 × [(Trp-D-Leu)4-Gln-D-Leu] into a fully hydrated membrane composed of DPPC/POPC/POPS/cholesterol and also the transport mechanism of ß-D-glucose through this nanotube was investigated. Our findings revealed that the CPNT was stable in the lipid bilayer during the simulation time and non-bonded interactions, especially hydrogen bonding have an important role to form a stable CPNT in the membrane. The glucose jumps from a Cα-region into the mid-Cα region and spends more time in this region because of its more desirable interactions with water molecules and the CPNT. In the transport pathway, non-bonded interactions between glucose, water molecules and the CPNT facilitate the transport of the glucose through the CPNT. The collaboration of hydrogen bonds, electrostatic and van der Waals interactions change the pulling force and lead to transport glucose through the CPNT. Potential of mean force (PMF) calculations revealed that the glucose has a minimum value of binding free energy and maximum configurational entropy in MPR regions. These findings can be used to design more CPNTs with different goals such as drug delivery.Communicated by Ramaswamy H. Sarma.

The trypsin inhibitor pro-peptide induces toxic effects in Indianmeal moth, Plodia interpunctella.

The inhibitory potential of an inhibitor peptide based on the pro-region of trypsin zymogen was investigated in Indianmeal moth, P. interpunctella, which is a world-wide insect pest of stored food. Five peptides were designed based on molecular docking simulations. The designed peptide with the best score was selected and synthesized for further screening in vitro and in vivo. The peptide was characterized and its inhibitory effects towards the insect trypsin were evaluated and the kinetic analysis revealed a competitive type of inhibition against the target enzyme. The results showed that the peptide could successfully suppress the pest midgut trypsin, and more interestingly, it did not show considerable inhibitory effects on a mammalian trypsin. We also aimed to assess the effect of dietary insect meal treated with different concentrations of the peptide and observed a significant growth and development retardation in pupa and adult insects fed with the inhibitor peptide. The outcomes of the present study suggest an efficient inhibitor peptide that could specifically bind the P. interpunctella trypsin and inhibit its activity, which would be safe against human being health and environment. Notably, this is the first report on in vivo assessment of the direct effect of a pro-region as the specific inhibitor in development as well as survival of the pest insect. Furthermore, our findings could be a promising for future designed pesticides used in pest management.

Reaction mechanism of the bioluminescent protein mnemiopsin1 revealed by X-ray crystallography and QM/MM simulations.

Bioluminescence of a variety of marine organisms, mostly cnidarians and ctenophores, is carried out by Ca2+-dependent photoproteins. The mechanism of light emission operates via the same reaction in both animal families. Despite numerous studies on the ctenophore photoprotein family, the detailed catalytic mechanism and arrangement of amino acid residues surrounding the chromophore in this family are a mystery. Here, we report the crystal structure of Cd2+-loaded apo-mnemiopsin1, a member of the ctenophore family, at 2.15 Å resolution and used quantum mechanics/molecular mechanics (QM/MM) to investigate its reaction mechanism. The simulations suggested that an Asp-156-Arg-39-Tyr-202 triad creates a hydrogen-bonded network to facilitate the transfer of a proton from the 2-hydroperoxy group of the chromophore coelenterazine to bulk solvent. We identified a water molecule in the coelenteramide-binding cavity that forms a hydrogen bond with the amide nitrogen atom of coelenteramide, which, in turn, is hydrogen-bonded via another water molecule to Tyr-131. This observation supports the hypothesis that the function of the coelenteramide-bound water molecule is to catalyze the 2-hydroperoxycoelenterazine decarboxylation reaction by protonation of a dioxetanone anion, thereby triggering the bioluminescence reaction in the ctenophore photoprotein family.

Exploring single-domain antibody thermostability by molecular dynamics simulation.

Single-domain antibodies also known as nanobodies are recombinant antigen-binding domains that correspond to the heavy-chain variable region of camelid antibodies. Previous experimental studies showed that the nanobodies have stable and active structures at high temperatures. In this study, the thermal stability and dynamics of nanobodies have been studied by employing molecular dynamics simulation at different temperatures. Variations in root mean square deviation, native contacts, and solvent-accessible surface area of the nanobodies during the simulation were calculated to analyze the effect of different temperatures on the overall conformation of the nanobody. Then, the thermostability mechanism of this protein was studied through calculation of dynamic cross-correlation matrix, principal component analyses, native contact analyses, and root mean square fluctuation. Our results manifest that the side chain conformation of some residues in the complementarity-determining region 3 (CDR3) and also the interaction between α-helix region of CDR3 and framework2 play a critical role to stabilize the protein at a high temperature. Communicated by Ramaswamy H. Sarma.

A combination of bioactive and nonbioactive alkyl-peptides form a more stable nanofiber structure for differentiating neural stem cells: a molecular dynamics simulation survey.

Self-assembling alkyl-peptides are important molecules due to their ability to construct nano-level structures such as nanofibers to be utilized as tissue engineering scaffolds. The bioactive epitope of FAQRVPP which acts as neural stem cells (NSCs) outgrowth inducing factor is used in nanofiber structures. Based on previous experimental studies the density and distribution pattern of the epitopes on the surface of the nanofibers plays an important role in the differentiation function efficiency. We decided to survey and compare the stability of two pre-constructed fiber structures in the forms of all-functionalized nanofiber (containing only bioactive alkyl-peptides) and distributed functionalized nanofiber (a combination of nonbioactive and bioactive alkyl-peptides with ratio 2:1). Our findings reveal that the all-functionalized fiber shows an unstable structure and is split into intermediate micelle-like structures to reduce compactness and steric hindrance of functional epitopes whereas the distributed functionalized fiber shows an integrated stable nanofiber with a more amount of beta sheets that are well-organized and oriented around the hydrophobic core. The hydrogen bonds and energy profiles of the structures indicate the role of hydrophobic interactions during the alkyl-chain core formation and the important role of electrostatic interactions and hydrogen bond network in the stability of the final structures. Finally, it seems that the possibility of the presence of intermediate structure is increased in the all-functionalized nanofiber environment, and it can reduce functional efficiency of the scaffolds. These findings can help to design more efficient nanofiber structures with different goals in scaffolds for tissue engineering. Abbreviations MD Molecular Dynamics NSCs Neural Stem Cells PME Particle mesh Ewald RDF Radial Distribution Function RG Radius of gyration RASA Relative Accessible Surface Area RMSD Root Mean Square Deviations SASA Solvent Accessible Surface Area. Communicated by Ramaswamy H. Sarma.

Structural and functional consequences of EF-hand I recovery in mnemiopsin 2.

Mnemiopsin 2 from Mnemiopsis leidyi is a calcium-regulated photoprotein which has luminescence properties in the presence of calcium and coelenterazine. All calcium-regulated photoproteins contain EF-hand loops consisting of 12 individual residues in which the 6th position is occupied by Gly. However, the 6th residue in mneniopsin 2 is Glu rather than Gly. Here, we investigated the structural and functional consequences of substitution of Glu by Gly (E50G variant) using site-directed mutagenesis and spectroscopic procedures. It was revealed that the luminescence activity of the variant was about 17 times greater than that of wild-type (WT) photoprotein. In comparison with WT protein, our variant showed higher optimum temperature and calcium sensitivity as well as slower rate of luminescence decay. Homology modeling and sequence analysis with other known photoproteins showed that EF-hand I loop can affect the luminescence activity of E50G variant. Structural studies using circular dichroism and fluorescence spectroscopy revealed that mutation leads to the reduction in secondary structural content and local structural alterations. Finally, it can be concluded that the activity of E50G variant increases as a result of more flexibility that brought about by Gly essential for adopting the correct conformation for functional activity.

Renin inhibition by soyasaponin I: a potent native anti-hypertensive compound.

One way to control hypertension is inactivation of the Renin- Angiotensin- Aldosterone System (RAAS). Inhibition of renin as a rate-limiting step of this system is an effective way to stop up RAAS. It has been proved that soyasaponin I, an herbal compound obtained from soybeans, has anti-hypertensive effect via renin inhibition, so it has the potential of being an anti-hypertensive drug. Herein, some theoretical approaches such as Docking Simulation, Molecular Dynamics (MD) Simulation and MMPBSA analysis have been used to study how soyasaponin I inhibits renin at the structural level. The results of docking simulation and hydrogen bond pattern show that this ligand is able to bind to the active site of renin and a region near the active site. Results of MD simulation for renin - soyasaponin I complexes confirm that soyasaponin I binds to the active site of renin and has inhibition effect on it via competing with the substrate. Besides, according to MMPBSA analysis, the binding free energy for renin - soyasaponin I complex is -42.61 kcal/mol when it binds to the active site. Comparing to the peptide obtained from angiotensinogen, ΔG = -74.96 kcal/mol, it may inferred that although binding of soyasaponin I to the active site of renin does not have a complete competition with the substrate, it might attenuate the formation of renin - angiotensinogen complex and have partial non-competitive effect. The results of this survey might be helpful to design partial non - competitive renin inhibitors with pharmaceutical capability.

Understanding the inhibitory mechanism of BIT225 drug against p7 viroporin using computational study.

P7 is the only viral channel encoded by the Hepatitis C Virus (HCV) genome. It is a small, highly hydrophobic protein containing 63 amino acids. Structural studies have shown that p7 has two transmembrane (TM) α helices linked by a short dibasic cytoplasmic loop. P7, mostly placed in the endoplasmic reticulum (ER), is a membrane-associated protein. The results obtained from different studies revealed that p7 is a polytopic membrane protein that could oligomerize in membrane bilayer to create ion channels with cation selectivity. In addition, p7 is highly conserved and plays an important role in the assembly and release of mature viral particles. Thus, it can be considered as a potential target for anti-HCV drugs. It has been found that several compounds (amantadine, rimantadine, hexamethylene amiloride (HMA) and long-alkyl-chain iminosugar (IS) derivatives) inhibit p7 channel ability. Another new inhibitor identified as BIT225, a derivative of amiloride, also inhibits the viroporin function of HIV-1 Vpu and HCV p7. In the present study, molecular dynamics simulations were applied to get insights into molecular details of a BIT225 binding site. In addition, the g_mmpbsa approach was employed to calculate the binding free energy and free energy decomposition per residue. MD simulation results in the p7-BIT225 complex revealed that drug binding to hydrophobic pocket can allosterically inhibit ion conduction via the funnel tip by restricting significant intrinsic channel breath at the tip of the funnel. Based on the molecular dynamics simulation (MD) analysis and the energy profiles, the hydrophobic interactions were the main driving force for BIT225 binding.

Molecular cloning, prokaryotic expression, purification, structural studies and functional implications of Heat Shock Protein 70 (Hsp70) from Rutilus frisii kutum.

A novel Hsp70 chaperone from Rutilus frisii kutum was identified, cloned, expressed, purified and its functional characteristics revealed. The 3D structure of Hsp70 from Rutilus kutum was constructed using the crystal structure of E. coli Hsp70 as the template, with 47% sequence identity. The in vitro ATPase activity assay after 60min, ATP hydrolysis of purified recombinant Hsp70 (8µM) was improved by binding to denatured thermally luciferase (3µM) about 2.5-fold compared with that of Hsp70 alone. Based on the results, it was found that the purified Hsp70 chaperone was able to considerably suppress heat-induced aggregation of luciferase by binding to DnaJ co-chaperone (5µM) more than 70% after 10min at 42°C. In addition, Hsp70 DnaJ complex improved the refolding of heat-shocked luciferase nearly 40% after 60min at 25°C. It was concluded that Hsp70 protein from Rutilus frisii kutum has the critical role in preventing heat-induced aggregation of luciferase and refolding of heat-denatured luciferase was strictly dependent on the activity of Hsp70, thus, this protein can potentially be used for improving the functional properties of luciferase in various applications.

QM/MM simulations provide insight into the mechanism of bioluminescence triggering in ctenophore photoproteins.

Photoproteins are responsible for light emission in a variety of marine ctenophores and coelenterates. The mechanism of light emission in both families occurs via the same reaction. However, the arrangement of amino acid residues surrounding the chromophore, and the catalytic mechanism of light emission is unknown for the ctenophore photoproteins. In this study, we used quantum mechanics/molecular mechanics (QM/MM) and site-directed mutagenesis studies to investigate the details of the catalytic mechanism in berovin, a member of the ctenophore family. In the absence of a crystal structure of the berovin-substrate complex, molecular docking was used to determine the binding mode of the protonated (2-hydroperoxy) and deprotonated (2-peroxy anion) forms of the substrate to berovin. A total of 13 mutants predicted to surround the binding site were targeted by site-directed mutagenesis which revealed their relative importance in substrate binding and catalysis. Molecular dynamics simulations and MM-PBSA (Molecular Mechanics Poisson-Boltzmann/surface area) calculations showed that electrostatic and polar solvation energy are +115.65 and -100.42 kcal/mol in the deprotonated form, respectively. QM/MM calculations and pKa analysis revealed the deprotonated form of substrate is unstable due to the generation of a dioxetane intermediate caused by nucleophilic attack of the substrate peroxy anion at its C3 position. This work also revealed that a hydrogen bonding network formed by a D158- R41-Y204 triad could be responsible for shuttling the proton from the 2- hydroperoxy group of the substrate to bulk solvent.

Photoinactivation related dynamics of ctenophore photoproteins: Insights from molecular dynamics simulation under electric-field.

Photoinactivation is a common phenomenon in bioluminescence ctenophore photoproteins (e.g mnemiopsin, berovin and BfosPP) with still unknown mechanism. The activity of coelenterate photoproteins (e.g aequorin), which has high structural similarity with ctenophore photoproteins, is not affected by light. Recently, we have characterized the effects of light on ctenophore photoprotein mnemiopsin, in different conformations, which has demonstrated light induced structural changes, uniquely secondary structures, of both apo and holo mnemiopsin. This paper is further expansion of our previous work, by applying molecular dynamics simulations to investigate photoinactivation related dynamics of berovin at atomistic level, in comparison with aequorin, under the influence of electric component of electromagnetic field. The results have indicated that the intense electric filed could influence structure of both berovin and aequorin but in different manner, whereas moderate electric field only effects on berovin's structure remarkably. In this case, increased helicity of residues E180-M193 and decreased helical contents of L38-D46 and L125-D138 segments are considerable in berovin as well as flexibility elevation of calcium binding loops. These changes cause structural expansion of berovin, especially at N-terminal domain, in direction of electric field. In conclusion, the induced structural changes of mentioned helical parts together with elevated fluctuation of their adjacent segments, N26-D46 and M193-Y206, indicate the influence of light on substrate stabilizing residues, Arg41 and Y204. This condition could presumably leads to inactivation of bioluminescence reaction due to separation of substrate from the cavity of the protein.

Ca2+ Binding and Conformational Switch of the Photoprotein Mnemiopsin.

BACKGROUND: Bioluminescence in Ca2+-binding photoproteins is an intramolecular reaction triggered by the addition of Ca2+. A comparative study has been done on Ca2+-depleted and Ca2+-loaded apo-mnemiopsin to understand the structural transition of the photoprotein by Ca2+ binding. Ca2+ is removed by TCA (trichloroacetic acid) precipitation to obtain Ca2+-depleted apomnemiopsin. METHOD: UV-visible, CD and fluorescence spectroscopic studies demonstrate that the addition of Ca2+ is brought about by the overall structure of apo-mnemiopsin becomes more open in a concentration- dependent manner without significantly influencing the secondary structure and indicate that the Ca2+-depleted form of apo-mnemiopsin, in contrast to most other EF-hand calcium binding proteins, adopt a closed conformation when compared to the Ca2+-loaded form. On the other hand, dynamic quenching and limited proteolysis analysis revealed that Ca2+-loaded apo-mnemiopsin became much more flexible than Ca2+ free apo-mnemiopsin. RESULTS: It seems that increased flexibility of the protein, which occurs due to calcium binding, is a critical factor in oxidative decarboxylation reaction on coelenterazine and consequently light emission.